Botany Pro (1)

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ANTIBACTERIAL ACTIVITY IN MEDICINAL PLANTS

Dissertation

Submitted to the University of Kerala

For the partial fulfillment of the requirements for the award of

Bachelor’s degree in Biotechnology

By

Iype Praveen Thomas , Unnimaya .S , Vandana Balakrishna , NeethuNath.R.S , Sreelekshmi.S , Laya Johnson ,Sherin .P.L, Sumita Muralidhar.

Department of Botany and Biotechnology

Mar Ivanios College

Bethany Hills

Trivandrum – 15

2011-2012

Thiruvananthapuram-15

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CERTIFICATE

This is to certify that this dissertation entitled

“ANTIBACTERIAL ACTIVITY IN MEDICINAL PLANTS” is an authentic record of

the studies and research work carried out by Mr. /Ms ……………… under my supervision

and guidance for the partial fulfillment of the requirements for the degree of Bachelor of

Science in Biotechnology (Restructured) of the University of Kerala and that no part of this

dissertation has been presented earlier for any degree or diploma in this or any Universities.

MrsThressiamma.Mathew Dr.Bindhu.T.Alex

Head of the department Supervising teacher

Examiners:

1.

2.

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Declaration

We hereby declared that this dissertation entitled “ANTIBACTERIAL ACTIVITY IN

MEDICINAL PLANTS” is an authentic record of the orginal research work carried out by

us under the supervision of Dr. Bindhu . T. Alex, Department of Botany & Biotechnology,

Mar Ivanios College, Thiruvananthapuram and that not part of this dissertation has been

previously presented for any degree or diploma or similar title of this or any other

Universities.

Iype Praveen Thomas (62436)

Unnimaya.S (62434)

Vandana Balakrishna (62413 )

Neethu Nath .R.S (624 )

Sreelekshmi.S (62435)

Laya Johnson (624 )

Sherin .P.L (62425)

Sumita Muralidhar (62412 )

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ACKNOWLEDGEMENT

We are very much obliged to Dr. Bindhu .T.Alex , Assistant Professor, Department of

Botany & Biotechnology, Mar Ivanios College, Nalanchira for suggesting this topic and also

for guiding us throughout the course of this dissertation. We also have the good fortune to be

able to draw upon the devotion, deep knowledge and mature experience of his for the

completion of this study.

We wish to express our heartfelt gratitude and indebtedness to Mrs. Thressiamma Mathew,

Head of the Department, Department of Botany & Biotechnology, Mar Ivanios College,

Nalanchira for providing the infrastructure facilities for this dissertation. We take this

occasion to thank Ms. Fathima Zehra (Department of Botany & Biotechnology) for being

with us during this work.

We express our heartfelt thanks to Mr. R.Surendran, Laboratory Assistant (Department of

Botany & Biotechnology for the help he had rendered in the laboratory. We are grateful to all

our classmates for their co-operation and suggestion. Moreover we are greatly indebted to our

parents for their encouragement and support.

Above all we are deeply indebted to “The God Almighty” who always remains as the

helping hand behind us in all crises.

Iype Praveen Thomas (62436)

Unnimaya.S (62434)

Vandana Balakrishna (62413 )

Neethu Nath .R.S (624 )

Sreelekshmi.L (62433)

Laya Johnson (624 )

Sherin .P.L (62425)

Sumita Muralidhar (62412 )

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CONTENTS

INTRODUCTION

OBJECTIVE

REVIEW OF LITERATURE

MATERIALS AND METHODS

RESULTS AND DISCUSSION

SUMMARY AND CONCLUSION

BIBLIOGRAPHY

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INTRODUCTION

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Medicinal plants are an integral component of ethenoveterinary

medicine. Medicinal plants, since time immemorial, have been used virtually all cultures as a

source of medicine. The widespread use of herbal remedies and healthcare preparations, as

those described in ancient texts, 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.

As defined by WHO, health is a state of complete physical, mental

and social wellbeing and not merely the absence of disease or infirmity.Medicine, in several

developing countries, using local traditions and beliefs, is still the mainstay of healthcare. The

practice of traditional medicine is widespread in China, India, Japan, Pakistan, Srilanka and

Thailand. In China, about 40% of the total medicine consumption is attributed to traditional

tribal medicines. In Thailand, herbal medicines make use of legumes encountered in the

Caesalpiniaceae, the Fabaceae and the Mimosaceae.

Ethno botanical information is leading to the discovery of novel

phytopharmaceuticals and other phytoproducts.This trend has made their commercialization a

necessity.Therfore industries based on medicinal and aromatic plants have been established

allover the world with a view to manufacture the so-called green products to satisfy the

growing demand. The development of formulation of drugs of plant origin involves botanical

identification of the vegetable drug,cultivation and post harvest procedures, extraction

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procedures, standardization of extracts and pharmaceutical formulations.This means that the

phytotherapeutics are in the hands of personnel from different disciplines. The production of

phytotherapeutics and drugs from the plants needs the cooperation of a big team of

horticulturists, botanists,

ecologists,taxonomists,phytochemists,pharmacists,pharmocologists,pharmaceutical

specialists, marketing and distribution specialists.

HISTORY OF MEDICINAL PLANTS

As early as 3000BC,the ancient Egyptians put much confidence in

plants for curing many diseases.Until now the same confidence is still existing among the

contemporary Egyptians and a “turn back”to “remedy by herbs”is now becoming global

rather than regional or national request.This is strongly favoured,since the natural drugs have

little more or no side effects as do the chemically synthesized medications.

Pharmacopoeial wild medicinal plants: These plants are used in folk medicine

since a long time ago.Recent and modern studies on these plants proved the occurance of

active principles in them. Their pharmacological activity has been investigated.They are

among the pharmacopoeial drugs in in different pharmaopoeias;either in thevArab countries

or abroad.

Plants used in folkmedicine:There are numerous plant species which are

collected from the field to be sold in the “Attarin:”or the herb seller shops.

Plants of potential medicinal value:Many plant species were investigated for

their active constituents.These has been done depending on the information of the folk use of

these plants,or in species with relatives of species,genera or the same family,known from

other countries to have active constituents.

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Medicinal plants are an important health and economic component of the

floras in the developed as well as developing countries.Increasing worldwide interest in

herbal remedies expanding reliance of local health care of traditional remedies,and a renewed

interest in the development of pharmaceuticals from plant sources have greatly increased

trade in medicinal plant materials.Important populations of medicinal plants are found not

only in the regions and ecosystems with high biological diversity but also in less diverse

floras and in floristic communities that are not a common focus of conservation efforts.

The production of medicinal and aromatic plants requires an

inderstanding of plant growth,ecology,business,economics,law,conservation and a lot of

subjects related to tillage and gathering plants.While development such as

machinery,fertilizers and pesticides have helped farmers meet demands for quality materials

at affordable prices,the balance with farming costs,and labour compel societies to set

directions and establish limits.

Plants produce a diverse range of bioactive molecules,making them a

rich source of different types of medicines.Higher plants,as sources of medicinal compounds,

have continued to play a dominant role in the maintainance of human health in ancient times.

Over 50% of all modern clinical drugs are of natural product origin and natural products play

an important role in drug development programs in the pharmaceutical industry. The

relatively lower incidence of adverse reactions to plant preparations compared to modern

conventional pharmaceuticals,coupled with their reduced cost, is encouraging,both the

consuming public and national health care institutions to consider the plant medicines as

alternatives to synthetic drugs. Plants with possible antimicrobial activity should be tested

against an appropriate microbial model to confirm the activity to ascertain the parameters

associated withit.The effects of plant extracts on bacteria have been studied by a very large

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number of researchers in different parts of the world. Much work has been done on ethno

medicinal plants in India.

Infectious diseases account for approximately one half of all deaths

in tropical countries. In industrialized nations, despite the progress made in the understanding

of microbiology and their control,incidents of epidemics due to drug resistant micro

organisms and the emergence of hither to unknown disease causing microbes,pose enormous

public health concerns.Historically plants have provided a good source of antiinfective

agents;emetine,quinine,and berberine remain highly effective instruments in the fight against

microbial infections.Phytomedicines derived from plants have shown great promise in the

treatment of intractable infectious diseases including opportunistic AIDS.

Historically,plants have provided a source of inspiration for novel

drug compounds,as plant derived medicines have made large contributions to human health

and well being.Their role is twofold in the development of new drugs: (1)they may become

the base for the development of a medicine,a natural blueprint for the development of new

drugs or (2) a phytomedicine to be used for the treatment of disease.

The first generation of plant drugs were usually simple botanicals

employed in more or less their crude form.Several effective medicines used in their natural

stale such as chinchona,opium,belladonna and were selected as therapeutics agents based on

empirical evidence of their clinical application by traditional societies from different parts of

the world.Folllowing the industrial revolution,a second generation of plant based drugs

emerged based on scientific processing of the plant extracts to isolate “their active

constituents.”

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The second generation phytopharmaceutical agents were pure

molecules and some of the compounds were even more pharmacologically active than their

synthetic counterparts. Notable examples were Quinine from Chinchona,reserpine from

Rauvolfia and more recently taxol from Taxus species.These compounds differed from the

synthetic therapeutic agents only in their origin. They follow the same method of

development and evaluation as other pharmaceutical agents.In the development of “Third

Generation”phytotherapeutic agents a top-bottom approach is usually adopted.This consists

of conducting a clinical evaluation of the treatment modalities and therapy as administrated

by traditional doctors or as used by the community as folk medicine.This evaluation is

followed by acute and chronic toxicity studies in animals.Studues should,when

applicable,includes cytotoxicity studies. It is only if the substance has an acceptable safety

index would it be necessary to conduct detailed pharmacological/biochemical studies.

PRESENT USE OF PLANTS

It is estimated that today,plant materials are present,in or have provided

the models for 50%Western drugs.Many commercially proven drugs used in modern

medicine were initially used in crude form in traditional or folk healing practices,or for other

purposes that suggested potentially useful biological activity.The primary benefits of using

plant derived medicines are that they are relatively safer than synthetic alternatives,offering

profound therapeutic benefits and more affordable treatment.

NEEM

For thousands of years the beneficial properties of Neem (Azadirachta indica A. Juss) have

been recognized in the Indian tradition. Each part of the neem tree has some medicinal

property. Biswas et al (2002) have recently reviewed the biological activities some of the

neem compounds, pharmacological actions of the neem extracts, clinical study and plausible

medicinal applications of neem along with their safety evaluation.

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Neem has two closely related species: A. indica A. Juss and M. azedarac, the former

is popularly known as Indian neem (margosa tree) or Indian lilac, and the other as the Persian

lilac. Neem has been extensively used in ayurveda, unani and homoeopathic medicine.The

Sanskrit name of neem tree is Arishtha meaning 'reliever of sickness' and hence is considered

asSarbaroganibarini. The tree is still regarded as 'village dispensary' in India. The

importance of the neem tree has been recognized by US National Academy of Sciences,

which published a report in 1992 entitled 'Neem – a tree for solving global problems'.

More than 135 compounds have been isolated from different parts of neem and several

reviews have also been published on the chemistry and structural diversity of these

compounds. The compounds have been divided into two major classes: isoprenoids (like

diterpenoids and triterpenoids containing protomeliacins, limonoids, azadirone and its

derivatives, gedunin and its derivatives, vilasinin type of compounds and C- secomeliacins

such as nimbin, salanin and azadirachtin ) and non-isoprenoids, which are proteins (amino

acids) and carbohydrates (polysaccharides), sulphurous compounds, polyphenolics such as

flavonoids and their glycosides, dihydrochalcone, coumarin and tannins, aliphatic

compounds, etc.

Various parts of the neem tree have been used as traditional Ayurvedic medicine in India.

Neem oil and the bark and leaf extracts have been therapeutically used as folk medicine to

control leprosy, intestinal helminthiasis, respiratory disorders, constipation and also as a

general health promoter. Its use for the treatment of rheumatism, chronic syphilitic sores and

indolent ulcer has also been evident. Neem oil finds use to control various skin infections.

Bark, leaf, root, flower and fruit together cure blood morbidity, biliary afflictions, itching,

skin ulcers, burning sensations and pthysis.

Oil from the leaves, seed and bark possesses a wide spectrum of antibacterial action against

Gram-negative and Gram-positive microorganisms, including M. tuberculosis and

streptomycin resistant strains. In vitro, it inhibits Vibrio cholerae ,Klebsiella pneumoniae, M.

tuberculosis and M. pyogenes. Antimicrobial effects of neem extract have been demonstrated

against Streptococcus mutans and S. faecalis.

Neem extract 

Its effective to cure ringworm, eczema and scabies. Lotion derived from neem leaf, when

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locally applied, can cure these dermatological diseases within 3-4 days in acute stage or a

fortnight in chronic case. A paste prepared with neem and turmeric was found to be effective

in the treatment of scabies in nearly 814 people100.

Neem leaf extract has been prescribed for oral use for the treatment of malaria by Indian

ayurvedic practitioners from time immemorial. Recently, a clinical trial has been carried out

to see the efficacy of neem extract to control hyperlipidemia in a group of malarial patients

severely infected with P. falciparum. The lipid level, especially cholesterol, was found to be

lower during therapy when compared to non-malaria patients. Reports are available regarding

the use of neem to treat patients suffering from various forms of cancer. One patient with

parotid tumour and another with epidermoid carcinoma have responded successfully when

treated with neem seed oi1.

NIM- 76, a refined product from neem oil, was studied in 10 human volunteers, where intra-

vaginal application before sexual intercourse could prevent pregnancy with no adverse effect

on vagina, cervix and uterus. The data suggested that intrauterine treatment is safe.

KACHOLAM

Kacholam ( Kaempferia galanga L). is a highly useful medicinal plant. Its rhizomes (tubers)

are used in indigenous medicines as stimulant, expectorant, diuretic, carminative etc.

Decoction of rhizomes is useful for curing dyspepsia, headache, rheumatism and malaria. For

clearing nasal obstructions, rhizomes may be boiled in oil may be applied externally.

Digestive power can be enhanced by using kacholam tubers. Larvicidal and anticancerous

principles are extracted from tubers. The tubers enhance digestion, remove bad odour of

mouth and are used as masticatory for chewing along with betel and arecanut .

Kacholam, also known as Sugandhavacha, Chandramulika or Sidhul is a rhizomatous

perennial plant, the rhizomes of which yield an essential oil.  The oil is utilized in the

manufacture of perfumes and in curry flavouring. It is also employed in cosmetics, mouth-

washes, hair tonics and toiletries. The pungent, hot, sharp, bitter and aromatic rhizomes find

an important place in indigenous medicine as stimulant, expectorant, diuretic and

carminative. It promotes digestion and cures skin diseases, piles, phantom tumors, coughs,

oedema, fever, epilepsy, splenic disorders, wounds, asthma and rheumatism. The rhizomes

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are used for protecting clothes against insects and are eaten along with betel and arecanut as a

masticatory. The rhizomes and leaves are attached to neck- laces and added to bath water for

perfume.Karcura (Kacholam) is a reputed remedy for all diseases caused by the morbidity of

vata and kaphaand is especially useful in respiratory ailments like cough, bronchitis and

asthma. The drug is reported to be acrid, hot, bitter and aromatic. It cures skin diseases,

wounds and splenic disorders. It promotes digestion, removes bad odour of the mouth and

destroys pathogenic organisms (Aiyer and Kolammal, 1964). The officinal part is the rhizome

and it is a constituent of a variety of Ayurvedic preparations like Dasamularista, Valiya

rasnadi kasaya, kaccoradi churna, Asanaeladi taila. Valiya narayana taila, etc. The identity of

the plant sources of the drug Karcura is a matter of debate. The source of Karcura in Kerala

in the recent times has been Kaempferia galanga (Sivarajan and Balachandran,

1994).                     

The rhizomes and root-stocks are good for dyspepsia, leprosy, skin diseases, rheumatism,

asthma, cough, bronchitis, ulcers, helminthiasis, fever, malarial fever, splenopathy,

cephalalgia, inflammatorytumour, nasal obstruction, halitosis, strangury, urolithiasis, and

haemorrhoids. The leaves are used for pharyngodynia, ophthalmia, swellings, fever and

rheumatism . The tubers reduced to powder and mixed with honey are given in case of

coughs and pectoral infections. The oil in which they are boiled is useful in applying to the

stoppages of the nasal organs . Kaempferia galanga is reported to have great export

potential . The tuberous rhizome of kacholam contains an alkaloid, starch, gum, fatty matter

with a fragrant liquid essential oil and a solid white crystalline substance and mineral matter.

The rhizome possesses a camphoraceous odour with somewhat bitter aromatic taste

resembling that of Hedychium spicatum.The essential oil is reported to contain over 54

components of which the major constituents are ethyl-trans-p-methoxy, cinnamate 16.5%,

pentadecane 9%, 1,8-cineole 5.7%,  g-carene 3.3%, and borneol .Insectididal constituents 

from Kaempferia galanga were isolated by .Cyclohexane oxide derivatives and diterpenes

were isolated by . Rhizome yields essential oil, which has antifungal activity. Ethyl-p-Methyl

O-trans-cinnamate is the main compound in root . p-Methoxycinnamic acid and its methyl

and ethyl esters have been isolated from the essential oil . Essential oil from rhizomes contain

npentadecane, ethyl-p-methoxy cinnamate, ethyl cinnamate, carene, camphene, borneol, p-

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methoxystyrene, pmethoxy cinnamate, p-methoxy- trans-cinnamic acid and cinnamaldehyde.

Rhizome is cytotoxic. Tuber is stimulant, expectorant, diuretic and carminative . 

TULSI

Tulasi is perhaps the most sacred plant of India nest to kamala – the lotus. Particularly, the

leaves of Tulasi are used to worship Visnu. It is interesting to note that the plants sacred to

Sankara, Ganesha and Visnu are bilva, durva and tulasi, which alleviate vata, pitta and kapha

doshas, respectively. There are innumerable references of Tulasi in Indian mythology. The

ancient Ayurvedic scriptures have mentioned the plant in the management of several

diseases. The plant is supposed to have a purifying influence by liberating ozone and also is

said to repel the mosquitoes. Thus, in many parts of India, the plant is grown in the courtyard,

traditionally and worshipped daily as a necessary ritual for family will-being. Ayurvedic texts

categorise it as kasaghna – alleviates cough, svedala – induces sweating, ajirna nasaka –

mitigates indigestion and agnimandya nasaka – alleviates anorexia (Bhavaprakas)

The plant grows all over India up to 2000 meters height. It is grown in houses, temples and

gardens. An erectannual grows 0.5-1.5 meters in height and has red or purple quadrangular

branches. The leaves are opposite, about 2-4 cm long, margins entire or toothed, hairy on

both the surfaces, dotted with minute glands and are aromatic. The flowers tiny, purple and

inflorescence is a long spike or 12-14 cm in length. The fruits are small, smooth nut lets,

reddish grey in color.

Botanically, Tulasi is known as Ocimum sanctum and it belongs to family Lamiaceae. The

leaves contain an essential oil, which contains eugenol, eugenal, carvacrol, methylchavicol,

limatrol and caryophylline. The seeds contain oil composed of fatty acids and sitosterol. The

roots contain sitosterol and three triterpenes A, B, and C. The leaves also contain a steroid

ursolic acid and n-triacontanol. Eugenol (70.5), its methyl ether (4.8), nerol (6.4),

caryophyllene (7.5), terpinen –4-(0.4), decylaldehyde (0.2), selinene (0.4), pinene (0.4),

camphene (2.0) and a-pinene (3.5%) identified in essential oil by GC.

Tulasi is pungent and bitter in taste, pungent in the post digestive effect and has hot potency.

It alleviates kapha and vata doshas, but slightly aggravates the pitta dosha. It possesses light

and dry attributes. On the contrary the seeds are oily and slimy in attributes and have a cold

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potency. Tulasi is a stimulant, aromatic herb and effectively reduces the fever.

(Bhavaprakash)

The seeds, leaves and the roots of Tulasi have great medicinal value. It is used both,

internally as well as externally. Tulasi has mild antiseptic, analgesic properties and it relieves

the swellings also. Hence, it beneficial, externally, in various skin diseases. The paste of

leaves works well, with marica powder, when applied topically in ringworm infestations. The

dressing with the pulp of its leaves effectively controls the infections and hastens the healing

of chronic infected wounds. The leaves when chewed mitigate the infections of the gums.

Instillation of fresh juice of the leaves into ears is an effective domestic medicament for ear

aches. The massage with the leaves juice improves the circulation beneath the skin and

augments the sensation in the skin. In the headache due to sinusitis, the instillation of juice in

the nose facilitates the secretions of kapha and relieves the headache. The dried powder of the

leaves can be inhaled, like a snuff, for the same purpose.

Internally, Tulasi is used on many occasions, as a vehicle or an adjuvant. It enhances the

bioavailability of the medicine or acts synergistically, respectively. It is also used in number

of Ayurvedic proparations for bhavana, to potentate their effects, namely in Tribhuvana kurti,

Caturbhuja rasa etc. Tulsi is salutary to increase the appetite and improve digestion. It has a

mild laxative as well as vermicidal action, hence is rewarding in worm infestations. It is a

good blood purifier and is helpful in the diseases of the blood and heart. A tea prepared with

the leaves of Tulasi is a common domestic remedy for cold, cough, milk indigestion,

diminished appetite and malaise.

Tulasi has specified actions on the respiratory system-pranavaha srotasa. It effectively

liquefies the phlegm due to its hot and sharp attributes. It gives excellent results in cough due

to kapha, allergic bronchitis, asthma and eosinophilia. Combined with honey, the juice works

well to control the hiccup. In tubercular cough, Tulasi is also beneficial. It is an effective

panacea for fever, especially of kapha type, while given with honey and marica fruit powder.

In such conditions it effectively controls colds and reduces pain. Tulasi juice works as

amapacana, meaning it digests and destroys ama – the toxins.

The seeds of tulasi being cool and sweet along with its slimy-(picchila) attribite, are

beneficial in dysuria. The khira or pudding, prepared from the seeds, is useful to boost the

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energy in debility, especially due to pitta dosha. In diarrhea associated with bleeding, the

seeds soaked overnight in water to which a teaspoon of sugar is added, and taken in the

morning.

ANTIMICROBIAL ACTIVITY

There is a continuous and urgent need to discover new antimicrobial compounds with diverse

chemical structures and novel mechanisms of action because there has been an alarming

increase in the incidence of new and re-emerging infectious diseases. Another big concern is

the development of resistance to the antibiotics in current clinical use. In recent years, drug

resistance to human pathogenic bacteria has been commonly reported from all over the world.

In the present scenario of emergence of multiple drug resistance to human pathogenic

organisms, this has necessitated a search for new antimicrobial substances from other sources

including plants. Higher plants produce hundreds to thousands of diverse chemical

compounds with different biological activities. The antimicrobial compounds produced by

plants are active against plant and human pathogenic microorganisms. It is expected that

plant extracts showing target sites other than those used by antibiotics will be active against

drug-resistant microbial pathogens.

Antibiotics provide the main basis for the therapy of microbial (bacterial and fungal)

infections. Since the discovery of these antibiotics and their uses as chemotherapeutic agents

there was a belief in the medical fraternity that this would lead to the eventual eradication of

infectious diseases. However, over use of antibiotics has become the major factor for the

emergence and dissemination of multidrug resistant strains of several groups of

microorganisms. The worldwide emergence of multidrug resistant Escherichia coli and many

other ß-lactamase producers has become a major therapeutic problem Thus, in light of the

evidence of rapid global spread of resistant clinical isolates, the need to find new

antimicrobial agents is of paramount importance. However, the past record of rapid,

widespread emergence of resistance to newly introduced antimicrobial agents indicates that

even new families of antimicrobial agents will have a short life expectancy .For this reason,

researchers are increasingly turning their attention to herbal products, looking for new leads

to develop better drugs against MDR microbe strains The antimicrobial efficacy attributed to

some plants in treating diseases has been beyond belief. It is estimated that local communities

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have used about 10% of all flowering plants on Earth to treat various infections, although

only1% have gained recognition by modern scientists. During the last ten years the pace of

development of new antimicrobial drugs has slowed down while the prevalence of

resistance(especially multiple) has increased astronomically .The increase in number of

antibiotic resistant bacteria is no longer matched by expansion in the arsenal of agents

available to treat infections. Literature reports and ethnobotanical records suggest that plants

are the sleeping giants of pharmaceutical industry. They mayprovide natural source of

antimicrobial drugs that will/or provide novel or lead compounds that may be employedin

controlling some infections globally.

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Review OF

LITERATURE

Ethanol extracts of 19 Malaysian traditional vegetables (locally known as ‘ulam’)

belonging to 15 families were screened for antimicrobial and cytotoxic activities. Seven

extracts, i.e. Anacardium occidentale, Garcinia atroviridis, Sesbania grandiflora, Barringtonia

racemosa, Polygonum minus, Kaempferia galanga and Etlingera elatior displayed cytotoxic

activity against the HeLa (human cervical carcinoma) cell line with CD 50 values in the

range of 10–30 µg/ml.( M.M. Mackeen et al ., 1997). Ocimum sanctum the sacred 'Tulsi'

finds diverse uses in the indigenous system of Medicine. The leaves of the plant have been

used as an expectorant, diaphoretic, anticancer, antihelminthic, antiseptic, analgesic and tonic

rejuvinator. Dry leaves are used in fungal infections, the fresh juice of the leaves are used in

the treatment of bronchitis, otitis media, and skin diseases.   Antibacterial activity of ether

extract of the leaves against   Escherichia  coli, Staphylococcus aureus, and   Mycobacterium

tuberculosis have been reported.( Geeta et al .,2001). The antimicrobial effects of extracts

of neem seed (Azadirachta indica A. Juss.) were investigated using microbial growth

inhibition assays.( E. Coventry  et Al.,2001). The antibacterial activity of Karanj (Pongamia

pinnata) and Neem (Azadirachta indica) seed oil in vitro against fourteen strains of

pathogenic bacteria was assessed (BASWA Manjula et al.,2001 ). Fifty four plant extracts

(methanol and aqueous) were assayed for their activity against multi-drug resistant

Salmonella typhi. Strong antibacterialactivity was shown by the methanol extracts of Aegle

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marmelos, Salmalia malabarica, Punica granatum, Myristica fragrans, Holarrhena

antidysenterica, Terminalia arjuna and Triphal (mixture of Emblica of fi cinalis, Terminalia

chebula and Terminalia belerica) ( Rani P et al .,2004 )

Ocimum sanctum fixed oil showed good antibacterial activity against Staphylococcus

aureus, Bacillus pumilus and Pseudomonas aeruginosa, where S. aureus was the most

sensitive organism.(Singh PP et al ., 2005). Screening was done of some plants of importance

in the Ayurvedic system of traditional medicine used in India to treat enteric diseases.

Volatile oil of dried rhizome of Kaempferia galanga obtained by water distillation was

determined for its chemical components using gas . Antimicrobial activity of the volatile oil

was tested against various microbes using agar disc diffusion method (S Tewtrakul et

al.,2005). The immunomodulatory activity of the neem-bark extract has been reported. The

medicinal and industrial uses of various parts of neem tree and the compounds isolated have

been reviewed. (Wafaa A. Helmy et al ., 2007 ). .. The antibacterial activity of guava

(Psidium guajava) and neem (Azadirachta indica) extracts against 21 strains of foodborne

pathogens were determined. Guava and neem extracts showed higher antimicrobial activity

against Gram-positive bacteria compared to Gram-negative bacteria except for V.

parahaemolyticus, P. aeroginosa, and A. hydrophila.( M.D. Mahfuzul Hoque et al ., 2007).

Basil or tulasi (Ocimum sanctum L.) is considered as a sacred plant and its various medicinal

properties have been mentioned in ancient medicinal text, Ayurveda. Different parts of this

plant are used for treatment of various ailments. The activity of essential oils obtained from

fresh and dried leaves of O. sanctum against clinical isolates of enteric bacteria

i.e., Escherichia coli, Shigella sp. and Salmonella typhi and yeast (Candida albicans) was

compared.( S. Mondal et al .,2007)

For evaluating antibacterial activity, both aqueous and organic solvent methanol was used.

The plants screened were Ocimum sanctum, Jatropha gossypifolia, Boerhavia diffusa,

Azadirachta indica, Solidago virgaurea, and Commelina benghalensis. The antibacterial

activity was assessed against six bacterial strains--Pseudomonas testosteroni, Staphylococcus

epidermidis, Klebsiella pneumoniae, Bacillus subtilis, Proteus morganii, Micrococcus flavus.

( Nair R et al .,2007). Essential oil obtained by hydrodistillation and two different solvent

21

extractions(petroleum ether and ethanol) from five Zingiberaceae species: ginger (Zingiber

officinaleRoscoe.), galanga (Alpinia galanga Sw.), turmeric (Curcuma longa L.), kaempferia

(Boesenbergia pandurata Holtt.) and bastard cardamom (Amomum xanthioides Wall.) was

characterized,(Krittika Norajit et al.,2007). Based on local use of common diseases and

Ethnobotanical knowledge, an attempt has been made to assess the antibacterial properties of

selected medicinal plants.VIZ. Ocimum sanctum (Tulsi), Origanum majorana (Ram Tulsi),

Cinnamomum zeylanicum (Dalchini), and Xanthoxylum armatum (Timur), for potential

antibacterial activity against 10 medically important bacterial strains, namely Bacillus

subtilis, Bacillus cereus, Bacillus thuringiensis, Staphylococcus aureus, Pseudomonas spp,

Proteus spp, Salmonella Typhi, Escherichia coli, Shigella dysentriae, Klebsiella

pneumoniae. The antibacterial activity of ethanol extracts was determined by agar well

diffusion method.( Bishnu joshi et al ., 2008). The enhancement of

hydrophilicity of DC air and oxygen plasma treated cotton fabric and its

effect on the antimicrobial efficacy when treated with neem leaf extract (K

Vaideki et al .,2009).

Silver nanoparticles are known to have bactericidal effects. A new generation of dressings

incorporating antimicrobial agents like silver nanoparticles is being formulated to reduce or

prevent infections. Apart from being environmentally friendly process, use of Neem leaves

extract might add synergistic antibacterial effect of Neem leaves to the biosynthesized

nanoparticles. With this hypothesis the biosynthetic production of silver nanoparticles by

aqueous extract of Neem leaves and its bactericidal effect in cotton cloth against E. Coli were

studied (Tripathi et al.,2009).Neem (Azadirachta indica) is an Indian tree well known for its

several pharmacological activities, including antimicrobial activity. More than 300

composites have already been isolated and azadirachtin (AZA) is its main active component.

Neem leaves hydroalcoholic extracts were prepared by percolation in 96% ethanol different

concentrations (50%, 60%, 70%, 80% and 90% (v/v)).( Priscila D. Alves et al .,2009).

In recent years this interest to evaluate plants possessing antibacterial activity for various

diseases is growing . Based on local use of common diseases and Ethnobotanical knowledge,

22

an attempt has been made to assess the antibacterial properties of selected medicinal

plants.VIZ. Ocimum sanctum (Tulsi), Origanum majorana (Ram Tulsi),Cinnamomum

zeylanicum (Dalchini), and Xanthoxylum armatum (Timur), for potential antibacterial activity

against 10 medically important bacterial strains, namely Bacillus subtilis, Bacillus cereus,

Bacillus thuringiensis, Staphylococcus aureus,

Pseudomonas spp, Proteus spp, Salmonella Typhi, Escherichia coli, Shigella dysentriae,

Klebsiella pneumonia (Bishnu Josh et al.,2009). The ethnobotanical efficacy of Indian

medicinal plants; Achyranthes aspera, Artemisia parviflora, Azadirachta indica, Calotropis

gigantean, Lawsonia inermis, Mimosa pudica, Ixora coccinea, Parthenium hysterophorus

and Chromolaena odorata were examined using agar disc diffusion method against clinical

bacteria (Escherichia coli and Staphylococcus aureus) and phytopathogenic bacteria

(Xanthomonas vesicatoria and Ralstonia solanacearum). Leaves were extracted using

different solvents such as methanol, ethanol, ethyl acetate and chloroform.( S. L. Sukanya et

al.,2009).  Tulsi (Ocimum sanctum) extract has an antimicrobial activity against

Streptococcus mutans and the concentration of Tulsi (Ocimum sanctum) extract among the

15 concentrations investigated has the maximum antimicrobial activity was determined .

(Agarwal P et al.,2010).

. Tulsi has been tested against a variety of microorganisms like Candida albicans,

Staphyloccus aureus, enteric pathogens, Klebisella,  Escherichia  coli and Proteus. It has also

demonstrated antigonorrheal efficacy against multiresistant strains of  Neisseria   gonorrhea

and clinical isolates of beta lactamase-producing methicillin-resistant Staphylococcus aureus.

( Pooja Agarwal et al .,2010). Shoots and roots were induced from axillary buds of

Kaempferia galanga when cultured on Murashige and Skoog (MS) medium supplemented

with NAA + BAP (0.1mg + 1.0 mg/l). Liquid detergent (2%) + Alcohol (70%) + Mercuric

chloride (0.1%) used for surface sterilization of explants.Rhizomes were developed after four

months of transferring in to earthen pots. The methanolic extracts of both micropropagated

plant rhizome and natural plant rhizome were screened for antimicrobial activities against

two gram ‐ve and two gram +ve pathogenic bacteria i.e. Escherichia coli,Salmonella typhi,

23

Bacillus subtilis and Staphylococcus aureus.( HANUMANTHARAJU N et al.,2010). Four

medicinal plants (Quercus infectoria, Kaempferia galanga, Coptis chinensis and Glycyrrhiza

uralensis) as well as one traditional Thai treatment for aphthous ulcers based on these four

plants were tested for antimicrobial activity.( Mekseepralard C et al.,2010). Four medicinal

plants (Quercus infectoria, Kaempferia galanga, Coptis chinensis and Glycyrrhiza glabra) as

well as one traditional Thai treatment for aphthous ulcers based on these four plants were

tested for antimicrobial activity. MIC values for a range of bacteria and Candida albicans

were determined, with both type strains and clinical isolates being used. Antioxidant activity

was determined using the ABTS radical scavenging assay (Kamkaen et al.,2010).

Neem has been extensively used in Ayurveda, Unani and homoeopathic medicine and has

become a cynosure of modern medicine. The antibacterial activity of neem seed oil was

assessed in vitro against 14 strains of pathogenic bacteria. The present study was to find out

the antibacterial activity of neem leaves against human pathogenic bacteria, including

Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Micrococcus luteus, Bacillus

subtilis, Enterococcus faecalis and Streptococcus faecalis.( Saradhajyothi KOONA et

al.,2011). The antibacterial activity of aqueous extracts , chloroform extract ,alcohol extract

and oil obtained from leaves of Ocimum sanctum against the selected bacteria was observed (

Poonam Mishra et al .,2011). Antimicrobial activity in leaf extract of neem (Azadirachta

indica) against human pathogenic bacteria. E.coli, Staphylococcus aureus, Pseudomonas

aeruginosa , Salmonella typhimurium, Bacillus pumilus. Antimicrobial activities of alcoholic

extracts of neem leaves were used.( Maragathavalli, S et al.,2012). Kaempferia galanga L.

(KG), commonly known as cekor is one of those precious medicinal herbs of Zingiberaceae

that are still included in un-utilized herbs inspite of the variety of useful pharmacological

properties it possesses. Extracts of KG have anti-inflammatory, analgesic, nematicidal,

mosquito repellent, larvicidal, vasorelaxant, sedative, antineoplastic, antimicrobial, anti-

oxidant, antiallergic and wound healing properties.( Muhammad Ihtisham Umar et al.,2011).

Volatile oil from the rhizomes of Kaempferia galanga was isolated and characterized by Gas

Chromatography–Mass Spectroscopy. The antimicrobial activity of oil was tested against

human and plant pathogenic bacteria and fungi. (P.A. Mary Helen et al .,2011).

24

25

MATERIALS AND

METHODS

MATERIALS

COLLECTION OF PLANT MATERIAL

Fresh leaves of three different plants Neem ( Azadirachta indica A. Juss ) , Kacholam (

Kaempferia galanga L ), Tulsi ( Ocimum sanctum ) free from disease were collected from

different areas. The leaves were washed thoroughly 2-3 times with running water and once

26

with sterile distilled water. The material was chopped into small pieces and then air dried on a

sterile blotter under shade for 20-30 days.

PREPARATION OF PLANT EXTRACTS

10 g of each powdered plant material of Neem , Kacholam and Tulsi were defatted with

petroleum ether and extracted in a soxhlet extraction apparatus with 100% ethanol. The

solvent extracts were concentrated under reduced pressure and preserved at 5°C in airtight

bottle until further use.

TEST MICROORGANISMS USED FOR ANTIMICROBIAL STUDIES

Bacterial cultures of of both gram negative and gram positive - were obtained from the

culture collection centre. Different bacterial strains used for the antimicrobial studies were ,

Staphylococcus aureus (S.aureus) - It is a facultative anaerobic Gram -

positive coccal bacterium. It is frequently found as part of the normal skin flora on the skin

and nasal passages. S. aureus can cause a range of illnesses, from minor skin infections, such

as pimples, impetigo, boils(furuncles), cellulitis folliculitis, carbuncles, scalded skin

syndrome, and abscesses, to life-threatening diseases such

as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock

syndrome (TSS), bacteremia, andsepsis. Its incidence ranges from skin, soft tissue,

respiratory, bone, joint, endovascular to wound infections. It is still one of the five most

common causes of nosocomial infections and is often the cause of postsurgical wound

infections. 

Escherichia coli (E.coli) – it is a Gram-negative, rod-shaped bacteriumthat is commonly found

in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are

harmless, but some serotypes can cause serious food poisoning in humans, and are

occasionally responsible for people making product recalls. The harmless strains are part of

the normal flora of the gut, and can benefit their hosts by producing vitamin K2, and by

preventing the establishment of pathogenic bacteria within the intestine.

27

Klebsiella pneumoniae – is a Gram-negative, non-

motile, encapsulated, lactose fermenting, facultative anaerobic, rod shaped bacterium found

in the normal flora of the mouth, skin, and intestines. It is clinically the most important

member of the Klebsiella genus of Enterobacteriaceae. K. pneumoniae can cause the

disease Klebsiella pneumonia. They cause destructive changes to human

lungs inflammation and hemorrhage with cell death (necrosis) that sometimes produces a

thick, bloody, mucoid sputum (currant jelly sputum). Typically these bacteria gain access

after a person aspirates colonizing oropharyngeal microbes into the lower respiratory tract.

Pseudomonas fluorescens (P.fluorescens) -  is a common Gram-negative, rod-

shaped bacterium. It belongs to the Pseudomonas genus. P. fluorescens is an unusual cause of

disease in humans, and usually affects patients with compromised immune systems (e.g.,

patients on cancer treatment). 

. Muller – Hinton agar

Beef infusion - 300 ml

Caesin hydrolysate - 17.5 g

Starch - 1.5g

Agar - 25 g

Distilled water – 1000 ml

The above constituents werw added one by one and the final volume was made up to 1000 ml

with distilled water . Then the pH was adjusted to 7 at 25◦Ccusing 0.1 Naoh/HCL . It was

autoclaved and was poured into sterile petridishes and was allowed to solidify . It was stored

at 4◦C.

Nutrient Broth

Nutrient broth was used for the preparation of inoculum

Peptone – 5g

28

Beef extract – 3g

Nacl – 5g

The above constituents were added one by one and the final volume was made up to 1000 ml

with distilled water . Then the pH was adjusted to 7 at 25◦c using0.1 NaoH / HCL. The

bacteria were maintained on nutrient broth (NB) at 37°C.

Streptomycin was used as a reference . It was purchased and was dissolved in 2ml of

distilled water.

DETERMINATION OF ANTIMICROBIAL ACTIVITY WITH WELL DIFFUSION

METHOD

In order to determine the biological significance and ability of the plant part, the antibacterial

activity was determined by agar well diffusion method. Muller Hinton agar plates (MHA)

were prepared as follows :

Overnight grown different species of bacteria such as E-coli, Staphylococcus aureus,

Klebsiella pneumoniae and Pseudomonas fluorescens were swabbed. Wells of approximately

10mm was bored used a well cutter and di concentrations of plant extracts (30,60µl ) was

added, the zone of inhibition was measured after overnight incubation and compared with

that of standard antibiotics, DMSO(Dimethylsulfoxide) was used as negative control and

streptomycin was used as reference.

The antimicrobial activity was determined by measuring the zone of inhibition after overnight

incubation and compared with standards.

29

RESULTS AND

DISCUSSION

30

RESULTS

The antimicrobial activity of the extracts and their potency was quantitatively assessed by the

presence or absence of inhibition zone and zone diameter,

respectively as given in Table 1.

Organism Sample Measurement of zone of

Inhibition(cm)

E Coli

Streptomycin

DMSO

2.1

1.1

31

Kacholam

Tulsi

Neem

1.3

1.4

1.2

Streptococcus

Streptomycin

DMSO

Kacholam

Tulsi

Neem

5.5

1.5

1.7

1.5

2.0

Staphylococcus

Streptomycin

DMSO

Kacholam

2.7

1.2

1.3

32

Tulsi

Neem

1.2

1.3

Klebsiella

Streptomycin

DMSO

Kacholam

Tulsi

Neem

5.5

1.5

1.7

1.5

2.0

33

DISCUSSION

34

Plants are important source of potentially useful structures for the development of new

chemotherapeutic agents. Many reports are available on the antiviral, antibacterial, antifungal,

anthelmintic, antimolluscal and anti-inflammatory properties of plants. Some of these

observations have helped in identifying the active principle responsible for such activities and

in the developing drugs for the therapeutic use in human beings. Emergence of multi-drug

resistance in human and animal pathogenic bacteria as well as undesirable side effects of

certain antibiotics has triggered immense interest in the search for new

antimicrobial drugs of plant origin..

In the present study , antimicrobial activity of plant extracts was determined

by agar well diffusion assay using Streptomycin as a positive control. The antimicrobial

spectrum was evaluated against E. coli, Pseudomonas, Staphylococcus, Klebsiella. The zone

of inhibition was more in wells corresponding to extracts of Neem (Azadirachta indica )

when compared with other two extracts (Kaempferia galanga and Ocimum sanctum) . Study

suggests potent antibacterial activity of all three plant extract which can find its application in

therapeutic regimens, confirming the great potential of bioactive compounds and are useful for

rationalizing the use of this plant in primary health care. In vivo data may be helpful in determining

the real potential usefulness of this plant for the treatment of infectious diseases.

35

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