Doss - PhD Thesis - Feb 2014.pdf

PHYTOPHARMACOLOGICAL AND ANTIDIABETIC ACTIVITY OF ASTERACANTHA LONGIFOLIA (LINN.) NEES. AND

PERGULARIA DAEMIA (FORSSKAL) CHIOV.

Thesis submitted to the Bharathidasan University, Tiruchirappalli

for the award of the degree of

DOCTOR OF PHILOSOPHY IN BOTANY

Submitted by

A. DOSS, M.Sc., M.Phil. (Ref. No. 5543/Ph.D.1/Botany/Full Time/Apr. 2011, Dt 26-11-2012)

Under the guidance of

Dr. S. P. ANAND, M.Sc., M.Phil., Ph.D. Assistant Professor

PG & RESEARCH DEPARTMENT OF BOTANY NATIONAL COLLEGE (AUTONOMOUS)

College with Potential for Excellence Nationally Re-Accreditted with ‘A’ Grade by NAAC

Affiliated to Bharathidasan University TIRUCHIRAPPALLI - 620 001.

TAMIL NADU, INDIA

FEBRUARY 2014

Dr. S. P. ANAND, M.Sc., M.Phil., Ph.D. Assistant Professor PG & Research Department of Botany National College (Autonomous) Tiruchirappalli, Tamil Nadu, India ________________________________________________________________

CERTIFICATE

Certified that this thesis entitled ‘Phytopharmacological and Antidiabetic

Activity of Asteracantha longifolia (Linn.) Nees. and Pergularia daemia

(Forsskal) Chiov.’ is a record of research work done by A. DOSS

(Ref. No.5543/Ph.D.1/Botany/Full Time/April 2011/ dated 26.11.2012) in the PG

and Research Department of Botany, National College (Autonomous),

Tiruchirappalli - 620 001, Tamil Nadu, South India and it has not previously been

formed the basis for the award of any Degree, Diploma, Associateship, Fellowship

or other similar titles.

Place: Tiruchirappalli - 1 (S. P. ANAND) Date:

A. DOSS, M.Sc., M.Phil. Research Scholar PG & Research Department of Botany (Ref. No. 5543/PhD.1/Bot/FT/Apr. 2011 National College (Autonomous) Dated 26 -11-2012 Tiruchirappalli - 620 001 _______________________________________________________________________

DECLARATION

I hereby declare that the thesis entitled ‘Phytopharmacological and

Antidiabetic Activity of Asteracantha longifolia (Linn.) Nees. and Pergularia

daemia (Forsskal) Chiov.’ has been originally carried out by me under the

guidance and supervision of Dr. S. P. Anand, M.Sc., M.Phil., Ph.D., Assistant

Professor, PG and Research Department of Botany, National College

(Autonomous), Tiruchirappalli - 620 001 and this work has not been submitted

elsewhere for any other Degree, Diploma or other similar titles.

Place: Tiruchirappalli-1 A. DOSS Date: Research Scholar

_____________________________________________________________ Phytopharmacological and antidiabetic activity of A.longifolia and P. daemia

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ACKNOWLEDGEMENT

At the outset, I thank and praise God Almighty for his immense grace and

blessings that sustained me to complete this piece of work successfully.

I am extremely grateful and deeply indebted to my guide

Dr. S. P. Anand, M.Sc., M. Phil., Ph.D., Assistant Professor, PG & Research

Department of Botany, National College (Autonomous), Tiruchirappalli, for his

precious and expert guidance by giving valuable suggestions, constant

encouragements, critical comments and fruitful discussions from time to time

throughout the period starting from the inception to the end which led to the

success of this project.

I owe my sincere thanks to Dr. Ragunathan, Secretary, Dr. K. Anbarasu,

Principal and other College Staff Members for their encouragement and the

opportunities given in carrying out my research work from this prestigious

institution.

I wish to express my sincere gratitude to Dr. V. Kannan, M.Sc., M.Phil.,

Ph.D., Head & Associate Professor, PG & Research Department of Botany,

National College (Autonomous), Tiruchirappalli, for providing all facilities to

carry over this work. I am also indebted to Dr. M. N. Abubacker, M.Sc.,

M.Phil., Ph.D., Former Head, PG & Research Department of Botany, National

College (Autonomous), for the encouragement he gave and the interest he showed

in carrying out my research work.

I am thankful to Dr. B. Muthukumar, Associate Professor,

Dr. S. Srinivasan, Associate Professor, Dr. V. Nandagopalan, Associate


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Professor, Dr. E. Natarajan, Assistant Professor, Dr. K. Ramar, Assistant

Professor and Dr. P. Ananthi, Assistant Professor who form the Teaching staff

members of PG & Research Department of Botany, National College

(Autonomous), Tiruchirappalli.

I also extend my thanks to the Non-teaching Staff of Botany department

for their parental encouragement in doing my research work.

I express my sincere and heartfelt gratitude to Dr. H. Muhamed

Mubarack, Secretary, RVS Educational Institutions, Coimbatore. He infused the

necessary blood and flesh into my dissertation and enhanced its practical value for

future scholars.

I express my cheerful thanks to Ms. M. Vijayasanthi, Reserach Scholar,

PG & Research Department of Botany, National College (Autonomous),

Tiruchirappalli, for their valuable suggestions and timely help during my research

work.

I am also thanks to Dr. M. Sekar, Head & Assistant Professor, Department

of Management, RVS College of Arts and Science, Coimbatore and

Dr. M. Shummugasundram, Assistant Professor, Department of Management

Studies, PSN College of Engineering and Technology, Melathediyoor,

Tirunelveli, for their support and help in carrying out this project work.

I acknowledgement with special gratitude to Mr. A. Chinnappar Prabhu,

Teacher, Annai Velankkani School, Tiruchirappalli, Mr. G. Mohan, Teacher,

Salem, Ms. M. Keerthiga, Research Scholar, PG & Research Department of


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Botany, National College, Tiruchirappalli and Mr. G. Velmurugan, Research

Scholar, PG & Research Department of Botany, National College, Tiruchirappalli,

for their timely help and encouragement which enabled me to complete the work

within the time.

I express my genuine thanks to Doctoral Committee Members

Dr. R. Jeyachandran, Head & Associate Professor, Department of Botany,

St. Joseph’s College (Autonomous), Tiruchirappalli and Dr. R. Ravikumar,

Associate Professor, Department of Botany, Jamal Muhamed College

(Autonomous), Tiruchirappalli.

I appreciate the services of Mr. S. Jesudoss for the fine execution of typing

the manuscript at a short notice.

Last but not least I would like to express my love and affection to my

Parents, Sisters, Friends, and all my relatives who inspired and encouraged me

that made me more confident to complete this project successfully.

A. DOSS


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LIST OF ABBREVIATIONS

AI : Activity Index BHA : Butylated Hydroxy Anisole BHT : Butylated Hydroxy Toluene BSA : Bovine Serum Albumin CAT : Catalase CFU : Colony Forming Unit DTNB : 2-Nitrobenzoic acid DMSO : Dimethyl sulfoxide DPPH : 1, 1,-Diphenyl 2, picryl hydrazyl EDTA : Ethylene diamine tetra acetate GST : Glutathione-S-transferase GSH : Reduced Glutathione GPx : Glutathione Peroxidase GC-MS : Gas Chromatography-Mass Spectrum GDM : Gestational Diabetes Mellitus HCL : Hydrochloric acid HPLC : High Performance Liquid Chromatography HDL : High Density Lipoprotein H2O2 : Hydrogen peroxide H2SO4 : Sulfuric acid Hb A1c : Glycosylated Haemoglobin IDDM : Insulin-dependent diabetes mellitus IZ : Inhibition Zone KOH : Potassium Hydroxide KCL : Potassium chloride LD : Lethal Dose LDL : Low Density Lipoprotein LPO : Lipid Peroxidation MRSA : Methicillin Resistant Staphylococcus aureus MIC : Minimum Inhibitory Concentration MBC : Minimum Bactericidal Concentration MDA : Malondialdehyde M : Molar NIDDM : Non-Insulin Dependent Diabetes Mellitus NADPH : Nicotinamide adenine dinucleotide phosphate


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NaOH : Sodium hydroxide NaH2 : Sodium hydrogen N : Normality OD : Optical Density ROS : Reactive Oxygen Species SGPT : Serum Glutamic Pyruvic Transaminase SGOT : Serum Glutamic Oxalocetic Transaminase SOD : Superoxide Dismutase TCA : Trichloroacetic acid VLDL : Very Low Density Lipoprotein V/V : Volume/Volume W/V : Weight/Volume WHO : World Health Organization µg : microgram µM : micromolar mg/ml : microgram/ milliliter g : gram g/l : gram/litre mm : millimeter mmol : millimolar min : minutes


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CONTENTS

Chapter Title Page

I INTRODUCTION 1

II OBJECTIVES 5

III MATERIALS AND METHODS 7

IV ANTIMICROBIAL ACTIVITY 13

V ANTIOXIDANT ACTIVITY 52

VI ANTIDIABETIC ACTIVITY 84

VII PHYTOCHEMICAL SCREENING 173

VIII SUMMARY AND CONCLUSION 188

REFERENCES 193

APPENDICES


1

Chapter-I

INTRODUCTION . .

Plant and plant produce are being used as a source of medicine since long.

According to WHO, more than 80% of the world’s population, are in poor and

from under developed countries so that they mainly depend on traditional plant

based medicines for their primary healthcare needs (WHO, 1993). The efficacy

and safety of herbal medicine have turned the major pharmaceutical population

towards medicinal plant’s research. Owing to the global trend towards improved

‘quality of life’, there is considerable evidence of an increase in demand from

medicinal plants (Kotnis et al., 2004). Use of plants for treating various ailments

of both man and animal is as old practice as man himself. India is richly endowed

with a wide variety of plant shaving medicinal value. These plants are widely used

by all sections of the society whether directly as folk remedies or indirectly as

pharmaceutical preparation of modern medicine (Bhagwati Uniyal, 2003). In

recent times, focus on plant research has increased all over the world and a large

body of evidence has collected to show immense potential of medicinal plants

used in various traditional systems (Ayurveda, Siddha and Unani) (Dahanukar

et al., 2000) and also major source of biodynamic compounds of therapeutic

values (Harsha et al., 2002).

Exploration of the chemical constituents of the plants and pharmacological

screening may provide us the basis for developing the lead for development of

novel agents. Herbs have provided us some of the very important life saving drugs

Chapter - I Introduction


2

used in the armamentarium of modern medicine. Among the estimated 400,000

plant species, only 6% have been studied for biological activity, and about 15%

have been investigated phytochemically (Cragg et al., 1997). This shows a need

for investigation of various chemical constituents, its activity and

phytopharmacological evaluation of herbal drugs.

The Indian herbal medicines industry has been growing for the last several

years. Continuous cultivation and utilization of natural resources in India,

especially, medicinal plants, have been observed and will remain in place due to

the strong influence of cultural tradition for using traditional medicines in Indian

society, and the general trend in medication to go back to nature. Hence, some

natural products have become prospective export commodities from India, in the

forms of finished products. Many countries have imported Indian natural products,

a phenomenon that confirmed the widespread trend of medication using natural

medicines in almost all over the world. For India, this trend opens the opportunity

for Indian medicinal plants to be the leading natural medicines in the world

market. It is estimated that 37% of sales of the world’s pharmaceutical products

utilized or originated from natural raw materials, and thus global pharmaceutical

industry is growing. So development program for Indian medicinal plants is

gaining its momentum and has commercial potentials to fulfill the global needs of

natural medicines.

In India, there are five well-recognized systems of traditional medicine

namely, Ayurveda, Unani, Siddha, Yoga and Naturopathy. In Ayurveda, about

2000 plant species are considered to have medicinal value, while the Chinese



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pharmacopeias list over 5,700 traditional medicines, most of which are of plant

origin. In Indian medicinal plants have made a good contribution to the

development of ancient Indian Materia medica. India with a large reservoir of

medicinal and aromatic plants are a versatile botanical garden of the world. India

has a rich diversity of flora and fauna of about 45,000 species of plants identified;

nearly 15,000 species are flowering plants. About 166 species of crops and 320

species of wild varieties seem to have originated here.

Overall, 119 plants derived prescription drugs are commonly used in

different countries, 74% of which discovered due to chemical isolation of active

compounds used in traditional systems of medicines. For examples cardiac

glycosides Digoxin from Digitalis purpurea, used in traditional medicines,

Reserpine, a tranquilizer and anti-hypertensive agent from Rauwolfia serpentine

known for its value in Ayurveda and Vinblastin from Catharanthus roseus used as

anticancer drug. However, our knowledge of medicinal plants has mostly been

inherited traditionally. Use of plants for curing various ailments are not confined

to the doctors only but is well known to several households as well. There is

growing tendency all over the world to shift from synthetic to natural based

products including medicinal plants. It is also timely now to consider neglected

and little known medicinal plants.

In modern medicine also plants occupy a very significant place as raw

material for some important drugs. Although synthetic drugs as antibiotics brought

about a revolution in controlling different diseases which were thought to be fatal

in past centuries. But these synthetic drugs are out of reach to majority of the



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world population. The tribal and rural people who live in remote places, dense

forests, and small villages or Dhanis mostly depend on traditional healers whom

they know and trust. Judicious, accurate, and timely use of medicinal plants can

even cure fatal or deadly, and incurable diseases that have long defined synthetic

drugs.

A tremendous change has been taking place globally in the last two decades

in favour of the increased use of medicinal plant products in pharmaceutical and

related industries. India, one among the twelve mega biodiversity centres have

more than 1, 44,000 plant species and a recent survey shows that only about

10,000 species of plants are medicinally active. Information regarding the

identification and utilization of medicinal plants are available in plenty. But the

efforts to collect and propagate such information are to be strengthened. In this

direction this research work has been carried out.


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Chapter - II

OBJECTIVES . .

“Plants that are recognized by people to have reliable and effective

medicinal values, are commonly used in treating and preventing specific ailments

and diseases, and play an essential role in health care.” Herbal formulations are

getting more importance in the treatment of diabetes, cancer and hepatic disorder

because of the hazardous adverse effects of the current therapy. Especially

diabetes can be controlled by Allopathic medicine as well as Herbal medicine. In

case of Allopathic medicine, complete cure and tolerance are major problems.

Herbal drugs are considered to be less toxic and free from side-effects compared

to synthetic drugs. Wide arrays of plant-derived active principles representing

numerous chemical compounds have demonstrated activity consistent with their

possible use in the treatment of various ailments/diseases. Recently, a search for

appropriate drugs has focused on plants used in traditional medicine because

natural products may be a better option than currently used drugs. Plants have

always been an exemplary source of drugs and many of the currently available

drugs were derived directly or indirectly from them. With this background, present

research work was framed with the following objectives:

1. To prepare of extracts from Asteracantha longifolia and Pergularia daemia

with methanol and water.

2. To screen the antimicrobial effects of crude extracts on clinically important

microbial pathogens by Disc diffusion method, Minimum Inhibitory

Chapter - II Objectives


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Concentration, Minimum Bactericidal Concentration, Total activity and

Activity Index assays.

3. To investigate the effects of Antioxidant activity on crude extracts by DPPH

and Reducing power assays.

4. To analyze the total phenol and flavonoids of the above mentioned crude

extracts.

5. To compare the effects of Asteracantha longifolia and Pergularia daemia

extracts in normal and diabetic rats.

6. To evaluate the effects of crude extracts on liver and pancreas function by

assessing the respective organ marker enzymes along with histological

study.

7. To detect the presence of the active compounds like alkaloids, saponins,

terpenoids, glycosides and steroids etc. by phytochemical analysis of

alcoholic and aqueous extracts.


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Chapter-III

MATERIALS AND METHODS . .

Based on the objectives, the following plants are selected for the present

research work.

3.1 Asteracantha longifolia (Linn.) Nees.

Synonyms

Hygrophila auriculata (Schumach.) Heine., Hygrophila spinosa T. Anders.

(Plate 3.1 a & b).

Vernacular names

Sanskrit: Iksura; Bengali: Kuliyakhara; Gujarati: Ekharo; Hindi:

Talmakhana; Malyalam: Nirmuli; Marathi: Talimakhana; Tamil: Golmidi and

Urdu: Talmakhana.

Taxonomical information

Class : Equisetopsida

Family : Acanthaceae

Genus : Asteracantha

Species : longifolia

Description

The plant is a sub-shrub, usually growing in marshy places along water

courses. The stem is reddish brown and the shoot has 8 leaves and six thorns at

each node. The leaves occur in whorls, the outer pair of leaves are larger,

Chapter - III Materials & Methods


8

lanceolate, scalerous, margins are minutely dentate, subsessile, and the thorns are

strong, straight or curved. Flowers occur in axillary whorls, bract and bracteoles

are leafy. The calyx is four-lobed, and the lobes are unequal. Corolla, 5 petals

gamopetalous, unequally 2-lipped, middle lobe of the lower lip with a yellow

palate; corolla purple coloured. Stamens - four, in two pairs, filaments unequal;

anthers divergent; ovary two celled; four ovules in each cell. The fruit is in the

form of dehiscent capsules.

3.1.1 Pharmacological Properties

Asteracantha longifolia is an important medicinal herb widely distributed in

Indian subcontinent and is used by local population for different medicinal

purposes. The plant is known to possess antitumor (Ahmed et al., 2001),

hepatoprotective (Anubha singh and Handa, 1995; Shanmugasundram and

Venkatraman, 2005), antibacterial (Doss and Anand, 2013a), free radical

scavenging and lipid peroxidation activities (Doss and Anand, 2013b). The roots,

seeds and ashes of the plant are extensively used in traditional system of medicine

for various ailments like jaundice, hepatic obstruction, rheumatism, inflammation,

pain, urinary infection, edema, gout, malaria, and impotence and also as an

aphrodisiac (Jain, 1991).

3.1.2 Phyochemical constituents

The plant contains a diversity of biologically compounds such as alkaloids,

waxy substances, gum, phytosterols, fatty acids, minerals, polyphenols,

proanthocyanins, mucilage, alkaloids, enzymes, amino acids, carbohydrates,



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hydrocarbons, flavonoids, terpenoids, vitamins, glycosides, etc. (Sarfaraj Hussain

et al., 2010).

3.2 Pergularia daemia (Forsskal) Chiov.

Synonyms

Pergularia extensa (Jacq.) N.E.Br., Daemia extensa (Jacq.) R.Br., Daemia

scandens, Pergularia berbata Klotzsch, Daemia cordifolia, Daemia garipensis,

Asclepias daemia Forssk, Cynanchum extensum Jacquin, Daemia extensa (Plate

3.2 a & b).

Vernacular names

Sanskrit: Uttamarani, Kurutakah, Visanika, Kakajangha; Bengali:

Chagalbati, Ajashringi; Gujarati: Chamardudhi; Hindi: Utaran, Sagovani,

Aakasan, Gadaria Ki bel, Jutak; Malyalam: Veliparatti; Marathi: Utarn; Tamil:

Uttamani, Seendhal kodi, Veliparithi, English: Hariknot plant; Punjab: Karial,

Silai, Trotu and Oriya: Utrali, Uturdi.

Taxonomical information

Class : Magnoliopsida

Family : Asclepiadaceae

Genus : Pergularia

Species : daemia

Description

Pergularia daemia is a perennial twining herb, foul-smelling when bruised;

Stems bears milky juice and covered with longer stiff erect hairs 1 mm; Leaves



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are thin, broadly ovate and heart-shaped 2-12 cm long, covered with soft hairs;

Greenish yellow or dull white, sweet-scented flowers born in auxillary, double

white corona at the base of a stamina column, long-peduncled, umbellate or

corymbose clusters tinged with purple; Fruits paired with follicles 5.8 cm long and

1 cm in diameter, reflexed, beak long, covered with soft spinous outgrowth and

release many seeds with long white hairs when they split open. Seeds are densely

velvety on both sides. The entire plant constitutes the drug and is used as a

medicine.

3.2.1 Pharmacological Properties

Aerial parts of the plant used for snake bite. Entire plant used as an

anthelmintic (Dutta and Ghosh, 1947), antimicrobial (Doss and Anand, 2013a),

antioxidant (Doss and Anand, 2013b), antidiabetic (Doss and Anand, 2014),

antifertility activity (Golam sadik et al., 2001), hepatoprotective (Suresh kumar

and Mishra, 2007), anticancer (Khorombi et al., 2006), antiinflammatory,

analgesic and antipyretic (Hukkeri et al., 2001), central nervous system

depreesant activity (Lokesh, 2009), treat high levels of homocysteine, heart

disease and liver disease, rheummatid arthritis, thyroid problems, tic colours,

vitiglio, gall stones, indigestion (Stuart, 2004), snake bite, malaria, fever, catarrhal

infection, infantile diarrhea, rheumatism, uterine and menstrual disorders and

facilitating parturition, gastric ulcers, expectorant, emetic, anthehelmintic,

leucoderma, amenorrhea, dysmenorrheal, asthma, healing cuts and wounds and

dysentery (Karthishwaran and Mirunalini, 2010).



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3.2.2 Phyochemical constituents

The plant contains a diversity of biologically active compounds such as

flavonoids, alkaloids, terpenoids, tannins, steroids, carbohydrates, cardenolides,

triterpenes (lupeol), saponins and steroidal compounds. The seeds contain

uzarigenin, coroglaucigenin, calactin, calotropin, other cardenolides and a bitter

resin, Pergularin (Karthishwaran and Mirunalini, 2010).

3.3 Plant Materials

Fresh plant parts (Asteracantha longifolia and Pergularia daemia) were

collected randomly from the gardens and villages of Trichy district, Tamil Nadu

from the natural stands. The botanical identity of these plants was confirmed by

Dr. V. Sampath Kumar, Scientist - C, Botanical Survey of India (Southern Circle),

Coimbatore, Tamil Nadu. A voucher specimen has been deposited at the

Department of Botany, National College (Autonomous), Tiruchirapalli-620 001,

Tamil Nadu, India.

3.4 Preparation of extracts

3.4.1 Aqueous extraction

100 grams of dried powder were extracted in distilled water for 6 h at slow

heat. Every 2 h it was filtered through What man no.1filter paper and centrifuged

at 5000 g for 15 min. The supernatant was collected. This procedure was repeated

twice and after 6 h the supernatant was concentrated to make the final volume

one-fifth of the original volume.



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3.4.2 Solvent extraction

100 grams of dried plant powdered samples were extracted with 200 ml of

methanol kept on a rotary shaker for 24 h. Thereafter, it was filtered and

centrifuged at 5000 g for 15 min. The supernatant was collected and the solvent

was evaporated to make the final volume one-fifth of the original volume. It was

stored at 4oC in airtight bottles for further studies, viz. antimicrobial, antioxidant,

antidiabetic and phytochemical analysis.


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Chapter-IV

ANTIMICROBIAL ACTIVITY . .

4.1 INTRODUCTION

Infectious diseases are the leading cause of death world-wide. The

emergence of multi-drug resistant bacterial strains throughout the globe limits the

effectiveness of current drugs and significantly limits treatment, leading to

prolonged infections (Hancock, 2005). The increasing resistance of bacteria to

antibiotics is kindled due to the misuse and over prescription of the drugs. As

resistance to antibiotics spreads, the development of new antimicrobial agents has

to be expedited if the problem is to be contained. Thus there is a need to develop

new antibiotics to delay or prevent the arrival of a post-antibiotic era. Thus the

search for newer sources of antibiotics is a global challenge preoccupying research

institutions, pharmaceutical companies and academia (Latha and Kannabiran,

2004). However, the past record of rapid, widespread and emergence of resistance

to newly introduced antibiotics indicates that even new families of antibiotics are

expected to have a short life (Coates et al., 2002).

The problem posed by the high cost, adulteration and increasing toxic side

effects of these synthetic drugs coupled with their inadequacy in diseases

treatment found more especially in the developing countries should also be

emphasized (Shariff, 2001). Many plants possess antimicrobial activities and are

used for the treatment of different diseases.

Chapter - IV Antimicrobial Activity


14

Medicinal plants have continued to attract attention in the global search for

effective antimicrobial agents that can combat resistant pathogens that have been

rendering many conventional drugs obsolete in the treatment of infections. Many

important drugs used in medicine today are directly or indirectly derived from

plants (Idris et al., 2009). Plant based drugs have been used worldwide in

traditional medicines for treatment of various diseases. The WHO has estimated

that 80% of the populations of developing countries still rely on traditional

medicines, mostly plant drugs, for their primary health care needs. Demand for

medicinal plant is increasingly felt, in both developing and developed countries

due to growing needs of natural products being non-toxic and benefit of side

effects, apart from availability at affordable prices. The medicinal plant sector has

traditionally occupied a pivotal position in the socio cultural, spiritual and

medicinal areas of rural and tribal families (WHO, 2002).

India is a varietal emporium of medicinal plants and is one of the richest

countries in the world in regard to genetic resources of medicinal plants. It

exhibits a wide range in topography and climate, which has a bearing on its

vegetation and floristic composition. Moreover, the agro-climatic conditions are

conducive for introducing and domesticating new exotic plant varieties (Parekh

and Chanda, 2007). In recent years, secondary plant metabolites (phytochemicals),

previously with unknown pharmacological activities, have been extensively

investigated as a source of medicinal agents (Krishnaraju et al., 2005). Thus, it is

anticipated that phytochemicals with adequate antibacterial efficacy will be used

for the treatment of bacterial infections. Since time immemorial, man has used



15

various parts of plants in the treatment and prevention of various ailments (Tanaka

et al., 2007).

4.2 REVIEW OF LITERATURE

Literature is replete with reference of studies relating to the effect of plant

extracts on antimicrobial activities. In general, many focus on determining the

antimicrobial activity of the plant extracts found in folk medicine (Ngwendson et

al., 2003), essential oils (Alma et al., 2003) or isolated compounds such as

alkaloids (Klausmeyer et al., 2004), flavonoids (Sohn et al., 2004), sesquiterpene

lactones (Lin et al., 2003), diterpenes (El-Seedi et al., 2002), triterpenes (Katerere

et al., 2003), or napthoquinones (Machado et al., 2003), among others. Some of

these compounds were isolated or obtained by bio-guided isolation after

previously detecting antimicrobial activity on the part of the plant. A second block

of studies focus on the natural flora of a specific region or country. Examples of

such articles that have been published recently include studies of medicinal plants

from Thailand (Wannissorn et al., 2005), India (Jeevan Ram et al., 2004) and

Qatar (Mahasneh, 2002). The third relevant group of research is made up of

specific studies of the activity of a plant or principle against a concrete

pathological microorganism. These studies have in the past focused on activity

against Candida albicans, Escherichia coli (Voravuthikunchai et al., 2004),

bacteria resistant to known antibiotics such as Staphylococcus auereus (Machado

et al., 2003), as well as activity against multi-drug resistant bacteria such as

Salmonella typhi (Rani and Khullar, 2004). Another criterion is the study of plants

used for alimentary purposes, such as the study of spices to justify their use as



16

antimicrobial agents. While spices are thought to be antimicrobial agents against

human pathogenic bacteria and yeast and when Arora and Kaur (1999) tested

different spices, only garlic and clove were found to exhibit antimicrobial activity.

Indeed, some bacteria which showed resistance to certain antibiotics were

sensitive to extracts of both garlic and clove.

A good number of plant extracts have been tested for their antimicrobial

effects, such as Lantana indica (Verma et al., 1997), Azadirachta indica

(Manoharan et al., 1998), Psidium guajava (Anas et al., 2008), Cassia siamea

(Chandrasekaran and Venkatesalu, 2004), Ricinus communis (Parameswarai and

Tulsi Latha, 2001), Jatropa tanjorensis (Sekaran, 1998), Thuja orientalis (Rakesh

Kumar Jain and Garg, 1997), Curcuma longa (Singh et al., 2002) and Achyranthes

aspera (Bhoomica et al., 2007), Chromolaena odorata, Panicum maximum,

Barleria lupulina (Doss et al., 2011).

The in vitro efficacy of Canavalia ensiformis seed extracts were studied by

Pugalenthi et al. (2010), using filter paper disc method against several microbial

pathogens. In their investigation methanol extract showed significant activity

against all the tested bacterial pathogens, while other extracts showed mild to poor

activity. Anand et al. (2011) investigated the in vitro assessment of various

solvent extracts of Clitoria ternatea leaves against five bacterial organisms (two

gram positive and three gram negative bacterial species) and reported positive

antimicrobial activity. In their work except methanol extract, other extracts

showed no inhibitory activity.



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Antimicrobial potential of various extracts of Lantana indica have been

studied against different bacteria and fungi by Venkataswamy et al. (2010a). The

various extracts were inhibitory to Staphylococcus aureus, Escherichia coli,

Bacillus subtilis, Proteus vulgaris, Streptococcus pyogens, Klebsiella pneumoniae

and the fungi Aspergillus niger and Candida albicans. The antimicrobial activity

may be due to the presence of tannins. The leaves of Acacia nilotica was extracted

with methanol and aqueous extracts and studied for in vitro antimicrobial

property. The methanol extract was found to be most active against all the

bacterial species except Staphylococcus aureus (Venkataswamy et al., 2010b).

The leaves of Rheo discolor has been evaluated for its antimicrobial activity

(Parivuguna et al., 2008). The petroleum ether extract showed significant

antimicrobial activity. The extract was highly effective against bacteria isolated

from tooth tartar of dental patients. The chloroform and methanol extracts of the

leaves of Vitex trifolia exhibit a very good activity against gram negative

organisms (Geetha et al., 2004). The antimicrobial activity of Solanum trilobatum

indicated that all gram positive organisms were sensitive to the extracts but gram

negative bacteria were resistant to even a very low concentration of the extracts

(Doss and Dhanabalan, 2008).

The ethanolic and aqueous extracts of the leaves of Justicia beddomei

exhibit a very good activity against gram positive organisms (Srinivasa et al.,

2006). The antimicrobial activity of Alternanthra sessilis indicated that all gram

positive organisms were sensitive to the extracts but gram negative bacteria were

resistant to even a very high concentration of the extracts (Malayagupta et al.,



18

1992). The antimicrobial efficacy of Euphorbia hirta, Erythrophlem suaveolens

and Thevetia peruviana were tested against beta lactamase producing bacteria

(Escherichia coli, Pseudomonas sp., Klebsiella sp.) and methicillin resistant

bacterial species (Staphylococcus aureus). Methanolic extract showed significant

antibacterial activity against Klebsiella sp. and Escherichia coli, while aqueous

extracts revealed the least antibacterial activity against all the bacteria (Virender

singh et al., 2012).

The various solvent extracts of Mucuna pruriens var. pruriens were tested

against Staphylococcus aureus, Klebsiella pneumoniae, Bacillus subtilis,

Pseudomonas aeruginosa, Salmonella typhi and Escherichia coli using disc

diffusion method. All the solvent extracts revealed various degrees of significant

inhibitory effect against the tested organisms (Murugan and Mohan, 2011). The

antibacterial activity of alcoholic (methanol and ethanol) extracts of stem, berries

and whole plant of Solanum nigrum against Bacillus subtilis, Escherichia coli,

Klebsiella pneumoniae and Pseudomonas aeruginosa were screened. The

methanolic extracts showed highest antibacterial activity than ethanolic extracts

(Parameswari et al., 2012).

The methanol and aqueous extarcts of leaves of six different medicinal

plants, Acalypha indica, Aerva lanata, Phyllanthus amarus, Phyllanthus emblica,

Cassis auriculata and Caesalpinia pulcherrima, were used for the investigation of

antibacterial studies. The methanol extracts of Acalypha indica, Aerva lanata and

Phyllanthus amarus exhibited clear zone of inhibition against the tested

microorganisms (John De Britto et al., 2011). The aerial parts of Peumus boldus,



19

Agathosma betulina, Echinacea angustifolia, Humulus lupulus, Glycyrrhiza

glabra, Mahonia aquifolium, Usnea barbata and Anemopsis californica were

screened for their antibacterial activity against four gram positive and four gram

negative microbial pathogens. Plant extracts showed strong antibacterial action

against Staphylococcus aureus, Staphylococcus aureus (MRSA), Staphylococcus

epidermidis and Streptococcus pyogenes, while negligible to no inhibitory activity

against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa,

Staphylococcus aureus and Salmonella enteritidis was observed (Wendakoon et

al., 2012).

The crude extracts from leaf and stem of Cardiospermum halicacabum in

different solvents, were subjected to phytochemical and antimicrobial screening

against selected microbial pathogens. Among the different solvent extracts,

acetone and chloroform extract of leaf had higher inhibitory action against

Salmonella typhi and Streptococcus subtilis respectively. Acetone extracts of

stem showed maximum inhibitory action against S.typhi and benzene extracts of

stem had moderate inhibitory action against Escherichia coli (Viji and Murugesan,

2010). The ethanol extract of Bauhinia variegata leaves was tested against

clinically important microbial pathogens. Ethanol extracts showed varying degree

of inhibitory potential against all the tested bacterial species. Maximum inhibitory

activity against Salmonella typhi was observed and followed by Vibrio cholera,

Klebsiella pneumoniae, Escherichia coli and Staphylococcus aureus (Gunalan et

al., 2011).



20

The methanol leaf extracts of Acacia nilotica, Sida cordifolia, Tinospora

cordifolia, Withania somnifera and Ziziphus mauritiana were screened their

antibacterial activity against some clinically important microbial pathogens.

Acacia nilotica and Sida cordifolia leaf extract showed highest antibacterial

activity against B. subtilis and Z. mauritiana leaf extracts showed significant

activity against X.a.pv.malvacearum. Tinospora cordifolia recorded significant

antifungal activity against D.turcica. The methanol extract of Sida cordifolia

exhibited significant antifungal activity against F.verticillioides (Mahesh and

Satish, 2008).

The in vitro efficacy of Thuja orientalis leaf extract was studied by Sanjai

Sharma et al. (1990), using filter paper disc method against several human

pathogenic gram positive and gram negative bacteria and phyto pathogenic fungi.

In their investigation acetone, ethanol and methanol extracts showed significant

activity against some bacteria, while other extracts showed mild to poor activity.

The antimicrobial activity of different extracts from the aerial parts, except for the

stems of four Hypericum plants as well as the flower and leaf extracts of

Hypericum calycinum were evaluated by Sakar and Tamer (1990), against eight

microorganisms including Escherichia coli, Staphylococcus aureus and Candida

albicans. The acetone extract of Hypericum calycinum was found to show highest

antimicrobial activity. Studying the anti-inflammatory and antimicrobial activities

of the root, bark and leaves of Azadirachta indica by Manokaran et al. (1998),

concluded that 50% acetone extracts of the root, bark and leaves of Azadirachta

indica showed marked anti-inflammatory activity in carrageenin induced edema in



21

rats and the extracts were more active against gram negative organisms than the

positive organisms.

Raman et al. (1998) investigated the in vitro assessment of various solvent

extracts of Cardiospermum halicabacum leaves against ten bacterial organisms

and reported positive antimicrobial activity. In their work except ethyl acetate

extract, other extracts showed no inhibitory activity. Positive antibacterial activity

was found by Parameshwari and Tulasi Latha, (2001), when administered with

leaf extract of Ricinus communis. They noted maximum effect in the methanolic

extract. The ethanolic extract of leaves of Jatropha tanjorensis was detected to be

antimicrobial against the six organisms tested inclusive of Staphylococcus aureus

and Escherichia coli (Sekaran, 1998). Sesquiterpene lactones from Anamirta

cocculus seeds were found to exhibit antifungal activity. The methanolic extract

inhibited Candida albicans (Santhos et al., 1999). The chloroform and alcohol

extracts of Boerhaavia diffusa plants displayed antibacterial activity against six

test organisms (Hamsaveni et al., 1999). The chloroform extracts showed good

activity against Escherichia coli. Evaluating Pterocarpus santalinus extracts for

their antibacterial activity Sathyanarayana and Srinivas (1998), found more

activity with alcoholic extract of heart wood than sap wood. The essential oil

obtained from rhizomes of Luvunga scandens were found to show antifungal

activity against four test fungi (Garg and Jain, 1999).

The essential oil from seed coats of Thuja orientalis possessed

antimicrobial activity against six bacteria and five fungi (Rakesh Kumar Jain and

Garg, 1997), while essential oil from Anisomeles indica indicated activity against



22

seven organisms of the fourteen tested (Yadava and Deepak, 1998). Chaudry et al.

(1999) proved that turmeric leaf oil was bactericidal to four species of Shigella sp.

Rajakaruna et al. (2002) in their study found the plant extracts of Lantana camara

to be antibacterial against Staphylococcus aureus and Bacillus subtilis, besides

inhibiting the growth of gram negative Pseudomonas aureginosa but not

Escherichia coli. Antimicrobial properties of Calotropis gigantia, Cassia

auriculata and Lantana camara have been recorded by Tailor et al. (1996).

Valsaraj et al. (1997) and Srinivasan et al. (2001) have stated that alkaloids and

their derivatives have activities against Staphylococcus aureus. Kumar et al.

(2007) after the study of twelve medicinal plant extracts against

Propionibacterium acnes and Staphylococcus epidermidis detected successful

activity in the extracts of Hemidesmus indicus, Eclipta alba and Tinospora

cordifolia. Ray and Majumdar (1973) after investigating 105 medicinal plants

species for their antimicrobial activity found that only 30 of them showed

antibacterial activity, while 20 of these exhibited antifungal action as well. They

also reported that the Tamarindus indica exhibited considerable activity against

fungi apart from possession of antibacterial properties. However they concluded

that in general, extracts having considerable antibacterial activity posses less

antifungal action and vice versa. Verma et al. (1997) have attributed the

antimicrobial activity of Lantana species to the occurrence of triterpinoids and a

rare antibacterial flavone glycoside. Anas et al. (2008), analyzing the in vitro

antibacterial activity of Psidium guajava on clinical isolates of multidrug resistant

Staphylococcus aureus have found it to be differently active.



23

Khan et al. (2001) showed that the anthroquinone and flavonoid glycosides

isolated from Cassia alata inhibited many types of bacterial strains of Bacillus

subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae

and Escherichia coli. Solanum trilobatum one of the potential medicinal plants has

the presence of tannins, saponins, flavonoids and phenolic compounds (Amir and

Kumar, 2004) and exhibits an effective antibacterial activity against gram positive

and gram negative bacteria (Swapna Latha and Kannapiran, 2006). Purusothaman

et al. (1987) in his studies on Solanum trilobatum has reported that glyco-

alkaloidal mixture from the plant containing beta solamarine as the major

constituent has shown antibacterial, antifungal, and antitumour activities.

Achyranthes aspera leaves are reported to possess antimicrobial character due to

the presence of a chemical substance called betanine. This active substance when

treated to fabrics renders the fabric resistant to microbial attacks (Thilagavathi and

Kannaian, 2008).

Antimicrobial potential of various extracts of Cassia auriculata have been

studied against different bacteria and fungi by Surana et al. (2007). The various

extracts were inhibitory to Staphylococcus aureus, Escherichia coli, Bacillus

subtilis, Salmonella typhi and the fungi Aspergillus niger and Candida albicans.

The antimicrobial activity may be due to the presence of steroids. The

antimicrobial activity of the leaves of Moringa oleifera was studied by Edwin et

al. (2006). The aqueous extract of Moringa leaves exhibited very good activity

against all the microbes compared to the other extracts of Hibiscus rosasinensis.

The bark of Acacia nilotica has been evaluated for its antimicrobial activity



24

(Phadnis et al., 2006). The acetone extract showed very good antimicrobial

activity. The extract was highly effective against bacteria isolated from tooth tartar

of dental patients. The ethanolic and aqueous extracts of the leaves of Justicia

beddomei exhibit a very good activity against gram positive organisms (Srinivasa

et al., 2006). The antimicrobial activity of Alternanthra sessilis indicated that all

gram positive organisms were sensitive to the extracts but gram negative bacteria

were resistant to even a very high concentration of the extracts (Malayagupta et

al., 1992).

The aqueous extract of Tinospora cordifolia (stem) was found to be the

most potent antibacterial agent, successful in inhibiting Salmonella typhi and

Escherichia coli in dose dependent manner (Afaq et al., 2004). The leaf extract of

Cocculus hirsutus was found to be successful in its antibacterial activities

inhibiting both gram positive and gram negative strains and the phytochemical

analysis revealed the presence of carbohydrates / glycosides (Panda et al., 2007).

Appreciable antimicrobial activity against microbial strains by the extracts of

Helicteris isora root has been recorded by Venkatesh et al. (2007). Study at the

antibacterial activity of solvent fractions of crude water decoction of the twigs and

latex of Calotropis procera was found to produce greatest inhibitory zone on

Staphylococcus aureus, one of the major wound infectious bacterial strain (Farzin

et al., 2008). Porchezian and Ansari (2001) studying the antimicrobial potency of

the Alangium salvifolium found that the methanol extract at 60mg/disc showed

significant activity against the investigated bacterial organisms. Both the methanol

and chloroform extracts were devoid of antifungal activity even at the highest



25

concentration. These findings have been endorsed by the work of Ragasudha and

Gangarao (2008). Albizzia lebbeck bark extract was found to exhibit antimicrobial

activity, the active constituent of bark extract being anthroquinone glycosides. The

main constituent of the bark was active against aerobes and mechanism of action

was that the glycosides caused the leakage of the cytoplasmic constituents

(Ganguly and Bhatt, 1993).

Albizzia lebbeck is shown to be an important source of chemicals like

melacacidin, D-catachin, B-sitosterol, Albizziahexoside and betulinicacid which

are effective as antiseptic, antidysentric and antitubercular and used in bronchitis,

leprosy, paralysis and helmenth infection (Arvind Kumar et al., 2007). The

antimicrobial activities of extracts of the stem bark and leaves of Tamarindus

indica was evaluated by Doughari (2006) against some common gram positive

and gram negative bacteria and fungi. The study also investigated the chemical

constituents of the plants on their antimicrobial activity. Results of phytochemical

studies revealed the presence of tannins, saponins, sesquiterpenes, alkaloids and

phlobactamins as active against both gram positive and gram negative bacteria.

Tamarindus indica has broad spectrum antibacterial activity and is a potential

source of new classes of antibiotics that could be useful for infectious disease,

chemotherapy and control. The fresh extract of Tephrosia purpurea roots have

been detected to show antibacterial activity against Staphylococcus aureus,

Escherichia coli and Bacillus subtilis (Shrikalp Deshpande et al., 2005).

Sabeetha and Suryanarayana (2006) have evaluated the antifungal activity

of different concentrations of Spilanthes acumella head extract. The fungi chosen



26

were the potential human pathogens Aspergillus flavus and Aspergillus paraciticus

besides the agricultural pathogens Fusarium oxisporium and Fusarium

moniliformis. In all the organisms the maximum zone of inhibition was observed

at 2000 µg concentration. Five among twelve medicinal plants used by Himalayan

people were found to possess antifungal activity by Sanjay Guleria and Ashok

Kumar (2006). Clear inhibition zones were observed for extracts of Vitex

negundo, Xanthoxylum alatum, Ipomoea carnea, Thuja orientalis and

Cinnamomum camphora. Parekh and Chanda (2006) studying in vitro

antimicrobial activity of extracts of Launaea procumbens, Vitis vinifera and

Cyperus rotandus found that the ethanolic extracts of all the plants were active

against all the investigated bacterial strains while all the aqueous extracts were

inactive.

In a study for screening antimicrobial activity of 10 medicinal plants used

in Colombian folkloric medicine, water extracts of Bidens pilosa, Jacaranda

mimosifolia and Piper pulchrum showed a higher activity against Bacillus cereus

and Escherichia coli. Similarly ethanolic extracts of all species were active against

Staphylococcus aureus, Bixa orellana, Justicia secunda and Piper pulchrum

presented the lowest MICs against Escherichia coli compared to Gentamicin

sulphate. Likewise Justicia secunda and Piper pulchrum showed an analogous

MIC against Candida albicans compared to Nystatin (Rojas et al., 2006). In a

study on the antimicrobial activity of methanol extract of Coccinia grandis leaves,

major activity was seen against Staphylococcus aureus, Escherichia coli, Shigella

dysentriae, Shigella sonic and Psuedomonas aeruginosa. Against fungal strains,



27

extracts were found effective at higher concentrations and Candida albicans had

shown higher sensitivity (Dewanjee et al., 2007).

Parekh and Chandra (2007) studied the antimicrobial activities of

Woodfordia fruticosa (Lythraceae) against an array of human pathogens. The

crude methanol extract of Woodfordia fruticosa flower exhibited antibacterial

activity against Bacillus subtilis, Micrococcus flavus and was most active against

Psuedomonas sp. The extract was more active against gram negative as compared

to gram positive bacteria. Raghavendra et al. (2006) had tested Oxalis corniculata

for antibacterial activity. In the five solvents tested, methanol and ethanol extracts

showed significant antibacterial activity.

Phytochemical analysis of the leaf material revealed that the antibacterial

activity was due to the presence of phenolic compounds. Panthi and Chaudhary

(2006) selected 18 folklore medicinal plants and tested their antibacterial activity.

Extracts of eight plants showed encouraging results against 3 strains of bacteria

while others showed activity against 1 (or) 2 strains. The extract of Phyllanthus

emblica showed activity against Staphylococcus aureus only. Sida cordata and

Terminalia chebula were active against S.aureus and S.boydii. Justicia adathoda

showed activities against S.aureus, P. aeruginosa and S. boydii.

4.3 MATERIALS AND METHODS

The antimicrobial (antibacterial & antifungal) screening was carried out in

the Department of Microbiology, RVS College of Arts and Science, Sulur,

Coimbatore, Tamil Nadu, India. The standard glass wares, equipmens and discs



28

were used for the present study. The antimicrobial activity of crude extracts of

Asteracantha longifolia and Pergularia daemia were determined by various

methods such as, Disc diffusion, MIC, MBC, TA & AI.

Screening for Antibacterial Activity

Medium Preparation

The ingredients (Muller Hinton Agar - Hi-media) were dissolved in

distilled water with the aid of heat and pH was adjusted to 7.0 using dilute alkali

or dilutes acid. Mueller-Hinton agar was prepared and autoclaved at a pressure of

15 psi (121ºC) for not less than 15 minutes. The sterilized medium was transferred

into sterile petridish.

Microorganisms

Microorganisms were obtained from the Microbial Type Culture Collection

Centre (MTCC), Chandigarh, India.

Amongst eleven microorganisms investigated, nine bacterial species were

Staphylococcus aureus MTCC 3160, Bacillus cereus MTCC 442, Streptococcus

pneumoniae MTCC 655, Escherichia coli MTCC 598, Pseudomonas aeruginosa

MTCC 42642, Klebsiella pneumoniae MTCC 7407, Salmonella typhi MTCC

3917, Proteus vulgaris MTCC 742, Shigella flexneri MTCC 1457 while two

fungal species were Aspergillus niger MTCC 2546, Candida albicans MTCC

183. All the microorganisms were maintained at 4C on nutrient/ Potato dextrose

agar slants.



29

Working Conditions

The entire work was done using horizontal laminar flow hood so as to

provide aseptic conditions. Before commencement of the work air sampling was

carried out using a sterile nutrient agar plate and exposing it to the environment

inside the hood. After incubation it was checked for the growth of the

microorganism and absence of growth confirmed aseptic working conditions.

Preparation of Inoculum

The inoculum for the experiment was prepared fresh in nutrient broth from

preserved frozen slant culture. It was incubated at 37ºC for 24 hours and used after

standardization.

Disc diffusion method

Antimicrobial activity was carried out by the disc diffusion method. The

antimicrobial assays of aqueous and methanolic extracts were performed by Bauer

et al. (1966). Each plant extracts were tested at two different concentrations (100

& 200 mg/ml) to see their inhibitory effects against microbial pathogens. Sterile

paper discs (6 mm in diameter) prepared from Whatman No. 1 filter paper was

impregnated with drug, containing solution placed on the inoculated agar.

Negative and Positive controls used DMSO and Chloroamphenical. The

inoculated plates were incubated at 37C for 24 h. The antibacterial activity was

evaluated by measuring the diameter of the inhibition zone for the test

microorganisms.



30

MIC

For determination of MIC, 1 ml of broth medium was taken into 10 test

tubes for each bacteria. Different concentrations of plant extracts ranging from

0.125-8 mg/ml-1 concentration were incorporated into the broth and the tubes were

then inoculated with 0.1 ml of inoculum of respective bacteria (105 CFU ml-1) and

kept at 37°C for 24 h. The test tube containing the lowest concentration of extract

which showed reduction in turbidity when compared with control was regarded as

MIC of that extract (Muhamed Mubarack et al., 2011).

MBC

The MBC is defined as the lowest concentration where no bacterial growth

is observed (bacteriocidal concentration). In this technique, the contents of the test

tubes resulting from MIC was streaked using a sterile wire loop on agar plate free

of bacteria and incubated at 37ºC for 18 hours. The lowest concentration of the

extract which showed no bacterial growth was noted and recorded as the MBC.

Total activity

Total activity is the volume at which test extract can be diluted with the

ability to kill microorganisms. It is calculated by dividing the amount of extract

from 1 g plant material by the MIC of the same extract or compound isolated and

is expressed in ml/g (Sharma and Kumar, 2009).

AI = IZ developed by extract/IZ developed by standard



31

Screening for Antifungal activity

Preparation of Inoculum

The inoculum for the experiment was prepared fresh in Potato Dextrose

broth from preserved frozen slant culture. It was incubated at 25ºC for 24-48 hrs,

and used after standardization.

Disc diffusion method

The potato dextrose agar plates were inoculated with each fungal culture by

point (10 days old cultures) inoculation. The filter paper discs impregnated with

100 and 200 mg/ml concentrations of the extracts were placed on test organism

seeded plates. The activity was determined after 72 h of incubation at 28C. The

diameters of the inhibition zones were measured in mm (Taylor et al., 1995). The

antifungal activity was evaluated by measuring the diameter of the inhibition zone

for the test microorganisms.

4.4 RESULTS

Antimicrobial activity

The antimicrobial activity have been screened because of their great

medicinal relevance with the recent years, infections have increased to a great

extent and resistant against antibiotics, becomes an ever increasing therapeutic

problem. The results revealed variability in inhibitory concentrations of each

extract against a given bacteria. The inhibition of bacterial growth was dose

dependent since the inhibitory action of the extract was found to increase with an

increase in concentration against all bacterial strains as evidenced by the higher

zone of inhibitions at higher concentrations of each extract. Antimicrobial activity



32

(assessed in terms of inhibition zone, total activity and activity index) of the crude

extracts, tested against selected microorganisms were recorded.

Antibacterial activity

The zone of inhibition values ranged from the lowest of 8 mm recorded in

the methanol extract of both plants at 100 and 200 mg/ml concentration to the

maximum zone of inhibition 18 & 15 mm found in the methanol extracts of

A.longifolia and P.daemia respectively, at 200 mg/ml concentrations (Plate 4.1

and 4.2). Standard Chloramphenicol exhibited maximum zone of inhibition 22

mm at 10mcg concentration against Staphylococcus aureus (Fig. 4.1; Table 4.1).

In the case of Staphylococcus aureus the zone of inhibition values were from

10mm in the aqueous extract of A.longifolia at 200 mg/ml concentration to 22 mm

recorded in the methanol extract of A.longifolia at 200 mg/ml concentration as

well in the methanol extract of P.daemia (15 mm) at 200 mg/ml concentration.

Standard Chloramphenicol exhibited maximum zone of inhibition (22 mm) at

10mcg concentration (Plate 4.1; Fig. 4.2; Table 4.2).

Against Escherichia coli zone of inhibition ranged from 8 mm (recorded in

aqueous extract of A.longifolia at 200 mg/ml concentration) to a maximum 12 mm

recorded in methanol extract of A.longifolia at 200 mg/ml concentration. Standard

Chloramphenicol exhibited maximum zone of inhibition 21mm at 10 mcg

concentration. With regard to Proteus vulgaris, highest zone of inhibition was 10

and 9 mm recorded in the methanol and aqueous extracts of A.longifolia at 200

mg/ml concentration respectively, while minimum zone of inhibition 8 mm was

seen in aqueous extract of A.longifolia at 100 mg/ml concentration. Standard

Chloramphenicol exhibited maximum zone of inhibition (20 mm) at 10 mcg



33

concentration. Against Klebsiella pneumoniae minimum zone of inhibition 8 mm

was recorded in the methanol extract of P.daemia at 200 mg/ml concentration

(Plate 4.2). Highest zone of inhibition 10 mm was recorded in the methanol

extract of A.longifolia at 200 mg/ml concentration. Standard Chloramphenicol

exhibited maximum zone of inhibition 17 mm at 10 mcg concentrations (Plate 4.1;

Fig. 4.1; Table 4.1).

The zone of inhibition ranged from 10 mm to maximum 12 mm recorded

against Escherichia coli in methanol extract of P.daemia at 200 mg/ml

concentration. Standard Chloramphenicol exhibited maximum zone of inhibition

21mm at 10 mcg concentration (Plate 4.2 & 4.3). With regard to Proteus vulgaris,

highest zone of inhibition was 9 mm recorded in the methanol extract of P.daemia

at 200 mg/ml concentration. Standard Chloramphenicol exhibited maximum zone

of inhibition (20 mm) at 10 mcg concentration (Table 4.2; Fig. 4.2).



34

Table 4.1

Antimicrobial activity of Methanol and Aqueous extracts of A. longifolia (Linn.) Nees. by Disc diffusion method

S.No. Name of the Strains

Zone of Inhibition (mm) Methanol (mg/ml)

Aqueous (mg /ml) Synthetic drug

(Chloramphenicol) 100 200 100 200

1. Staphylococcus aureus 11 18 8 10 22 2. Streptococcus pneumonia 10 12 - - 20 3. Bacillus cereus 9 11 - 9 17 4. Escherichia coli 10 12 - 8 21 5. Pseudomonas aeruginosa 8 12 - 8 18 6. Klbseillae pneumonia - 10 - - 17 7. Salmonella typhi - 9 - - 16 8. Proteus vulgaris 8 10 - - 20 9. Shigella flexneri - 10 - 9 16

Antifungal activity Synthetic drug (Fluconazole)

10. Candida albicans - 9 - - 15 11. Aspergillus niger - 9 - - 17



35

Fig. 4.1 Antimicrobial activity of Methanol and Aqueous extracts of A. longifolia (Linn.) Nees. by Disc diffusion method



36

Table 4.2 Antimicrobial activity of Methanol extract of P. daemia (Forsskal) Chiov. by Disc diffusion method


Zone of Inhibition (mm) Methanol (mg/ml)

Aqueous (mg /ml) Synthetic drug

(Chloramphenicol) 100 200 100 200

1. Staphylococcus aureus 10 15 10 12 22 2. Streptococcus pneumonia 10 11 - 9 20 3. Bacillus cereus 9 10 - 10 17 4. Escherichia coli 10 12 - - 21 5. Pseudomonas aeruginosa 8 10 - 8 18 6. Klbseillae pneumonia - 8 - - 17 7. Salmonella typhi - 10 - 8 16 8. Proteus vulgaris - 9 - - 20 9. Shigella flexneri 8 10 - - 16


10. Candida albicans - - - - 15 11. Aspergillus niger - - - - 17



37

Fig. 4.2 Antimicrobial activity of Methanol extract of P. daemia (Forsskal) Chiov. by Disc diffusion method



38

The results of Pseudomonas aeruginosa exhibited a minimum zone of

inhibition value of 8mm in the methanol extract of A.longifolia at 200 mg/ml

concentration and maximum of 10 and 12mm in the aqueous and methanol

extracts of P.daemia and A.longifolia at 200 mg/ml concentration respectively

(Plate 4.1 and 4.3). Standard Ciprofloxacin exhibited maximum zone of inhibition

(18 mm) at 10mcg concentration. In the antibacterial screening of methanol and

aqueous extracts of tested two plants, higher zone of inhibitions against the

bacteria were recorded at 200 mg/ml concentration levels. Highest zone of

inhibition (18 mm) was recorded in the methanol extract of A.longifolia at 200

mg/ml concentration, against Staphylococcus aureus. 100 mg/ml exhibited

comparatively lesser zone of inhibitions ranging from 8mm to 11 mm. Highest

zone of inhibition (11mm) was recorded in the methanol extract of A.longifolia, at

100 mg/ml concentration, against Staphylococcus aureus (Plates 4.1 & 4.2;

Fig. 4.1; Tables 4.1 & 4.2).

MIC & MBC

Methanol extract of P.daemia showed least MIC value i.e. 0.500 mg/ml

(MBC = 0.250 mg/ml) against S.aureus while aqueous extract has shown

moderate activity against S.aureus at 0.500 mg/ml (MBC = 1.0mg/ml)

concentration (Fig. 4.4; Table 4.4). Similarly A.longifolia methanol extract was

found to be highly susceptible as it has shown very low MIC value 0.125 mg/ml

against S.aureus. Aqueous extract has shown highest activities against S.aureus,

B.cereus and P.vulgaris at 4 mg/ml concentration (Fig. 4.3; Table 4.3). TA was

also calculated and recorded (Fig. 4.5 & 4.6; Tables 4.5 & 4.6).



39

Table 4.3 Antimicrobial activity of Methanol and Aqueous extracts of A. longifolia (Linn.) Nees. by Broth dilution method

S.No. Name of the Strains Methanol Aqueous

MIC (mg /ml)

MBC (mg /ml) MICindex MIC

(mg /ml) MBC

(mg /ml) MICindex

1. Staphylococcus aureus 0.125 0.250 2 4 4 1

2. Streptococcus pneumonia 0.250 0.500 2 - - -

3. Bacillus cereus 0.500 0.500 1 4 4 1

4. Escherichia coli 0.500 0.500 1 - - -

5. Pseudomonas aeruginosa 2 2 1 - - -

6. Klbseillae pneumonia 4 4 1 - - -

7. Salmonella typhi 2 2 1 - - -

8. Proteus vulgaris 0.500 1 2 4 4 1

9. Sheigella flexneri 0.500 0.500 1 - - -

10. Candida albicans 2 2 1 - - -

11. Aspergillus niger 2 2 1 2 2 1



40

Fig. 4.3 Antimicrobial activity of Methanol and Aqueous extracts of A. longifolia (Linn.) Nees. by Broth dilution method



41

Table 4.4 Antimicrobial activity of methanol and aqueous extract of P. daemia (Forsskal) Chiov. by Broth dilution method

S.No. Name of the Strains Methanol Aqueous

MIC (mg /ml)

MBC (mg /ml) MICindex MIC

(mg /ml) MBC

(mg /ml) MICindex

1. Staphylococcus aureus 0.500 0.250 0.500 0.500 1.0 2

2. Streptococcus pneumonia 1.0 2.0 1 2.0 2.0 1

3. Bacillus cereus 0.500 1.0 1 1.0 1.0 1

4. Escherichia coli 2.0 2.0 1 - - -

5. Pseudomonas aeruginosa - - - - - -

6. Klbseillae pneumonia - - - - - -

7. Salmonella typhi - - - - - -

8. Proteus vulgaris - - - - - -

9. Sheigella flexneri 4.0 2.0 0.5 - - -

10. Candida albicans - - - - - -

11. Aspergillus niger - - - - - -



42

Fig. 4.4 Antimicrobial activity of methanol and aqueous extract of P. daemia (Forsskal) Chiov. by Broth dilution method



43

Table 4.5

Activity index and Total activity of Methanol and Aqueous extracts of A. longifolia (Linn.) Nees.


Methanol Aqueous Activity index Total

activity (ml/g)

Activity index Total activity ( ml/g) 100 200 100 200

1. Staphylococcus aureus 0.500 0.818 4 0.363 0.454 0.175

2. Streptococcus pneumoniae 0.500 0.600 2 - - -

3. Bacillus cereus 0.529 0.647 1 - 0.529 0.175

4. Escherichia coli 0.476 0.571 1 - 0.380 -

5. Pseudomonas aeruginosa 0.444 0.666 0.250 - 0.444 -

6. Klbseillae pneumoniae - 0.588 0.125 - - -

7. Salmonella typhi - 0.562 0.250 - - -

8. Proteus vulgaris 0.400 0.500 1 - - -

9. Sheigella flexneri - 0.625 1 - 0.562 0.175

10. Candida albicans - 0.600 0.250 - - -

11. Aspergillus niger - 0.529 0.250 - - -



44

Fig. 4.5 Activity index and Total activity of Methanol and Aqueous extracts of A. longifolia (Linn.) Nees.



45

Table 4.6 Activity index and Total activity of Methanol and Aqueous extract of P. daemia (Forsskal) Chiov.


Methanol Aqueous

Activity index Total activity (ml/g)

Activity index Total activity ( ml/g) 100 200 100 200

1. Staphylococcus aureus 0.454 0.681 1.100 0.454 0.545 1

2. Streptococcus pneumoniae 0.500 0.550 0.550 - 0.450 0.25

3. Bacillus cereus 0.529 0.588 1.100 - 0.588 0.50

4. Escherichia coli 0.476 0.571 0.275 - - -

5. Pseudomonas aeruginosa 0.444 0.555 - - 0.444 -

6. Klbseillae pneumoniae - 0.470 - - - -

7. Salmonella typhi - 0.625 - - 0.500 -

8. Proteus vulgaris - 0.450 - - - -

9. Sheigella flexneri 0.500 0.625 0.137 - - -

10. Candida albicans - - - - - -

11. Aspergillus niger - - - - -



46

Fig. 4.6 Activity index and Total activity of Methanol and Aqueous extract of P. daemia (Forsskal) Chiov.



47

TA was highest for methanol extracts of both plants (4.0 & 1.1 ml/g)

against S. aureus. On a general note, methanolic extracts exhibited higher degree

of antibacterial activities than the aqueous extracts. Amongst the gram-positive

and gram-negative bacteria, gram-positive bacterial strains were more susceptible

to the extracts when compared to gram negative bacteria. This may be attributed

to the fact that these two groups differ by its cell wall component and its

thickness. In the present study IZ, AI, MIC, MBC and TA have been evaluated for

each extracts. For methanol extracts of the tested plants MIC values recorded were

very low, indicating strong bioefficacy of the plant.

Both crude methanolic and aqueous forms of the extracts of A.longifolia

exhibited varying degrees of antimicrobial activities against the test organisms. At

200 mg/ml, methanolic extract had higher antibacterial activity with zone of

inhibition 18 mm (AI = 0.818), 12 mm (AI = 0.6), 12 mm (AI = 0.571) and 12

mm (AI = 0.666) than aqueous extract with zone of inhibition 10 mm (AI = 0.454)

against S.aureus, S.pneumoniae, E.coli and P.aeruginosa, respectively (Fig. 4.5 &

4.6; Table 4.5 & 4.6). In presence of P.daemia methanol extract inhibition zone

diameter was obtained 15 mm (AI = 0.818) in S.aureus and 12 mm (AI = 0.571)

in E.coli at 200 mg/ml concentration. P.daemia aqueous extract has shown highest

inhibition zone diameter i.e. 12 mm in (AI = 0.454) in S.aureus and 10 mm (AI =

0.529) in B.cereus.

Antifungal activity

The zone of inhibition of leaf extracts was found to be 9 mm for Aspergillus

niger and Candida albicans. The widest zone of inhibition was produced by



48

methanol extract of A.longifolia while methanol and aqeous extracts of P.daemia

had the no zone of growth inhibition (Fig. 4.1 & 4.2; Tables 4.1 & 4.2).

4.5 DISCUSSION

Many medicinal plants are considered to be store houses of potential

antimicrobial crude drugs as well as sources for novel compounds with

antimicrobial activity, with possibly new modes of action. This expectation that

some naturally occurring plant compounds can kill antibiotic-resistant strains of

bacteria such as Bacillus cereus, Escherichia coli, Micrococcus luteus and

Staphylococcus aureus has been confirmed (Friedman et al., 2006). In the past

few decades, the search for new anti-infection agents has engaged many research

groups in the field of ethnopharmacology. A Pubmed search for the antimicrobial

activity of medicinal plants has produced 115 articles during the period between

1966 and 1994. However, in the following decade between 1995 and 2004, this

number has more than doubled, to 307. In these studies one could find a wide

range of criteria related to the discovery of antimicrobial compounds in plants.

Staphylococcus aureus, a wound infecting pathogen which can cause

septicemia, endocarditis and toxic shock syndrome; Pseudomonas aeruginosa

which infects the pulmonary tract, urinary tract, burns and wounds.

Staphylococcus aureus infections can spread through contact with pus from an

infected wound, skin to skin contact with an infected person by producing

hyaluronidase that destroy tissues. Salmonella are closely related to the

Escherichia coli genus and are found worldwide in warm-and cold-blooded

animals, in humans, and in non-living habitats. They cause illness in humans and



49

many animals, such as typhoid fever, paratyphiod fever and the food borne illness

salmonellosis (Ryan and Ray, 2004).

The antibacterial assays in this study were performed by the disc diffusion

and the broth microdilution methods as they could be qualified and quantified by

inhibition zone diameters, MIC, MBC, MICindex, TA and AI values as summarized

in Tables 4.1- 4.6. The susceptibility of the bacteria to the extract, on the basis of

inhibition zone diameters varied according to microorganisms, but globally, the

highest inhibition zone diameters have been recorded with Gram-positive bacteria

(Scherrer and Gerhardt, 1971). However, in this study both gram positive and

gram negative organisms were found to be active against plant extracts. According

to the antibacterial assay, among gram positive bacteria in A.longifolia, the highest

zone of inhibition was noted in the S.aureus (18 mm) followed by S.pneumoniae

(12 mm) and B.cereus (11 mm). At the same time moderate activity was noted in

P.daemia extracts against bacterial pathogens. Antifungal activity of plant extracts

showed the was highest activity against Aspergillus niger (9 mm) followed by

Candida albicans (9 mm).

It is reported that gram positive bacteria tend to be more susceptible since

they have only an outer peptidoglycan layer which is not an effective barrier

(Scherrer and Gerhardt, 1971). The gram-negative bacteria have an outer

phospholipidic membrane that make the cell wall impermeable to lipophilic

solutes, while the porines constitute a selective barrier to hydrophilic solutes with

an exclusion limit of about 600 Da (Nikaido and Vara, 1985). Many results have

confirmed these observations and some plant extracts have been found to be more



50

active against gram-positive bacteria than against gram-negative ones (Kelmanson

et al., 2000; Masika and Afolayane, 2002).

Fungal infections continue to be a significant cause of morbidity and

mortality despite advances in medicine and the emergence of new antifungal

agents. Candida albicans has a worldwide distribution due to the fact that it is a

frequent opportunistic pathogen in AIDS patients (De Pavia et al., 2003: Nolte et

al., 1997). It is a common commensal of the gastrointestinal and urogenital tracts

of human beings (Black, 1996) and is also the cause of Candiasis in women

(Parekh and Chanda, 2008). Candida tropicalis is one of the non-albicans candida

strains currently emerging in fungal infections (Powderly et al., 1999). Aspergillus

niger which causes Aspergillosis, a serious lung infection, if large amounts of

spores are inhaled.

The present study carried out on the plant samples revealed the presence of

medicinally active phytoconstituents. Analysis of plant extracts revealed the

presence of phenols, tannins, flavonoids, glycosides, saponins and steroids in most

of the selected plants which could be responsible for the observed antimicrobial

properties. Phytochemicals exert their antimicrobial activity through different

mechanisms; tannins for example act by iron deprivation, hydrogen bounding or

nonspecific interactions with vital proteins such as enzymes (Scalbert, 1991). The

antibacterial activity of flavonoids is probably due to their ability to complex with

extracellular and soluble proteins and to complex with bacterial cell walls (Doss et

al., 2011). Antimicrobial activity of phenolic compounds present in plants change

according its structure; flavone, quercetin and naringenin were effective in



51

inhibiting the growth of Aspergillus niger, Bacillus subtilis, Candida albicans,

Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa, Saccharomyces

cerevisiae, Staphylococcus aureus and Staphylococcus epidermidis while gallic

acid inhibited only P. aeruginosa; rutin as well as catechin did not show any effect

on the tested microorganisms.

Several reports are available in support of antimicrobial activity of saponins

against bacterial and fungal pathogens (Gopish khanna and Kannabiran, 2008).

The alkaloids are known to have antimicrobial and antiparasitic properties.

Verpoorte (1998) have reported about 300 alkaloids showing such activity.

Similar results on antibacterial activity were reported on related species of the

genus Mahonia by Duraiswamy et al. (2006), Livia et al. (2004) and Li et al.

(2007). Steroids have been reported to have antibacterial properties, the

correlation between membrane lipids and sensitivity for steroidal compound

indicates the mechanism in which steroids specifically associate with membrane

lipid and exerts its action by causing leakeages from liposomes (Raquel and

Epand, 2007). The solvents used in the extraction procedure were found to have

pronounced effect on the solubility of the antibacterial compounds. Several reports

have shown the antimicrobial properties of plant extracts under laboratory

conditions (Doss et al., 2009a; Doss et al., 2009b; Venkataswamy et al., 2010;

Anand et al., 2011).


52

Chapter - V

ANTIOXIDANT ACTIVITY . .

5.1 Introduction

Reactive oxygen species (ROS), such as superoxide anions, hydrogen

peroxide, hydroxyl, nitric oxide and peroxynitrite radicals, play an important role

in oxidative stress related to the pathogenesis of various important diseases. In

healthy individuals, the production of free radicals is balanced by the antioxidative

defense system; however, oxidative stress is generated when equilibrium favors

free radical generation as a result of a depletion of antioxidant levels. The

oxidation of lipid, DNA, protein, carbohydrate, and other biological molecules by

toxic ROS may cause DNA mutation or/and serve to damage target cells or

tissues, and this often results in cell senescence and death. Cancer

chemoprevention by using antioxidant approaches has been suggested to offer a

good potential in providing important fundamental benefits to public health, and is

now considered by many clinicians and researchers as a key strategy for

inhibiting, delaying, or even reversal of the process of carcinogenesis. Moreover,

knowledge and application of such potential antioxidant activities in reducing

oxidative stresses in vivo has prompted many investigators to search for potent

and cost-effective antioxidants from various plant sources. These research

activities have contributed to new or renewed public interests worldwide in herbal

medicines, health foods, and nutritional supplements (Lie-Fen Shyur et al., 2005).

Chapter - V Antioxidant Activity


53

Antioxidant substances block the action of free radicals which have been

implicated in the pathogenesis of many diseases including atherosclerosis,

ischemic heart disease, cancer, Alzheimer’s disease, Parkinson’s disease and in

the aging process. Currently available synthetic antioxidants like BHA, BHT,

tertiary butylated hydroquinon and gallic acid esters, have been suspected to cause

or prompt negative health effects. Hence, strong restrictions have been placed on

their application and there is a trend to substitute them with naturally occurring

antioxidants.

Moreover, these synthetic antioxidants also show low solubility and

moderate antioxidant activity. The therapeutic effects of several plants and

vegetables, which are used in traditional medicine, are usually attributed to their

antioxidant compounds. Antioxidants are also used to preserve food quality

mainly because they arrest oxidative deterioration of lipids. Plant-based

antioxidants are now preferred to the synthetic ones because of safety concerns

(Akinmoladun et al., 2007). These factors have inspired the widespread screening

of plants for possible medicinal and antioxidant properties, the isolation and



54

characterization of diverse phytochemicals and the development and utilization of

antioxidants of natural origin (Oluwaseun and Ganiyu, 2008).

Approximately 80% of the world population depends exclusively on plants

for their health and healing. Whereas in the developed world, reliance on surgery

and pharmaceutical medicine is more usual but in the recent years, more and more

people are complementing their treatment with natural supplements (Dursum et

al., 2004). Furthermore, motivation of people towards herbs are increasing due to

their concern about the side effect of drugs, those are prepared from synthetic

materials. The people want to concern their own health rather than merely

submitting themselves to impersonal health care system. Many botanical and some

common dietary supplements are good sources of antioxidants and anti-

inflammatory compounds (Khalil et al., 2007). To drive the maximum health

benefits, sufficient amounts of phytochemicals from a variety of plant source such

as fruits, vegetables and whole grain based foods are recommended. The

phytochemicals in fruits and vegetables are different from those in the grains,

which contain tocotrienols and tocopherol, while rice contains oryzanols. The

phenolic like ferulic acid and diferulate are predominant in grains but are not

significant in some fruits and vegetables (Sreeramulu et al., 2009).

Free radicals

Free radicals are highly reactive molecules or chemical species capable of

independent existence. Generation of highly ROS is an integral feature of normal

cellular function like mitochondrial respiratory chain, phagocytosis, arachidonic

acid metabolism, ovulation, and fertilization. Their production however,



55

multiplies several folds during pathological conditions. The release of oxygen free

radicals has also been reported during the recovery phases from many pathological

noxious stimuli to the cerebral tissues (Beal, 2000). Oxygen, because of its bi-

radical nature, readily accepts unpaired electrons to give rise to a series of

partially reduced species collectively known as (ROS) including, superoxide

(O2-), hydrogen peroxide (H2O2), hydroxyl (HO), peroxyl (ROO), alkoxy (RO),

and nitric oxide (NO), until it is itself completely reduced to water. Most of the

superoxide radicals are formed in the mitochondrial and microsomal electron

transport chain. Except for cytochrome oxidase, which retains the partially

reduced oxygen intermediates bound to its active site, all other elements in the

mitochondrial respiratory chain, e.g., ubiquinone, etc., transfer the electron

directly to oxygen and do not retain the partially reduced oxygen intermediates in

their active sites (Halliwell and Gutteridge, 1989). On the internal mitochondrial

membrane, the superoxide anion may also be generated by auto-oxidation of

semiquinones. The majority of superoxide radicals generated by mitochondrial

electron transport chain are enzymatically dismutated to H2O2. The hydroxyl

and alkoxy free radicals are very reactive species and rapidly attack the

macromolecules in cells (Hemnani and Parihar, 1998).

Damage due to free radicals caused by ROS leads to several damaging

effects as they can attack lipids, proteins/enzymes, carbohydrates, and DNA in

cells and tissues. They induce undesirable oxidation, causing membrane damage,

protein modification, DNA damage, and cell death induced by DNA

fragmentation and lipid peroxidation. This oxidative damage/stress, associated



56

with ROS is believed to be involved not only in the toxicity of xenobiotics but

also in the pathophysiological role in aging of skin and several diseases like heart

disease (atherosclerosis), cataract, cognitive dysfunction, cancer (neoplastic

diseases), diabetic retinopathy, critical illness such as sepsis and adult/acute

respiratory distress syndrome, shock, chronic inflammatory diseases of the

gastrointestinal tract, organ dysfunction, disseminated intravascular coagulation,

deep injuries, respiratory burst inactivation of the phagocytic cells of immune

system, production of nitric oxide by the vascular endotheliums, vascular damage

caused by ischaemia reperfusion known as ischaemia/reperfusion injury and

release of iron and copper ions from metalloprotein (Boveris et al., 1972). Iron

changes have been detected in multiple sclerosis, spastic paraplegia, and

amyotrophic lateral sclerosis, which reinforces the belief that iron accumulation is

a secondary change associated with neuro degeneration in these diseases, although

it could also be related to gliosis (glia might produce free radicals) in the diseased

area, or the changes in the integrity of the blood brain barrier caused by altered

vascularisation of tissue or by inflammatory events (Heinonen et al., 1998).

Antioxidant activity

Antioxidant is a substance that when present at a concentration low

compared to that of an oxidisable substrate, significantly delays or prevents

oxidation of that substrate. Even though plant phenols are not always treated as

real antioxidants in the literature, many in vitro studies have demonstrated the

antioxidant potential of phenols as direct aqueous phase radical scavengers and as

agents capable of enhancing the resistance to oxidation of low density lipoproteins



57

implicated in the pathogenesis of coronary heart disease (Rice-Evans et al., 1995).

It is admitted that a part of the antioxidant capacity of many fruits and berries is

derived from flavonoids (Heinonen et al., 1998) and, in fact, all the major

polyphenolic constituents of food show greater efficacy in these systems as

antioxidants on a molar basis than the antioxidant nutrients vitamin C, vitamin E,

and β-carotene. Differences between the antioxidant potential of selected

compounds can be measured using many different techniques. Because most

phytochemicals are multifunctional, a reliable antioxidant protocol requires the

measurement of more than one property relevant to either foods or biological

systems (Rice Evans et al., 1995).

Biological systems have evolved with endogenous defense mechanisms to

help protect against free radical induced cell damage. Glutathione peroxidase,

catalase, and superoxide dismutase are antioxidant enzymes, which metabolize

toxic oxidative intermediates. They require micronutrient as cofactors such as

selenium, iron, copper, zinc and manganese for optimum catalytic activity and

effective antioxidant defense mechanisms (Halliwell, 2001; Singh et al., 2003).

Superoxide dismutase, catalase, and glutathione peroxidase are three primary

enzymes, involved in direct elimination of active oxygen species (hydroxyl

radical, superoxide radical, hydrogen peroxide) whereas glutathione reductase,

glucose-6-phosphate dehydrogenase, and cytosolic GST are secondary enzymes,

which help in the detoxification of ROS by decreasing peroxide levels or

maintaining a steady supply of metabolic intermediates like glutathione and

NADPH necessary for optimum functioning of the primary antioxidant enzymes



58

(Vendemiale et al., 1999; Pietta, 2000). Glutathione, ascorbic acid, α-tocopherol,

β-carotene, bilirubin, selenium, NADPH, BHA, mannitol, benzoate, histidine

peptide, the iron-bonding transferrin, dihydrolipoic acid, reduced CoQ10,

melatonin, uric acid, and plasma protein thiol, etc., as a whole play a homoeostatic

or protective role against ROS produced during normal cellular metabolism and

after active oxidation insult. Glutathione is the most significant component which

directly quenches ROS such as lipid peroxides and plays major role in xenobiotic

metabolism. When an individual is exposed to high levels of xenobiotics, more

glutathione is utilized for conjugation making it less available to serve as an

antioxidant. It also maintains ascorbate (vitamin C) and alpha-tocopherol (vitamin

E), in their reduced form, which also exert an antioxidant effect by quenching free

radicals.

A number of other dietary antioxidants exist beyond the traditional vitamins

collectively known as phytonutrients or phytochemicals which are being

increasingly appreciated for their antioxidant activity, one example is flavonoids

which are a group of polyphenolic compounds. These are widely found in plants

as glucosylated derivatives. They are responsible for the different brilliant shades

such as blue, scarlet, and orange. They are found in leaves, flowers, fruits, seeds,

nuts, grains, spices, different medicinal plants, and beverages such as wine, tea,

and beer (Weisburger, 1997; Harborne, 1994).

Sources of Antioxidants

Plant cells normally contain a suite antioxidants localized in different

compartments. The antioxidant complement often changes as organs grows and



59

develop and is markedly affected by environmental stresses. Antioxidants are

broadly categorized into two groups; the enzymatic antioxidants and low

molecular weight (non-enzymatic) antioxidants. The enzymatic antioxidants like

catalases, superoxide dismutase peroxidases. Non enzymatic low molecular

weight antioxidants are vitamins, glutathione, and several plant pigments (Gould,

2003).

Flavonoids

Occurring naturally in foods and beverages from plant sources such as

fruits, vegetables, berries, tea, and wine, flavonoids are polyphenolic compounds

that provide an important dietary source of antioxidants (Hertog et al., 1993).

Flavonoids have the chemical structure C6-C3-C6, which includes two benzene

rings linked by three carbons. Flavonoids can be classified according to the

variations in the C3-ring as flavonols, flavones, flavanols (or catechins),

flavanones, anthocyanins, and isoflavonoids. More than 6000 different flavonoids

have been identified, having properties related to their chemical structures

(Harborne, 1993).

Tea is rich in catechins, and contains the flavonols quercetin, kaempferol,

and myricitin (Balentine et al., 1997). Catechins are oxidized during the

processing of black tea, which involves fermentation of tea leaves and forms

fermentation products such as thearubigens and theaflavins. Blueberries have

attracted interest because of their high anthocyanin content (Kahkonen et al.,

2001), which is reflected in their high antioxidant capacity. Lowbush "wild"



60

blueberries, bilberries (Vaccinium myrtillus) have been reported to have a higher

anthocyanin content and antioxidant capacity than cultivated high bush blueberry.

Poly phenols

Polyphenols are present in a variety of plants utilized as important

components of both human and animal diets (Crozier et al., 2000). These include

food grains such as sorghum, millet, barley, dry beans, peas, pigeon peas, winged

beans, and legumes; fruits such as apples, blackberries, cranberries, grapes,

peaches, pears, plums, raspberries, and strawberries; and vegetables such as

cabbage, celery, onion and parsley also contain a large quantity of polyphenols.

The presence of polyphenols in plants is largely influenced by genetic factors and

environmental conditions. Other factors, such as germination, degrees of ripening,

variety, processing and storage, also influence the content of plant phenolics.

Flavonoids represent the most common and widely distributed group of

plant phenolics. Their common structure is that of diphenylpropanes (C6-C3-C6)

and consists of two aromatic rings linked through three carbons that usually form

an oxygenated heterocycle. Structural variations within the rings subdivide the

flavonoids into several families: flavonols, flavones, flavanols, isoflavones,

antocyanidins and others. These flavonoids often occur as glycosides,

glycosylation rendering the molecule more water-soluble and less reactive toward

free radicals.

Phenolic plant compounds have attracted considerable attention for being

the main sources of antioxidant activity, in spite of not being the only ones. The



61

antioxidant activity of phenolics is mainly due to their redox properties, which

allow them to act as reducing agents, hydrogen donors, and singlet oxygen

quenchers. In addition, they have a metal chelation potential. The antioxidant

activities of phenolics play an important role in the adsorption or neutralization of

free radicals. Several synthetic antioxidants are commercially accessible but have

been reported to be toxic Plants have been reported to exhibit antioxidant activity

due to the presence of antioxidant compounds such as phenolics,

proanthocyanidins and flavonoids.

Typical structures of different groups of plant phenolics, flavonoids, anthocyanin



62


Bagul et al. (2006) have reported the free radical scavenging activity of the

alcoholic extract of Mesua ferrea. It was found to be a good scavenger of DPPH

radical due to the presence of Gallic acid. Gopinathan et al. (2004) have

investigated in vitro antioxidant activity of ethanol extract of Saccharum

spontaneum by DPPH free radical scavenging method. They attributed the

antioxidant activity to phenolic hydroxyl grouping in the Flavones. Baskar et al.

(2008) conducted a study to investigate the free radical scavenging activity of

polysaccharide fractions extracted from the fruit body of Ganoderma lucidum and

the results revealed that acetic acid and NaOH fractions exhibited potent

antioxidant activity and effectively scavenged the free radicals in a dose

dependent manner. The herbs consisting of flavonoids and polyphenols as active

constituents were evaluated for their antioxidant activity by Vijaya et al. (2002).

Even though the tested herbs had showed antioxidant activity, a positive

correlation between total phenolic content of the extracts and antioxidant activities

were noted denoting that phytoconsituents other than flavonoids and poly herbals

could have contributed to the antioxidant activity.

Desai et al. (2008) have investigated in vitro antioxidant activity of

aqueous extract of Baliospermum montanum by DPPH free radical scavenging

and Nitric oxide method. They attributed the antioxidant activity to phenolic

compounds in the flavones. Antioxidant activity of cereals, millets, pulses and

legumes were assessed by various methods such as DPPH radical scavenging

assay, ferric reducing antioxidant power and antioxidant assay. Even though the



63

tested crops had showed antioxidant activity, a positive correlation between total

phenolic content of the extracts and antioxidant activities were noted denoting that

phytoconsituents other than flavonoids and poly herbals could have contributed to

the antioxidant activity (Sreeramulu et al., 2009).

Umamaheshwari et al. (2007) evaluated the antioxidant activities of 70%

ethanolic extract of leaves of Jasminum grandiflorum using DPPH assay. The free

radical scavenging activity of the leaf extract of Jasminum grandiflorum depended

on the concentration levels. Free radical scavenging activity of selected essential

oils has been recorded by Ashok et al. (2004). Rashmi et al. (2007) have attributed

the antioxidant activities in Achyranthes aspera to the presence of 4 steroids in the

leaves, oleonolic glycosides in the roots and stem. Antiulcer and antioxidant

activity of Psidium guajava has been recorded by Edwin et al. (2007). DPPH

scavenging activity and super oxide scavenging activity studied have exhibited

very good antioxidant effect by preventing the formation of free radicals. They

have concluded that the presence of Flavonoids and Tannins might be the

antioxidants.

Gautham et al. (2007) have exhibited the in vitro antioxidant activity of the

extract of Tecomella undulata leaves. The aqueous extract of leaves showed

significant antioxidant activity by inhibiting DPPH, when compared with standard

ascorbic acid. The antioxidant activity may be due to the presence of alkaloids,

flavonoids and phenolic compounds. Weng & Wang (2000) isolated six

antioxidant compounds from Salvia plebeia and found that beta-sitosterol, 2`-

hydroxy-5 methoxybiochanin A and coniferyl aldehyde had strong antioxidant



64

activities. Of late more attention is paid to the role of natural oxidants especially

phenolic compounds which may act both by reducing the content of toxic

compounds in foods and by supplying the human body, exogenous antioxidants.

Al-Mamary (2002) studied the total antioxidant activity of 7 commonly

consumed vegetables in Yemen. The results showed that all juices increased

antioxidants or decreased pre-oxidant activity, increased with volume of juice with

addition of 50μl from each sample. Most vegetable juices showed antioxidant

activity. Coriander leaf extract had exceptionally high antioxidant activity (60%).

However garlic, red onion, parselay and Cissus rotundifolia showed considerable

high antioxidant activities (54 - 41%) when compared to the other vegetables.

Shrififar et al. (2003) found that the methanolic extract of the aerial parts of

Otostegia persica exhibited strong antioxidant activity due to the presence of two

compounds identified as Morin and Quercetin. Ansari et al. (2005) had tested five

vegetables traditionally consumed among the South Asian migrants in UK for

their free radical scavenging activity. They found strong activity in the extracts

derived from Abelmoschus esculentus fruits and aerial parts of Caralluma edulis.

Aiyegoro and Okoh (2010) have reported the antioxidant activity of the aqueous

extract of Helichrysum longifolium. It was found to be a good scavenger of DPPH

radical due to the presence of BHT and Gallic acid.



65



The antioxidant activity of Asteracantha longifolia and Pergularia daemia

were determined by two different in vitro methods such as, the DPPH free radical

scavenging assay and reducing power methods.

Preparation of solutions

(a) DPPH solution

0.1mM ethanolic DPPH was prepared.

(b) Sodium phosphate buffer

Solution -A

About 3.12 gm of sodium dihydrogen orthophosphate (NaH2PO4.2H20) was

dissolved in 50 ml of distilled water. Finally the volume was made up to 100 ml.

Solution-B

About 3.56 gm of disodium hydrogen phosphate dihydrate (Na2HPO4.2H2O)

was dissolved in 50 ml of distilled water and the final volume was made up to 100

ml. About 19.5 ml of the A solution was mixed with 30.5 ml of the B solution and

the final volume was made up to 100 ml, which constitute the sodium phosphate

buffer.

Solution A (gm /100 ml)

NaH2PO4.2H2O - 3.12

Distilled water - 100

Solution B (gm /100 ml)

Na2HPO4.2H2O - 3.56




66

Sodium phosphate buffer (ml)

Solution A - 19.5

Solution B - 30.5


1% Potassium ferric cyanide solution (gm /100 ml)

Potassium ferric cyanide - 1


10% TCA (Trichloroacetic acid) (gm /100 ml)

TCA - 10


0.1% Ferric chloride solution (mg /100 ml)

Ferric chloride - 100


DPPH Radical scavenging activity

DPPH scavenging activity was carried out by the method of Blois (1958).

Different concentrations (1000, 500, 250, 125, 62.5 and 31.2 µg/ml) of crude

extracts were dissolved in DMSO and taken in test tubes in triplicates. Then 5 ml

of 0.1mM ethanol solution of DPPH (1, 1, Diphenyl-2- Picrylhydrazyl) was added

to each of the test tubes and were shaken vigorously. They were then allowed to

stand at 37o C for 20 minutes. The control was prepared without any extracts.

Methanol was used for base line corrections in absorbance (OD) of sample

measured at 517nm. A radical scavenging activity was expressed as 1%

scavenging activity and was calculated by the following formula:

OD Control – OD Sample OD Control

100 Radical scavenging activity (%) =



67

Reducing power

Reducing activity was carried out by using the method of Oyaizu (1986).

Different concentrations (1000, 500, 250, 125, 62.5 and 31.2 µg/ml) of crude

extracts were dissolved in DMSO and taken in test tubes in triplicates. To the test

tubes 2.5 ml of sodium phosphate buffer and 2.5 ml of 1% potassium ferric

cyanide solution was added. These contents were mixed well and were incubated

at 50o C for 20 minutes. After incubation 2.5 ml of 10% TCA was added and were

kept for centrifugation at 3000 rpm for 10 minutes. After centrifugation 5 ml of

supernatant were taken and to this 5 ml of distilled water was added. To this about

1 ml of 1% ferric chloride was added and was incubated at 35o C for 20 minutes.

The OD (absorbance) was taken at 700 nm and the blank was prepared by adding

every other solution but without extract and ferric chloride (0.1%) and the control

was prepared by adding all other solution but without extract. The reducing power

of the extract is linearly proportional to the concentration of the sample.

Quantitative Estimation of Total Phenols and Flavonoids

Total phenolic content

Total phenolic contents were determined by Folin Ciocalteu reagent

(McDonald et al., 2001). A dilute extract of each crude extracts (0.5 ml of

1:10 g ml-l) or garlic acid (standard phenolic compound) was mixed with Folin

Ciocalteu reagent (5 ml, 1:10 diluted with distilled water) and aqueous sodium

carbonate (4 ml, 1 M). The mixtures were allowed to stand for 15 min and the

total phenols were determined by colorimetry at 765 nm. The standard curve was



68

prepared using 0, 50, 100, 150, 200, 250 mg/ml solutions of gallic acid in

methanol: water (50:50, v/v). Total phenol values are expressed in terms of gallic

acid equivalent (mg g-l of dry mass), which is a common reference compound.

Determination of Total flavonoids

Aluminum chloride colorimetric method was used for flavonoids

determination (Chang et al., 2002). Each crude extracts (0.5 ml of 1:10 g/ml) in

methanol were separately mixed with 1.5 ml of methanol, 0.1 ml of 10%

aluminum chloride, 0.1 ml of 1M potassium acetate and 2.8 ml of distilled water.

It remained at room temperature for 30 min; the absorbance of the reaction

mixture was measured at 415 nm with a double beam Perkin Elmer UV/Visible

spectrophotometer (USA). The calibration curve was prepared by preparing

quercetin solution at concentrations 12.5 to 100 g ml -1 in methanol.

5.4. RESULTS

DPPH Free Radical scavenging activity

The antioxidant activity of the extracts was determined using a DPPH

scavenging and reducing power assay. The DPPH assay is often used to evaluate

the ability of antioxidants to scavenge free radicals which are known to be a major

factor in biological damages caused by oxidative stress. This assay is known to

give reliable information concerning the antioxidant ability of the tested

compounds.

The effects of methanol and aqueous extracts of A.longifolia and P.daemia

was evaluated for its antioxidant activity on different in vitro models like DPPH

radical scavenging activity and ferric reducing assay in a concentration dependent



69

manner (Fig. 5.1 - 5.4; Tables 5.1 - 5.4). The extracts of all the tested extracts

possessed antioxidant properties, but to varying degrees, ranging from 6.41 to

83.90%. Using the crude extracts, generally methanol extract showed better DPPH

scavenging activity than aqueous extract. A maximum scavenging activity was

offered by methanol of A.longifolia (83.90 %) (Table 5.1; Fig. 5.1) and P.daemia

(81.61%) (Table 5.2; Fig. 5.2), followed by aqueous extracts of A.longifolia

(70.11 %) and P.daemia (65.06%).

Reducing power assay

The reducing power assay of methanol and aqueous extracts of A.longifolia

and P.daemia was given in Table 5.3 and 5.4, which was found to be ranged from

0.090 - 0.842%. The reducing power of the crude extracts increased in a

concentration dependent manner. Among the two different species the A.longifolia

(0.842 0.02) have exhibited the highest rate of reduction of ferric ions (Table 5.3

& 5.4; Fig. 5.3 & 5.4).

Quantitative estimation total phenols and flavonoids

The crude methanol and aqueous extracts were prepared to examine the

antioxidant activity and concentrations of phenols and flavonoids. The extraction

solvents of different polarity were used to extract the active substances of different

polarity. The concentration of phenols in the examined crude extracts using the

Folin-Ciocalteu reagent was expressed in terms of gallic acid equivalent

(Table 5.5; Fig. 5.5).



70

Table 5.1 Antioxidant activity of A. longifolia (Linn.) Nees. (DPPH assay)

Concentrations (µg/ml)

Antioxidant activity (%) Standard (Ascorbic acid) Methanol Aqueous

1000 83.90 0.030 70.11 0.010

79.0 0.03

500 70.80 0.050 64.05 0.350

250 58.33 0.025 50.47 0.030

125 43.27 0.020 34.91 0.050

62.5 31.49 0.017 20.31 0.011

31.25 16.70 0.120 6.41 0.030

Each Value is SEM 5 individual observations



71

Fig. 5.1 Antioxidant activity of A. longifolia (Linn.) Nees. (DPPH assay)



72

Table 5.2

Antioxidant activity of P. daemia (Forsskal) Chiov. (DPPH assay)


Antioxidant activity (%) Standard (Ascorbic acid) Methanol Aqueous

1000 81.61 0.07 65.06 0.15

79.0 0.03

500 69.51 0.17 49.51 0.01

250 56.08 0.02 35.25 0.01

125 41.83 0.05 29.68 0.01

62.5 32.45 0.03 19.51 0.015

31.25 17.86 0.05 10.96 0.17




73

Fig. 5.2 Antioxidant activity of P. daemia (Forsskal) Chiov. (DPPH assay)



74

Table 5.3 Ferric Reducing capacity of A. longifolia (Linn.) Nees.


Ferric Reducing activity (%) Standard (Ascorbic acid) Methanol Aqueous

1000 0.842 0.02 0.727 0.07

0.689 0.05

500 0.623 0.01 0.630 0.04

250 0.502 0.01 0.524 0.03

125 0.356 0.11 0.401 0.04

62.5 0.268 0.02 0.220 0.04

31.25 0.110 0.011 0.104 0.02




75

Fig. 5.3 Ferric Reducing capacity of A. longifolia (Linn.) Nees.



76

Table 5.4 Ferric Reducing capacity of P. daemia (Forsskal) Chiov.


Ferric Reducing activity (%) Standard (Ascorbic acid) Methanol Aqueous

1000 0.768 0.05 0.741 0.15

0.689 0.05

500 0.561 0.01 0.596 0.01

250 0.418 0.01 0.395 0.01

125 0.342 0.05 0.219 0.05

62.5 0.291 0.02 0.169 0.15

31.25 0.096 0.05 0.090 0.01




77

Fig. 5.4 Ferric Reducing capacity of P. daemia (Forsskal) Chiov.



78

Table 5.5 Total phenol and Flavonoids contents in the crude extracts of A. longifolia (Linn.) Nees.

and P. daemia (Forsskal) Chiov.

Medicinal plants Total phenols content* (mg/GAE/g dry material)

Methanol Aqueous

A. longifolia 246.14 0.01 168.01 0.29

P. daemia 218.40 0.12 144.61 0.21

Flavonoid content* (mg/QE/g dry material)

Methanol Aqueous A. longifolia 104.20 0.01 57.01 0.01

P. daemia 88.01 0.12 51.13 0.11 *Each value in the table was obtained by calculating the average of three analyses standard deviation.



79

Table 5.5

Total phenol and Flavonoids contents in the crude extracts of A. longifolia (Linn.) Nees. and P. daemia (Forsskal) Chiov.



80

The concentrations of phenols in the examined crude extracts ranged from

144.61 to 246.14 mg/g. The high concentration of phenols was measured in

methanol extracts of A.longifolia. The concentration of flavonoids in crude

methanol and aqueous extracts were determined using spectrophotometric method

with aluminium chloride. The summary of quantities of flavonoids identified in

the tested extracts is shown in Table 5.5; Fig. 5.5. The concentrations of

flavonoids in methanol and aqueous extracts ranged from 51.13 to 104.20mg/g.

High concentrations of flavonoids were measured in methanol extracts of

A.longifolia. The lowest flavonoid concentration was measured in aqueous of

P.daemia extract.

5.5 DISCUSSION

The various diseases in the world are due to the production of free radicals.

These free radicals exert harmful effects when it reacts with important cellular

components like proteins, DNA and cell membrane (Mantena et al., 2008).

However, an overload of these radicals had been linked to certain chronic diseases

of heart, liver and some form of cancers and diabetes (Prakash et al., 2007).

Antioxidants that can neutralize free radicals may therefore be used to protect the

human body from diseases and retard rancidity in foods consumed by humans

(Leong and Shui, 2002). It is believed that higher intake of antioxidant rich food is

associated with decreased risk of degenerative diseases particularly cardiovascular

disease and cancer. Several research studies have demonstrated that herbal plant

contain diverse classes of compounds such as polyphenols, alkaloids, tannins and

carotenoids (Zheng and Wang, 2001). In the present study, the antioxidant activity



81

of A. longifolia and P. daemia leaves were evaluated on different methods like

DPPH scavenging assay, reducing power assay, total phenol and flavonoids

content of the extracts.

DPPH assay

DPPH is a very stable free radical, unlike in vitro generated free radicals,

such as hydroxyl radical and superoxide anion, DPPH has the advantage of being

unaffected by certain side reactions, such as metal-ion chelation and enzyme

inhibition, brought about by various additives. Freshly prepared DPPH solution

exhibited a deep purple colour with an absorption maximum at 517 nm. This

purple colour generally fades/disappears when antioxidant molecules can quench

DPPH free radicals (that is by providing hydrogen atoms or by electron donation,

conceivably via a free-radical attack on the DPPH molecule) and converts them

into a colourless/bleached product (that is 2, 2-diphenyl-1- hydrazine, or a

substituted analogous hydrazine), resulting in a decrease in absorbance at 517 nm

band (Suhanya Parthasarathy et al., 2009). The extracts of all the tested extracts

possessed free radical scavenging properties, but to varying degrees, ranging from

6.41 to 83.90% DPPH scavenging. A maximum scavenging activity was offered

by methanol of A.longifolia (83.90 %) and P.daemia (81.61%), followed by

aqueous extracts of A.longifolia (70.11 %) and P.daemia (65.06%) (Table 5.1 -

5.2). The present result is in augment with that of the previous reports of Ruan et

al., 2008; Chatterjee et al., 2012 and Har & Ismail, 2012 on Syzygium cumini root,

Eugenia jambolana seed extract and S.polyanthum leaf extracts.



82

Ferric reducing capacity

Reducing power assay is often to evaluate the ability of natural antioxidant

to donate electron or hydrogen (Dorman et al., 2003). Samples with high reducing

power were reported to have a better ability to electrons. It has been widely

accepted that the higher level of absorbance at 700 nm indicates greater reducing

power of the test samples (Duh et al., 1997).

In the present study the reducing power of A.longifolia and P.daemia

extracts were compared with the ascorbic acid as reference antioxidant. The

reducing power is increased with increasing concentrations in all the samples but

the leaf methanolic extract has exhibited once again the significant effect in

comparison with aqueous extracts.

Many studies indicated that only polar extracts of plants showed effective

antioxidant activity and some researchers further proved that moderate polarity

extracts are more potent even if their total antioxidant recovery from the plant is

not high (Wangensteen et al., 2004). The reducing power assay of methanol and

aqueous extracts of A.longifolia and P.daemia was given in Tables 5.3 and 5.4,

which was found to be ranged from 0.090 - 0.842% of the dried powder. Both of

the plant extracts possessed the ability to reduce iron (III) ions. This suggests that

the leaf extracts are an electron donor and could neutralize the free radicals (Zhu

et al., 2001). Among the two different species the A.longifolia (0.842 0.02) have

exhibited the highest rate of reduction of ferric ions. These findings supported the

investigation of Ruan et al. (2008) on Syzygium cumini leaf extracts.



83

Phenols and Flavonoids

Plant materials containing phenolic constituents are increasingly of interest

for probing as they retard oxidative degradation of lipids and thereby improve the

quality and nutritional value of food. The importance of the antioxidant

constituents of plant material in the maintenance of health and protection from

coronary heart disease and cancer has been well recognized (Khalil et al., 2007).

It has been recognized that flavonoids show antioxidant activity and their effects

on human nutrition and health are considerable. The mechanisms of action of

flavonoids are through scavenging or chelating process (Kessler et al., 2003).

Phenolic compounds are a class of antioxidant agents which act as free radical

terminators (Shahidi and Wanasundara, 1992). The high phenol and flavonoids

contents of crude extracts of A.longifolia and P.daemia may cause high

antioxidant activity. The concentrations of phenols in the examined crude extracts

ranged from 144.61 to 246.14 mg/GAE/g dry material. The high concentration of

phenols was measured in methanol extracts of A.longifolia. The extracts obtained

using more polar solvents had higher concentrations of phenols while the extracts

obtained using low polar solvents contained small concentrations (Canadanovic et

al., 2008). The summary of quantities of flavonoids identified in the tested

extracts is shown in Table 5.5. The concentrations of flavonoids in methanol and

aqueous extracts ranged from 51.13 to 104.20 mg/QE/g dry material. The

concentration of flavonoids in the extracts depends on the polarity of solvents and

the type of plant material used for the extractions. The concentration of flavonoids

in plant extracts depends on the polarity of solvents used in the extract preparation

(Min and Chun-Zhao, 2005).


84

Chapter-VI

ANTIDIABETIC ACTIVITY

. .

6.1 INTRODUCTION

Diabetes mellitus (DM) is a common metabolic disease characterized by

elevated blood glucose levels, resulting from absent or inadequate pancreatic

insulin secretion, with or without current impairment of insulin action. Reports

from the WHO indicate that diabetes mellitus is one of the major killers of our

time, with people in Southeast Asia and the Western Pacific being most at risk

(Firdous et al., 2009).

There are an estimated 246 million people with diabetes in the world, of

whom about 80% reside in developing countries. Although diabetes is often not

recorded as the cause of death, globally, it is believed to be the fifth leading cause

of death in 2000 after communicable diseases, cardiovascular disease, cancer and

Chapter - VI Antidiabetic Activity


85

injuries (Khan et al., 2010). Almost 80% of deaths related to diabetes occur in low

and middle income countries. Although diabetes is sometimes considered a

condition of developed nations, the loss of life from premature death among

persons with diabetes is greatest in developing countries.

Types

Diabetes can be divided into two main groups based on their requirements

of insulin: insulin dependent diabetes mellitus (Type 1/IDDM) and non-insulin

dependent diabetes mellitus (Type 2/NIDDM). However, other types of diabetes

have also been identified. Maturity Onset Diabetes of the Young (MODY) is now

classified as Type 3 and gestational diabetes classified as Type 4.

Type 1 diabetes or IDDM

Type 1 diabetes mellitus is characterized by loss of the insulin-producing

beta cells of the islets of Langerhans in the pancreas leading to insulin deficiency.

This type of diabetes can be further classified as immune-mediated or idiopathic.

The majority of type 1 diabetes is of the immune-mediated nature, where beta cell

loss is a T-cell mediated autoimmune attack (Rother, 2007). There is no known

preventive measure against type 1 diabetes, which causes approximately 10% of

diabetes mellitus cases in North America and Europe. Most affected people are

otherwise healthy and of a healthy weight when onset occurs. Sensitivity and

responsiveness to insulin are usually normal, especially in the early stages. Type 1

diabetes can affect children or adults but was traditionally termed ‘juvenile

diabetes’ because it represents a majority of the diabetes cases in children.



86

In Type 1 diabetes, insulin deficiency provokes high blood glucose levels

and alterations in lipid metabolism. The evolution of this disease may be

associated with the development of premature micro and macrovascular

complications; the pathogenesis of which may be linked to oxidative stress

(Giugliano et al., 1996; Rabinovitch et al., 1996). Increased ROS generation may

contribute to beta cell damage and vascular dysfunction through various

mechanisms (Baynes, 1991; Tesfamariam, 1994). In diabetic children, puberty

may trigger microvascular complications that may later be the major cause of

tissue damage, disability and death. Although the mechanism of glucose toxicity is

unknown, recent in vitro and whole animal studies have implicated ROS, which

promote the formation of cytotoxic lipid peroxides (Bottino et al., 2002; Pileggi et

al., 2001). The beta cell destruction by ROS, whether induced by oxidants given

exogenously or elicited by cytokines, is a process that occurs through changes in

the apoptotic and antiapoptotic balance (Chervonsky et al., 1997; Itoh et al.,

1997). There appears to be an intrinsic cardiovascular sensitivity to oxidative



87

stress in diabetic rats and in nonobese diabetic (NOD) mice, a property that may

extend to human patients.

Type 2 Diabetes or NIDDM

Type 2 diabetes mellitus is a common disorder, characterized by

hyperglycemia, insulin resistance and relative impairment in insulin secretion.

The defective responsiveness of body tissues to insulin is believed to involve the

insulin receptor. However, the specific defects are not known. Diabetes mellitus

due to a known defect are classified separately. Type 2 diabetes is the most

common type. In the early stage of type 2 diabetes, the predominant abnormality

is reduced insulin sensitivity. At this stage hyperglycemia can be reversed by a

variety of measures and medications that improve insulin sensitivity or reduce

glucose production by the liver. As the disease progresses, the impairment of

insulin secretion occurs, and therapeutic replacement of insulin may sometimes

become necessary in certain patients.



88

Over seven percent of adults in the United States are known to have

diabetes and the number continues to rise every year (Abraham et al., 2008). The

spectacular increase in prevalence of type 2 diabetes in the past decade, in large

part is linked to the trends in obesity and physical inactivity (Sullivan et al., 2005).

Abdominal obesity, in particular, is associated with resistance to the effects of

insulin on peripheral glucose and fatty acid utilization. Insulin resistance plays a

major role in the pathogenesis of type 2 diabetes and is often accompanied by

other conditions, including hypertension, high serum LDL, low serum HDL and

high serum triglyceride levels, which promote the development of atherosclerotic

cardiovascular disease (DeFronzo and Ferrannini, 1991). The majority of patients

with diabetes die of cardiovascular events and controlling risk factors leading to

atherosclerosis is a major challenge in clinical practice (Fonseca, 2007).

Gestational diabetes mellitus (GDM)

GDM resembles type 2 diabetes in several respects, involving a

combination of relatively inadequate insulin secretion and responsiveness. It

occurs in about 2% -5% of all pregnancies and may improve or disappear after

delivery. Gestational diabetes is fully treatable but requires careful medical

supervision throughout the pregnancy. About 20% -50% of affected women

develop type 2 diabetes later in life. Even though it may be transient, untreated

gestational diabetes can damage the health of the fetus or mother. Risks to the

baby include macrosomia (high birth weight), congenital cardiac and central

nervous system anomalies, and skeletal muscle malformations. Increased fetal

insulin may inhibit fetal surfactant production and cause respiratory distress



89

syndrome. Hyperbilirubinemia may result from red blood cell destruction. In

severe cases, perinatal death may occur, most commonly as a result of poor

placental perfusion due to vascular impairment. Labor induction may be indicated

with decreased placental function. A cesarean section may be performed if there is

marked fetal distress or an increased risk of injury associated with macrosomia,

such as shoulder dystocia.

The Role of Insulin in Diabetes

Our body breaks food in to glucose and insulin in the blood stream helps

cells to take up glucose and use it as fuel. Glucose levels increase after a meal, but

quickly return to normal as cells remove excess glucose from the blood stream.

This normal process of using glucose for energy falls apart in diabetes. In a

diabetic person, either the pancreas cells do not make insulin or cells of the body

cannot use the insulin properly. Without insulin, cells are unable to take up

glucose. Instead, glucose builds up in the bloodstream where it can cause damage

to eyes, nerves and blood vessels as well as cause a person to feel thirsty or urinate

frequently.

Treatment of Diabetes

Control of diabetes normally involves exercise, diet and chemotherapy. At

present the treatment of diabetes mainly involves a sustained reduction in

hyperglycemia by the use of synthetic drugs. Based on the mechanism, anti-

diabetic drugs can be mainly divided into insulin, insulin-secretagogues, insulin

sensitivity improvement factor, insulin-like growth factor, aldose reductase



90

inhibitor, alpha-glucosidase inhibitors and protein glycation inhibitor (Li et al.,

2004). Insulin and various oral anti-diabetic agents such as sulfonylureas,

biguanides, thiazolidinediones, and D-phenylalanine derivatives, alpha glucoside

inhibitors are the olny option available for the management of diabetes mellitus.

Metformin, a less toxic biguanides and potent oral glucose-lowering agent, was

developed from Galega officianalis and used to treat diabetes. Out of dozens of

oral medications for diabetes, only one medication (metformin) is approved for

use in children and it has been originated from an herb (Jarald et al., 2008).

Conventional treatment

Diabetes is a multidimensional disorder and its management needs firm

adherence to the prescribed treatment plan. The contemporary treatment of

diabetes is focused on suppressing and controlling blood glucose to a normal

level. The common agreement on management of type II diabetes is

transformation in lifestyle along with appropriate diet and weight control.

However, antidiabetic drugs are needed as these measures cannot provide

satisfactory results. Antidiabetic drug therapy includes insulin injections and oral

hypoglycemic drugs. These drugs act by various mechanisms to control the blood

glucose level. However, many side-effects such as hypoglycemia, lactic acid

intoxication and gastrointestinal upset, etc. have been reported in patients

(Li et al., 2004). Because the antidiabetic medication may sometimes involve

prescribing more than one drug at the same time, which can augment the severity

of these side-effects, efforts are being made to find a suitable antidiabetic and

antioxidant therapy.



91

Need for natural antidiabetic study

Several hypoglycemic agents have been reported to produce serious adverse

side effects such as liver problems, lactic acidosis and diarrhoea (Rajalakshmi et

al., 2009). In addition, they are not suitable for use during pregnancy. Therefore,

the search for more effective agents with their ready availability, low cost and less

adverse side effects from plant source has continued to be an important area of

research. So there is a demand for new natural compounds for the treatment of

diabetes (Sherma et al., 2010).

Treatment of Diabetes in traditional medicine (Medicinal plants)

The control of blood glucose in diabetic patients was achieved mainly by

the use of oral hypoglycemic/antihyperglycemic agents and insulin. However, all

these treatments have limited efficacy and have been reported to be associated

with undesirable side effects (Gayathri and Kannabiran, 2008). In order to

overcome the side effects associated with diabetes, interest has been shifted to use

of other alternative medicine. Traditional medicines and extracts from medicinal

plants have been extensively used as alternative medicine for better control and

management of diabetes mellitus. Medicinal plants are continued to be a powerful

source for new drugs, now contributing about 90% of the newly discovered

pharmaceuticals (Mosh, 2005). Traditional medicine provides better health

coverage for 80% of the world population, especially in the developing countries

(Srinivasan, 2005).



92

Many ethnobotanical surveys on medicinal plants used by the local

population have been performed in different parts of the world including Morroco,

Saudi Arabia, India, Taiwan, and Trinidad and Tobogo. According to the WHO,

as many as 80% of the world's people depend on traditional medicine for their

primary healthcare needs. There are considerable economic benefits in the

development of indigenous medicines and in the use of medicinal plants for the

treatment of various diseases (Azaizeh et al., 2003).

Around 70,000 plant species, from lichens to flowering trees have been

used at one time or another for medicinal purposes. The earliest mentions of the

medicinal use of plants have been found in “Rig Veda”, which was written

between 4000 and 1600 BC. All five traditional system of medicine viz.,

Ayurveda, Siddha, Unani, Tibetan and Homeopathy mention about 200 plant

species of medicinal value. Chinese pharmacopoeia lists 5,700 traditional

medicines.

In India, it is reported that traditional healers use 2500 plant species and

100 species of plants serve as regular sources of medicine. During the last few

decades there has been an increasing interest in the study of medicinal plants and

their traditional use in different parts of the world (Muthu et al., 2006). Since

ancient times a number of herbal medicines have been used in the treatment of

diabetes. There is increasing demand by patients to use the natural products with

anti-diabetic activity. Herbal medicines for diabetes can be classified into four

categories according to their mode of action:

Drugs acting like insulin.



93

Drugs acting on insulin secreting beta cells.

Drugs acting by modifying glucose utilization.

Drugs acting by miscellaneous mechanisms.

Several plant species have been described as hypoglycaemic. These include

Opuntia streptacantha, Trigonella foenum graecum, Momordica charantia, Ficus

bengalensis, Polygala senega, Gymnema sylvestre, Allium sativum, Citrullus

colocynthis, Commiphora myrrha, Nigella sativa, Trigonella foenum, Aloe vera,

Artemisia absinthium and other species are less well known.

Action sites of herbs in diabetes treatment


Extracts of ripe leaves, tender leaves, fruits and flowers of Azadirachta

indica (neem) have been reported to possess anti-diabetic and antiviral activity



94

(Rao et al., 1994; Bhattacharji, 1953). The presence of hypoglycemic property in

leaves and flowers of Vinca rosea has been reported (Chopra, 1956). A mild

hypoglycemic effect of crude aqueous extract of Vinca rosea whole plant except

roots on alloxan induced diabetic rabbits was reported by Shrotri et al. (1963).

Barleria lupulina leaves are found to be used by the rural people of West Bengal

for the treatment of snake bites, diabetes mellitus and mental illness (Chopra,

1969).

There are reports about the hypoglycemic action of Allium cepa (Liliaceae),

Allium sativum (Liliaceae), Cuminum nigrum (Umbelliferae), Psidium guajava

(Myrtaceae) and Cucurbita ficifolia (Cucurbitaceae) (Akhtar and Ali, 1985;

Roman - Ramos et al., 1991). Alcoholic extract of Vinca rosea leaves cause

significant reduction in blood sugar level in streptozotocin induced diabetic rats

(Chakraberty and Poddar, 1984).

A promising hypoglycemic activity was observed in animal experiments

with an ethanolic extract of the root bark of Bumelia sartorum mart, a plant

belonging to the sapotaceae family (Almeida, 1982: Almeida et al., 1985).

Bauhinia candicans is used in Chilean folk medicine for the treatment of diabetes

mellitus (Fuentes et al., 2004). Ajabnoor (1990) and Ghannam et al. (1986) have

reported the antidiabetic activity of Alove extracts. It has been shown that fruits of

Momordica charantia controlled the hyperglycaemia in non-insulin dependent

diabetic patients (Welihinda et al., 1986). According to Baily and Flatt (1986), it

is possibly the world’s fastest growing metabolic disease and as knowledge of the



95

heterogeneity of this disorder increase, so does the need for more appropriate

therapies.

Artemisia herba-alba is reported to have hypoglycemic effect (Twaij and

Badr, 1998). Many plants have been studied for their hypoglycaemic and insulin

release stimulatory effects (Ivorra et al., 1989). There were positive results

concerning the decrease of glucose, triglycerides and cholesterol in blood caused

by Opuntia streptacantha (Cactaceae) (Ibangez Camacho and Roman Ramos,

1979), (Fratimunari et al., 1988). In hyperglycemic rabbits Salvia fruticosa

administration produces a statistically significant hypoglycemic effect (Perfumi et

al., 1991). The antidiabetic activity has also been recorded in Prunus davidi ana

(Choi et al., 1991) and Cinnamomum tamala (Sharma et al., 1996). Some

unsaturated triterpene acids isolated from Bumelia sartorum have been reported to

exhibit significant hypoglycemic activity (Naik et al., 1991).

Phyllanthus amarus has been used as a hypoglycemic agent in traditional

medicine to control non-insulin dependent diabetes mellitus (Sivarajan and

Balachandran, 1994). The volatile oil extracted from the leaves of Rosmarinus

officinalis produce a significant change in plasma glucose and serum insulin levels

(Al-Hader et al., 1994). The powder of Caesalpinia bonducella seeds form a

common household remedy for treatment of diabetes in the Nicobar Island of

India (Rao et al., 1994). Ragunathan and Sulochana (1994) reported that the

Hibiscus vitifolius flowers have hypoglycemic activity in female rats. The aerial

parts of Artemisia pallens (endemic to south India) have antidiabetic properties in

alloxan- induced diabetic rats (Subramaniam, 1996). Orthosiphon stamineus



96

exhibited the anthihyperglycemic property in alloxan induced diabetic rats

(Mariam, 1996).

Caesalpina bonducella seeds possess antidiabetic principle and can be

useful for treatment of diabetes. Both aqueous extract and ethanolic extract of

Caesalpinia bonducella seeds exhibited significant hypoglycaemic and

antihyperglycaemic activities in normal and STZ hyperglycaemic rats (Sharma,

1997). Preliminary investigation of Hibiscus rosa sinensis ethanolic leaf extract

showed hypoglycemic activity in male albino rate (Sachdewa and Khemani,

1999). Globularia alypum showed the hypoglycemic activity in alloxan rats (Skim

et al., 1999).The leaves of Gymnema sylvestre were studied for its hypoglycaemic,

hepatoprotective and antiviral properties (Siddiqui et al., 2000).

In a study by Pepato et al. (2001), streptozotocin-diabetic rats were treated

for 17 days with a decoction of Eugenia jambolana (Myrtaceae) leaves (15%,

w/v) as a substitute for water. Body weight, food and fluid intake, urine volume,

glycemia, urinary glucose and urea were evaluated every 5 days. The animals

were sacrificed by decapitation and blood samples collected for the determination

of glycemia, serum cholesterol, HDL-cholesterol, triglycerides and angiotensin-

converting enzyme. The weights of adipose and muscle tissues were also

determined. There were no statistically significant differences between treated and

untreated rats for any of the biochemical or physiological parameters. They

concluded finally that, at least in this experimental model, Eugenia jambolana leaf

decoction has no antidiabetic activity. The aqueous leaf extract of Hibiscus rosa-

sinensis showed hypoglycemic effect in glucose and streptozotocin induced



97

hyperglycemic rats. Ghosh and Suryawanshi (2001) reported that the Vinca rosea

extracts controlled the diabetic state; the aqueous extract of Vinca rosea leaf and

flower has prophylactic activity against necrotic actions of alloxan monohydrate.

However treatment of diabetes required the use of these extract for a longer time.

In the light of 222 citations from the Medline database, this is an exhaustive

presentation of research into 45 plants used in traditional Indian medicine that

have been found to have potential in treating diabetes mellitus. Research also

includes complications such as diabetic nephropathy and neuropathy, fructose

induced insulin resistance, and cataract. Ethno botanical information identifies

about 800 Indian plants (of more than 45,000 species found in India), which may

have antidiabetic potential. For each plant, the authors discuss botanical

characteristics, range, and habitat; traditional uses in India and other parts of the

world; and research findings, including plant parts used, methods of extraction and

preparation, and specific compounds isolated and tested for their antidiabetic

effects (Grover et al., 2002).

Studies showed that Phyllanthus amarus extract reduces blood sugar in

alloxan diabetes rats and the extract was found to scavenge oxygen free radicals

in vitro (Raphacl et al., 2002). Alcohol extract of Cassia kleinii is an attractive

material for the development of a potent phytomedicine for diabetes. The extract

showed marked antihyperglycaemic effect in the alloxan - induced severe diabetic

rats (Babu et al., 2002). The significant and consistent hypoglycaemic effect of

Artemisia herba alba in diabetic rabbits and rats within 2-4 weeks, indicates that

the plant extract acts by stimulating glucose utilization by peripheral tissues (Loai



98

Al-Shamanoy et al., 1994). The plant Abroma augusta is effective in the treatment

of diabetes and in amenorrhoea (Eshrat Halim, 2001).

Crude saponins fractions from Gymnema sylvestre (Gurmar in Hindi) and

other saponins from several plant extract have been shown to possess potent S-

GLUT-I-mediated inhibition of glucose and antihyperglycemic activity. Extracts

of this plant have been reported to possess variety of actions related to the

antidiabetic properties such as reducing insulin requirements by possibly

enhancing endogenous insulin availability, improving vitiated blood glucose

homeostasis, better controlling of hyperlipidemia associated with diabetes, and

reducing amylase activity (Agarwal, 2003). Antidiabetic property is proved in

some medicinal plants like Aegle marmels, Gymnema sylvestre, Eugenia

jambolana, Momordica charantia, Azadirachta indica, Cassia auriculata,

Withania somnifera and Curcuma longa, a polyherbal formulation “Dianex”

(Mutalik, 2003).

Oral administration of 1g/kg stem bark extract of Bauhinia monandra to

groups of glucose - loaded and alloxan diabetic rats gave a significant antidiabetic

activity (P<0.05) compared to control rats. Peak effect (35.6% inhibition) was

observed at 180mins. A peak response of 36.1% reduction in blood glucose was

observed on 6th day of treatment, which was significant at P<0.05 (Abo and

Jimoh, 2004). Regarding reported antidiabetic effect of Satureja khuzestanica

essential oil (SKEO) and important role of liver on body glucose metabolism by

glycogenolysis and gluconeogenesis, effect of SKEO treatment on rat hepatic key



99

enzymes of glycogenolysis and gluconeogenesis in vivo was examined by Saadat

et al. (2004).

Annona squamosa (Annonaceae) is a popularly known medicinal plant. The

water extract of A. squamosa leaves seems to be useful in controlling elevated

blood glucose levels in diabetes induced by both the agents, alloxan and STZ in

two species of animals, namely rabbits(non-rodents)and rats (rodents) (Gupta,

2005). The leaf extract of Boerhaavia diffusa L. possess hypoglycemic effects

(Chude, 2001). The alkaloid and sterols are responsible for the antioxidant and

antidiabetic activity of B. diffusa leaves (Amarnath Satheesh and Pari, 2004). The

methanolic extract of Bauhinia candicane showed hypoglycemic activity in

normal and alloxan induced diabetic rabbits (Fuentes, 2004).

The methanolic extract of aerial parts of Barleria lupulina exhibited

antidiabetic potential in rats (Suba et al., 2004). The hypoglycaemic effect of

different compounds, obtained from Ficus bengalensis has been reported by

different workers (Geetha et al., 1994; Cherian and Augusti, 1993). The water-

soluble fraction of the ethanol extract of Fiscus hispida decreases blood glucose

level in normal and alloxan induced diabetic albino rats (Ghosh et al., 2004).

Shankar et al. (2005) evaluated the antidiabetic activity of Ginkgo biloba to probe

into its mechanism of action. Albino Wister rats with streptozotocin-induced

diabetes were divided into 4 groups of 10 each. Gum acacia, troglitazone 36

mg/kg, Ginkgo biloba 50 mg/kg and 100 mg/kg, were administered to group I

(control), group II (standard), group III and group IV respectively. After 10 and 15

days of drug administration fasting blood sugar (FBS), blood glutathione (GSH)



100

and serum ceruloplasmin were estimated. Ginkgo biloba in a high dose of 100

mg/kg produced a significant reduction in FBS by 31% and increase in blood GSH

(57.6%) that is however much less than the fall in FBS produced by troglitazone

(47%). However treatment with troglitazone and Ginkgo biloba at both doses did

not alter the serum ceruloplasmin levels significantly.

Antia et al. (2005), reported that Adult albino Wistar rats (150-180 g) of

either sex obtained from University of Uyo, animal house, Uyo, Nigeria were used

for the antidiabetic study. Alloxan monohydrate (BDH) 150 mg/kg, body weight

was dissolved in normal saline and injected intraperitoneally after 18 h fasting to

induce hyperglycemia. The result of the phytochemical screening of the aqueous

leaf extract of Persea americana revealed that the extract sustained significant

(P < 0.01) reduction in the blood glucose levels of the treated rats throughout the

period of treatment. Saravanan and Pari (2005), investigated the effect of Diasulin,

a poly herbal drug composed of ethanolic extract of ten medicinal plants on blood

glucose, plasma insulin, tissue lipid profile, and lipidperoxidation in alloxan-

induced diabetes. Treatment with Diasulin and glibenclamide resulted in a

significant reduction of blood glucose and increase in plasma insulin. Diasulin

also resulted in a significant decrease in tissue lipids and lipid peroxide formation.

The effect produced by Diasulin was comparable with that of glibenclamide.

There is a growing interest in herbal remedies because of their effectiveness

minimal side effects in clinical experience and relatively low costs. Herbal drugs

or their extracts are prescribed widely, even when their biological active

compounds are unknown. Even the WHO approves the use of plant drugs for



101

different diseases, including Diabetes mellitus. Therefore, studies with plant

extracts are useful to know their efficacy and mechanism of action and safety

(Gupta 2005).

Active hypoglycemic constituents from plants

Many active compounds have been isolated from the plant and herb species

of India. These active principles are dietary fibres, alkaloids, flavonoids, saponins,

amino acids, steroids, peptides, terpenoids, glycosides and poly saccharides. These

have produced potent hypoglycemic, anti-hyperglycemic and glucose suppressive

activities (Saxena et al., 2006; Dineshkumar et al., 2010). Particularly, schulzeines

A, B, and C, radicamines A and B, 2,5-imino-1,2,5-trideoxy-L-glucitol,

homofuconojirimycin, myrciacitrin IV, dehydrotrametenolic acid, corosolic acid

(Glucosol™), 4-(α-rhamnopyranosyl)ellagic acid, and 1,2,3,4,6- pentagalloyl

glucose have shown significant antidiabetic activities (Jung et al., 2006).

The above effects achieved by either insulin release from pancreatic β-cells,

inhibited glucose absorption in gut, stimulated glycogenesis in liver or increased

glucose utilization by the body (Grover et al., 2002; Saxena and Vikram, 2004).

These compounds also exhibited their antioxidant, hypolipidemic, anticataract

activities, restored enzymatic functions, repair and regeneration of pancreatic

islets and the alleviation of liver and renal damage (Mukherjee et al., 2006). Some

active constituents obtained from plants possess insulin like activity and could

provide alternate for insulin therapy.



102

Flavonoids, especially quercetin, have been reported to possess antidiabetic

activity. Some flavonoids, most of which have quercetin and kaemferol as their

parent nucleus, such as rutin (quercetin-3-O-rutinoside), kaemferol-3-O-glucoside

were isolated from Folium eriobotryae. Some reports showed that Folium

eriobotryae has hypoglycemic action (Kawahara et al., 2002; Louati et al., 2003).

Vessal et al. (2003) reported that quercetin brings about the regeneration of

pancreatic islets and proprably increases insulin release in strptozotocin-induced

diabetic rats. Hif and Howell (1985) reported that quercetin stimulate insulin

release and enhanced Ca2+ uptake from isolated islets cell which suggest a place

for flavonoids in noninsulin- dependent diabetes. Recent studies showed that the

flavonoid fraction has the highest hypoglyceamic activity. The value of any

hypoglycaemic agent depends not only on its hypoglyceamic potency but also on

its lack of toxicity (Chandrika et al., 2006). A sulphur containing amino acid, S-

methyl cystein sulphoxide from onion showed potent hypoglycemic activity in

alloxan induced diabetic rats (Kumari et al., 1995).


Antidiabetic activity of Asteracantha longifolia and Pergularia daemia

were screened with the following standard methods.

Acute toxicity test

Acute toxicity study was carried out on both plant extracts using Swiss

Albino rats. The rats were fasted overnight and the weight of each rat was

recorded just before use. Animals were divided rando mly into a control and six



103

treatment groups, each group consisting of five rats. Control group received only

the vehicle & each treatment group received orally the methanol and aqueous

extracts of the studied plants in a dose of 100, 200, 400, 800, 1200 and 1600

mg/kg. Animals were kept under close observation for 4 hours after administering

the extract (Burger et al., 2005), and then they were observed daily for three days

for any change in general behaviour and/or other physical activities.

Oral glucose tolerance test (OGTT)

After one week of treatment with the plant extracts, the animals were made

to fast for 12-14 hours. Their blood glucose level was measured and glucose

solution (2 g/kg body weight) was administered orally in a volume of 1 ml. Blood

samples were collected 30, 60 120 and 180 minutes after administration of glucose

in order to evaluate their blood glucose level (Kumar et al., 2006).

Analysing the antidiabetic effect using experimental animals

Chemicals used

Alloxan monohydrate was purchased from Sigma Chemical Company,

USA, and all the other chemicals and reagents used in the experiments were of

analytical grade and were obtained from BDH (England and India), E.Merck

(Germany), Sigma Chemical Company (U.S.A.), Sarabhai, M. Chemicals (India),

LOBA - Chemie Indo Austranol Co., (India).

Experimental fauna

Healthy adult Swiss Albino rats (both sexes) (Plates 6.1 & 6.2) weighing

about ~160-175 g were used, which were adapted to metabolic cages for 2 or 3



104

days maintained under standard conditions (12 h light/12 h dark cycle; 25 3°C;

35-60% humidity), and were fed with a standard rat pellet diet and water ad

libitum and their mean weight was observed 158 2 g. The study was conducted

at RVS Pharmaceutical College, Coimbatore Distrcit, Tamil Nadu. The animals

were used for this experiment with the permission of Institutional Animal Ethical

Committee, RVS College of Pharmaceutical Sciences, Sulur, Coimbatore, Tamil

Nadu,India(R.No.IAEC1012/C/06/CPSEA-Corres-2008-2009) (Refer Appendice).

Experimental Design

Rats, weighing 160-175 g, fasted overnight was used for induction of

diabetes. Rats were divided into two sets: Diabetic and non-diabetic. In the

diabetic set, 2 days after the induction of diabetes, animals were divided into

seven groups:

Group I : Normal control (Saline) (by using an intragastric catheter tube (IGC)

Group II : Diabetic control

Group III : Diabetic rats received A.longifolia Methanol extract (250 mg/kg bw)

for 21 days by IGC

Group IV : Diabetic rats received A.longifolia Aqueous extract (250 mg/kg bw)

for 21 days by IGC

Group V : Diabetic rats received P.daemia Methanol extract (250 mg/kg bw)

for 21 days by IGC

Group VI : Diabetic rats received P.daemia Aqueous extract (250 mg/kg bw) for

21 days by IGC



105

Group VII : Diabetic rats received Glibenclamide (10 mg/kg bw) daily orally for

for 21 days by IGC

Induction of Diabetes

The Group II, Group III, Group IV, Group V, Group VI and Group VII

animals were then anesthetized with Alloxan monohydrate, which was dissolved

in saline immediately before use, and injected intraperitoneally in a dose of

150mg/kg body weight. After 2 days, rats with moderate diabetes having

glycosuria indicated by Benedict’s qualitative test and moderate hyperglycemia

(200-280 mg/dl) were used for the experiment.

Oral drugs administration

Group III, IV, V, VI (Diabetes treated group) rats were assigned to be

treated with crude methanol and aqueous extracts (A.longifolia and P.daemia)

(250 mg/kg of bw) and the other to an untreated Group I (control group) every day

unto the final day of the experiment. Body weight, food and liquid intake, urine

volume, plasma and urinary glucose were measured every 7 days at about 9 a.m.

The rats were sacrificed by decapitation 21 days after Alloxan injection and

treatment with plant drugs (A.longifolia and P.daemia) when free running blood

was collected for the determination of plasma glucose, serum cholesterol, total

protein, urea, catalase, peroxidase, glycogen, SGOT and SGPT.

Estimation of Insulin (Anderson et al., 1993)

Serum Ultra-Sensitive Mouse Insulin was estimated by using commercial

diagnostic Kits (Crystal Chem, USA).



106

Principle

It is a solid phase enzyme linked immunosorbant assay (ELISA). The wells

are coated with monoclonal antibody with higher activity for insulin. When the

samples, and controls are incubated in the wells with enzyme conjugate, which is

another antibodies linked to horse radish peroxidase to form a sandwich complex

bound to the well. Unbound conjugate are then washed off with wash buffer. The

amount of bounded peroxidase is proportional to the concentration of insulin

present in the sample. Upon addition of the substrate and chromogen, intensity of

the color developed is proportional to the concentration of insulin in the samples.

Assay procedure

Secured the designed number of coated wells in the holder. Marked data sheet

with sample identification.

Dispensed 25 μl of serum sample, control and reference into the assigned

wells.

Dispensed 100 μl of enzyme conjugate into each well and mixed for 5 secs.

Incubated for 30 min. at 25º C.

Removed incubation mixture and rinsed the wells five times with washing

buffer.

Dispensed 100 μl of solution A and then 100 μl of solution B in to each well.

Incubated for 15 min. at room temperature.

Stop reaction by adding 50 μl of 1N sulphuric acid or 2N HCl to each well

and read O.D at 450 nm with a micro well reader.



107

Estimation of Blood Glucose

The blood glucose was estimated by the method of Sasaki et al. (1972).

Principle

Ortho toludine reacts with glucose in hot acetic acid solution to produce

blue color, which is measured at 630nm.

Reagents

1. Ortho toludine boric acid reagent: This reagent consists of 2.5g of thiourea

and 2.4g of boric acid in 100 ml of a mixture of water, acetic acid and ortho

toludine (distilled) in the ratio of 10:75:15.

2. Standard glucose: 100mg of glucose in 0.1% benzoic acid. 10 ml of the

above solution was diluted to 100 ml to give 100μg of glucose per ml.

Procedure

To 0.2 ml of serum added to 0.8 ml of 10% TCA mixed well and

centrifuged. 0.5 ml of the supernatant was taken. To this 2.0 ml of ortho toludine

reagent was added and heated in a boiling water bath for 15min along with

standard solution containing 20-100 μg of glucose. The blue colour developed was

read at 640nm. The result was expressed as mg/dl in serum.

Estimation of Serum Protein

The protein concentration of the serum was estimated by the method of

Lowry et al. (1951).



108

Principle

In alkaline solutions, protein forms a complex with copper ions and this

copper - protein complex reacts with Folin ciocalteau reagent to give a blue colour

due to the reduction of phosphomolybdate by thyrosine and tryptophan present in

the protein. The intensity of the colour is proportional to the concentration of

protein.

Reagents

Sodium hydroxide: 0.4 gm of sodium hydroxide was dissolved in 100 ml of

distilled water.

Reagent A: 2% sodium carbonate in 0.1 N sodium hydroxide (W/V) (2gm of

sodium carbonate was dissolved in 100 ml of 0.1N sodium hydroxide).

Reagent B: 0.5% copper sulphate in 1.35 % sodium potassium tartarate, 5 mg

of copper sulphate was dissolved in 1 ml of 0.35% sodium potassium tartarate

solution (1.35 gm of sodium potassium tartarate dissolved in 100 ml (W/V) of

water). This was prepared just before use.

Reagent C: This was prepared just before use by mixing 50 ml of reagent A

with 1 ml of reagent B.

Folin - Ciocalteau phenol reagent: 1 ml of Folin’s phenol reagent was

dissolved in 2 ml of distilled water. This was prepared just before use.

Standard: A standard solution of BSA containing 250 mg/ml was prepared in

0.1 N sodium hydroxide (12.5 mg of BSA was weighed and dissolved in 50

ml of 0.1 N sodium hydroxide using 50 ml standard flask).



109

Determination of standard curve

For plotting the standard curve, a set of standards were run, 25, 50, 75 to

250 mg of standard solutions were taken in a series of test tubes. The volume in

each tube was made up to 1 ml with distilled water. 5 ml of alkaline copper

reagent was added, mixed and allowed to stand for 10 minutes at room

temperature. 0.5 ml of Folin Ciocalteau Phenol reagent was then added to each

tube and was shaken well. The blue colour developed was read at 720 nm after 20

min. against a reagent blank in a spectrophotometer. The standard graph was

drawn by plotting the concentration of the standard solution on the y-axis and the

optical density on the x axis. A set of standards was run along with each set of

sample assays.

Procedure

0.01 ml of sample was taken and made up to 1.0 ml with distilled water. 5

ml of alkaline copper reagent was added, mixed and allowed to stand for 10

minutes at room temperature. 0.5 ml of Folin Ciocalteau Phenol reagent was then

added to each tube and was shaken well. The blue colour developed was read at

720 nm after 20 min. against a reagent blank in a spectrophotometer. The amount

of protein present in the sample by referring the standard graph and expressed as

gm/dl.



110

Estimation of Albumin and Globulin (Wolfson et al., 1948)

Principle

The blue colour developed by the aminoacids tyrosine and tryptophan

present in the protein by the biuret reaction of the protein with the alkaline cupric

tartarate are measured at 555 nm.

Reagents

Sulphate-sulphite solution: Weighed 208 gm of sodium sulphate (anhydrous)

and 70 gm of sodium sulphite (anhydrous) and dissolved with stirring in about

900 ml of distilled water, to which 2.0 ml of concentrated sulphuric acid was

added. Transferred to one-litre volumetric flask and made up to the mark with

distilled water. The pH as adjusted to 7.0.

Stock biuret reagent: Dissolved 45gm of Rochelle salt in 400 ml of

200 mmol/l NaOH and added 15gm of copper sulphate and mixed. 5gm of

potassium iodide was added and made up to a litre with 200 mmol/l NaOH.

Working biuret reagent: Diluted 20 ml of stock reagent to 100 ml with

200mmol/l NaOH.

Ether.

Stock standard solution: 100mg of bovine serum albumin was dissolved in 100

ml of saline.

Working standard: 1 ml of stock was diluted to 5 ml with distilled water.

Therefore 1 ml of this solution contains 200μg of protein.



111

Procedure

Total protein

Pipetted out 6 ml of sulphate-sulphite solution in a test tube and onto it

layered 0.4 ml of serum and mixed well. From the mixture, 2.0 ml were taken and

to it 5.0 ml of biuret reagent was added.

Albumin

Added about 3.0 ml of ether to the rest of the serum-sulphate mixture and

shaken 40 times, twice each second for 20 seconds. The tube was centrifuged for

5 min. After centrifuging, the tube was tilted and inserted a pipette into the clear

solution below the globulin layer and pipette out 2.0 ml. To this, 5.0 ml of biuret

reagent was added. A set of standards were taken, to this 6.0 ml of sulphate-

sulphite solution was added and mixed well. To 2.0 ml of this mixture, 5.0 ml of

biuret reagent was added. All the tubes were warmed at 37 ºC for 10 min. Allowed

to cool for 5 min. at room temperature and colour was read at 555 nm. The

difference between the amount of total protein and albumin gives the globulin.

The values in the serum were expressed as gm/dl.

Estimation of Urea

The serum urea was estimated by Varley (1976).

Principle

Diacetyl monoxime in the presence of acid, hydrolysis to produce the

unstable compound diacetyl. This reacts with urea to produce a yellow diazone



112

derivative. The color of this product becomes pink by addition of

thiosemicarbazide which is measured colorimetrically at 520nm.

Reagents

TCA, 10%

Stock Diacetylmonoxime, 25g/l

Stock Thiosemicarbazide 2.5g/l

Acid ferric chloride solution: Added 1.0 ml sulphuric acid to 100 ml of ferric

chloride solution containing 50 g/l in water.

Acid reagent: Added 10 ml of ortho phosphoric acid, 80 ml sulphuric acid and

10 ml acid ferric chloride solution to one litre of water and mixed.

Color reagent: To 300 ml acid reagent added 200 ml water, 10 ml stock

diacetylmonoxime and 2.5 ml thiosemicarbazide.

Stock urea standard: 5, 10, 15,20,30,40, and 50 mmol/l (30, 60, 90, 120, 180,

240 and 300 mg/100 ml).

Procedure

To 0.2 ml of serum added 1.0 ml water and 1.0 ml of 10%TCA. Mixed well

and centrifuged. 0.2 ml of the supernatant was taken and added 3.0 ml of color

reagent. At the same time took 0.2 ml of water bath for 20 min. Cooled to room

temperature and read the color developed at 520 nm within 15 min. The result was

expressed as mg/dl in serum.



113

Estimation of Glycosylated Haemoglopbin

Plasma was separated and cells were washed twice (0.154 M saline) and

stored at 20° C until Hb A1c concentrations were determined by the method of

Karunayake and Chandrasekharan (1985).

Estimation of Creatinine

The serum creatinine was determined using the Jaffe’s reaction by the

method of Owen et al. (1954).

Principle

Creatinine in a protein free supernatant of plasma or serum reacts with

alkaline picrate to form a colour complex whose intensity is measured at 510 nm.

Reagents

Sodium tungstate solution (5%): 5 gm of sodium tungstate was dissolved in

water and made up to 100 ml.

Sulphuric acid (2/3N): This is prepared by diluting 2 ml of conc. sulphuric acid

to 100 ml with distilled water. It was standardized against known sodium

hydroxide solution and adjusted, if necessary.

0.04 M picric acid solution

0.75 N sodium hydroxide

Standard solution: Pure, dry creatinine - 1 mg/ml in 0.1 N HCl served as the

stock working standards corresponding to 1 - 1.5 mg/dl were prepared.



114

Procedure

To 2 ml of serum in a test tube, added 2 ml of distilled water and mixed

well. To this 2 ml of 5% sodium tungstate and 2 ml of 2/3 N sulphuric acid were

added drop by drop, mixed well and allowed to stand for 10 min. At the same

time, 2 ml of water (blank) and 2 ml of the standards were treated in the same

way, centrifuged at 3000 rpm for 10 min.

To 3 ml of filtrate from each tube, added 1 ml picric acid, mixed thoroughly

and added 1 ml of 0.75 N sodium hydroxide. Exactly after 15 min. the absorbance

of each tube was read at 510 nm against the blank set to zero absorbance. The

serum creatinine values were expressed as mg/dl of serum.

Estimation of Alanine Transaminase (SGPT) (Reitman and Frankel, 1957)

Principle

The enzyme catalyses the following reaction:

L-Alanine + α-oxoglutarate Pyruvate +L-glutamate

The oxaloacetate is measured by the reaction with 2, 4-dinitrophenyl-

hydrazine giving a brown coloured hydrazone after the addition of sodium

hydroxide. The colour developed is read at 520 nm.

Reagents

Phosphate buffer: 0.1M, pH 7.5

Substrate: Dissolved 146 mg of β- ketoglutarate and 17.8 g of L-alanine in 1N

NaOH with constant stirring. Adjusted the pH to 7.4 and made up to 1000 ml

with phosphate buffer.



115

Standard pyruvate, 2 mM: Dissolved 22 mg of sodium pyruvate in 100 ml of

phosphate buffer, 0.2 ml of standard contained 0.4 μM of sodium pyruvate.

Dinitrophenyl hydrazine reagent, l mmol/l: 200 mg/l in l mol/l HCl.

0.4 N NaOH: Dissolved 16 g of NaOH in 1000 ml water.

Procedure

0.2 ml of sample and 1.0 ml of the buffer substrate were incubated for 30

min. at 37 C. To the control tubes, enzyme was added after arresting the reaction

with 1.0 ml of DNPH and the tubes were kept at room temperature for 20 min.

Then 10 ml of 0.4 N NaOH was added. A set of standard pyruvate was also

treated in a similar manner. The colour developed was read at 520 nm. The

enzyme activities were expressed as units/L in serum and units/protein in tissues.

Estimation of Aspartate Transaminase (SGOT) (Reitman and Frankel, 1957)

Principle

The enzyme catalyses the following reaction:

L-Aspartate +α-oxoglutarate Oxaloacetate + L-glutamate

The oxaloacetate is measured by the reaction with 2, 4-dinitrophenyl-

hyrdrazine giving a brown coloured hydrazone after the addition of sodium

hydroxide. The colour developed is read at 520 nm.

Reagents

Phosphate buffer 0.1 M, pH 7.5.

Solution A: 0.1 M solution of monobasic sodium phosphate (13.9 g/l).

Solution B: 0.1 M solution of dibasic sodium phosphate (6.8 g/l).



116

16 ml of A and 84 ml of B, diluted to a total of 200 ml.

Substrate: Dissolved 146 mg of α-Ketoglutarate and 13.3 g of aspartic acid in

l N NaOH with constant stirring. Adjusted the pH to 7.4 and made up to 1000

ml with phosphate buffer.

Standard pyruvate, 2 mmol/l: Dissolved 22 mg of sodium pyruvate in 100 ml

of phosphate buffer. 0.2 ml of standard contained 0.4 μM of sodium pyruvate.

Dinitrophenylhydrazine (DNPH) reagent, l mmol/l: 200 mg in l mol/l HCl.

0.4 N NaOH: Dissolved 16 g of NaOH in 1000 ml water.

Procedure

0.2 ml of sample and 1.0 ml of the buffer substrate was incubated for 60

min. at 37º C. To the control tubes, enzyme was added after arresting the reaction

with 1.0 ml of DNPH and the tubes were kept at room temperature for 20 min.

Then 10 ml of 0.4 N NaOH was added. A set of standard pyruvate was also

treated in a similar manner. The colour developed was read at 520 nm. The

enzyme activity was expressed as units/L in serum and units/protein in tissue.

Estimation of Alkaline Phosphatase (King and Armstrong, 1934)

Principle

The method used was that of King and Armstrong in which disodium

phenyl phosphate is hydrolysed with the liberation of phenol and inorganic

phosphate. The liberated phenol is measured at 700 nm with Folin-Ciocalteau

reagent.



117

Reagents

Sodium carbonate-sodium bicarbonate buffer (100mmol/l): Dissolved 6.36 g

anhydrous sodium carbonate and 3.36 g sodium bicarbonate in water and made

to a litre.

Disodium phenyl phosphate (100 mmol/l): Dissolved 2.18 g in water, heated to

boil, cooled and made to a litre. Added 1.0 ml of chloroform and stored in the

refrigerator.

Buffer substrate: Prepared by mixing equal volume of the above two solutions.

This has a pH of 10.

Sodium carbonate solution (15%): Dissolved 15 g of anhydrous sodium

carbonate in 100 ml of water.

Standard phenol solution (l g/l): Dissolved 1g pure crystalline phenol in

100mmol/l HCl and made to a litre with the acid.

Working standard solution: Added 100 ml dilute phenol reagent to 5.0 ml of

stock standard and diluted to 500 ml with water. This contained 10μg

phenol/ml.

Procedure

Pipetted 4.0 ml of the buffer substrate into a test tube and incubated at 37oC

for 5 min. added 0.2 ml of serum or tissue homogenate and incubated further for

exact 15min. removed and immediately added 1.8 ml of diluted phenol reagent. At

the same time a control was set up containing 4.0 ml buffer substrate and

0.2 ml sample to which 1.8 ml phenol regent was added immediately. Mixed well

and centrifuged. To 4.0 ml of supernatant added 2.0 ml of sodium carbonate. Took



118

4 .0 ml of working standard solution and for blank taken 3.2 ml water and 0.8 ml

of phenol regent. Then added 2.0 ml of sodium carbonate. Incubated all the tubes

at 37oC for 15 min. Read the colour developed at 700 nm. The activity was

expressed as units/l in serum and units/protein in tissue.

Estimation of Total Cholesterol (Parekh and Jung, 1970) Principle

Cholesterol reacts with ferric chloride in the presence of concentrated

sulphuric acid to give a pink color. The intensity of color developed is directly

proportional to the amount of cholesterol present and is read at 540 nm in a

colorimeter.

Reagents

Stock ferric chloride: 840 mg of pure dry ferric chloride was weighed and

dissolved in 100 ml of glacial acetic acid.

Ferric Chloride precipitation reagent: 10 ml of stock ferric chloride reagent

was taken in 10 ml of standard flask and made upto the mark with pure glacial

acetic acid.

Ferric chloride diluting reagent: 8.5 ml of stock ferric chloride is diluted to 100

ml with pure glacial acid.

Standard cholesterol solution: 100 mg of cholesterol was dissolved in 100 ml

of glacial acetic acid.

Working standard: 10 ml of stock was dissolved in 0.85 ml of stock ferric

chloride reagent and made up to 100 ml with glacial acetic acid. The

concentration of working standard is microgram/ml.



119

Procedure

To 0.1 ml of plant extract added 4.9 ml of ferric chloride precipitating

reagent. Centrifuged and to 2.5 ml of supernatant added 2.5 ml of ferric chloride

diluting agent. Added 4.0 ml of concentrated sulphuric acid. A blank was prepared

simultaneously by taking 5.0 ml of diluting reagent and 4.0 ml of concentrated

sulphuric acid. A set of standards (0.5-2.5 ml) were taken and made up to 5.0 ml

with ferric chloride diluting reagent. Then added 4.0 ml of concentrated sulphuric

acid. After 30 min. the intensity of color developed was read at 540 nm against a

reagent blank. The amount of cholesterol in the sample is expressed as mg/dl.

Estimation of Triglycrdies (Rice, 1970)

Principle

The glycerol moiety is oxidized to formaldehyde and the later condensed

with ammonia and 2, 4-pentanedione (acetyl acetone) to produce 3, 5- diacetyl 1,

4-dihydrotoludine, which is yellow in color and has absorption at 450 nm.

Reagents

Chloroform - methanol mixture (2:1)

Activated silicic acid: It was activated by washing silicic acid with 4N or 2N

HCl and then with water until the washings become natural. After drying ether

was added. Silicic acid was then dried at 60⁰C and activated at 100⁰ C over

night prior to use.

0.2 N H2SO₄



120

Saponification reagent: Dissolved 5g of KOH in 60 ml water and added 40 ml

of isopropanal.

Sodium-metaperiodate reagent: To 77 g of anhydrous ammonium acetate in

700 ml water, added 60 ml acetic acid and 650 mg of sodium metaperiodate.

Dissolved and diluted in 1 litre with distilled water.

Acetyl acetone reagent: Added 0.75 ml of acetyl acetone to 20 ml of

isopropanol and mixed well. Added 80 ml of distilled water and mixed.

Tripalmitin standard was containing 100µg/ml in chloroform.

Procedure

Take 0.1 ml of the serum or dried lipid extract. Makeup the volume to 4.0

ml with isopropanol. Mixed well and added 400 mg of silicic acid placed them in

a mechanical shaker and centrifuged. To 2.0 ml of the supernatant added 0.6 ml of

saponification reagent and incubated at 60-70⁰ C for 15 min. After cooling added

1.0 ml of sodium metaperiodate and mixed well. Then added 0.5 ml of acetyl

acetone reagent and mixed again. Incubated the tubes at 50⁰C for 30 min. After

cooling read the color at 405 nm. Standard tripalmitin (20-100µg) were taken in

tubes and treated similarly. Triglycerides are expressed as mg/100 ml in serum.

Estimation of HDL Cholesterol (Warnick et al., 1985)

Cholesterol reacts with hot solution of ferric per chlorate, ethyl acetate and

sulphuric acid (Cholesterol reagent) and gives lavender colored complex which is

measured at 560 nm. High density lipoproteins (HDL) are obtained in the



121

supernatant after centrifugation. The cholesterol in the HDL fraction is also

estimated by this method.

Procedure

HDL-Cholesterol separation: Mixed well, kept at room temperature for 10

min. and then centrifuged at 2000 rpm for 15 min. to obtain a clear supernatant.

Proceed to step II.

HDL - Cholesterol estimation

Reagent Blank (B) Standard (S) Test (T)

Reagent 1: Cholesterol reagent 3.0 ml 3.0 ml 3.0 ml

Reagent 2: Working cholesterol standard

(200 mg %)

- 0.015 ml -

15 µl 0.12 ml

Supernatant from step-1 - - 120 µl

Mixed well and kept the tubes immediately in the boiling water bath exactly

for 90 seconds (1 ½ min.). Cooled them immediately to room temperature under

running tap water. Measured the OD of standard (S) and test (T) against blank (B)

on a calorimeter with a yellow green filter or on a spectrophotometer at 560 nm.

Determination of LDL Cholesterol

LDL cholesterol level in serum was calculated by Friedwald et al. (1972)

formula.



122

Estimation of Lipid Peroxidation (Uchiyama and Mihara, 1978)

Principle

Malondialdehyde has been identified as the product of lipid peroxidation

that reacts with thiobarbituric acid to give a red colour absorbing at 535 nm.

Reagents

15% KCl

1% Phosphoric acid

n-butanol

0.6% thiobarbituric acid

10 mM ferrous sulphate

0.2 mm ascorbate

Procedure

Tissue slices were homogenized in ice cold 1.15% KCl. 0.5 ml of aliquot of

the homogenate was mixed with 3.0 ml of phosphoric acid. The mixture was

heated for 45 min. in a boiling water bath and after addition of 4.0 ml of n-butanol

vigorously, vortexed and centrifuged at 2000 rpm for 20 min. The absorbance of

the upper organic layer at 535 nm was measured in a spectrophotometer and

compared with a standard of freshly prepared 1,1,3,3 teraethoxy propane

concentration of 5.125, 10.25 and 20.5nmol ml-1using an extinction coefficient of

chromophore 1.56 x 10-5 m-1 cm-1 and the results were expressed as n moles of

MDA formed / mg protein.



123

Estimation of Superoxide Dismutase (Das et al., 2000)

Principle

The methods involve generation of superoxide radical of riboflavin and its

detection by nitrite formation from hydroxylamine hydrochloride. The nitrite

reacts with sulphanilic acid to produce a diazonium compound, which

subsequently reacts with naphtylamine to produce a red azo compound whose

absorbance is measured at 543 nm.

Reagents

50 mM phosphate buffer, pH 7.4

20 mM - methionine

1% (v/v)Triton X-100

10 mM hydroxylamine hydrochloride

50 µM EDTA

50 µM Riboflavin

Griess reagent: 1 % of Sulphanilamide, 2% phosphoric acid and 0.1 %

Napthylethylene diamine dihydrochloride.

Procedure

Pippeted 1.4 ml of aliquot of the reaction mixture in a test tube. 100µl of

the sample was added followed by pre incubation at 37⁰C for 5 min. 80µl of

riboflavin was added and the tubes were exposed for 10 min to 200 W Philips

fluorescent lamp. The control tube contained equal amount of buffer instead of

sample. The sample and its respective control were run together. At the end of

exposure time, 0.1 ml of Griess reagent was added to each tube and the



124

absorbance of the color formed was measured at 543 nm. One unit of enzyme

activity was defined as the amount SOD capable of inhibiting 50% of nitrite

formation under assay condition.

Estimation of Catalase (Sinha, 1972)

Principle

Catalase causes rapid decomposition of hydrogen peroxide to water. The

method is based on the fact that dichromate in acetic acid reduces to chromatic

acetate when heated in the presence of H₂O₂ with the formation of perchloric acid

as an unstable intermediate. The chromic acetate thus produced is measured

calorimetrically at 610 nm. Since dichromate has no absorbancy in this region, the

presence of compound in the assay mixture does not interfere with calorimetric

determination of chromic acetate. The catalase preparation is allowed to split

H₂O₂ for different periods of time. The reaction is stopped at specific time

intervals by the addition of dichromate /acetic acid mixture and remaining H₂O₂ is

determined by measuring chromatic acetate calorimetrically after heating the

reaction.

Reagents

1M phosphate buffer pH 7.02

2.02M Hydrogen peroxide

Stock dichromate/acetic acid solution: Mixed a 5% potassium dichromate with

glacial acetic acid (1:3 by volume).

Working dichromate /acetic acid solution: The stock was diluted to 1:5 with

water to make the working dichromate/acetic acid solution.



125

Procedure

The assay mixture contained 0.5 ml of H₂O₂, 10 ml of buffer and 0.4 ml

water. 0.2 ml of the enzyme was added to initiate the reaction. 2.0 ml of

dichromate /acetic acid reagent was added after 0, 30, 60, 90 seconds of

incubation. To control tube the enzyme was added and. read at 610nm. The

activity of catalase was expressed as µ mole of H₂O₂ decomposed /min/mg

protein.

Estimation of Glutathione Peroxidase (Beutler et al., 1963)

Reagents

0.4 M sodium phosphate buffer , pH 7.0

10 mM sodium azide

2.5 mM hydrogen peroxide

4 mM reduced glutathione

10% TCA

0.3 M phosphate solution

0.04% DTNB in 1% sodium citrate

Reduced glutathione standard : 20 mg

10% TCA

0.3 M phosphate solution

0.04% DTNB in 1% sodium citrate

Reduced glutathione standard: 20 mg reduced glutathione was dissolved in 100

ml of water.



126

Procedure

0.4 ml of buffer, 0.1 ml of sodium azide, 0.2 ml of reduced glutathione, 0.1

ml of H₂O₂, 0.2 ml of enzyme and 1.0 of water were added to a final incubation

volume of 2.0 ml. The tubes were incubated for 0, 30, 60, 90 seconds. The

reaction was terminated by addition of 0.5 ml TCA. To determine the glutathione

content, 2.0 ml of the supernatant was removed by centrifugation and added 3.0

ml disodium hydrogen phosphate solution and 1.0 of DTNB reagent. The colour

developed was read at 412 nm. Standards in the range of 200-1000 µg were taken

and treated in the similar manner. The activity was expressed in terms of µg of

glutathione utilized mg/protein.

Estimation of Reduced Glutathione

Total reduced glutathione was estimated by the method of Moron et al.

(1979).

Reagents

Phosphate buffer : 0.2 M, pH 8.0

DTNB: 0.6mM in 0.2 M phosphate buffer, pH 8.0

Trichloro acetic acid: 5%

Procedure

0.5 ml of liver homogenate was precipitated with 5% TCA. The contents

were mixed well for complete precipitation of proteins and centrifuged. To an

aliquot of clear supernatant was added 2.0 ml of DTNB reagent and 0.2 M

phosphate buffer to a final volume of 4.0 ml. The absorbance was read at 412 nm



127

against a blank containing TCA instead of sample. A series of standards treated in

a similar way were also run to determine the glutathione content. The amount of

glutathione is expressed as n moles / g wet tissue.

Estimation of Liver Glycogen (Anthrone method)

Reagents

Glucose stock standard: 100 mg of glucose was dissolved in 100 ml of water in

a standard flask

Working standard: 10 ml of the stock was diluted to 100 ml. 1 ml of this

solution contains 100 mg of glucose

Anthrone reagent: 0.2% anthorne in concentrated sulphuric acid

45% ethanol & 5 % TCA

Sample extraction

Excised liver is washed in ice cold saline to remove any blood quickly blots

between folds of the filter paper. Weighed 10 g of liver tissue and is mixed

thoroughly in a beaker surrounded by ice. The mixed meat is then homogenized in

a homogenizer or blender for 2 min. after adding 5% TCA (2.3 ml/g). This

homogenate is centrifuged at 3000 rpm for 10 min. in ice cold. Rehomogenise the

sediment with half the volume of 5% TCA and centrifuged in cold.

To the combined supernatant added twice the volume of 45% ethanol, mix

well and left in a refrigerator overnight. The precipitate is collected by

centrifugation and dissolved in minimal volume of water. This is then repeated as

before by adding twice the volume of ethanol. The precipitate obtained is again



128

washed with ethanol once and then with ethyl acetate and is made up to 25 ml

water.

Procedure

Pipette out 0.2 to 1 ml of working standard glucose solution into a series of

test tubes corresponding to mg values 20-100 mg values. 1 ml of extracted sample

and 0.5 ml of given unknown solution was pipetted out. The volume was made up

to 1 ml in all tubes with distilled water. Set up a blank along with the working

standard. Added 4 ml of anthrone reagent to all the tube and heated in a boiling

water bath for 8 min. Cool the tubes and read the colour developed at 620 nm in a

spectrophotometer. A standard graph was drawn by taking concentration of

glucose on X-axis and optical density on Y-axis. From the graph, concentration of

glucose present in sample and unknown solution was calculated.

Histopathology studies

The histopathological studies were carried out at Lakshme Pathology

Laboratories, Peelamedu, Coimbatore. At the end of the experimental period, the

animals were killed by cervical decapitation. The liver and pancreas tissues were

excised immediately and washed with chilled physiological saline. The liver and

pancreas was fixed in 10% formalin for histopathological observations.

Microscopic and macroscopic observations were examined.

Statistical analysis

All results are expressed as mean S.D. Statistical evaluation was done

using one-way analysis of variance (ANOVA), followed by Student’s t- test.



129

6.3 RESULTS

Experimental animals and design

Thirty five Swiss albino rats weighing 160-175 g of body weight were used

for the study. The animals were divided into seven groups of five animals each

(Plates 6.1 & 6.2) and treatment schedule were mentioned in materials and

methods.

Assessment of toxicity study

The methanol and aqueous extracts of A.longifolia and P.daemia was

administrated orally to the rats at the doses of 100, 200, 400, 800, 1200 and 1600

mg/kg bw did not produce any significant changes in the autonomic, behavioural

or neurological alteration. Acute toxicity studies revealed the non-toxic nature of

both extracts of A.longifolia and P.daemia. The signs and symptoms in all groups

were found to be normal (Tables 6.1 – 6.4).

Oral Glucose Tolerance Test (OGTT)

A dose-dependent reduction in blood glucose levels was observed in

alloxan induced diabetic rats treated with aqueous and methanol extracts of

A.longifolia. After a single dose of the extract give to the alloxan induced diabetic

rats, there was a significant p < 0.05 reduction in blood glucose levels of the

diabetic rats within the period of acute study compared to control. The maximum

effect was observed at 180 min. with the methanol extract exerting comparable to

effect of aqueous extract that exerted a more pronounced effect (Table 6.5;

Fig. 6.1).



130

Table 6.1 Acute Toxicity study on A. longifolia (Linn.) Nees.

A. longifoila (Methanol & Aqueous)

Group No. of rats Death Dose Difference (mg/kg bw.) Mean death Dose Difference X death

Group I 6 0 0 - -

Group II 6 0 100 - -

Group III 6 0 200 - -

Group IV 6 0 400 - -

Group V 6 0 800 - -

Group VI 6 0 1200 - -

Group VII 6 0 1600 - -



131

Table 6.2 Acute Toxicity study on P. daemia (Forsskal) Chiov.

P. daemia (Methanol & Aqueous)

Group No. of rats Death Dose Difference (mg/kg bw.) Mean death Dose Difference X death

Group I 6 0 0 - -

Group II 6 0 100 - -

Group III 6 0 200 - -

Group IV 6 0 400 - -

Group V 6 0 800 - -

Group VI 6 0 1200 - -

Group VII 6 0 1600 - -



132

Table 6.3 Signs and symptoms of A. longifolia (Linn.) Nees. extracts on rats

Group (Dose) Signs & symptoms (No. of animals) Score

Group I Iritability (0) Tremor (0) Laboured breathing (0)Staggering (0)Convulsion (0)Death (0) Normal

Group II Irritability (0)Tremor (0)Laboured breathing (0)Staggering (0)Convulsion (0)Death (0) Good / Normal activities seen

Group III Irritability (0)Tremor (0)Laboured breathing (0)Staggering (0)Convulsion (0)Death (0) Good/ Normal activities seen

Group IV Irritability (1)Tremor (0)Laboured breathing (1)Staggering (1)Convulsion (0)Death (0) Good / Normal activities seen

Group V Irritability (2)Tremor (2)Laboured breathing (3)Staggering (3)Convulsion (3)Death (0) Good / Normal activities seen

Group VI Irritability (2)Tremor (2)Laboured breathing (3)Staggering (3)Convulsion (3)Death (0) Poor / Normal activites seen

Group VII Irritability (2)Tremor (2)Laboured breathing (3)Staggering (3)Convulsion (3)Death (0) Poor / Normal activites seen



133

Table 6.4 Signs and symptoms of P. daemia (Forsskal) Chiov. extracts on rats

Group (Dose) Signs & symptoms (No. of animals) Score

Group I Iritability (0) Tremor (0) Laboured breathing (0) Staggering (0) Convulsion (0) Death (0) Normal

Group II Irritability (0) Tremor (0) Laboured breathing (0) Staggering (0) Convulsion (0) Death (0) Good/ Normal activities seen

Group III Irritability (0) Tremor (0)Laboured breathing (0)Staggering (0) Convulsion (0) Death (0) Good/ Normal activities seen

Group IV Irritability (1) Tremor (0) Laboured breathing (1) Staggering (1) Convulsion (0) Death (0) Good/ Normal activities seen

Group V Irritability (2) Tremor (2) Laboured breathing (3) Staggering (3) Convulsion (3) Death (0) Good / Normal activities seen

Group VI Irritability (2) Tremor (2) Laboured breathing (3) Staggering (3) Convulsion (3) Death (0) Poor / Normal activites seen

Group VII Irritability (2) Tremor (2) Laboured breathing (3) Staggering (3) Convulsion (3) Death (0) Very Poor / Normal activites seen



134

Table 6.5 Antidiabetic effect of methanol and aqueous extracts of A. longifolia (Linn.) Nees. and

P. daemia (Forsskal) Chiov. on blood glucose level of alloxan-induced rats during acute study

Treatments 0 min (mg/dl) 30 min (mg/dl) 60 min (mg/dl) 120 min (mg/dl) 180 min (mg/dl)

Control 102.06 0.40 144.90 0.48 157.86 0.41 173.16 0.47 195.10 0.26

Diab. control 182.03 0.75 215.80 0.43 225.03 0.25 240.63 0.70 256.86 0.61*

ALME (250 mg/kg bw) 194.90 0.26a 172.10 1.65a 160.33 0.70a 123.03 0.95a 115.20 2.10*a

ALAE (250 mg/kg bw) 192.43 0.45b 170.26 0.35b 159.13 0.90b 142.00 0.80b 120.76 0.76b

PDME (250 mg/kg bw) 197.93 0.30c 187.03 0.05c 152.13 0.79c 143.93 0.30c 118.33 1.82c

PDAE (250 mg/kg bw) 195.16 0.20d 183.20 0.91d 155.73 0.37d 147.80 0.43d 121.13 0.61d

Glibenclamide 193.90 0.36e 168.23 0.20e 142.13 0.32e 130.23 0.25*e 105.36 0.90*e

ALME: Astercantha longifolia Methanol Extract; ALAE: Astercantha longifolia Aqueous Extract

PDME: Pergularia daemia Methanol Extract; PDAE: Pergularia daemia Aqueous Extract

Value represent mean S.D. (n=5);

Comparisons between groups are as follows, a: Group III and II, b: Group IV and II, c: Group V and III, d: Group VI and IV, e: Group VII and III; Statistical significance is as follows * p < 0.05; ** p<0.01;*** p<0.001



135

Fig. 6.1 Antidiabetic effect of methanol and aqueous extracts of A. longifolia (Linn.) Nees. and

P. daemia (Forsskal) Chiov. on blood glucose level of alloxan-induced rats during acute study



136

Changes in serum glucose concentration

The daily administration of A.longifolia and P.daemia extracts (250 mg/kg

of bw) on alloxan induced diabetic rats caused a significant reduction in blood

glucose level when compared with the vehicle treated control (p <0.01) group and

day zero value (p <0.05). Similarly, repeated administration of glibenclamide

(10 mg/kg) twice a day for 7, 14 and 21 days caused a significant reduction

(p <0.01) in the blood glucose level in alloxan induced diabetic rats when

compared to vehicle and day zero values. Among the two plant extracts treated

groups, the methanolic extract of A.longifolia treated group have shown good

hypoglycaemic activity than P.daemia treated group followed by aqueous extracts

of both the plants (Table 6.6; Fig. 6.2).

Changes in non-protein compounds and glycosylated haemoglobin levels

Table 6.7 showed the effect of A.longifolia and P.daemia extracts and

glibenclamide treatment on plasma insulin, urea, creatinine and glycosylated

haemoglobin in normal and experimental animals. The levels of plasma insulin

was significantly decreased whereas, the level of urea, creatinine and glycosylated

haemoglobin levels were significantly increased in diabetic groups when

compared with normal group of animals. The oral administration of A.longifolia

and P.daemia crude extracts and glibenclamide to diabetic rats significantly

reversed all these changes to near normal levels. As expected the Hb A1 C level of

both the plant extracts and glibenclamide treated groups have shown significant

reduction when compared to the diabetic untreated groups. This effect was



137

bringing down to normal in methanol extract of A.longifolia and standard drug

treated groups. On the other hand, a moderate reduction (p <0.05) was noted in

methanol extract of P.daemia treated groups (Fig. 6.3).

Changes in lipid profile

Changes on the serum lipid profile in the non-diabetic control, alloxan

induced diabetic control and different drug treated diabetic rats was shown in the

Table 6.8. A significant elevation in the concentration of serum total cholesterol

(p<0.05), triglyceride (p <0.05), LDL- C (p <0.01), VLDL - C (p < 0.01) and

phospholipid (p <0.01) except HDL- C (p<0.01) were noted in the alloxan induced

diabetic control animal when compared to the normal non-diabetic control group.

Except aqueous extract of both the plant extracts treated group, the other entire

three drug treated groups (A.longifolia, P.daemia and standard drug) the lipid

profile was significantly reduced to near normal when compared to the non-

diabetic control (Fig. 6.4).

Changes in protein compounds and hepatic marker enzymes level

Effect of A.longifolia and P.daemia leaf extracts in the liver function

parameter in alloxan induced diabetic control, non- diabetic control and drug

treated diabetic group were presented in the Table 6.9. In alloxan induced diabetic

control, a significant reduction was noted in the serum protein, albumin and

globulin (Table 6.9; Fig. 6.5) and significant elevated level of SGPT and SGOT

and alkaline phosphatase levels (Table 6.10; Fig. 6.6). After treatment with

A.longifolia and P.daemia crude extracts, glibenclamide, protein, albumin,



138

globulin and liver marker enzymes were brought back to near normal levels. The

level of glycogen decreased significantly (p <0.01) in the alloxan induced diabetic

rats as compared to control (Table 6.10; Fig. 6.6). The oral administration of

A.longifolia and P.daemia crude extracts and glibenclamide to diabetic mice

significantly reversed all these changes to near normal levels.

Body weight changes

Normal control animals were found to be stable in their body weight but

diabetic rats showed significant reduction in body weight on day 7, 14 and 21.

Alloxan caused body weight reduction, which is reversed by methanol and

aqueous extracts of A.longifolia and P.daemia after 7, 14 and 21 days of

treatment. The same trend was noted in glibenclamide treated groups (Table 6.11;

Fig. 6.7). Significant weight loss was observed in diabetic rats compared to

control non‐diabetic rats. Treatment with A. longifolia and P.daemia extracts or

glibenclamide improved the body weight as compared to normal control rats.



139

Table 6.6 Change in Fasting Plasma Glucose Level of mice treated with A. longifolia (Linn.) Nees.

and P. daemia (Forsskal) Chiov. in normal and alloxan induced diabetic rats

Treatments 0 day (mg/dl) 7 day (mg/dl) 14 day (mg/dl) 21 day (mg/dl) Group I 94.16 2.35 99.46 2.40 90.16 1.40 95.26 2.55 Group II 241.33 3.98 262.23 1.51 294.16 2.30* 310.13 4.50* Group III 248.30 1.45a 190.63 3.32a 143.10 1.36*a 115.26 6.30*a Group IV 260.46 1.30b 194.50 1.45b 158.06 2.35*b 129.16 2.28*b Group V 264.66 1.49c 192.23 2.40c 154.50 4.40*c 118.06 1.25*c Group VI 259.23 2.35d 196.30 4.20d 160.10 4.75*d 125.50 2.30*d Group VII 278.23 3.32e 183.50 6.20e 141.40 3.55*e 111.16 1.15*e

Value represent mean S.D. (n=5); Comparisons between groups are as follows, a: Group III and II, b: Group IV and II, c: Group V and III, d: Group VI and IV, e: Group VII and III Statistical significance is as follows * p < 0.05; ** p<0.01;*** p<0.001

Group I : Normal control (Saline) (by using an intragastric catheter tube (IGC). Group II : Diabetic control Group III : Diabetic rats received A.longifolia Methanol extract (250 mg/kg) for 21 days by IGC Group IV : Diabetic rats received A.longifolia Aqueous extract (250 mg/kg) for 21 days by IGC Group V : Diabetic rats received P.daemia Methanol extract (250 mg/kgw) for 21 days by IGC Group VI : Diabetic rats received P.daemia Aqueous extract (250 mg/kg) for 21 days by IGC Group VII : Diabetic rats received Glibenclamide (10 mg/kg bw) daily orally for 21 days by IGC



140

Fig. 6.2 Change in Fasting Plasma Glucose Level of mice treated with A. longifolia (Linn.) Nees.

and P.daemia (Forsskal) Chiov. in normal and alloxan induced diabetic rats



141

Table 6.7 Effect of A. longifolia (Linn.) Nees. and P.daemia (Forsskal) Chiov. extracts on non-protein compounds and

glycosylated haemoglobin levels in normal and alloxan induced diabetic rats

Treatments Insulin (Iu/L) Urea (mg/dl) Creatinine (mg/dl) Hb A1c (%) Group I 0.656 0.36 12.20 2.25 0.61 0.03 3.71 0.27 Group II 0.155 0.24** 30.45 1.35* 1.27 0.75* 9.49 0.20** Group III 0.562 0.06*a 15.89 2.02a 0.75 0.35a 3.86 0.98a Group IV 0.497 0.23b 17.21 4.17b 0.83 0.25b 4.13 0.51b Group V 0.545 0.04*c 15.65 3.23c 0.79 0.11c 3.88 0.10c Group VI 0.492 0.02d 16.92 1.02d 0.92 0.02d 5.29 0.07d Group VII 0.578 0.32e 14.55 3.01e 0.62 0.02e 3.76 0.20e





142

Fig. 6.3 Effect of A. longifolia (Linn.) Nees and P. daemia (Forsskal) Chiov. extracts on non-protein compounds and

glycosylated haemoglobin levels in normal and alloxan induced diabetic rats



143

Table 6.8 Effect of A. longifolia (Linn.) Nees. and P.daemia (Forsskal) Chiov. extracts on serum lipid profiles in normal and

Alloxan induced diabetic rats

Treatments TC

(mg/dl) TG

(mg/100ml) HDL-C

(mg/dl) LDL-C

(mg/dl) VLDL-C

(mg/dl) PL

(mg/dl) Group I 72.22 3.90 62.07 1.16 32.10 0.43 26.31 0.31 12.11 0.20 130.23 1.90

Group II 120.07 1.06** 91.20 3.54* 28.84 0.67** 50.99 1.26** 18.58 0.42* 170.22 2.90**

Group III 76.22 6.80a 65.25 8.78a 35.48 1.93a 26.89 0.33a 12.43 0.54a 132.63 1.81a

Group IV 79.06 1.05b 70.72 1.01b 32.44 0.61b 32.31 1.97b 13.41 1.49b 137.21 1.77b

Group V 79.35 2.76c 69.56 2.74c 34.06 1.76c 31.25 0.35c 13.17 2.21c 136.75 1.52c

Group VI 81.41 1.10d 72.70 1.89d 31.72 0.45d 34.15 0.61d 14.54 0.49d 139.85 1.32nsd

Group VII 74.70 1.27e 68.25 1.66e 34.72 1.83e 26.33 1.18e 13.65 0.23e 130. 91 0.77e Value represent mean S.D. (n=5); Comparisons between groups are as follows, a: Group III and II, b: Group IV and II, c: Group V and III, d: Group VI and IV, e: Group VII and III Statistical significance is as follows * p < 0.05; ** p<0.01;*** p<0.001




144

Fig. 6.4 Effect of A. longifolia (Linn.) Nees. and P. daemia (Forsskal) Chiov. extracts on Serum lipid profiles in normal and




145

Table 6.9 Effect of A. longifolia (Linn.) Nees. and P.daemia (Forsskal) Chiov. extracts on protein profiles in normal and


Treatments Total Protein (g/dl)

Albumin (g/dl)

Globulin (g/dl) A/G ratio

Group I 7.25 0.12 4.06 0.08 3.19 0.40 0.785 Group II 6.59 0.83* 3.71 0.12* 2.88 0.61 0.776 Group III 7.49 0.36a 3.98 0.15a 3.51 0.55a 0.881 Group IV 7.27 0.14b 3.93 0.11b 3.34 0.11b 0.849 Group V 7.50 0.10c 3.97 0.76c 3.53 0.21c 0.889 Group VI 7.22 0.47d 3.95 0.65d 3.27 0.42d 0.827 Group VII 8.26 0.85e 4.42 0.55e 3.84 0.65e 0.868





146

Fig. 6.5 Effect of A. longifolia (Linn.) Nees. and P.daemia (Forsskal) Chiov. extracts on protein profiles in normal and




147

Table 6.10 Effect of A. longifolia (Linn.) Nees. and P.daemia (Forsskal) Chiov. extracts on hepatic marker enzymes levels and

liver glycogen contents in normal and Alloxan induced diabetic rats

Treatments SGOT (u/l)

SGPT (u/l)

Liver glycogen (mg/100g)

ALP (u/l)

Group I 11.33 2.86 15.74 2.47 42.13 1.60 95.60 1.17 Group II 25.67 3.065 32.48 2.51 23.40 0.60** 147.69 0.86* Group III 13.62 2.08a 15.78 3.10a 39.10 0.45a 95.43 1.27a Group IV 14.43 2.12b 16.21 4.43b 34.36 0.47b 97.05 0.05b Group V 13.59 1.17c 18.42 2.01c 36.06 0.30c 97.34 1.43c Group VI 17.43 4.11d 20.63 1.28d 31.23 0.49d 101.97 1.04d Group VII 12.46 1.66e 14.79 1.51e 43.76 0.49e 91.42 0.25e





148

Fig. 6.6 Effect of A. longifolia (Linn.) Nees. and P. daemia (Forsskal) Chiov. extracts on hepatic marker enzymes levels and

liver glycogen contents in normal and Alloxan induced diabetic rats



149

Table 6.11 Effect of A. longifolia (Linn.) Nees. and P.daemia (Forsskal) Chiov. extracts on body weight changes in normal

and Alloxan induced diabetic rats

Treatments Day 0 (g) Day 7 (g) Day 14 (g) Day 21 (g) Group I 172.20 2.07 165.25 0.45 169.68 2.15 170.44 3.55 Group II 164.66 1.28 159.37 2.23 158.65 0.05** 158.18 1.28** Group III 161.15 1.07a 164.88 2.25a 167.54 4.01a 170.16 0.36a Group IV 164.32 1.75b 164.93 1.35b 165.22 1.28b 166.94 2.36b Group V 164.58 2.23c 165.94 0.03c 167.52 0.25c 169.07 2.83c Group VI 161.35 1.05d 164.80 1.45d 165.17 3.20d 165.91 0.56d Group VII 164.63 1.02e 165.48 0.032e 165.95 0.45e 171.47 1.25e





150

Fig. 6.7 Effect of A. longifolia (Linn.) Nees. and P.daemia (Forsskal) Chiov. extracts on body weight changes in normal

and Alloxan induced diabetic rats



151

Assessment of serum, liver and kidney functions

Changes in lipid peroxidation and antioxidant enzymes

The activities of serum, liver and kidney functions were assessed by lipid

peroxidation and antioxidant enzymes such as superoxide dismutase, catalase,

glutathione peroxidase and glutathione reductase in normal, alloxan induced and

drug treated groups were illustrated in Table 6.12 - 6.14. In the present study, the

alloxan induced diabetic rats had shown increased activities of LPO. The levels of

SOD, CAT, GPx and GSH in the serum liver and kidney were significantly

reduced in alloxan induced rats. Treatment with A.longifolia and P.daemia crude

extracts and glibenclamide showed reversal of all these parameters to near normal

levels (Fig. 6.8 - 6.10).

Effect of Treatment on Histology of the Pancreatic Tissues

The histology of normal, diabetic control and drug treated are depicted in

the Plate 6.3. In untreated alloxan diabetic rats represented with damaged islets

markedly reduced (shrunken) in mass, less than 20% in the few surviving islets.

There was also observed infiltration of lymphocytes - general fibrosis, whereas the

non-diabetic mice showed preserved (numerous) islets, with cell mass devoid of

fibrosis, the islets widely distributed throughout the exocrine pancreas and

demonstrated well stained nuclei. Treatment with P. daemia extract caused a

partial recovery in damage to islet cell - moderate reduction in islet cell mass and

fibrosis, whereas more prominent recovery was produced by treatment with

A. longifolia extract. This was not significantly different from non diabetic

control. Treatment with glibenclamide did not affect the damaged islets and was

similar to diabetic control.



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Effects of Treatment on Histology of the Liver Tissue

The histopathological changes were observed in control and experimental

group rats. Results of the cellular architecture and integrity of the hepatocytes as

examined in this study revealed cell sequestration, indistinct cell nuclei and

outline the sinusoids were non-radiating and tend to be wider and interrupted in

the untreated alloxan diabetic rats. Also the hepatocytes were degenerated and

number of nuclei reduced. On the other hand, non-diabetic control liver histology

showed distinct lobulation with a central vein. Sinusoids radiate out from the

central vein; hepatocytes were distinct, well stained and showing distinct single or

polynuclei. Treatment with extracts of A.longifolia and P.daemia caused partial

reversal in the lesions observed with alloxan treatment (Plate 6.4). The nuclei of

the outlined hepatocytes were rather faint. There was better improvement with

A.longifolia extracts treatment compared to P.daemia extract. The hepatocytes of

A.longifolia treated rats were distinctly outlined together with their nuclei,

implying an increase in activity of the cells. These features were similar to those

of glibenclamide treated mice and both compared well with the nondiabetic

control histology. Administration of P.daemia extract to non-diabetic rats showed

features of mild injury - fairly indistinct cell outlines and non-prominent nuclei.

This was however not the case with A.longifolia extract treatment, which

presented a histological architecture even better than non-diabetic control.

Treatment with glibenclamide showed no features of injury; although the nuclei

were not in all cases conspicuous. The extent of reversal and recovery was partial

with A.longifolia better with P.daemia.



153

Table 6.12 Effect of A. longifolia (Linn.) Nees. and P.daemia (Forsskal) Chiov. extracts on Serum LPO, GPX, GSH, SOD

and CAT in normal and Alloxan induced diabetic rats

Treatments LPO (nanomol/ mg protein)

GPX (/mg protein)

GSH (m/mg protein)

SOD (m/mg protein)

CAT (m/mg protein)

Group I 1.12 0.02 605.55 3.62 29.47 1.07 411.74 3.11 66.57 0.89 Group II 3.14 0.03** 327.78 2.39*** 18.69 0.60* 296.09 6.0** 28.55 0.51** Group III 1.71 0.01a 591.00 3.21a 28.42 0.50a 402.61 6.81a 68.56 0.46a Group IV 1.52 0.01b 572.01 1.90b 26.32 0.39b 391.28 4.63b 63.63 0.85b Group V 1.19 0.01c 583.14 2.24c 28.68 0.51c 394.87 4.41c 67.69 0.73c Group VI 1.04 0.02d 577.61 2.93d 26.18 0.93d 388.63 1.67d 65.34 0.51d Group VII 1.92 0.25e 597.58 1.88e 30.52 0.71e 408.31 2.40e 70.01 0.78e





154

Fig. 6.8 Effect of A. longifolia (Linn.) Nees. and P.daemia (Forsskal) Chiov. extracts on Serum LPO, GPX, GSH, SOD




155

Table 6.13 Effect of A. longifolia (Linn.) Nees. and P.daemia (Forsskal) Chiov. extracts on Liver LPO, GPX, GSH, SOD



GPX (/mg protein)

GSH (m/mg protein)

SOD (m/mg protein)

CAT (m/mg protein)

Group I 0.083 0.005 8.78 0.12 43. 43 0.77 5.47 0.08 85.83 0.53 Group II 0.168 0.002** 3.75 0.17** 13.77 0.49*** 2.11 0.03* 62.46 0.44* Group III 0.102 0.002a 6.95 0.19a 42.53 0.62a 3.83 0.032a 81.66 1.29a Group IV 0.114 0.001b 5.79 0.14b 39.38 0.45b 3.12 0.02b 75.79 0.36b Group V 0.106 0.001c 6.58 0.39c 40.59 0.50c 3.71 0.08c 76.89 0.32c Group VI 0.122 0.002d 5.78 0.13d 35.02 0.35d 3.42 0.02d 72.69 0.35d Group VII 0.096 0.002c 7.51 0.39e 55.25 0.36e 4.61 0.05e 83.58 0.34e


Group I : Normal control (Saline) (by using an intragastric catheter tube (IGC). Group II : Diabetic control Group III : Diabetic rats received A.longifolia Methanol extract (250 mg/kg) for 21 days by IGC Group IV : Diabetic rats received A.longifolia Aqueous extract (250 mg/kg) for 21 days by IGC Group V : Diabetic rats received P.daemia Methanol extract (250 mg/kgw) for 21 days by IGC Group VI : Diabetic rats received P.daemia Aqueous extract (250 mg/kg) for 21 days by IGC Group VII : Diabetic rats received Glibenclamide (10 mg/kg bw) daily orally for 21 days by IGC Each Value is SEM 5 individual observations



156

Fig. 5.9 Effect of A. longifolia(Linn.) Nees. and P.daemia (Forsskal) Chiov. extracts on Liver LPO, GPX, GSH, SOD




157

Table 6.14 Effect of A. longifolia (Linn.) Nees. and P.daemia (Forsskal) Chiov. extracts on Kidney LPO, GPX, GSH, SOD and CAT

in normal and Alloxan induced diabetic rats


GPX (/mg protein)

GSH (m/mg protein)

SOD (m/mg protein)

CAT (m/mg protein)

Group I 0.06 0.003 5.48 0.03 32.00 0.2 18.16 0.20 38.03 0.55 Group II 1.62 0.015* 2.27 0.04** 14.00 0.3** 9.13 0.32*** 13.23 0.25** Group III 0.99 0.015a 4.20 0.05a 21.10 0.45a 15.53 0.35a 29.03 0.35a Group IV 1.21 0.03b 3.92 0.02b 18.93 0.40b 11.80 0.3b 24.06 0.40b Group V 1.18 0.02c 4.08 0.011c 22.96 0.25c 13.20 0.4c 27.16 0.02c Group VI 1.26 0.01d 3.90 0.045d 18.16 0.37d 12.86 0.20d 22.96 0.55d Group VII 0.95 0.025e 4.96 0.005e 29.93 0.30e 16.06 0.30e 36.9 0.45e





158

Table 6.10 Effect of A. longifolia (Linn.) Nees. and P. daemia (Forsskal) Chiov. extracts on Kidney LPO, GPX, GSH, SOD




159

6.4 DISCUSSION

Diabetes mellitus is one of the most serious, chronic diseases that are

developing along with an increase in both obesity and ageing in the general

population. Insulin-dependent (Type I, IDDM) diabetes is characterized by

juvenile onset and by absolute insulin deficiency. Non-insulin-dependent (Type II,

NIDDM) diabetes is characterized by mature onset, by varying basal insulin levels

and a frequent association with obesity. It is likely that further heterogeneity exists

within these two basic types. Currently available drugs for treatment of Diabetes

mellitus have a number of limitations, such as adverse effects and high rate of

secondary failure (Koski, 2004). As there is a growing trend towards using natural

remedies as adjuncts to conventional therapy, traditionally used plants might

provide a useful source of new hypoglycemic compounds. A number of plants

have been reported to possess hypoglycemic effects and the possible mechanism

suggested for such hypoglycemic actions could be through an increased insulin

secretion from β-cells of islets of Langerhans or its release from bound insulin or

such hypoglycemic effects of plant extracts could also be because of their insulin-

like actions (Pradeep kumar et al., 2010).

Alloxan became the first diabetogenic chemical agent when Dunn and

Letchie accidentally produced islet-cell necrosis in rabbits while researching the

nephrotoxicity of uric acid derivatives. Alloxan is a specific toxic sub-stance that

destroys the β cells provoking a state of primary deficiency of insulin without

affecting other islet types (Prince and Menon, 2000; Jeldor et al., 2007). Insulin

deficiency leads to various metabolic alterations in the animals viz increased blood



160

glucose, increased cholesterol, increased levels of alkaline phosphate and

transaminases. Glibenclamide is often used as a standard antidiabetic drug in

alloxan induced diabetes to compare the efficacy of variety of hypoglycemic

compounds. The present study was conducted to assess the hypoglycemic activity

A.longifolia and P.daemia leaves in alloxan-induced diabetic rats.

Blood glucose level

The present investigation shows that in A. longifolia and P.daemaia alloxan

diabetic rats, methanolic extracts of both the plants caused significantly reductions

of blood glucose levels after 21 days of extracts administration, while

glibenclamide exhibited maximum hypoglycemic activity in these animals. Both

the plant extracts showed activity at the tested dose level by decreasing glucose

level when compared to control group animals in acute and prolong treatment. The

methanol extracts of both plants significantly decreased the glucose level at 7th,

14th and 21st days in hyperglycemic animals (p <0.01) than aqueous extracts.

Glibenclamide reduces the elevated blood glucose level from 278 to 111 mg/dl.

The possible mechanism by which the plant extracts decrease the blood sugar

level may be by potentiation of insulin effect either by increasing the pancreatic

secretion of insulin from beta-cells of islets of Langerhans or by increasing the

peripheral glucose uptake (Aybar et al., 2001). In this context a number of other

plants have also been observed to have hypoglycemic and insulin release

stimulatory effects (Ayoola et al., 2009; Awobajo and Olatunji, 2010; Nikhil

K.Sachan et al., 2009). Several controlled clinical trials of trace element

supplements for glycemic control revealed the beneficial role for supplementation



161

for the control and management of diabetes (Halberstam et al., 1996; Anderson et

al., 1987; Paolisso et al., 1992).

In the present study, we also investigated glucose tolerance test in normal

rats. The extracts of A. longifolia and P.daemia significantly decreased the serum

glucose level in glucose loaded rats and this information could be endorsed to the

potentiation of the insulin effect of blood by increasing the pancreatic secretion of

insulin from existing beta cells or its release from bound insulin (Kasiviswanath et

al., 2005). In this context, a number of other plants have been observed to have

similar pattern of hypoglycemic effects. Results of the insulin release from

pancreas directly that the anti-diabetic activity of A.longifolia may be through the

release of insulin from the pancreas.

Protein compounds

Table 5.9 shows significant reduction in serum albumin and globulin were

observed in alloxan induced diabetic group (Group II), when compared to control

(Group I) and glibenclamide treated rats (Group VII). On administration of leaf

extracts of A.longifolia and P.daemia to the diabetic group restored the protein,

albumin and globulin levels to normal. These results were in consistent with the

result of Wattakaka volubilis leaf in diabetic rats (Maruthupanadiyan et al., 2000).

The increased level of serum protein, albumin and globulin in alloxan induced

diabetic rats are presumed to be due to increased protein catabolism and

gluconeogenesis during diabetes (Palanivel et al., 2001). The protein oxidation in

insulin dependent diabetic mellitus subject was increased with decreasing the

plasma levels of total protein, albumin, globulin and to non-diabetic subjects.



162

Generally hyperglycemia is characterized by alternations in the metabolism of

carbohydrate, protein and lipids. Hyperglycemia induces the over production of

oxygen free radicals and consequently increases the protein oxidation and lipid

oxidation. Among the parameters of protein metabolism, the present study showed

a slight decline in total protein, sharp fall in serum albumin and globulin in

diabetic rats. This is in agreement with hypoalbuminia observed in diabetes (Porte

and Halter, 1971). On the other hand, the extracts treated rats protein metabolism

never deviated from normal range. Hypoalbuminemia is common problem in

diabetic animals and generally attributed in the presence of nephropathy. An

overall reduction in serum total protein in diabetic animal and consequence in

albumin were observed in the present study. This corroborates earlier reports

(Soon and Tan, 2002). The reversal of these changes by methanol and aqueous

extracts of A.longifolia and P.daemia proved that insulin deficiency had been

grassly corrected.

SGOT & SGPT

The levels of SGOT and SGPT are increased in the diabetic induced rats.

Table 6.10 summarized the effect of alloxan on the activity of the hepatic marker

enzymes in serum. Our results stay in touch with (Ghosh and Suryawanshi, 2001),

who has reported that transaminase activity is increased in serum of diabetics. It

may be due to leaking out of enzymes from the tissues and migrating into the

circulation by the adverse effect of alloxan (Stanely et al., 1999). The increased

levels of transaminases, which are active in absence of insulin, because of the

availability of amino acids in the blood of diabetes, are responsible for the



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increased gluconeogenesis and ketogenesis observed in diabetics. In diabetic

animals, the changes in level of serum enzymes are directly related to changes in

the metabolism in which the enzymes are involved (Felig et al., 1970). In this

study, the crude extracts of A.longifolia and P.daemia regulated the activity of

SGPT and SGOT in liver of rats intoxicated with alloxan. The effect of

glibenclamide on the recovery of hepatic enzyme activity in serum was very

similar to that of the earlier study (Preethi and Kuttan, 2009). The restorations of

SGPT and SGOT to their respective normal levels after treatment with both

glibenclamide and extracts of A.longifolia and P.daemia, further strengthen the

antidiabetic effect of this extract. Moreover SGOT levels also act as indicators of

liver function and restoration of normal levels of these parameters indicate normal

functioning of liver. Since the alloxan can also affect the liver by free radical

mechanism. In addition to the assessment of SGPT and SGOT levels during

diabetes the measurement of enzymatic activities of phosphatases such as acid

phosphatase (ACP) and alkaline phosphatase (ALP) is of clinical and

toxicological importance as changes in their activities are indicative of tissue

damage by toxicants.In the present investigation, serum ALP increased in alloxan

induced diabetic rats. Treatment with the extracts of A.longifolia and P.daemia in

alloxan induced diabetic rats produces a decline in ALP level.

Non-protein compounds

Insulin, urea and creatinine were significantly increased, whereas plasma

insulin was decreased rats (Group II) as compared to control rats (Group I). The

status of urea, creatinine and plasma insulin levels were restored in diabetic rats



164

after treated with crude extracts of A.longifolia and P. daemia at 250 mg/kg bw

respectively. The diabetic groups treated with glibenclamide showed a comparable

effect to that of extracts. The diabetic hyperglycemic induced by alloxan produces

elevation of plasma levels of urea and creatinine, which are considered as

significant markers of renal dysfunction (Alarcon et al., 2005). Our results showed

significant increase in the level of plasma urea and creatinine in the diabetic

groups compared to control level. These results indicated that diabetes might lead

to renal dysfunction. While, after treatment with extracts, the level of urea and

creatinine were significantly decreased compared to the mean value of diabetic

group. This further confirms the utility of these plants in diabetes-associated

complications (El-Demerdash et al., 2005). And these results were consistent with

the result of Eugenia jambolana on diabetic rats (Srivastava et al., 2012).

Glycosylated haemoglobin

Glycosylated haemoglobin determinations are self-monitoring of blood

glucose therefore, it play an important complementary role for the management of

diabetes mellitus (Thai et al., 1983). The observed increase in the levels of

glycosylated haemoglobin (HbA1c) in diabetic control group of rats is due to the

presence of excessive amounts of blood glucose. During diabetes the excess of

glucose present in blood react in the haemoglobin to form glycosylated

haemoglobin (Alyassin & Ibrahim, 1981; Sheela & Augusti, 1992). Mechanisms

by which increased oxidative stress is involved in the diabetic complications are

partially known, including activation of transcription factors, advanced glycated

end products (AGEs), and protein kinase C. Glycosylated hemoglobin has been



165

found to be increased over a long period of time in the diabetic mellitus (Bunn et

al., 1978). There is an evidence that glycation may itself induce the generation of

oxygen derived free radicals in diabetic condition (Gupta et al., 1997). Treatment

with A.longifolia and P.daemia extracts showed a decrease in the glycosylated

hemoglobin with a concomitant increase in the level of total hemoglobin in the

diabetic rats standard drug glibenclamide also showed the same results.

Lipid Profiles

The levels of serum lipids are usually elevated in diabetes mellitus and such

as elevation represent a risk factor for coronary heart disease. The abnormally

high concentration of plasma and hepatic lipids in diabetes is mainly due to an

increase in the mobilization of free fatty acids from the peripheral depots, since

insulin inhibits hormone sensitive lipase (Al-Shamony et al., 1994). The marked

hyperlipidemia that characterizes the diabetic state is regarded as a consequence of

the uninhibited actions of lipolytic hormones (glucagon and catecholamines) on

the fat depots (Ravi et al., 2005). On the other hand, increased LDL-cholesterol

may arise from glycosylation of the lysyl residues of apoprotein B (Ravi et al.,

2005). The ability of LDL-cholesterol to form lipid peroxides was found to be

specifically responsible for the atherogenesis in diabetic patients (Kondo et al.,

2001). It is reported that a deficiency in lipoprotein lipase activity in diabetics may

contribute to significant elevation of triglycerides in blood and with insulin

administration; lipoprotein lipase activity is elevated and leads to lowering of

plasma triglyceride concentrations (Lopes-Virella, 1983; Braun and Severson,

1992). The A.longifolia and P.daemia administration almost reversed these effects



166

as it reduced total cholesterol and triglyceride concentrations (plasma), LDL

concentration and increased HDL notably in combination. In this context,

combination of A.longifolia and P.daemia was found to be as effective as

glibenclamide in reducing the plasma lipid profiles in diabetic rats.

Body weight changes

During the 21-day experimental period the body weight is reduced in

diabetic rats, whereas there was a significant gain of body weight in treated rats.

The failure of alloxan-induced diabetic rat to gain weight has already been

reported. The administration of crude extracts of both plants restored these levels

significantly (p <0.001) towards normal. The ability of the methanol and aqueous

extracts to restore body weight seems to be a result of its ability to reduce

hyperglycemia (Santhosh et al., 2007). Diabetic rats treated with the aqueous

extract showed and slightly increase in body weight compared to diabetic control.

The hypoglycaemic activity was compared with glibenclamide a sulphonylurea,

stimulate insulin secretion pancreatic beta cell (Tiedge and Lenzen, 1995). Our

results are supported by Esharat (2002), who reported that diabetic untreated

animals showed loss in body weight. This may also be due to the protective effect

of the extract in controlling muscle wasting i.e. reversal of gluconeogenesis. The

significant body weight gain after treatment with the plant extracts revealed the

previous report of Eugenia jambolana in diabetic albino rats (Srivastava et al.,

2012).



167

Liver glycogen

From the table 5.10, it is seen that diabetic induced Group II animals

showed a sharp decline in liver glycogen level when compared to control Group I

animals. Our results are supported by Babu et al. (2002) who stated that there was

a significant reduction in liver glycogen levels in alloxan diabetic rats. In the

treated Groups the glycogen concentration in the liver was significantly increased

after the treatment. This may be due to the enhancement of glycogen synthesis by

the extracts of A.longifolia and P.daemia leaves. Previous result corroborated

with Ficus hispida and Cassia kleini by Babu et al. (2002). So the A.longifolia and

P.daemia extracts could have stimulated glycogenesis and / or inhibited

glycogenolysis in the diabetic rat liver. The Glibenclamide treated Group VII rats

and plant extract treated Groups III - VI showed a similar result which proved that

the plant drug has the insulin potency.

Assessment of serum liver and kidney functions

SOD and CAT

Oxidative stress in diabetes is coupled to a decrease in the antioxidant

status, which can increase the deleterious effects of free radicals. The SOD and

CAT are the two major scavenging enzymes that remove free radicals in vivo

(Jin et al., 2008). A decreased activity of these antioxidants can lead to an excess

availability of superoxide anion (O2) and hydrogen peroxide (H2O2) which in turn

generate hydroxyl radicals (OH), resulting in initiation and propagation of LPO.

The SOD can catalyze dismutation of O2 into H2O2 by catalase or SOD works in



168

parallel with selenium-dependent glutathione peroxidase, which plays an

important role in the reduction of H2O2 in the presence of reduced glutathione

forming oxidized glutathione, and it protects cell protein and cell membrane

against oxidative stress (Jin et al., 2008; Tuzun et al., 1999). In our study, the

SOD and CAT enzyme were significantly (p< 0.001) decreased in alloxan induced

diabetic control rats, may be due to inactivation caused by free radicals.

Lipid peroxidation

Increased lipid peroxidation (LPO) in diabetes can be due to enhanced

oxidative stress in the cells as a result of depletion of antioxidant scavenger

systems. The results showed increased LPO of alloxan induced diabetic rats.

Earlier reports have revealed that there was an increased LPO in liver, kidney and

brain of diabetic rats (Latha and Pari, 2003; Ananthan et al., 2004). Increasing

evidence in both experimental and clinical studies suggests that oxidative plays a

major role in the development and progression of both types of diabetes mellitus.

Diabetes is usually accompanied by impaired antioxidant defenses (Senthilkumar

et al., 2006). In the present study, an increase in the levels of LPO was found and

these levels were significantly reduced after the supplementation of the crude

extracts of A.longifolia and P.daemia and glibenclamide treated rats.

Blood Glutathione

Table 5.12 - 5.14 showed that oral administration of extracts significantly

increased (p <0.05) the depressed blood glutathione (GSH) level of chemically

induced diabetic rats. The elevation of glutathione level by extract has important



169

implications because of the fact that the tripeptide, in addition to many of its

important physiological/biochemical functions such as maintenance of the

integrity of the red cell, detoxification of hepatic xenobiotics, transport of amino

acids in the γ-glutamyl cycle or Alton Meister’s cycle, plays a very important

protective role against the damaging effect of toxic oxides radicals such as

superoxide (O-2), hydroxyl radical (OH) and toxic peroxides such as hydrogen

peroxide (H2O2), and other peroxides (R-OOH). These highly reactive species are

thought to be partly responsible for the destruction of the β- cells of the pancreas

in diabetes. The diabetogenic drug alloxan has been reported to give rise to

superoxide (O-2) whereby the drug destroys the islet of Langerhans of the pancreas

and precipitate diabetes mellitus. Oral administration of A.longifolia, P.daemia

and glibenclamide to alloxan treated rats showed the activity of GSH, which was

significantly increased pancreas. The decrease in GSH concentration in the early

stage of diabetes is probably due to reactive oxygen species (ROS) generation by

mechanisms such as glucose autooxidation, advanced glycation endproduct (AGE)

pathway, polyol pathway and activation of protein kinase C (PKC) (Cooper et al.,

2007).

Glutothione peroxidase

GPx plays a pivotal role in H2O2 catabolism and in the detoxification of

endogenous metabolic peroxides and hydroperoxides which catalyses GSH

(Sozmen et al., 2001). Decreased activity of GPx may result from radical induced

inactivation and glycation of the enzymes (Meister, 1984). In diabetic rats treated

with the crude extracts of A.longifolia and P.daemia significant increase in GPx



170

was observed. This might reflect the antioxidant potency of the plant extracts

which by reducing glucose levels, prevented glycation and inactivation of GPx.

The effect of these plant extracts on the recovery of hepatic enzyme activity in

serum was very similar to that of the earlier study (Preethi and Kuttan, 2009;

Sajeeth et al., 2011).

Histology of the Liver Tissues

The liver is one of the tissues that bear the brunch of chronic

hyperglycaemia, since glucose is freely permeable to its cells (Meyes, 2003). This

unrestrained entry, in the presence of excess and sustained glucose in blood, is

bound to cause metabolic derangements which would express themselves on the

gross architecture of the tissues. In this study, untreated diabetic rats presented

with a sequestestered and disoriented cellular architecture in accordance with the

earlier report (Atangwho et al., 2007). Administration of extracts of A.longifolia

and glibenclamide ameliorated or reversed these features. This obviously results

from the antihyperglycemic action of these treatments. As pathogenic factor -

hyperglycaemia is ameliorated or reversed normal metabolism gradually becomes

reestablished, so also the gross architecture of the tissues, as was the case with

administration of Eugenia jambolana extracts in the earlier report of Ravi et al.

(2004). A.longifolia extracts could only cause partial recovery in treated diabetic

rats and even in non-diabetic rats caused features of mild injury, but not the case

of with P.daemia extracts. This may imply that at certain concentrations and

duration of treatment A.longifolia extracts might become toxic to the hepatocytes.

The absence of these features in rats that received P.daemia extracts may imply



171

that P.daemia extarct is more protective to the hepatocytes. The present result is

parallel to the antidiabetic effects of Azadirachta indica and Vernonia amygdalina

(Ebong et al., 2008).

Histology of the Pancreas Tissue

The present result therefore supports and corroborates the synergistic action

of A.longifolia and P.daemia extracts in protecting the hepatocytes against

possible hyperglycemia induced damage. Pancreatic lesions induced by alloxan

leading to full blown diabetes were reversed upon treatment with extracts in this

study revealed the possible pancreatic islets cell regeneration. Alloxan is known to

induce chemical diabetes by selective destruction of pancreatic beta cells through

three processes viz; DNA alkylation, nitric oxide production and free radical

generation (Szkudelski, 2001). This was observed in this study as diabetic control

rats pancreas showed markedly reduced and shrunken islets mass, infiltered by

lymphocytes-general fibrosis. This observation agree with several reports in

literature of alloxan damage to pancreas (Noor et al., 2008; Soto et al., 2004;

Ahmed et al., 2005; Jelodar et al., 2007). As with these cited works, treatment

with our extracts ameliorated and reversed the lesions. A plant extract with the

ability to ameliorate this type of diabetes will necessarily address in part or whole,

the reactive oxygen species generation process or their concentration both in

serum and the tissues. This implies that the plant must be endowed with

antioxidants which could reverse the cytotoxic cycle of alloxan in the pancreas, or

at least mop up or quench the potentiation of reactive oxygen species in

circulation. The extent of recovery and reversal in this study was partial with



172

A.longifolia extracts treatment, better with P.daemia treatment. This may imply

the relative abundance of the antioxidant components in these two plants.

A possible synergistic interaction between and among components from these two

plants with respect to pancreatic cell recovery has been demonstrated from this

result, as this has not been previously reported.


173

Chapter-VII

PHYTOCHEMICAL SCREENING

. .

7.1 INTRODUCTION

Phytochemicals are as antimicrobial compounds & pesticides of

antimicrobial agents which found in aromatic and essential oil plants which have

made great contribution for quick and effective management of human health,

plant disease and microbial contamination in several agricultural conditions.

Phytochemicals preserved by screening of plant parts eg. leaves, roots, stems,

fruits etc., reveals the presence of carbohydrates (free reducing starch, sugar etc.)

saponins, steroids, tannins, phenols and terpenoids. The compounds that are

responsible for therapeutic effect are usually the secondary metabolites. A

systematic study of a crude drug signifies a thorough consideration of both

primary and secondary metabolites derived as a result of plant metabolism. The

plant material is subjected to preliminary phytochemical screening for the

detection of various plant constituents (Sazada Siddiqui et al., 2009).

Plant secondary metabolites are a generic term used for more than 30,000

different substances which are exclusively produced by plants. The plants form

secondary metabolites e.g. for protection against pests, as colouring, scent, or

attractants and as the plant's own hormones. Phytochemical studies on medicinal

plants are required for the following reasons:

Phytochemical information on a species of medicinal plant is an essential

basis for a fine chemical analysis to follow by in vitro and clinical studies.

Chapter - VII Phytochemical Screening


174

Almost every species of medicinal plants contain more than one actual

compound and it is necessary to know this composition before other studies

were undertaken.

A phytochemical survey would provide information on the distribution of

certain chemical compounds in different species, to offer a wider choice of

material for the work of other scientists.

Plant Secondary Metabolites: Sources and Effects

Carotenoids

Carotenoids are organic pigments occurring in plants and are mostly found

in red, orange and yellow fruits and vegetables. Other vegetables such as broccoli,

spinach or curly kale also contain carotenoids. Carotenoids have antioxidative

effects and prevent cancer. In addition to this they boost the immune system and

reduce the risk of getting heart attacks.

Phytosterols

Phytosterols are found in plant foods such as sunflower seeds, sesame, nuts

and soya beans. Phytosterols protect against colon cancer and lower cholesterol

levels. Phytosterols are chemically similar to cholesterol and therefore they

compete against each other for absorption in the body.

Saponins

Saponins are flavour additives, which are found in legumes and spinach.

Saponins boost the immune system, lower the cholesterol levels in the blood and

reduce the risk of getting intestinal cancer.



175

Glucosinolates

Glucosinolates are flavour additives, which are found in all types of

cabbages, mustard, radish and cress. Glucosinolates prevent infections and inhibit

the development of cancer.

Flavonoids

Flavonoids are organic pigments occurring in plants which give plants a

red, violet or blue colour. Flavonoids have a particularly broad spectrum of

efficacy. Flavonoids inhibit the growth of bacteria and viruses, protect the cells

against the damages of free radicals, protect against cancers and heart attacks,

have a repressive effect against inflammations and they influence blood

coagulation.

Protease-inhibitors

Protease-inhibitors are found in plants that are rich in protein such as

legumes, potatoes and wheat and they inhibit the decomposition of protein.

Protease inhibitors protect the body against cancers and regulate the blood sugar

levels.

Terpenes

Terpenes are plant flavours for e.g. the menthol in peppermint oil or the

essential oils in herbs and spices. Terpenes decrease the risks of cancer.

Phytoestrogens

Phytoestrogens are natural plant hormones which are similar to the sexual

hormones. Phytoestrogens are mostly found in wheat, legumes and wheat



176

products. Phytoestrogens protect the body against hormonal dependant cancers

such as breast, uterine and prostate cancer.

Sulphides

Sulphides are compounds containing sulphur which are mostly found in

plants that belong to the lily family such as onions, leeks, asparagus and garlic.

Sulphides inhibit the growth of bacteria, lower cholesterol levels, protect the body

from free radicals and have preventive effects against cancer.

Phytic acid

Phytic acid is found in wheat, legumes and flaxseeds. Phytic acid was

considered undesirable for a long time because it binds trace elements such as iron

and zinc and it also affects various digestive enzymes. However new studies have

proved that phytic acid has an antioxidant effect in the large intestine.


Fakkim and Sewraj (1972) screened some medicinal plants occurring in

Maturities. Tanira et al. (1994) carried out phytochemical of some medicinal

plants from United Arab Emirates. Tripathi et al. (1996) review the occurrence of

secondary metabolites in pipper sp. Anuba and Bharagava (1996) carried out

phytochemical investigation of twenty two multipurpose tree species with a view

to decipher the economically important bioactive compounds. Coe and Anderson

(1996) screened 229 plant species used for medicinal purpose to assess the

chemical nature of bioactive compounds. Mandi and Sharma (1994) investigated

1500 species of Zingiberaceae for their chemical constituents.



177

Several other investigations like, Fransworth (1996), Bhakuni et al. (1969),

Das and Battarjee (1970), Joshi et al. (1974), Odebiyi and Sofowora (1978), Mosa

et al. (1983), Nautiyal and Semwal (1984), Nandi et al.(1985), Rao et al. (1985)

and Reddy (1995) have undertaken phytochemical screening of several medicinal

plants.

Study of plant constituents generally involves two processes. 1. Extraction

using either one of the following solvents namely Petroleum ether/ Benzene/

Chloroform/ Acetone/ Ethanol/ Methanol/ Water. 2. Qualitative chemical

examination for the identification of various plant constituents such as Alkaloids,

Glycosides, Carbohydrates, Phytosterols, Saponins, Phenolic compounds,

Tannins, Gums, Mucilage, Proteins and Free amino acids and detection of Fixed

oils and Fats. Phytochemical study is useful to isolate the pharmacologically

active principles present in the drug. Subjecting the leaf extracts of Pergularia

extensa (= P. daemia (Forssk.) Chiov.) by Jalalpure et al. (2002) have found the

presence of Flavonoids, Steroids and Saponins. Extracts from leaves of Carissa

carandas L. revealed the presence of Phenolic compounds, Tannins and Alkaloids

indicating antipyretic activity in rats (Rajasekaran et al., 1999).

Chakraborthy and Patil (1997) showed positive antibacterial activity from

different plant extracts of Plumbago zeylanica. The antibacterial nature is

attributed to the presence of Napthoquinone derivatives, tannins and flavonoids.

Sterols, alkaloids, glycosides and flavonoids were found to be present in the root

extracts of Swertia chirata. The wound healing activity in mice was ascribed to

the presence of sterols and flavonoids (Manjunath et al., 2006). Sapkale et al.



178

(2006) have showed the presence of Alkaloids, Glycosides, Flavonoids, Tannins,

Proteins, Amino acids, Sterols, Triterpenoids, Fixed oils and Saponins in the fruit

extracts of Solanum torvum. The histo-pathological studies of the alcoholic

extracts indicated significant hepato-protective activity in rats. Antiviral

Isoflavonoid-sulphate and Steroidal-glycosides have also been isolated from the

fruits of Solanum torvum (Arthan et al., 2002).

Sahare et al. (2008) have found out the antimicrofilarial activity of

methanolic extract of Vitex negundo and Aegle marmels. Phytochemical screening

revealed the presence of Glycosides, Saponins and Flavonoids in Vitex negundo L.

and Coumarins in Aegle marmelos (L.) Correa ex Roxb. Krupavaram et al. (2007)

have found out that alcoholic and chloroform extracts obtained from the roots of

Boerhavia diffusa L. had significantly increased the anoxious test tolerance time

in mice. The alcoholic extracts contained Alkaloids, Glycosides, Carbohydrates,

Flavonoids, Tannins, Triterpenoids and Saponins, whereas the chloroform extracts

contained the same components of alcoholic extracts except Tannins. Among the

various root extracts of Moringa oleifera Lam. the aqueous extract indicated the

presence of Carbohydrates, Glycosides, Saponins and Triterpenoids. Alcoholic

extracts indicated the presence of Alkaloids and Glycosides only. These two

exrtracts exhibited antiarthritic property in albino rats (Karadi et al., 2006).

Phytochemical observation of aerial extracts of Marsilea minuta L. by

Nagavalli et al. (2008) has revealed the presence of all the phytochemical

constituents except Saponins, Triterpenoids and Cardiac glycosides. Patil et al.

(2004) screening for phytochemical constituents of the roots of Eclipta alba (L.)



179

Hassk. ( = E.prostrata (L.) L.) showed the presence of Sterols, Alkaloids, Proteins

and Tannins. The ethanol extracts of roots of Eclipta alba (L.) Hassk. ( =

E.prostrata (L.) L.) and its fractions of petroleum ether, diethyl ether and ethyl

acetate were found to contain the Sterols, but Butanol fraction was not found to

contain Sterols. Ethanol extracts and its fractions exhibited wound healing activity

in albino mice. This property is attributed to the presence of sterols endorsing

observation of Irvin (1981).

Gabhe et al. (2006) explored the immunomodulatory potential of aerial

parts of Ficus benghalensis L. The methanolic extract was found to stimulate cell

mediator and antibody mediator immuno responses in rats. The methanolic and

aqueous extracts were found to contain Flavonoids, Phenolics, Steroids,

Glycosides, Carbohydrates and Proteins. Alkaloids were totally absent.

Sugumaran et al. (2008) conducting preliminary phytochemical study on different

extracts of Pithecellobium dulce (Roxb.) Benth. found the presence of

Phytosterols, Triterpenoids, Flavonoids, Phenolics, Tannins and Saponins in huge

amounts. Alkaloids, Aromatic acids, Volatile oils and Fixed oils were totally

absent in this plant.

Soetan et al. (2006) have found the antimicrobial activity of saponin

extracted from Sorghum bicolor (L.) Moench against Escherichia coli,

Staphylococcus aureus and Candida albicans. The extract inhibited the growth of

Staphylococcus aureus. It was concluded that saponins had inhibitory effects on

gram positive bacteria but not on gram negative bacteria and fungi. El-Desouky et

al. (2007) were able to isolate new pyrrole alkaloids along with other known



180

compounds, from Arum palaestinum Boiss. Investigation on the antioxidant

against DPPH radicals exhibited a string radical scavenging activity. However

they have attributed this capacity to the phenolic compounds such as caffeic acid,

luteline and iso-orientin which have strong free radical scavenging activities.

Srinivas Koduru et al. (2007) have isolated two steroid glycosides namely

tomatidine and solasodine from berries of Solanum aculeastrum Dunal and

studied their antioxidant activities. The study has revealed strong antioxidant

activity and synergistic effect of the isolated compounds. Sauvain and Moretti

(1996) isolated 3 alkaloids from the medicinal plants Pogonopus tubulosus

(A. Rich. ex. DC.) K. Schum. and found them to be antimalarial in nature. Tsuzuki

et al. (2007) have reported the antifungal activity of the extracts and saponins

from Sapindus saponaria L. fruits that provide preliminary scientific validation

for the traditional medicinal use of the plants, against some fungal diseases and

control of fungi in the environment. Du et al. (2003) and Lee et al. (2001) have

reported that triterpenoid saponins having oleonolic acid as aglycone posses

antifungal activity against several fungi.


Phytochemical screening

Phytochemical screening of Asteracantha longifolia and Pergularia daemia

was carried out according to the methods described by Trease and Evans (1997).

Qualification phytochemicals analysis of the crude powder of the samples for the

identification of phytochemicals like as a tannins, alkaloid, steroid, phenols and

terpenoid, flavonoid etc.



181

Test for alkaloids

Five ml of the extract was added to 2 ml of HCL. To this acidic medium,

1 ml of Dragendroff’s reagent was added. An orange or red precipitate produced

immediately indicates the presence of alkaloids.

Test for flavonoids

One ml of the extract, a few drops of dilute sodium hydroxide was added.

An intense yellow color was produced in the plant extract, which become

colorless on addition of a few drops of dilute acid indicates the presence of

flavonoids.

Test for Cardiac glycosides

The extract was hydrolysed with HCL for few hours on a water bath. To the

hydrolysate, 1 ml of pyridine was added and a few drops of sodium nitroprusside

solutions were added and then it was made alkaline with sodium hydroxide

solution. Appearance of pink to red color shows the presence of glycosides.

Test for saponins

The extract was diluted with 20 ml of distilled water and it was agitated in

a graduated cylinder for 15 minutes. The formation of 1 cm layer of foam showed

the presence of saponins.

Test for steroids

One ml of the extracts was dissolved in 10 ml of chloroform and equal

volume of concentrated sulphuric acid was added by sides of the test tube. The



182

upper layer turns red and sulphuric acid layer showed yellow with green

fluorescence. This indicated the presence of steroids.

Test for tannins

Five ml of the extract and a few drops of 1% lead acetate were added.

A yellow precipitate was formed, indicates the presence of tannins.

Test for triterpenoids

Ten mg of the extract was dissolved in 1 ml of chloroform; 1 ml of acetic

anhydride was added following the addition of 2 ml of Conc.H2SO4. Formation of

reddish violet color indicates the presence of triterpenoids.

Test for Phenolic compounds

To 2 ml of filtered solution of the aqueous macerate of the plant material, 3

drops of a freshly prepared mixture of 1 ml of 1% ferric chloride and 1 ml of

potassium ferrocyanide was added to detect phenolic compounds. Formation of

bluish-green colour was taken as positive.

Anthraquinones (Borntrager’s test)

The hydro-alcoholic extract of the plant material (equivalent to 100 mg)

was shaken vigorously with 10 ml of benzene, filtered and 5 ml of 10% ammonia

solution added to the filtrate. Shake the mixture and the presence of a pink, red or

violet color in the ammonia (lower) phase indicated the presence of free

anthraquinones.



183

7.4 RESULTS

Methanol and aqueous extracts of the selected medicinal plants were

subjected to qualitative phytochemical analyses. Extracts of A.longifolia and

P.daemia answered positively tannins, flavonoids, protein & amino acids and

cardiac glycosides. Methanol extract of A.longifolia positively answered for

alkaloids (Tables 7.1 & 7. 2).

Saponins were identified in the aqueous extract of P.daemia when tested for

foam tests. Steroids were identified in the methanol extracts of A.longifolia and

P.daemia. Terpenoids were identified in the methanol extract of A.longifolia

(Tables 7.1 & 7. 2).



184

Table 7.1 Phytochemical screening of Asteracantha longifolia (Linn.) Nees.

S.No. Phytoconstituents Methanol Aqueous

1. Alkaloids + – 2. Saponins – + 3. Steroids + – 4. Phenolic compounds + – 5. Tannins + + 6. Flavonoids + + 7. Terpenoids – – 8. Cardiao Glycosides + + 9. Protein & Amino acids + +

10. Anthraquinones – –

Table 7.2 Phytochemical screening of Pergularia daemia (Forsskal) Chiov.


1. Alkaloids + + 2. Saponins – – 3. Steroids + – 4. Phenolic compounds + – 5. Tannins + + 6. Flavonoids + + 7. Terpenoids + – 8. Cardiao Glycosides + + 9. Protein & Amino acids + +

10. Anthraquinones – –



185

7.5 DISCUSSION

All plant parts synthesize some chemicals in themselves which metabolize

their physiological activities. In our present study, the selected plants have

exhibited different kinds of secondary metabolites. The medicinal value of these

secondary metabolites is due to the presence of chemical substances that produce

a definite physiological action on the human body. The most important of these

include: alkaloids, glucosides, steroids, flavonoids, fatty oils, resins, mucilages,

tannins, gums, phosphorus and calcium for cell growth, replacement, and body

building (Kubmarawa et al., 2008). A variety of herbs and herbal extracts contain

different phytochemicals with biological activity that can be of valuable

therapeutic index. It is due to the presence of non-nutritive plant phytochemicals.

Different phytochemicals have been found to possess a wide range of activities,

which may help in protection against chronic diseases. For example, the phenolics

such as flavonoids, tannins are the group of compounds that act as primary

antioxidant or free radical scavengers (Hada et al., 2001).

Alkaloids have been well investigated for many pharmacological properties

including antiprotozoal, cytotoxic, antidiabetic (Oliver, 1980; Cherian and

Augusti, 1995) and anti-inflammatory (Liu, 2003) properties but there are only

few reports about their antimicrobial properties. The phenolics such as flavonoids,

tannins are the group of compounds that act as primary antioxidant or free radical

scavengers (Hada et al., 2001). Flavonoids are referred as natures biological

response modifiers, because of their inherent ability to modify the body’s reaction

to allergies, anti-inflammatory, antimicrobial, anticancer activities. Apart from the



186

flavonoids shows potent antihyperglycemic activity in animal studies

(Chakravarth, 1980; Geetha et al., 1994).

Tannins were reported to exhibit antidiabetic (Oliver, 1980; Cherian and

Augusti, 1995), anti-inflammatory (Latha et al., 1998), antibacterial and antitumor

activities. It has also been reported that certain tannins were able to inhibit HIV

replication selectively besides use as diuretics. Plant tannins have been widely

recognized for their pharmacological properties and are known to make trees and

shrubs a different meal for many caterpillars (Haslem, 1989). Saponins are

glycosides occurring widely in plants. They are abundant in many foods

consumed by animals and man. Saponin is used as mild detergents and in

intracellular histochemstry staining to allow antibody access to intracellular

proteins. In medicine, it is used in hypercholesterolemia, hyperglycemia

(Rupasinghe et al., 2003), antioxidant, anti-cancer, anti-inflammatory (Manach et

al., 1996), central nervous system activities (Argal and Pathak, 2006) and weight

loss etc. It is also known to have antifungal properties (Rupasinghe et al., 2003).

Plant steroids are known to be important for their cardiotonic activities,

possession of insecticidal, anti-inflammatory (Akindele and Adeyemi, 2007; Ilkay

Orhan et al., 2007), analgesic properties (Sayyah et al., 2004; Malairajan et al.,

2006), central nervous system activities (Argal and Pathak, 2006) and

antimicrobial properties. They are also used in nutrition, herbal medicine and

cosmetics. Glycosides were reported to exhibit anti diabetic characteristics

(Oliver, 1980; Cherian and Augusti, 1995).



187

Besides the above said phytochemicals terpenes were found to stimulate

secretion or possess insulin - like effect (Marles and Fransworth, 1995) and

decrease blood sugar level in animal studies (Luo et al., 1999). Similarly,

terpenoids and vitamins acts as regulators of metabolism and play a protective role

as antioxidants. Steroids and triterpenoids showed the analgesic properties

(Sayyah et al., 2004 and Malairajan et al., 2006).

Cardiac glycosides on the other hand are known to hamper the Na + / K +

pump. This results in an increase in the level of sodium ions in the myocytes

which then enhance in the level of calcium ions. This consequently increases the

amount of Ca 2+ ions available for contraction of the heart muscle, which improves

cardiac output and reduces distention of heart and thus are used in the treatment of

congestive heart failure and cardiac arrhythmia. Phytochemical screening of the

methanol and aqueous extracts of Asteracantha longifolia and Pergularia daemia,

leaves used in this study revealed that the crude extracts contained alkaloids,

flavonoids, phenols, tannins, saponins, glycosides, triterpenoids, fats and fixed oils

(Table 7.1 & 7.2). Asteracantha longifolia and Pergularia daemia can also have

various medicinal values such as antioxidant, anti-diabetic and antimicrobial

activities etc. Although, the presence of active constituents in all the extracts, the

leaf methanolic extracts showed higher amount of these phytoconstituents. From

the qualitative and quantitative analysis it is confirmed that the leaf methanolic

extract contains maximum phytochemicals than the aqueous extracts.

_____


188

Chapter-VIII

SUMMARY AND CONCLUSION . .

Medicinal plants are an important source of indigenous traditional

medicinal system in our country. The wide spectrum of disease and resistance

caused by microbes has led to research for remedy in the roots of our ancient

traditional medical practices. The widespread use of herbal remedies and

healthcare preparations, as those described in ancient texts such as the Vedas and

the Bible, and obtained from commonly used traditional herbs and medicinal

plants, has been traced to the occurrence of natural products with medicinal

properties. The use of traditional medicine and medicinal plants in most

developing countries, as a normative basis for the maintenance of good health, has

been widely observed. Furthermore, an increasing reliance on the use of medicinal

plants in the industrialised societies has been traced to the extraction and

development of several drugs and chemotherapeutics from these plants as well as

from traditionally used rural herbal remedies. The present research work focuses

on Asteracantha longifolia (Linn.) Nees. and Pergularia daemia (Forsskal) Chiov.

because it has huge pharmacological properties, as used by rural communities for

various treatments such as Snake bite, Head ache, Skin diseases, and Fever etc. It

acts as an important fodder for livestock. Studies on antimicrobial, antioxidant,

antidiabetic and phytochemical properties have not yet been carried out in these

plants.

Chapter - VIII Summary & Conclusion


189

Antimicrobial activity

The antimicrobial activity was done by agar disc diffusion method for nine

bacterial and two fungal strains, viz., Staphylococcus aureus, Bacillus cereus,

Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Klebsiella

pneumoniae, Salmonella typhi, Proteus vulgaris, Shigella flexneri, Aspergillus

niger and Candida albicans. The plant material was extracted with distilled water

(aqueous) and methanol.

The preliminary screening of the methanol extracts of both the plants

showed more potent than the aqueous extracts. The plant extracts were more

active against gram-positive bacteria than gram-negative bacteria. The activity of

aqueous and methanol leaf extracts of A.longifolia and P.daemia exhibited

significant activity against S. aurues and moderate inhibition against

P. aeruginosa.

The most susceptible bacteria was noted as S.aureus for the plant extracts of

A.longifolia and P.daemia. For this study, it is cleared that the aqueous as well as

the methanol extracts produced form the plant A.longifolia revealed the presence

of more bioactive medicinal components which have suppressed almost all the

pathogenic bacteria and fungal used in the study than P.daemia.


In the present study, the total phenols, flavonoids, DPPH assay and

reducing power assays of leaf extracts of two different species were evaluated.

The extracts of both plants possess significant activity in DPPH scavenging and



190

reducing power assays. Thus, antioxidant potential of extracts of A. longifolia and

P.daemia may be attributed to the presnce of phenols, flavonoids and saponins.

The analysis of antioxidant properties of two different species showed that

the A.longifolia exhibited higher levels of antioxidant and reducing power

properties. Out of the two plant species viz A.longifolia and P.daemia screened for

antioxidant activities, the extracts of A.longifolia containing highest amount of

flavonoid and phenolic compounds, exhibited the greatest antioxidant activity.

Antidiabetic activity

The methanol and aqueous extracts of A.longifolia and P.daemia was

evaluated for their antidiabetic activity in alloxan induced diabetic rats. Diabetic

rats were assigned to be treated with crude methanol and aqueous extracts

(A.longifolia and P.daemia) (250 mg/kg of b.w) and untreated rats were assigned

to the treated with saline. At the end of the 21st day the animals were sacrificed

and serum kept from them were analyzed for histopathological and biochemical

studies.

Acute toxicity study in A.longifolia and P.daemia were done in Swiss

albino rats by oral administration of crude extracts of the plants in the

concentration of 100 - 1600 mg/kg of body weight. The medium lethal

concentration dose (LD50) of the extracts in 1600 mg/kg of the animal body

weight showed no adverse effects.

The methanol and aqueous extracts of both the plants have significantly

reduced the elevated blood glucose level. The reduced level of insulin in the



191

diabetic rats was also found to be significantly secreted after the administration of

the extracts.

The extracts of these palnts have remarkabley decreased the urea, creatinine

and glycosylated haemoglobin levels in the diabetic rats were detected in the

study. The serum lipid profiles of Total cholesterol, Triglycerides, Low density

lipoprotein, High density lipoprotein and very low density lipoprotein were

expressively decreased diabetic rats when compared to non-treated rats of the

palnt extracts. However, high density lipoprotein cholesterol level was lowered in

the diabetic control and restored after the administration of the extracts.

The crude extracts of the plants significantly improved the protein, albumin

and globulin levels of the diabetic induced rats. The marker enzymes like SGPT,

SGOT and ALP levels were also significantly controlled in the diabetic rats. The

result showed that the protective role of A.longifolia and P.daemia were confined

the extracts against the organ damage of the tesed animals so that the plants

extracts were proved to be prevented the enzymes leakage from the cellular site

because of the any damage in the organs of the tested animals.

Alloxan was induced lipid lipid peroxidation in the tested animals along

with diabetes. This effect was also inhibited by the palnt extracts when

administration to the tested animals. This is one of the remarkable role of the plant

extracts in the study. As the phytochemical analysis of the plants detected the

presence of phenolic and flavonoid derivatives and ferric reducing assay indicated

the electron trapping capacity in a significant manner.



192

As the comparision of control rats with the alloxan induced diabetic rats

there is a reduction in the SOD, CAT, GPx and GSH activities which were

restored with the administration the plant extracts.The reduction in the body

weight due to the alloxan chemical in the rats were significantly controlled and the

increase in body weight were observed by treating them with the plant extracts

especially methanolic and aqueous extracts. Histopathology of liver and pancreas

showed disoriented cellular architecture. But after treatment with A. longifolia and

P.daemia extracts showed all the disorientation to normal levels. The

normalization was more in A.longifolia extracts.

Phytochemical screening

The plant extracts showed the presence of some secondary metabolites such

as alkaloids, flavonoids, steroids, terpenoids, glycosides, saponins and phenolic

compounds which were determined by the preliminary qualitative phytochemical

analysis. In conclusion, it is apparent that the pharmacological activity of

A.longifolia and P.daemia reflects its uses in traditional medicine. Thus, this study

showed that the administration of A.longifolia and P.daemia exhibited better

antidiabetic activity. Further, it can be suggested tha the active antihyperglycemic

agents present in the extracts can be help to overcome the diabetic complications

by increasing the insulin secretion or by scavenging free radical and preventing

the depletion of endogenous antioxidants. Thus, the present study, concluded that

there is a need of further advancement in the pharmacological investigations of the

plants in the field of clinical study of diabetes is essential and encourageable.

_____


193

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241

LIST OF PUBLICATIONS

1. A.Doss and S.P.Anand, 2012. Preliminary phytochemical screening of

Asteracantha longifolia and Pergularia daemia. World Applied Sciences

Journal. 18(2): 233 – 235.

2. A.Doss and S.P.Anand, 2013. Antimicrobial activity of Hygrophila auriculata

(Schumach.) Heine and Pergularia daemia Linn. African Journal of Plant

Science. 7(4): 137-142.

3. A.Doss and S.P.Anand, 2013. Evaluation of antioxidant activity of

Hygrophila auriculata (Schumach.) Heine and Pergularia daemia Linn.

Wudpecker Journal of Medicinal Plants. 2(4): 74-79.

4. A.Doss and S.P.Anand, 2014. Antihyperglycemic activity of methanol and

aqueous extracts of Pergularia daemia Linn. African Journal of

Biotechnology, 13(1): 170-174. (Impact factor: 0.619)

5. A.Doss and S.P.Anand, 2014. Evaluation of antidiabetic activity of

Asteracantha longifolia (Linn.) Ness. Research Journal of Pharmacology.

Accepted for Publication.

_____

Plate 3.1. Asteracantha longifolia (Linn.) Nees.

a. Natural Habitat

b. Closer view with flower

c. Plate

Plate 3.2. Pergularia daemia (Forsskal) Chiov.

a. Natural Habitat

a. Closer view of Flower and Fruit

b. Closer view of flower and fruit

Plate 4.1

Zones of Inhibition with methanol and aqueous extract of Asteracantha longifolia

i. Staphylococcus aureus (a: 100mg/ml, b: 200 mg/ml, c: chloramphenicol, d: DMSO)

ii. Streptococcus pneumoniae (a: 100mg/ml, b: 200mg/ml, c: chloramphenicol,

d: DMSO)

iii. E.coli (a: 100mg/ml, b: 200 mg/ml, c: chloramphenicol, d: DMSO)

iv. Salmonella typhi (a: 100mg/ml, b: 200 mg/ml, c: DMSO, d: chloramphenicol)

v. Shigella flexneri (a: 100mg/ml, b: 200 mg/ml, c: chloramphenicol, d: DMSO)

vi. Staphylococcus aureus (a: 100mg/ml, b: 200 mg/ml, c: DMSO, d: chloramphenicol)

Plate 4.1

Zones of Inhibition with methanol and aqueous extract of Asteracantha longifolia

i ii

iii iv

v vi

Plate 4.2

Zones of Inhibition with methanol extract of Pergularia daemia

i. Streptococcus pneumonia (a: 100mg/ml, b: 200 mg/ml, c: chloramphenicol, d: DMSO)

ii. E.coli (a: 100mg/ml, b: 200 mg/ml, c: chloramphenicol, d: DMSO)

iii. Bacillus cereus (a: 100mg/ml, b: 200 mg/ml, c: DMSO, d: chloramphenicol)

iv. Staphylococcus aureus (a: chloramphenicol, b: DMSO, c: 100mg/ml, d: 200 mg/ml)

v. Klebsiella pneumonia (a: 100mg/ml, b: 200 mg/ml, c: DMSO, d: chloramphenicol)

vi. Salmonella typhi (a: 100mg/ml, b: 200 mg/ml, c: DMSO, d: chloramphenicol)

Plate 4.2 Zones of Inhibition with methanol extract of Pergularia daemia

i ii

iii iv

v vi

Plate 4.3

Zones of Inhibition with aqueous extract of Pergularia daemia

i. Staphylococcus aureus (a: 100mg/ml, b: 200 mg/ml, c: chloramphenicol, d: DMSO)

ii. E.coli (a: 100mg/ml, b: 200 mg/ml, c: DMSO, d: chloramphenicol)

iii. Klebsiella pneumonia (a: 100mg/ml, b: 200 mg/ml, c: chloramphenicol, d: DMSO)

iv. Proteus vulgaris (a: 100mg/ml, b: 200 mg/ml, c: chloramphenicol, d: DMSO)

v. Salmonella typhi (a: 100mg/ml, b: 200 mg/ml, c: DMSO, d: chloramphenicol)

Plate 4.3 Zones of Inhibition with aqueous extract of Pergularia daemia

i ii

iii iv

v

Plate 6.1

Experimental animals

a-d : Swiss albino rats with cages

e & f : Sacrifice methods

Plate 6.1

Experimental animals

a b

d

e

c

f

Plate 6.2

Animals handling methods

a, b, c : Induction of diabetes by Intraperitonial method

d : Oral drug administration

e : Collection of blood by Retinoorbital method

f : Dissection of animals

Plate 6.2 Animals handling methods

a b

d

e

c

f

Plate 6.3

Histopathological studies on Asteracantha longifolia and Pergularia daemia Pancreas

a. Pancreatic sections of normal rat show cells with well-preserved cytoplasm and nucleus.

b. In the pancreatic sections of alloxan intoxicated rats, the cells are irregular, not well defined and defect in cell membrane. Necrosis of the cells is very clear.

c. ALME treatment was improved restored the altered histopathological changes.

d. PDME treatment was improved restored the altered histopathological changes.

e. The damage is recovered with the treatment of ALAE

f. The damage is recovered with the treatment of PDAE

Plate 6.3

Histopathological studies on Asteracantha longifolia and Pergularia daemia Pancreas

a b

d

e

c

f

Plate 6.4

Histopathological studies on Asteracantha longifolia and Pergularia daemia Liver

a. The normal histological section shows the well-arranged cells and clear central

vein.

b. Section shows the complete destruction of hepatocytes degeneration of central vein, fatty degeneration and neutrophil distribution. Section shows the damaged hepatocytes and various size vacuoles

c. Histopathological changes are restored near to normal in the ALME treated group.

d. Histopathological changes are restored near to normal in the PDME treated group.

e. The damage is recovered with the treatment of ALAE

f. The damage is recovered with the treatment of PDAE

Plate 6.4

Histopathological studies on Asteracantha longifolia and Pergularia daemia Liver

p

a b

d

e

c

f

Chapter - I

INTRODUCTION

Chapter - II

OBJECTIVES

Chapter - III

MATERIALS AND METHODS

Chapter - IV

ANTIMICROBIAL ACTIVITY

Chapter - V

ANTIOXIDANT ACTIVITY

Chapter - VI

ANTIDIABETIC ACTIVITY

Chapter - VII

PHYTOCHEMICAL SCREENING

Chapter - VIII

SUMMARY AND CONCLUSION

REFERENCES

APPENDICES

Dedicated to

my beloved Family Members

and Friend s

Vol. 7(4), pp. 137-142, April 2013

DOI: 10.5897/AJPS12.193

ISSN 1996-0824 ©2013 AcademicJournals

http://www.academicjournals.org/AJPS

African Journal of Plant Science

Full Length Research Paper

Antimicrobial activity of Hygrophila auriculata (Schumach.) Heine and Pergularia daemia Linn.

A. Doss* and S.P. Anand

PG and Research Department of Botany, National College (Autonomous), Tiruchirappalli – 620 001 Tamilnadu, India.

Accepted 22 March, 2013

The antimicrobial efficiency and minimum inhibitory concentration of the extracts of Hygrophila auriculata (Schumach.) Heine (Acanthaceae) and Pergularia daemia Linn. (Apocyanaceae) were evaluated against nine bacterial species like (Bacillus cereus, Staphylococcus aureus, Streptococcus pneumonia, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Salmonella typhi, Proteus vulgaris and Shigella flexneri) and two fungal species (Aspergillus niger and Candida albicans). The susceptibility of the microorganism to the extracts of these plants were compared with each other and with selected antibiotics. All these plants were effective against three or more of the pathogenic microorganisms. This in vitro study corroborated the antimicrobial activity of the selected plants used in folklore medicine. Key words: Activity index (AI), total activity (TA), disc diffusion methods, microbial pathogens, folklore medicine.

INTRODUCTION Infectious diseases are the world's leading cause of premature deaths, killing almost 50,000 people every day (Yadav and Khan, 2012). Several synthetic antibiotics and drugs are employed in the treatment of the microbial infections and communicable diseases; but, the microbial pathogens develop resistance to the synthetic antibiotics. The increasing incidence of resistance to antibiotics and their side effects on the functioning of different parts of the body organ systems necessitate to finding out substitutes for the antibiotics (Sasikumar et al., 2007). In addition, in developing countries, synthetic drugs are not only expensive and inadequate for the treatment of diseases but are often found with adulterations and side effects. Therefore, there is need to search new infection-fighting strategies to control microbial infections. Due to a rapid increase in the rate of infections, antibiotic resistance in microorganisms and due to side effects of synthetic antibiotics, medicinal plants are gaining

popularity over these drugs (Babu and Subhasree, 2009). Natural products are important sources for biologically

active drugs. There has been an increasing interest in the study of medicinal plants as natural products in different parts of the world. Medical plants contain large varieties of chemical substances which possess important therapeutic properties that can be utilized in the treatment of human diseases (Panchavarnakili et al., 2012). Many medicines like strychnine, aspirin, vincristine and taxol are of plant origin. According to World Health Organization, more than 80% of the world's population relies on traditional medicine for their primary healthcare needs. In developing countries, people of low income group such as farmers, inhabitants of hamlets and native communities use folk medicine for the treatment of common infectious diseases (Ratha et al., 2012). Among the estimated 2,50,000 to 5,00,000 plant species, only a small percentage has been investigated phytochemically

*Corresponding author. E-mail: [email protected].

138 Afr. J. Plant Sci. and the fraction submitted to biological or pharmacological screening is even smaller (Mahesh and Satish, 2008). Rural communities in particular tribes of Trichy District, Tamilnadu, depend on plant resources mainly for herbal medicines, food, forage, construction of dwellings, making household implements, sleeping mats, and for fire and shade.

Hygrophila auriculata, a perennial angiosperm of Acanthaceae, widely distributed semi-aquatic herb in India, is being used as vegetable in some states like Odisha, Chhattisgarh and West Bengal. The pre-flowering or flowering succulent aerial parts are boiled and consumed by the rural people of these states to increase the haemoglobin level. This herbal remedy does not have any side effects with proven effectiveness. This plant contains various groups of phyto-constituents viz. phytosterols, fatty acids, minerals, polyphenols, proanthocyanins, mucilage, alkaloids, enzymes, amino acids, carbohydrates, hydrocarbons, flavonoids, terpenoids, vitamins, glycosides, etc. and is useful in the treatment of anasaraca, diseases of urinogenital tract, dropsy of chronic Bright’s disease, hyperdipsia, vesical calculi, flatulence, diarrhea, dysentery, leucorrhoea, gonorrhea, asthma, blood diseases, gastric diseases, painful micturition, menorrhagea, etc. (Rastogi and Mehrotra 1993; Annonymous, 2002; Sharma et al., 2002; Asolkar et al., 2005; Nadkarni, 2007).

Pergularia daemia (Forsk.) Chiov (Apocyanaceae), commonly known as utaran (Hindi), Dustapuchettu (Telugu), Uttamarani (Sanskrit) is a slender, hispid, fetid smelling laticiferous twiner found in the plains throughout the hot parts of India. P. daemia is said to have more magical application than medical application as it posses diverse healing potential for a wide range of illnesses. Some of the Folklore people use this plant to treat jaundice, as laxative, anti-pyretic, expectorants and also in infantile diarrhea. The leaf latex is locally used as pain killer killer and for relief from toothache (Hebbar et al., 2010), the sap expressed from the leaves are held to cure sore eyes in Ghana. The plant reduces the incidence of convulsion and asthma. It is used to regulate the menstrual cycle and intestinal functions. The root is useful in treating leprosy, mental disorders, anemia and piles (Omale et al., 2011). We report here the results of the antimicrobial properties of extracts from the leaves of H. auriculata A.longifolia and P. daemia.

MATERIALS AND METHODS Plant materials

Fresh plant leaves were collected randomly from the gardens and villages of Trichy district, Tamilnadu from the natural stands. The botanical identity of these plants was confirmed by Dr.V.Sampath Kumar, Scientist – C, Botanical Survey of India (Southern Circle), Coimbatore, Tamilnadu. The voucher specimens are deposited at the Department of Botany, National College (Autonomous), Tiruchirapalli-620 001, Tamilnadu, India.

Preparation of extracts Aqueous extraction Hundred grams of dried powder were extracted in distilled water for 6 h at slow heat. Every 2 h it was filtered through eight layers of muslin cloth and centrifuged at 5000 rpm 5000 g for 15 min. The supernatant was collected. This procedure was repeated twice and after 6 h the supernatant was concentrated to one-fifth of the original volume. Solvent extraction

Hundred grams of dried plant powdered samples were extracted with 200 ml of methanol kept on a rotary shaker for 24 h. Thereafter, it was filtered and centrifuged at 5000 rpm for 15 min. The supernatant was collected and the solvent was evaporated to make the final volume one-fifth of the original volume. It was stored at 4°C in airtight bottles for further studies. Antimicrobial activity

Microorganisms Microorganisms were obtained from the Microbial Type Culture Collection centre (MTCC), Chandigarh, India. Amongst eleven microorganisms investigated, nine were bacterial strains viz.,Staphylococcus aureus MTCC 3160, Bacillus cereus MTCC 442, Streptococcus pneumonia MTCC 655, Escherichia coli MTCC

598, Pseudomonas aeruginosa MTCC 42642 Klebsiella

pneumoniae MTCC 7407, Salmonella typhi MTCC 3917, Proteus

vulgaris MTCC 742 and Shigella flexneri MTCC 1457, while the other two were fungal strains viz. Aspergillus niger MTCC 2546, Candida albicans MTCC 183. All the microorganisms were maintained at 4°C on nutrient and potato dextrose agar slants.

Disc diffusion method Antimicrobial activity was carried out by the disc diffusion method. The antimicrobial assays of aqueous and methanolic extracts were performed by Bauer et al. (1966). Each plant extract was tested at two different concentrations (100 and 200 µg/ml) to see their inhibitory effects against microbial pathogens. Sterile paper discs (6 mm in diameter) prepared from Whatman No. 1 filter paper was impregnated with drug, containing solution placed on the inoculated agar. The inoculated plates were incubated at 37°C for 24 h. The antibacterial activity was evaluated by measuring the diameter of the inhibition zone for the test microorganisms.

The potato dextrose agar plates were inoculated each with fungal culture by point (10 days old cultures) inoculation. The filter paper discs loaded with 100 and 200 µg/ml concentrations of the extracts were placed on test organism- seeded plates. The activity was determined after 72 h of incubation at 28°C. The diameters of the inhibition zones were measured in mm (Taylor et al., 1995). Chloramphenicol and Fluconazole are used as standard antibiotics. Minimum inhibitory concentration (MIC) For determination of MIC, 1 ml of broth medium was taken into 10 test tubes for each bacterium. Different concentrations of plant extracts ranging from 0.125 to 8 µg/ml

-1 concentration were

incorporated into the broth and the tubes were then inoculated with 0.1 ml of inoculums of respective bacteria (10

5 CFU ml

-1) and kept

at 37°C for 24 h. The test tube containing the lowest concentration

Doss and Anand 139

Table 1. Effect of methanol and aqueous extracts of H. auriculata.

S/N Name of the strain

Zone of Inhibition (mm)

Methanol (µg/ml) Aqueous (µg/ml) Synthetic drug (Chloramphenicol)

100 200 100 200

1 Staphylococcus aureus 11 18 8 10 22

2 Streptococcus pneumoniae 10 12 - - 20

3 Bacillus cereus 9 11 - 9 17

4 Escherichia coli 10 12 - 8 21

5 Pseudomonas aeruginosa 8 12 - 8 18

6 Klbseillae pneumoniea - 10 - - 17

7 Salmonella typhi - 9 - - 16

8 Proteus vulgaris 8 10 - - 20

9 Shigella flexneri - 10 - 9 16

Antifungal activity

Synthetic drug (Fluconazole)

10 Candida albicans - 9 - - 15

11 Aspergillus niger - 9 - - 17

Table 2. The MIC index of methanol and aqueous extracts of H. auriculata.

S/N Name of the strain Methanol Aqueous

MIC (µg/ml) MBC (µg/ml) MICindex MIC (µg/ml) MBC (µg/ml) MICindex

1 S. aureus 0.125 0.250 2 4 4 1

2 S. pneumoniae 0.250 0.500 2 - - -

3 B. cereus 0.500 0.500 1 4 4 1

4 E. coli 0.500 0.500 1 - - -

5 P. aeruginosa 2 2 1 - - -

6 K. pneumoniae 4 4 1 - - -

7 S. typhi 2 2 1 - - -

8 P. vulgaris 0.500 1 2 4 4 1

9 S. flexneri 0.500 0.500 1 - - -

10 C. albicans 2 2 1 - - -

11 A. niger 2 2 1 2 2 1

of extract which showed reduction in turbidity when compared with control was regarded as MIC of that extract (Muhamed et al., 2011).

Total activity (TA) determination

Total activity is the volume at which test extract can be diluted with the ability to kill microorganisms. It is calculated by dividing the

amount of extract from 1 g plant material by the MIC of the same extract or compound isolated and is expressed in ml/g (Sharma and Kumar, 2009). AI = Activity Index (IZ developed by extract/IZ developed by standard).

RESULTS AND DISCUSSION

The results reveal variability in inhibitory nature of each

extract against specific bacteria. The inhibition of bacterial growth was dose dependent since the inhibitory action of the extract was found to increase with an increase in concentration against all bacterial strains as evidenced by the higher zone of inhibitions at higher concentrations of each extract. Antimicrobial activity (assessed in terms of inhibition zone, total activity and activity index) of the crude extracts, tested against selected microorganisms are recorded.

Both crude methanol and aqueous extracts of A. longifolia exhibited varying degrees of antimicrobial activities against the test organisms. The 200 µg/ml crude methanol extract showed higher inhibition zone than crude aqueous extract against S. aureus, S. pneumoniae, E. coli and P. aeruginosa, respectively (Tables 1 and 3). Similarly 200 µg/ml methanol extract of P. daemia exhibited inhibition zone of 15 mm (AI = 0.818) for

140 Afr. J. Plant Sci.

Table 3. Antimicrobial activity index of crude extracts of H. auriculata.

S/N Name of the strain

Methanol Aqueous



100 200 100 200

1 S. aureus 0.5 0.818 4 0.363 0.454 0.175

2 S. pneumoniae 0.5 0.6 2 - - -

3 B. cereus 0.529 0.647 1 - 0.529 0.175

4 E. coli 0.476 0.571 1 - 0.380 -

5 P. aeruginosa 0.444 0.666 0.25 - 0.444 -

6 K. pneumoniae - 0.588 0.125 - - -

7 S. typhi - 0.562 0.25 - - -

8 P. vulgaris 0.4 0.5 1 - - -

9 S. flexneri - 0.625 1 - 0.562 0.175

10 C. albicans - 0.6 0.25 - - -

11 A. niger - 0.529 0.25 - - -

Table 4. Effect of methanol and aqueous extracts of P. daemia on microbes.

S/N Name of the Strains

Zone of Inhibition (mm)

Methanol (µg/ml) Aqueous (µg/ml) Synthetic drug (Chloramphenicol)

100 200 100 200

1 Staphylococcus aureus 10 15 10 12 22

2 Streptococcus pneumoniae 10 11 - 9 20

3 Bacillus cereus 9 10 - 10 17

4 Escherichia coli 10 12 - - 21

5 Pseudomonas aeruginosa 8 10 - 8 18

6 Klbseillae pneumoniae - 8 - - 17

7 Salmonella typhi - 10 - 8 16

8 Proteus vulgaris - 9 - - 20

9 Sheigella flexneri 8 10 - - 16


10 Candida albicans - - - - 15

11 Aspergillus niger - - - - 17

S. aureus and 12 mm (AI = 0.571) for E. coli respectively. The aqueous extract showed highest inhibition zone of 12 mm in (AI = 0.454) for S. aureus and 10 mm (AI = 0.529) for B. cereus (Tables 4 and 6).

Antibiotics chloramphenicol and fluconazole have shown moderate inhibition zone diameter than that of plant extracts. It had the inhibition zone in the range of 15 to 22 mm. The zones of inhibition produced by the tested extracts against Aspergillus niger and Candida albicans ranged between 8 to 9 mm. The highest zone of inhibition was produced by methanol extracts of H. auriculata while that of P. daemia did not inhibit the growth (Tables 1 and 4).

Methanol extract of P. daemia showed least MIC value that is, 0.500 µg/ml (MBC = 0.250 µg/ml) against S. aureus while aqueous extract had moderate activity at

0.500 µg/ml (MBC = 1.0 µg/ml) concentration (Table 5). Similarly the H. auriculata methanol extract was found to be highly effective as it has shown very low MIC value (0.125 µg/ml) against S. aureus (Table 2). The total activity was highest for methanol extracts of both plants (4.0 and 1.1 ml/g) against S. aureus (Tables 3 and 6). Our results support this view as methanol extracts had comparatively more inhibition action than aqueous extracts (Hugo et al., 2005).

Several reports have shown the antimicrobial properties of plant extracts under laboratory conditions (Doss et al., 2009a; Doss et al., 2009b; Venkataswamy et al., 2010; Anand et al., 2001). Normally Gram-positive bacterial strains are found to be more susceptible to the extracts than Gram negative bacteria. This is attributed to the fact that these two groups differ by their cell wall

Doss and Anand 141

Table 5. The MIC index of methanol and aqueous extracts of P. daemia.

S/N Name of the strain Methanol Aqueous

MIC (µg/ml) MBC (µg/ml) MICindex MIC (µg/ml) MBC (µg/ml) MICindex

1 S. aureus 0.500 0.250 0.500 0.500 1.0 2

2 St. pneumoniae 1.0 2.0 1 2.0 2.0 1

3 B. cereus 0.500 1.0 1 1.0 1.0 1

4 E. coli 2.0 2.0 1 - - -

5 P. aeruginosa - - - - - -

6 K. pneumoniae - - - - - -

7 S .typhi - - - - - -

8 P. vulgaris - - - - - -

9 S. flexneri 4.0 2.0 0.5 - - -

10 C. albicans - - - - - -

11 A. niger - - - - - -

Table 6. Antimicrobial activity index of crude extracts of P. daemia.

S/N Name of the Strains

Methanol Aqueous



100 200 100 200

1 S. aureus 0.454 0.681 1.1 0.454 0.545 1

2 St. pneumoniae 0.5 0.55 0.55 - 0.45 0.25

3 B. cereus 0.529 0.588 1.1 - 0.588 0.5

4 E. coli 0.476 0.571 0.275 - - -

5 P. aeruginosa 0.444 0.555 - - 0.444 -

6 K. pneumoniae - 0.470 - - - -

7 S. typhi - 0.625 - - 0.5 -

8 P. vulgaris - 0.45 - - - -

9 S. flexneri 0.5 0.625 0.137 - - -

10 C. albicans - - - - - -

11 A. niger - - - - -

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Wudpecker Journal of Medicinal Plants ISSN 2315-7275 Vol. 2(4), pp 074 - 079, August 2013 2013 Wudpecker Journals

Evaluation of Antioxidant activity of Hygrophila auriculata (Schumach.) Heine and Pergularia damia

Linn.

A. Doss and S.P Anand

PG & Research Department of Botany, National College (Autonomous) Trichy, Tamilnadu, South India.

*Corresponding author E-mail: [email protected].

Accepted 15 July 2013

Cellular damage or oxidative injury arising from free radicals or reactive oxygen species (ROS) now appears the fundamental mechanism underlying a number of human neurodegenerative disorders, diabetes, inflammation, viral infections, autoimmune pathologies and digestive system disorders. Antioxidants are the compounds which terminate the attack of reactive species and reduce the risk of diseases. The study was conducted to determine the antioxidant activity of two folklore medicinal plants H. auriculata and P. daemia. The methanolic and aqueous extracts of H. Auriculata and P. daemia were screening their free radical scavenging and Ferric reducing properties using ascorbic acid as standard antioxidant. H. Auriculata and P. daemia exhibited varying degrees of antioxidant activity ranged between 6.41 to 83.90%. The methanolic extract of H. Auriculata showed significantly higher antioxidant activity than the P. daemia. These results suggested the potentials of H. auriculata as a medicine against free-radical-associated oxidative damage. Key words: Antioxidant activity, 1,1 Diphenyl-2- picryl hydrazyl, folklore, flavonoids, total phenols.

INTRODUCTION Reactive oxygen species (ROS), such as superoxide anions, hydrogen peroxide, and hydroxyl, nitric oxide and peroxynitrite radicals, play an important role in oxidative stress related to the pathogenesis of various important diseases (Davis, 2000). In healthy individuals, the production of free radicals is balanced by the antioxidative defense system; however, oxidative stress is generated when equilibrium favors free radical generation as a result of a depletion of antioxidant levels (Maria Kratchanova et al., 2010).

Antioxidant substances block the action of free radicals which have been implicated in the pathogenesis of many diseases including atherosclerosis, ischemic heart disease, cancer, Alzheimer’s disease, Parkinson’s disease and in the aging process. Currently available synthetic antioxidants like butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), tertiary butylated hydroquinon and gallic acid esters, have been suspected to cause or prompt negative health effects (Vinay et al., 2010).

Hence, strong restrictions have been placed on their application and there is a trend to substitute them with naturally occurring antioxidants. Natural sources of antioxidants have gained increasing interest in the safety and no toxic side effects as compared with the synthetic

antioxidants (Liu et al., 2011). Medicinal plants are used in many domains, including

medicinae, nutrition, flavouring, beverages, dyeing, repellents, fragrances and cosmetics. Many species have been recognized to have medicinal properties and beneficial impact on health, eg. antioxidant activity, antimicrobial, anti-diabetic, hypolipidemic, digestive stimulation action, anti-inflammatory, antimutagenic effects and anticarcinogenic potential.

The role of medicinal plants in disease prevention or control has been attributed to antioxidant properties of their constituents, usually associated to a wide range of amphipathic molecules, broadly termed polyphenolic compounds (Ivanova et al., 2005). The number of reports on the isolation of natural antioxidants mainly of plant origin, has increased immensely during the last decade (Djeridane et al., 2007).

The genus Asterocantha, perennial angiospermic plant of family Acanthaceae, is a commonly found herb in India being used as vegetable in some states like Odisha, Chhattisgarh and West Bengal. Boiled aerial parts of succulent plant of pre-flowering and flowering stages are used extensively to increase the haemoglobin status by the rural people of these states. This herbal remedy is devoid of any side effects with proven effectiveness.

075 Wudpecker J. Med. Plants Hygrophila auriculata (Schumach.) Heine Nees [Synonym(s) Hygrophila spinosa T. Anders] contains various groups of phyto-constituents viz. phytosterols, fatty acids, minerals, polyphenols, proanthocyanins, mucilage, alkaloids, enzymes, amino acids, carbohydrates, hydrocarbons, flavonoids, terpenoids, vitamins, glycosides, etc. and is useful in the treatment of anasaraca, diseases of urinogenital tract, dropsy of chronic Bright’s disease, hyperdipsia, vesical calculi, flatulence, diarrhea, dysentery, leucorrhoea, gonorrhoea, asthma, blood diseases, gastric diseases, painful micturition, menorrhagea, etc (Rastogi and Mehrotra, 1993; Annonymous, 2002; Sharma et al., 2002; Asolkar et al., 2005; Nadkarni, 2007).

Pergularia damia Linn (Asclepiadaceae), commonly known as utaran (Hindi), Dustapuchettu (Telugu), Uttamarani (Sanskrit) is a slender, hispid, fetid smelling laticiferous twiner found in the plains throughout the hot parts of India. Pergularia daemia is said to have more magical application than medical application as it posses diverse healing potential for a wide range of illnesses. Some of the Folklore people use this plant to treat Jaundice, as laxative, anti-pyretic, expectorants and also in infantile diarrhea. The leaf latex is locally used as pain killers and for relief from toothache (Hebbar et al., 2012), the saps expressed from the leaves are held to cure sore eyes in Ghana.

The plant reduces the incidence of convulsion and asthma. It is used to regulate the menstrual cycle and intestinal functions. The root is useful in treating leprosy, mental disorders, anemia and piles (Omale James et al., 2011). We report here the results of the antioxidant properties of extracts from the leaves of H. auriculata and P.daemia. MATERIALS AND METHODS Plant materials Fresh plant parts (Hygrophila auriculata and Pergularia daemia) were collected randomly from the gardens and villages of Trichy district, Tamilnadu from the natural stands. The botanical identity of these plants was confirmed by Dr. V. Sampath Kumar, Scientist – C, Botanical Survey of India (Southern Circle), Coimbatore, Tamilnadu. A voucher specimen has been deposited at the Department of Botany, National College (Autonomous), Tiruchirapalli-620 001, Tamilnadu, India. Preparation of extracts Aqueous extraction 100 grams of dried powder were extracted in distilled water for 24 h at Room Temperature. Every 2 h it was

filtered through whatman no1 filter paper and centrifuged at 5000 g for 15 min. The supernatant was collected. This procedure was repeated twice and after 6 h the supernatant was concentrated to make the final volume one-fifth of the original volume. Solvent extraction 100 grams of dried plant powdered samples were extracted with 200 ml of methanol kept on a rotary shaker for 24 h. Thereafter, it was filtered and centrifuged at 5000 g for 15 min. The supernatant was collected and the solvent was evaporated to make the final volume one-fifth of the original volume. It was stored at 4oC in airtight bottles for further studies. Chemicals 1, 1-Diphenyl-2-picryl hydrazyl (DPPH) was purchased from Sigma Chemical Co. (St., Louis, USA). Ascorbic acid, Folin Ciocalteu reagent, and methanol were purchased from Merck Co. (Germany). In vitro antioxidant activity DPPH radical scavenging activity DPPH scavenging activity was carried out by the method of Blois (1958). Different concentrations (1000, 500, 250, 125, 62.5 and 31.2 mg/ml) of H. Auriculata and P.daemia extracts were dissolved in DMSO (dimethyl sulfoxide) and taken in test tubes in triplicates. Then 5 ml of 0.1mM ethanol solution of DPPH (1, 1, Diphenyl-2- Picrylhydrazyl) was added to each of the test tubes and were shaken vigorously. They were then allowed to stand at 370 C for 20 minutes. The control was prepared without any extracts. Methanol was used for base line corrections in absorbance (OD) of sample measured at 517nm. A radical scavenging activity was expressed as 1% scavenging activity and was calculated by the formula: Control O.D – Sample O.D% radical scavenging activity = ----------------------------- Control O.D Determination of reducing power assay Reducing activity was carried out by using the method of Oyaizu (1986). Different concentration (1000, 500, 250,125, 62.5 and 31.2 mg/ml) of H. Auriculata and P.daemia extracts were prepared with DMSO and taken in test tube as triplicates. To test tubes 2.5 ml of sodium phosphate buffer and 2.5 ml of 1% Potassium ferric

cyanide solution was added. These contents were mixed well and were incubated at 500 C for 20 minutes. After incubation 2.5ml of 10% Trichloroacetic acid (TCA) was added and were kept for centrifugation at 3000rpm for 10 minutes. After centrifugation 5ml of supernatant were taken and to this 5ml of distilled water was added. To this about 1ml of 1% ferric chlorite was added and was incubated at 350 C for 20 minutes. The O.D (absorbance) was taken at 700nm and the blank was prepared by adding every other solution but without extract and ferric chloride (0.1%) and the control was prepared by adding every other solution but without extract. The reducing power of the extract is linearly proportional to the concentration of the sample. Total phenolic content Total phenolic contents were determined by Folin Ciocalteu reagent (McDonald et al., 2001). A dilute extract of each crude extracts (0.5 ml of 1:10g ml –l) or gallic acid (standard phenolic compound) was mixed with Folin Ciocalteu reagent (5ml, 1:10 diluted with distilled water) and aqueous sodium carbonate (4ml, 1 M). The mixtures were allowed to stand for 15 min and the total phenols were determined by colorimetry at 765 nm. The standard curve was prepared using 0, 50, 100, 150, 200, 250 mg/ml solutions of gallic acid in methanol: water (50:50, v/v). Total phenol values are expressed in terms of gallic acid equivalent (mg g-l of dry mass), which is a common reference compound. Determination of total flavonoids Aluminum chloride colorimetric method was used for flavonoids determination (Chang et al., 2002). Each crude fruit extracts (0.5ml of 1:10 g/ml) in methanol were separately mixed with 1.5 ml of methanol, 0.1ml of 10% aluminum chloride, 0.1ml of 1M potassium acetate and 2.8ml of distilled water. It remained at room temperature for 30 min; the absorbance of the reaction mixture was measured at 415nm with a double beam Perkin Elmer UV/Visible spectrophotometer (USA). The calibration curve was prepared by preparing quercetin solution at concentrations 12.5 to 100g ml -1 in methanol. RESULTS AND DISCUSSION Natural antioxidants that are present in herbs and medicinal plants are responsible for inhibiting or preventing the deleterious consequences of oxidative stress. Medicinal plants contain free radical scavengers like polyphenols, flavonoids, tannins and phenolic compounds. In the present paper, we have evaluated the free radical scavenger activity of methanolic and aqueous

Doss and Anand 076 extracts of A. longifolia and P.daemia. The antioxidant properties of A. longifolia and P.daemia have been evaluated by measuring their DPPH and reducing ability contents using crude methanolic and aqueous extract of aerial parts of these plants. The plants, A. longifolia as well as P.daemia, exhibited an antioxidant activity in a dose-dependent manner. The methanolic extract of A. longifolia leaf at different doses exhibited significantly higher antioxidant activity as compared to P.daemia leaf. The α, α,diphenyl-β-picrylhydrazyl (DPPH) a stable nitrogen centered free radical, has been used to evaluate the antioxidant activity of natural products by measuring the radical quenching capacity in a relatively short period of time (Rastogi and Mehrotra, 1993). DPPH radicals react with suitable reducing agent as a result of which electron become paired off forming the corresponding hydrazine. The solution therefore looses colour stoichometrically depending on the number of electrons consumed which is measured spectrometrically at 517 nm (Bhatia et al., 2011). The extracts of all the tested extracts possessed free radical scavenging properties, but to varying degrees, ranging from 6.41 to 83.90% DPPH scavenging. Using the alcoholic extraction, generally methanol extract showed better DPPH scavenging activity. A maximum scavenging activity was offered by methanol of H. auriculata (83.90 %) and P.daemia (81.61%), followed by Aqueous extracts of H. auriculata (70.11 %) and P.daemia (65.06%) (Tables 1 and 2).

The reducing power of A. longifolia, P.daemia and the standard drug (Ascorbic acid) is shown in Tables 3 & 4. The extract of A. longifolia leaf had shown significantly higher reducing power than the extract of P.daemia leaf in a dose-dependent manner. Absorbance of solution was increased with concentration of plant extract, indicating the concentration of hydrogen donating compounds present in the extracts was increased or reducing power of extracts was increased. Tanaka et al. (1998) have reported that the antioxidant activity is concomitant with the reducing power.

The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. However, the activity of antioxidants has been assigned to various mechanisms such as prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity and radical scavenging. Samples with high reducing power were reported to have a better ability to electrons. It has been widely accepted that the higher level of absorbance at 700 nm indicates greater reducing power of the test samples (Rastogi and Mehrotra, 1993).

The crude methanol and aqueous extracts were prepared to examine the antioxidant activity and concentrations of Phenols and flavonoids. The extraction solvents of different polarity were used to extract the active substances of different polarity. The concentration

077 Wudpecker J. Med. Plants

Table 1. Antioxidant activity of Hygrophila auriculata (Schumach.) Heine (Linn. ) Nees. (DPPH assay).

Concentrations (mg/ml) Extracts Standard (Ascorbic acid) Methanol Aqueous 1000 83.90 ± 0.03 70.11 ± 0.01

79.0 ± 0.03

500 70.80 ± 0.05 64.05 ± 0.35 250 58.33 ± 0.025 50.47 ± 0.03 125 43.27 ± 0.02 34.91 ± 0.05 62.5 31.49 ± 0.017 20.31 ± 0.011 31.25 16.70 ± 0.12 6.41 ± 0.03

Table 2. Antioxidant activity of Pergularia damia Linn. (DPPH assay)


79.0 ± 0.03

500 69.51 ± 0.17 49.51 ± 0.01 250 56.08 ± 0.02 35.25 ± 0.01 125 41.83 ± 0.05 29.68 ± 0.01 62.5 32.45 ± 0.03 19.51 ± 0.015 31.25 17.86 ± 0.05 10.96 ± 0.17

Table 3. Ferric reducing capacity of Hygrophila auriculata (Schumach.) Heine (Linn. ) Nees.


0.689 ± 0.05

500 0.623 ± 0.01 0.630 ± 0.04 250 0.502 ± 0.01 0.524 ± 0.03 125 0.356 ± 0.11 0.401 ± 0.04 62.5 0.268 ± 0.02 0.220 ± 0.04 31.25 0.110 ± 0.011 0.104 ± 0.02

Table 4. Ferric reducing capacity of Pergularia damia Linn.


0.689 ± 0.05

500 0.561 ± 0.01 0.596 ± 0.01 250 0.418 ± 0.01 0.395 ± 0.01 125 0.342 ± 0.05 0.219 ± 0.05 62.5 0.291 ± 0.02 0.169 ± 0.15 31.25 0.096 ± 0.05 0.090 ± 0.01

of phenols in the examined fruit extracts using the Folin-Ciocalteu reagent was expressed in terms of gallic acid equivalent (Table 5). The concentrations of phenols in the examined crude extracts ranged from 144.61 to 246.14 mg GAE/g dry material. The high concentration of phenols was measured in methanol extracts of A. longifolia. The extracts obtained using more polar solvents had higher concentrations of phenols while the extracts obtained using low polar solvents contained

small concentrations (Table 2) (Canadanovic et al.,2008). The concentration of flavonoids in crude methanol and aqueous extracts were determined using spectrophotometric method with aluminium chloride. The summary of quantities of flavonoids identified in the tested extracts is shown in Table 5. The concentrations of flavonoids in methanol and aqueous extracts ranged from 51.13 to 104.20 mg QE/g dry material (Table 5). High concentrations of flavonoids were measured in methanol

Doss and Anand 078

Table 5. Total phenol and Flavonoids contents in the crude extracts of Hygrophila auriculata (Schumach.) Heine Linn. ) Nees.and Pergularia damia Linn.*

Medicinal plants Total phenols content (mg GAE/g dry material) Methanol Aqueous

Hygrophila auriculata 246.14 ± 0.01 168.01 ± 0.29 Pergularia daemia 218.4 ± 0.12 144.61 ± 0.21 Flavonoid content (mg QE/g dry material) Methanol Aqueous Hygrophila auriculata 104.20 ± 0.01 57.01 ± 0.01 Pergularia daemia 88.01 ± 0.12 51.13 ± 0.11

*Each value in the table was obtained by calculating the average of three analyses ± standard deviation. extracts of A. longifolia. The lowest flavonoid concentration was measured in aqueous of P.a daemia extract. The concentration of flavonoids in the extracts depends on the polarity of solvents and the type of plant material used for the extractions. The concentration of flavonoids in plant extracts depends on the polarity of solvents used in the extract preparation (Min and Chun-Zhao, 2005). Our results showed that methanolic extracts of H. auriculata leaves were showed good antioxidant activity, whereas aqueous extracts were found to be moderate in antioxidant capacity. Obviously, to confirm the beneficial effects of these extracts, it is necessary to carry out further studies about their in vivo activity and bioavailability. REFERENCES

Annonymous (2002). The Wealth of India- A Dictionary of Indian Raw Materials and Industrial Products, Ist Supplement Series. Raw Materials. Volume III. NISCOM, CSIR, New Delhi, p. 319.

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McDonald S, Prenzler P.D, Autolovich M and Robards K (2001). Phenolic content and antioxidant activity of olive extracts. Food Chemistry, 73:73-84. [15]. Oyaizu M (1986). Studies on product of browning reaction prepared from glucose amine. J Pn J. Nutr., 44: 307-315.

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Omale James, Ebiloma Godwin Unekwojo, Agbaji Ann Ojochenemi, (2011). Assessment of Biological Activities: A Comparison of Pergularia daemia and Jatropha curcas Leaf Extracts. British Biotech. J., 1(3): 85-100

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Vol. 13(1), pp. 170-174, 1 January, 2014

DOI: 10.5897/AJB2013.12479

ISSN 1684-5315 ©2014 Academic Journals

http://www.academicjournals.org/AJB

African Journal of Biotechnology

Full Length Research Paper

Antihyperglycemic activity of methanol and aqueous extracts of Pergularia daemia Linn.

A. Doss* and S. P. Anand

Department of Botany, National College (Autonomous), Trichy, Tamilnadu, South India.

Accepted 12 December, 2013

The objective of this study was to evaluate the antidiabetic activities of methanol and aqueous extracts of Pergularia daemia in alloxan induced diabetic rats. Methods used was methanol and aqueous extracts (250 mg/kg b.w.) of P. daemia was administered to alloxan induced diabetic mice for 21 days and blood glucose levels of the diabetic rats were monitored at intervals of hours and days throughout the duration of the experiments. Results shows that oral administration of alcoholic extract of P. daemia leaves to diabetic rats for 21 days significantly reduced the levels of blood glucose levels in both acute and sub acute study. In conclusion, these results suggest that the methanol extract of P. daemia possess antidiabetic effect on alloxan induced diabetic rats and it can be recommended for the prevention of diabetes mellitus. Key words: Alloxan monohydrate, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), urea, methanol, haemoglobin.

INTRODUCTION Diabetes mellitus is one of the most common chronic diseases in the whole world. It is a complex, multifactorial disease which affects the quality, quantity and style of an individual’s life. The fact confirmed by reports from the World Health Organization (WHO) shows that India has the largest number of diabetic subjects in the world (Anil et al., 2013). It has been suggested that diabetes is the third leading cause of death due to high incidence of morbidity and mortality after cancer and cardiovascular disorders. Complications such as renal failure, coronary artery disorder, cerebro-vascular disease, neurological complications, blindness, limb amputation, long term damage, dysfunctions and failure of various organs and eventually premature death are associated with chronic hyperglycemia (Kumudhavalli and Jaykar, 2012). The number of people suffering from the disease worldwide is increasing at an alarming rate with a projected 366 million peoples likely to be diabetic by the year 2030 as against 191 million estimated in 2000 (Wild et al., 2004). India

has today become the diabetic capital of the world with over 20 million diabetics and this number is set to increase to 57 million by 2025 (Sharma et al., 2010). The presently available synthetic drugs such as sulfonylureas and biguanides have serious side effects and thus searching for a new class of compounds is essential to overcome diabetic problem (Noor et al., 2008). Management of diabetes without any side effect is still a challenge to the medical community. There is continuous search for alternative drugs. For centuries, medicinal plants have been used to treat various human diseases. Herbal medicines are in great demand in the developed as well as developing countries for primary healthcare because of their wide biological activities, higher safety margins and lesser costs. After the recommendations made by WHO on diabetes mellitus, investigations on hypoglycemic agents from medicinal plants has become more important. According to world ethnobotanical information reports, almost 800 plants may possess anti-

*Corresponding author. E-mail: [email protected].

http://www.hindawi.com/86904024/

http://www.hindawi.com/86904024/

diabetic potential. In India, a number of plants are mentioned in ancient literature (Ayurveda) for the cure of diabetic conditions.

Medicinal plants like Trigonella foenum graecum, Allium sativum, Gymnema slyvestre and Syzigium cumini have been studied (Grover et al., 2002) for treatment of diabetes mellitus. However, a scientific proof of the anti-diabetic activity of medicinal plants and phytopharmaceu-ticals with fewer side effects is still lacking (Prasad et al., 2009). Pergularia daemia (forsk.) Chiov (Asclepiadaceae), commonly known as utaran (Hindi), Dustapuchettu (Telugu), Uttamarani (Sanskrit) is a slender, hispid, fetid smelling laticiferous twiner found in the plains throughout the hot parts of India. P. daemia is said to have more magical application than medical application as it possess diverse healing potential for a wide range of illnesses. Some of the folklore people use this plant to treat Jaundice, as laxative, anti-pyretic, expectorants and also in infantile diarrhea. The leaf latex is locally used as pain killers and for relief from toothache (Hebbar et al., 2010), the sap expressed from the leaves are held to cure sore eyes in Ghana. The plant reduces the incidence of convulsion and asthma. It is used to regulate the menstrual cycle and intestinal functions. The root is useful in treating leprosy, mental disorders, anemia and piles (Omale et al., 2011).

Therefore, the present study aimed to investigate the antidiabetic activity of methanolic and aqueous extracts of P. daemia. MATERIALS AND METHODS Collection of plant materials Fresh plant parts (P. daemia) were collected randomly from the gardens and villages of Trichy district, Tamilnadu from the natural stands. The botanical identity of this plant was confirmed by Dr. V. Sampath Kumar, Scientist - C, Botanical Survey of India (Southern Circle), Coimbatore, Tamilnadu. A voucher specimen has been deposited at the Department of Botany, National College (Autonomous), Tiruchirapalli-620 001, Tamilnadu, India. Preparation of extracts Aqueous extraction One hundred grams (100 g) of dried powder were extracted in distilled water for 6 h at slow heat. Every 2 h it was filtered through Whatman No. 1 filter paper and centrifuged at 5000 g for 15 min. The supernatant was collected. This procedure was repeated twice and after 6 h the supernatant was concentrated to make the final volume one-fifth of the original volume. Solvent extraction One hundred grams (100 g) of dried plant powdered samples were extracted with 200 ml of methanol kept on a rotary shaker for 24 h. Thereafter, it was filtered and centrifuged at 5000 g for 15 min. The supernatant was collected and the solvent was evaporated to make

Doss and Anand 171 the final volume one-fifth of the original volume. It was stored at 4°C in airtight bottles for further studies. Animals The animals of both sexes were used for these experiments. They were obtained from Animal House, RVS Pharmaceutical Sciences, Coimbatore, Tamilnadu. The animals were housed in standard cages and were maintained on a standard pelleted feed and water was given ad libitum. All the experiments were carried out according to the guidelines recommended by the Committee for the Purpose of Control and Supervision of Experiments of Animals (CPCSEA), Government of India. Induction of diabetes The animals were fasted for 24 h and diabetes was induced by a single intraperitoneal injection of a freshly prepared solution of alloxan monohydrate (150 mg/kg b.w.) in sterile normal saline. 72 h later, rats with blood glucose (BGL) levels above 200 mg/dl were considered diabetic and selected for the experiment. Experimental design: The animals were randomly divided into five groups with 6 rats in each group and treated as follows: Group I: Normal control (saline) (by using an intragastric catheter tube (IGC). Group II: Diabetic control. Group III: Diabetic rats received P. daemia methanol extract (250 mg/kg b.w.) for 21 days by IGC. Group IV: Diabetic rats received P. daemia aqueous extract (250 mg/kg b.w.) for 21 days by IGC. Group V: Diabetic rats received glibenclamide (2 mg/kg b.w.) daily orally for for 21 days by IGC. The change in body weight and fasting plasma glucose (FPG) levels of all the rats were recorded at regular intervals during the experimental period. For acute toxicity study, FPG were monitored after 30, 60, 120 and 180 min of administration of single dose of the extracts and at the end of 0, 7th, 14th and 21st days for sub acute study. Blood was drawn from the ventricles and centrifuged. Biochemical analysis Blood samples were taken into centrifuged at 3000 rpm for 15 min. Serum biochemical parameter such as blood glucose (Sasaki et al., 1972), plasma insulin (Anderson et al., 1993), glycosylated haemoglobin (Karunanayake and Chandrasekharan, 1985), creatine (Owen et al., 1954) and urea (Varley, 1976). Statistical analysis All the data were subjected to Duncan’s multiple range test (DMRT) was done by using the SPSS version 2007 WINSAT software.

RESULTS The methanol and aqueous extracts of P. daemia admini-strated orally to the rats at the doses of 100, 200, 400, 800, 1200 and 1600 mg/kg b.wt did not produce any sig-nificant changes in the autonomic, behavioural or neurolo-

172 Afr. J. Biotechnol.

Table 1. Effect of methanol and aqueous extracts of P. daemia on body weights of alloxan-induced diabetic mice.

Treatment Day 0 (g) Day 7 (g) Day 14 (g) Day 21 (g)

Normal control 172.2 ± 2.07 165.25 ± 0.45 169.68 ± 0.15 170.44 ± 3.55

Diabetic control 164.66 ± 1.28 159.37 ± 2.23 158.65 ± 0.05** 158.18 ± 1.28**

PDME (250 mg/kg b.w.) 164.58 ± 2.23 165.94 ± 0.03 167.52 ± 0.25a 169.07 ± 2.83

aa

PDAE (250 mg/kg b.w.) 161.35 ± 1.05 164.8 ± 1.45 165.17 ± 3.20a 165.91 ± 0.56

a

Glibenclamide (10 mg/kg b.w.) 164.63 ± 1.02 165.48 ± 0.032 165.95 ± 0.45a 171.47 ± 1.25

aa

Each value is SEM ± 5 individual observations; * P < 0.05, ** P<0.01, *** P<0.001 compared normal control versus diabetic mice; a -P < 0.05, aa - P<0.01 compared -diabetic mice versus drug treated.

Table 2. Antidiabetic effect of methanol and aqueous extracts of P. daemia on blood glucose level of alloxan-induced mice during acute study.

Treatment 0 min 30 min 60 min 120 min 180 min

Normal control 102.06 ± 0.40 144.9 ± 0.48 157.86 ± 0.41 173.16 ± 0.47 195.1 ± 0.26

Diabetic control 182.03 ± 0.75 215.8 ± 0.43 225.03 ± 0.25 240.63 ± 0.70 256.86 ± 0.61*

PDME (250 mg/kg b.w.) 197.93 ± 0.30 187.03 ± 0.05 152.13 ± 0.79 143.93 ± 0.30 118.33 ± 1.82

PDAE (250 mg/kg b.w.) 195.16 ± 0.20 183.2 ± 0.91 155.73 ± 0.37 147.8 ± 0.43 121.13 ± 0.61

Glibenclamiide (10 mg/kg b.w.) 193.9 ± 0.36 168.23 ± 0.20 142.13 ± 0.32 130.23 ± 0.25* 105.36 ± 0.90*

Each value is SEM ± 5 individual observations; * P < 0.05, aa - P<0.01 compared -diabetic mice versus drug treated.

Table 3. Effect of methanol and aqueous extracts of P. daemia on blood glucose level of alloxan-induced mice during sub-acute study.

Treatment 0 day 7 day 14 day 21 day

Normal control 94.16 ± 2.35 99.46 ± 2.40 90.16 ± 1.40 95.26 ± 2.55

Diabetic control 241.33 ± 3.98 262.23 ± 1.51 294.16 ± 2.30* 310.13 ± 4.50*

PDME (250 mg/kg b.w.) 264.66 ± 1.49 192.23 ± 2.40 154.5 ± 4.4* 118.06 ± 1.25*

PDAE (250 mg/kg b.w.) 259.23 ± 2.35 196.3 ± 4.2 160.1 ± 4.75* 125.5 ± 2.30*

Glibenclamide (10 mg/kg b.w.) 278.23 ± 3.32 183.5 ± 6.2 141.4 ± 3.55* 111.16 ± 1.15*

Each value is SEM ± 5 individual observations; * P < 0.05, - P<0.01 compared -diabetic mice versus drug treated.

logical alteration. Acute toxicity studies revealed the non-toxic nature of both extracts of P. daemia. The signs and symptoms in all groups were found to be normal. Normal control animals were found to be stable in their body weight but diabetic rats showed significant reduction in body weight on days 7, 14 and 21. There were obser-vable changes in the body weight of treated diabetic rats. Alloxan caused body weight reduction, which is reversed by alcoholic and aqueous extracts of P. daemia after 7, 14 and 21 days of treatment. The same trend was noted in glibenclamide treated groups (Table 1). A dose-dependent reduction in blood glucose levels was observed in alloxan induced diabetic rats treated with aqueous and methanol extracts of P. daemia. After a single dose-of the extract given to the alloxan induced diabetic rats, there was a significant P < 0.05 reduction in

blood glucose levels of the diabetic rats within the period of acute study compared to control. The maximum effect was observed at 180 min with the methanol extract exerting comparable to effect of aqueous extract that exerted a more pronounced effect (Table 3). The increased blood glucose level in alloxan induced diabetic rats was significantly P < 0.05 by crude extracts (methanol and aqueous) treatment and it was found to be lowered up to 118.33 and 121.13 at the dose of 250 mg/kg of body weight, respectively (Table 2). The insulin and glycosylated haemoglobin levels of diabetic rats treated with methanol and aqueous extracts of P. daemia and glibenclamide, a known hypoglycemic drug, resulted from a significant decrease in glycosylated haemoglobin; whereas increase insulin levels when compared with alloxan alone treated rats. The maximum reverse the

Doss and Anand 173

Table 4. Effects of P. daemia extracts on non-protein compounds and glycosylated haemoglobin levels in normal and alloxan induced diabetic mice.

Treatment Insulin (Iu/L) Urea (mg/dl) Creatinine (mg/dl) Hb A1c (%)

Normal control 0.656 ± 0.36 12.20 ± 2.25 0.61 ± 0.03 3.71 ± 0.27

Diabetic control 0. 155 ± 0.24** 30.45 ± 1.35* 1.27 ± 0.75* 9.49 ± 0.20**

PDME (250 mg/kg b.w.) 0.545 ± 0.04*aa

15.65 ± 3.23a 0.79 ± 0.11 3.88 ± 0.10

a

PDAE (250 mg/kg b.w.) 0.492 ± 0.02a 16.92 ± 1.02 0.92 ± 0.02 5.29 ± 0.07

a

Glibenclamide (10 mg/kg b.w.) 0.578 ± 0.32aa

14.55 ± 3.01a 0.62 ± 0.02 3.76 ± 0.20

aa

Each value is SEM ± 5 individual observations;* P < 0.05, ** P<0.01, *** P<0.001 compared normal control versus -diabetic mice; a -P < 0.05, aa - P<0.01 compared -diabetic mice versus drug treated.

trend of insulin and glycosylated haemoglobin levels against alloxan induced diabetic aberrations was achieved with the optimum dose 250 mg/kg body weight both (aqueous and methanol) extracts of P. daemia. Among the two extracts treated, the methanol extract showed significant changes in insulin and glycosylated haemoglobin.

The level of urea and creatine in normal, diabetic and treated animals are shown in Table 4. The normal function of the kidney was assessed as blood urea level. The urea level in diabetic was found to be 30.45 mg/dl, it was altered from treated animals of 15.85 mg/dl against 12.20 mg/dl (control). The level of creatine in groups II and V showed significant variation when compared to control. Among the two extracts treated, the methanol extract showed significant changes in urea and creatine. DISCUSSION Diabetes mellitus is ranked five among the leading causes of death and is considered third when its fatal complications are taken into account. Medicinal plants with a potential of decreasing the blood sugar have been tested in experimental animal models and their effects confirmed. Many unknown and lesser known plants are used in folk and tribal medicinal practices in India. The medicinal values of these plants are not much known to the scientific world.

The present study was carried out to evaluate the antidiabetic activity of methanol and aqueous extracts of P. daemia on alloxan induced diabetes in rats. Alloxan is known to induce free radical production and cause tissue injury, and the pancreas is especially susceptible to the action of alloxan induced free radical damage (Akah et al., 2011). Accordingly, significantly high levels (P<0.001) of FBG were observed in alloxan control group rats and remained high throughout the experimental period. Alloxan induced diabetic rats treated with the extract showed a significant reduction in blood sugar levels com-pared to alloxan control group. This decrease in blood sugar levels may be attributed to stimulation of the resi-dual pancreatic mechanism or to a probable increase in

the peripheral utilization of glucose (Akah et al., 2011). Normoglycemic studies, however, revealed P. daemia to have no effect on euglycemia. This implies that the extract is probably acting through any of the extra pancreatic mechanisms rather than stimulating insulin secretion from β cells and results in antihyperglycemic action rather than hypogly-cemic effect, that is, does not affect normal blood sugar level, which may be beneficial in case of mis-dosing.

Protein can universally bind non-enzymatically with glucose or other sugars present in the vicinity. The degree of glycation is directly proportional to the concen-tration of the sugar present in the surrounding medium. Therefore, estimation of glycosylated hemoglobin (HbA 1c) gives an accurate reflection of mean plasma glucose concentration over this period and correlates best with the degree of the glycemia (Danze et al., 1987). A change in HbA 1c of 1% would reflect a blood glucose alteration of about 30 mg%. A significant decrease with leaf extract (P<0.01) was observed in the treated rats as compared to alloxan-induced diabetic rats. On treatment with roots, the decrease was moderate. This is indicative of a better glycemic control for a longer period by the leaf sample.

A significant reduction in the body weight was observed in the alloxan-induced diabetic rats. The decrease in the weight in diabetes is due to continuous excretion of glucose and decrease in peripheral uptake of glucose and glycogen synthesis (Defronzo et al., 1992). The decrease in weight was arrested on admini-stration of alcoholic leaf extract to a greater extent as compared to root extract. All the aforementioned obser-vations sug-gest that the test drug that is, alcoholic leaf extract can be a promising antidiabetic.

In diabetic mellitus, due to persistent hyperglycemia, the excess blood glucose reacts with haemoglobin in a nonenzymatic process to form glycosylated haemoglobin. Since the glycation rate is directly proportional to blood glucose concentration, level of glycosylated haemoglobin indicates glycemic control in the diabetic state (Monnier and Cerami, 1982). Estimation of haemoglobin is a well established parameter useful in the 22 management and prognosis of the disorder (Chang and Nobel, 1979). In the

174 Afr. J. Biotechnol. present study, administration of methanol and aqueous extracts of P. daemia leaves significantly reduced the elevated glycosylated haemoglobin levels in alloxan-diabetic rats further substantiating its potential in long term glycemic control of diabetes mellitus. Urea and uric acid are organic waste products produced during the breakdown of amino acids. Creatinine is generated in the skeletal muscle tissue by the breakdown of creatinine phosphate. Their increased level in serum is an indication kidney disorder. It was found that urea and creatinine levels were normal in all the experimental groups. Many oral hypoglycemic agents are normally metabolized or cleared by the kidneys and so accumulate in uraemic patients thus increasing the risk of hypoglycemia and toxicity (Marlin Cynthia and Rajeshkumar, 2012). Our results on the creatinine and urea are very close to normal range and are significant. The remarkable hypo-glycaemic potential of P. daemia was quite competent with standard drug.

Further studies are necessary to elucidate details of active phytochemicals and their mechanism of hypogly-caemic action. Isolation and study of active principles are under process. REFERENCES Akah PA, Uzodinma SU, Okolo CE (2011). Antidiabetic activity of

aqueous and methanol extract and fractions of Gongronema latifolium (Asclepidaceae) leaves in Alloxan Diabetic Rats. J. Appl. Pharm. Sci. 01(09):99-102

Anderson L, Dinesen B, Jorgonsen PN, Poulsen F, Roder ME (1993). Enzyme immune assay for intact human insulin in serum or plasma. Clin. Chem. 39:578-582.

Anil K, Sunil K, Vipin K (2013). Evaluation of antidiabetic activity of hydroalcoholic extract of cestrum nocturnum leaves in streptozotocin-induced diabetic rats. Adv. Pharmacol. Sci. http://dx.doi.org/10.1155/2013/150401.

Chang AT, Nobel J (1979). Estimation of HbA1c like glycosylated proteins in kidneys of streptozotocin diabetes and controlled rats. Diabetes 28:408-415

Danze PM, Tarjoman A, Rousseaux J, Fossati P, Dautrevaux M (1987). Evidence for an increased glycation of IgG in diabetic patients. Clin. Chim. Acta. 166:143-53

Defronzo RA, Bonadonna RC, Ferrannini I (1992). Pathogenesis of type 2 (non-insulin dependent) diabetes mellitus: A balanced overview. Diabetologia 35:389-97

Grover JK, Yadav S, Vats V (2002). Medicinal plants of India with anti-diabetic potential. J. Ethanopharmacol. 81(1):81-100.

Hebbar SS, Harsha VH, Shripathi V, Hedge GR (2010). Ethnomedicine of Dharward district in Karnataka, India plants use in oral health care. J. Ethnopharmacol. 94:261-266.

Karunanayake EH, Chandrasekharan NV (1985). An evaluation of a

colorimetric procedure for the estimation of glycosylated haemoglobin and establishment of reference values for Sri lanka. J. Natl. Sci. Counc. Sri Lanka 13:235-258.

Kumudhavalli MV, Jaykar B (2012). Evaluation of Antidiabetic activity of Costus igneus(L) leaves on STZ induced diabetic rats. Der Pharmacia Sinica 3(1):1-4

Marlin Cynthia J, Rajeshkumar KT (2012). Effect of aqueous root extract of Aristolochia indica (Linn) on diabetes induced rats. Asian J. Plant Sci. Res. 2(4):464-467.

Monnier VK, Cerami A (1982). Non-enzymatic glycosylation and browning in diabetes and aging. Diabetes 31:57-66

Noor AS, Gunasekaran AS, Manickam, Vijayalakshmi MA (2008). Antidiabetic activity of Aloe vera and histology of organs in streptpzotocin induced diabetic rats. Curr. Sci. 94:1070-1076.

Omale J, Ebiloma GU, Agbaji AO (2011). Assessment of biological activities: A Comparison of Pergularia daemia and Jatropha curcas Leaf Extracts. Br. Biotechnol. J. 1(3):85-100

Owen JA, Iggo JB, Scangrett FJ, Steward IP (1954). Determination of creatinine in plasma serum, a critical examination. J. Biochem. 58:426-437.

Prasad SK, Kulshreshtha A, Taj N. Qureshi (2009). Antidiabetic activity of some herbal plants in streptozotocin induced diabetic albino rats. Pak. J. Nutr. 8:551-557.

Sasaki T, Matsy S, Sorae A (1972). Effect of acetic acid concentration on the colour reaction in the O-toluidine boric acid method for blood glucose estimation. Rinsho Kagarku 1:346-353.

Sharma VK, Suresh Kumar, Patel HJ, Shivakumar Hugar (2010). Hypoglycemic activity of Ficus glomerata in alloxan induced diabetic rats. Int. J. Pharm. Sci. Rev. Res. 1(2):18-22.

Varley H (1976). Practical clinical biochemistry, Arnold Heinemann Publication Pvt. Ltd. p. 452.

Wild SG, Roglic A, Green R, King H (2004). Global prevalence of diabetes. Estimated for the year 2000 and projection for 2030. Diabetes Care 27:1047-1054.

World Applied Sciences Journal 18 (2): 233-235, 2012ISSN 1818-4952© IDOSI Publications, 2012DOI: 10.5829/idosi.wasj.2012.18.02.1136

Corresponding Author: A. Doss, Department of Botany, National College (Autonomous), Tiruchirappalli, Tamilnadu, India.

233

Preliminary Phytochemical Screening of Asteracantha longifolia and Pergularia daemia

A. Doss and S.P. Anand

Department of Botany, National College (Autonomous), Tiruchirappalli, Tamilnadu, India

Abstract: The aim of the study was to analyze the phytochemical constituents of two potential folkloremedicinal plants such as Asteracantha longifolia and Pergularia daemia. Methanol and Aqueous extract ofthe dried leaves of these plants were collected and used for phytochemical analysis. The selected plants werefound to contain alkaloids, phenolic compounds, tannins and flavonoids except for the absence of terpenoidsin A. longifolia and saponin in P. daemia respectively. The significance of the plants in traditional medicineand the importance of the distribution of these chemical constituents were discussed with respect to the roleof these plants in ethnomedicine in Tamilnadu.

Key words: Secondary metabolites Alkaloids Saponins Phenolic compounds

INTRODUCATION identification of crude drugs [2]. The main purpose of the

Medicinal plants are an important source for the phytochemicals in two potential traditional medicinaltherapeutic remedies of various ailments. Scientific plants.experiments on the antimicrobial properties of plant Fresh plant samples were collected from differentcomponents were first documented in the late 19 century. agro-climatic regions of Trichy District, Tamilnadu fromth

Since time immemorial, different parts of medicinal plants the natural stands. The taxonomic identities of thesehave been used to cure specific ailments in India. Now-a- plants were determined by Dr. V. Sampath Kumar,days there is widespread interest in evaluating drugs Scientist-C, Botanical Survey of India (Southern Circle),derived from plant sources. This interest primarily stems Coimbatore, Tamilnadu, South India. Fresh plant materialsfrom the belief that green medicine is safe and were washed under running tap water, air dried anddependable, compared to costly synthetic drugs which then homogenized to fine powder and stored in airtightare invariably associated with adverse effects. Natural bottles. 25 g of air-dried powder was taken in 100 ml ofantimicrobials have been often derived from plants, animal water in a conical flask, plugged with cotton wool andtissues or microorganisms. The adverse effects of the they were shaken at room temperature for 2 days. Afterdrugs available today, necessitates the discovery of new 2 days hours the supernatant was collected and theharmless pharmacotherapeutic agents from medicinal solvent was evaporated to make the final volume oneplants [1]. fourth of the original volume (12) and stored at 4°C in

Phytochemicals are responsible for medicinal activity airtight bottles. 25 g of air-dried powder was taken inof plants [2], these are non-nutritive chemicals that have 100 ml of methanol in a conical flask, plugged with cottonprotected human from various diseases. Phytochemicals wool and they were shaken at room temperature forare basically divided into two groups that are primary and 2 days. After 2 days the supernatant was collected andsecondary metabolites based on the function in plant the solvent was evaporated to make the final volume onemetabolism. The major constituents are consists of fourth of the original volume (12) and stored at 4°C incarbohydrates, amino acids, proteins and chlorophylls airtight bottles. This was carried out according to thewhile secondary metabolites consist of alkaloids, methods described by Trease and Evans [4]. Qualificationsaponins, steroids, flavonoids, tannins and so on [3]. phytochemicals analysis of the crude powder of the threePhytochemical constituents are the basic source for the plants for the identification of phytochemicals like as aestablishment of several pharmaceutical industries. The tannins, alkaloid, steroid, phenols and terpenoid,constituents are playing a significant role in the flavonoid etc.

present study was to evaluate the presence of various

World Appl. Sci. J., 18 (2): 233-235, 2012

234

All plant parts synthesize some chemicals bythemselves, to perform their physiological activities. Inour present study, the investigated plants have exhibiteddifferent kinds of secondary metabolites. The medicinalvalue of these secondary metabolites is due to thepresence of chemical substances that produce a definitephysiological action on the human body. The mostimportant of these substances include, alkaloids,glucosides, steroids, flavonoids, fatty oils, resins,mucilages, tannins, gums, phosphorus and calcium for cellgrowth, replacement and body building [5]. Phytochemicalscreening and qualitative estimation of two medicinalplants studied showed that the leaves were rich inphenolic compounds followed by alakloids, tannins andsaponins, maximum number of secondary metaboliteswere found in Asteracantha longifolia followed byPergularia daemia. Alkaloids have been wellinvestigated for many pharmacological propertiesincluding antiprotozoal, cytotoxic, antidiabetic [6] andanti-inflammatory [7] properties, but there are only fewreports about their antimicrobial properties. Plants withalkaloids in the present study are Asteracantha longifoliaand Pergularia daemia is used to cure asthma.

Saponins are glycosides occurring widely in plants.They are abundant in many foods consumed by animalsand man. Saponin is used as mild detergents and inintracellular histochemistry staining to allow antibodyaccess to intracellular proteins. In medicine, it is used inhypercholesterolemia, hyperglycemia [8], antioxidant,anti-cancer, anti-inflammatory [9], central nervous systemactivities (Argal & Pathak, 2006) and weight loss etc. It isalso known to have antifungal properties [8]. The plantshaving saponins are Asteracantha longifolia. Plantsteroids are known to be important for their cardiotonicactivities, possession of insecticidal, anti-inflammatory[10], analgesic properties [11], central nervous systemactivities [12] and antimicrobial properties. They are alsoused in nutrition, herbal medicine and cosmetics. Out ofthe two plants, studied steroids are present inAsteracantha longifolia. Tannins were reported to exhibitantidiabetic [6], anti-inflammatory, antibacterial andantitumor activities. It has also been reported that certaintannins were able to inhibit HIV replication selectivelybesides use as diuretics. Plant tannins have been widelyrecognized for their pharmacological properties and areknown to make trees and shrubs a different meal formany caterpillars [13]. Glycosides were reported to exhibitanti-diabetic characteristics [6]. Cardiac glycosides on theother hand are known to hamper the Na /K pump. This+ +

results in an increase in the level of sodium ions in the

Table 1: Phytochemical screening of Pergularia daemia


1 Alkaloids + +2 Saponins - -3 Steroids + -4 Phenolic compounds + -5 Tannins + +6 Flavonoids + +7 Terpenoids + -8 Cardiao Glycosides + +9 Protein & Amino acids + +10 Anthraquinones - -

Table 2: Phytochemical screening of Asteracantha longifolia


1 Alkaloids + -2 Saponins - +3 Steroids + -4 Phenolic compounds + -5 Tannins + +6 Flavonoids + +7 Terpenoids - -8 Carbohydrate & Glycosides + +9 Protein & Amino acids + +10 Anthraquinones - -

myocytes which then enhance the level of calcium ions.This consequently increases the amount of Ca ions2+

available for contraction of the heart muscle, whichimproves cardiac output and reduces distention of heartand thus are used in the treatment of congestive heartfailure and cardiac arrhythmia.

The plant extractive studied could be an answer tothe people seeking for better therapeutic agents fromnatural sources which is believed to be more efficient withlittle or no side effects when compared to the commonlyused synthetic chemotherapeutic agents. The anti-inflammatory, antispasmodic, antianalgesic andantidiuretic can be attributed to their high steroids,tannins, terpenoids and saponins. Further studies areneeded with this plant to isolate, characterize andelucidate thestructure of the bioactive compounds of thisplant for industrial drug formulation.

REFERENCES

1. Venkataswamy, R., A. Doss, M. Sukumar andH.M. Mubarack, 2010. Preliminary phytochemicalscreening and antimicrobial studies of Lantanaindica roxb. Indian Journal of Pharmaceutical Sci.,72(2): 229-231.

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2. Savithramma, N., M. Linga Rao and D. Suhrulatha, 8. Rupasinghe, H.P., C.J. Jackson, V. Poysa, C. Di2011. Screening of medicinal plants for secondary Berado, J.D. Bewley and J. Jenkinson, 2003.metabolites. Middle-East Journal of Scientific Res., Soyasapogenol A and B distribution in Soybean8: 579-584. (Glycine max (L.) Merr.) in relation to seed

3. Kumar, A., R. Ilavarasan, T. Jayachandran, M. physiology, genetic variability and growingDecaraman, P. Aravindhan, N. Padmanaban, and location. Journal of Agricultural Food Chemistry,M.R.V. Krishna, 2009. Phytochemical investigation 51: 5888-5894.on a tropical plants. Pakisthan Journal of Nutrition, 9. Manach, C., F. Regerat and O. Texier, 1996.8: 83-85. Bioavailability, metabolism and physiological impact

4. Trease, G.E. and W.C. Evans, 1978. Pharmacology of 4-oxo-flavonoids. Nutritional Res., 16: 517-544.11 Ed. Bailliere Tindall Ltd, London, pp: 60-75. 10. Akindele, A.J. and O.O. Adeyemi, 2007. Anti-th

5. Kubmarawa, D., M.E. Khan, A.M. Punah and Hassan, inflammatory activity of the aqueous leaf extracts of2008. Phytochemical screening and antibacterial Byrsocarpus coccineus. Fitoterapia, 78: 25-28.activity of extracts from Parkia clappertoniana keay 11. Malairajan, P., G. Geetha, S. Narasimhan and K. Jessiagainst human pathogenic bacteria. Journal of Kala Veni, 2006. Analgesic activity of some IndianMedicinal Plant Res., 2(12): 352-355. medicinal plants. Journal of Ethnopharmacology,

6. Cherian, S. and K.T. Augusti, 1995. Insulin sparing 19: 425-428.action of leucopelargonidin derivative isolated from 12. Argal, A. and A.K. Pathak, 2006. CNS activity ofFicus benghalesis Linn. Indian Journal of Exerimental Calotropis gigantea roots. Journal ofBiology, 33: 608-611. Ethnopharmacology, 106: 142-145.

7. Liu, R.H., 2003. Health benefits of fruit and 13. Haslem, E., 1989. Plant polyphenols: vegetablevegetables are from additive and synergistic tannins revisited-chemistry and pharmacology ofcombinations of phytochemicals. American Journal natural products. Cambridge University Press,of Clinical Nutrition, 78: 517-520. pp: 169.

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