ANTIMICROBIAL, ANTIOXIDANT AND PROTECTIVE EFFICACY OF ...

ANTIMICROBIAL, ANTIOXIDANT AND

PROTECTIVE EFFICACY OF FLOWER AND

LEAF EXTRACTS OF CALOTROPIS PROCERA

AGAINST FREE RADICAL DAMAGE

By

ABID ALI

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF KARACHI

KARACHI

2015


PROTECTIVE EFFICACY OF FLOWER AND LEAF

EXTRACTS OF CALOTROPIS PROCERA AGAINST

FREE RADICAL DAMAGE

BY

ABID ALI

THESIS SUBMITTED

TO THE FACULTY OF SCIENCE, UNIVERSITY OF

KARACHI, IN FULFILLMENT OF THE REQUIREMENT

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF KARACHI

KARACHI-75270.

2015


PROTECTIVE EFFICACY OF FLOWER AND LEAF

EXTRACTS OF CALOTROPIS PROCERA AGAINST

FREE RADICAL DAMAGE

THESIS APPROVAL SHEET

SUPERVISOR_______________________________

DR. TABASSUM MAHBOOB

MERITORIUS PROFESSOR

EXTERNAL EXAMINER______________________

DECLARATION

It is declared that this research work entitled: “Antimicrobial, antioxidant and protective

efficacy of flower and leaf extracts of Calotropis procera against free radical damage”

is my own work and has not been submitted in any form for another degree at any

university. Information derived from the published or unpublished literature of other

workers is acknowledged in the text and a list of references.

ABID ALI

DEDICATION

This thesis is dedicated to

My beloved Parents

ACKNOWLEDGEMENTS

First and foremost, I am thankful to Almighty ALLAH who blessed and

gave me the power to complete this research work.

I am greatly indebted to my supervisor Dr. Tabassum Mahboob,

Meritorious Professor, Department of Biochemistry, University of

Karachi for her expert advice, generous guidance and encouragement

throughout the period of research work.

I owe a great debt of gratitude to Dr. Shah Ali Ul Qader, Associate

Professor, Khan Institute of Biotechnology and Genetic Engineering

(KIBGE), University of Karachi for his guidance, cooperation and

providing laboratory facilities during this research.

I am also thankful to Prof. Dr. Majid Mumtaz and Dr. Sumayya Saied

Department of Chemistry, University of Karachi for their kind help in

connection with the preparation of various extracts.

Special thanks are also for Mr. Qader, Mr. Safaraz and Mr. Aijaz for their

cooperation in various ways.

I cannot express enough thanks to all of my family members, especially

my wife for encouragement and moral support throughout the period of

this research work.

CONTENTS

Title Page No.

ABSTRACT

i. English…………………………………………………………….1-3

ii. Urdu ………………………………………………………………4-5

CHAPTER # 1

GENERAL INTRODUCTION………………………………………………….6-23

CHAPTER # 2

GENERAL MATERIAL AND METHODS …………………………………..24-64

CHAPTER # 3

PHYTOCHEMICAL STUDIES OF CALOTROPIS PROCERA………………...65-70

CHAPTER # 4

ANTIMICROBIAL EFFECT OF CALOTROPIS PROCERA………………….71-85

CHAPTER # 5

EFFECT OF CALOTROPIS PROCERA ON ENZYMES ACTIVITY………....86-97

CHAPTER # 6

IN VITRO ANTIOXIDANT PROPERTIES OF CALOTROPIS PROCERA…98-117

CHAPTER # 7

EFFECT OF CALOTROPIS PROCERA ON IBUPROFEN TREATED

RATS………………………………………………………………………….118-144

CHAPTER # 8

GENERAL DISCUSSION…………………………………………………...145-155

CONCLUSION…………………………………………………………………....156

REFERENCES………………………………………………………………..157-184

PUBLISHED RESEARCH PAPER…………………………………………..185-190

EXPANDED CONTENT

Title Page No.

ABSTRACT

i. English…………………………………………………………….1-3

ii. Urdu ………………………………………………………………4-5

CHAPTER # 1

GENERAL INTRODUCTION………………………………………………….6-23

CHAPTER # 2

GENERAL MATERIAL AND METHODS ………………………………..…24-64

2.1. Collection of plant material……………………………………………………24

2.2. Preparation of extract………………………………………………………….24

2.3. Preparation of fractions………………………………………………………..24

2.4. General chemicals and materials………………………………………………28

2.5. Estimation of total protein……………………………………………………..28

2.6. Estimation of carbohydrates…………………………………………………...31

2.7. Estimation of total reducing sugars……………………………………………34

2.8. Estimation of total non-reducing sugars……………………………………….37

2.9. Estimation of total amino acids………………………………………………..37

2.10. Estimation of amino acids by paper chromatography….…………………….39

2.11. Estimation of phenolic compounds…………………………………………..40

2.12. Detection of phenolic compounds by chromatography…….………………...43

2.13. Method for protein free filtrate……………………………………………….44

2.13.1. Plasma urea estimation……………………………………………………..44

2.13.2. Methodology for plasma urea estimation…………………………………..45

2.13.3. Plasma creatinine estimation……………………………………………….45

2.13.4. Kidney homogenate………………………………………...........................48

2.13.5. Malonyldialdehyde estimation……………………………………………..48

2.13.6. 4 hydroxyl 2-nonenal estimation…………………………...........................50

2.13.7. Catalase estimation…………………………………………………............52

2.13.8. Superoxide dismutase estimation…………………………………………..54

2.13.9. Glutathione estimation………………………………………......................55

2.14. Lipid peroxidation inhibition method……………………………………….56

2.14.1. Preparation of tissue homogenate…………………………………………56

2.14.2. Procedure for lipid peroxidation inhibition………………………………..56

2.14.3. DPPH free radical scavenging method……………………………………57

2.14.4. Method for determining reducing power assay…………………………...58

2.15.1. Estimation of glucoamylase……………………………………………….59

2.15.1.1. Glucoamylase activity assay…………………………………………….59

2.15.1.2. GOD – PAP method…………………………………………………….59

2.15.1.3. Reducing sugar estimation for alpha amylase activity………………….60

2.15.1.4. Alpha amylase enzyme activity assay..………………………………….62

2.15.1.5. Urease activity…………………………………………………………...63

2.16. Statistical analysis…………………………………………………………....64

CHAPTER # 3

PHYTOCHEMICAL STUDIES OF CALOTROPIS PROCERA…………….....65-70

3.1. Introduction…………………………………………………………….….….65

3.2. Material and methods………………………………………………….….…..65

3.2.1. Total proteins estimation………………………………………….….……..65

3.2.2. Carbohydrates estimation………………………………………….………..66

3.2.3. Total reducing sugars………………………………………………….……66

3.2.4. Total non reducing sugars……………………………………………….….66

3.2.5. Total amino acids estimation………………………………………….….....66

3.2.6. Amino acid estimation by paper chromatography…………………….…….66

3.2.7. Phenolic compounds estimation…………………………………………….66

3.2.8. Estimation of phenolic compounds by chromatography…………………...66

3.3. Results………………………………………………………………………...67

3.4. Discussion…………………………………………………………………….69

CHAPTER # 4

EFFECT OF CALOTROPIS PROCERA AS ANTIMICROBIAL AGENT…..71-85

4. Introduction…………………………………………………………………….71

4.1. Material and methods………………………………………………………...75

4.2. Preparation of extract………………………………………………………...75

4.3. Preparation of fractions………………………………………………………75

4.4. Experimental microbes……………………………………………………….75

4.5. Culture media………………………………………………………………...75

4.6. Procedure for antimicrobial activity………………………………………….76

4.7. Results………………………………………………………………………...83

4.8. Discussion………………………………………………………………….…84

CHAPTER # 5

EFFECT OF CALOTROPIS PROCERA ON ENZYMES ACTIVITY……….86-97

5. Introduction……………………………………………………………………..86

5.1. Material and methods…………………………………………………………88

5.1.1. Preparation of aqueous extract……………………………………………...88

5.1.2. Enzyme activity for glucoamylase, alpha amylase and urease……………...88

5.1.3. Estimation of glucoamylase…………………………………………………88

5.1.4. Glucoamylase activity assay………………………………………………...88

5.1.5. Estimation of reducing sugar for alpha-amylase………………………….....88

5.1.6. Estimation of urease activity………………………………………………...89

5.1.7. Alpha amylase activity assay……………………………………………….89

5.2. Results and discussion………………………………………………………...96

CHAPTER # 6

IN VITRO ANTIOXIDANT PROPERTIES OF CALOTROPIS PROCERA..98-117

6. Introduction……………………………………………………………………...98


6.1.1. Preparation of extract………………………………………………………100

6.1.2. Preparation of fractions…………………………………………………….100

6.1.3. Preparation of tissue homogenate………………………………………….100

6.2. Lipid peroxidation inhibition ………………………………………………..101

6.3. 1,1-diphenyl -2-picrylhydrazyl (DPPH) radical scavenging assay………....101

6.4. Reducing power assay………………………………………………………..101

6.5. Results………………………………………………………………………..114

6.6. Discussion…………………………………………………………………….115

CHAPTER # 7

EFFECT OF CALOTROPIS PROCERA ON IBUPROFEN TREATED

RATS…………………………………………………………………………118-144

7. Introduction…………………………………………………………………….118

7.1. Nephrotoxic agents…………………………………………………………...119

7.2. Nsaids………………………………………………………………………...119

7.3. Ibuprofen…………………………………………………………………......120

7.4. Mechanism of action of Ibuprofen…………………………………………...120


7.5.1. Preparation of extract………………………………………………………121

7.5.2. Preparation of fractions…………………………………………………..121

7.5.3. Experimental animals and diet……………………………………….…..121

7.6. Proper recommendations…………………………………………………...122

7.7. Preparation of drug……….………………………………………………...122

7.8. Study protocol and drug administration plan…………………………….....122

7.9. Collection of samples…………………………………………………….....123

7.9.1. Blood sample……………………………………………………………...123

7.9.2. Kidney sample………………………………………………………….....123

7.10. Analytical methods……………………………………………………......124

7.10.1. Preparation of protein free filtrate……………………………………….124

7.10.2. Estimation of plasma urea……………………………………………….124

7.10.3. Estimation of plasma creatinine…………………………………………124

7.10.4. Preparation of kidney homogenate……………………………………...124

7.10.5. Estimation of malonyldialdehyde (MDA)………………………………125

7.10.6. Estimation of 4-hydroxyl-2-nonenal (4-HNE) ………………………….125

7.10.7. Estimation of catalase …………………………………………………...125

7.10.8. Estimation of superoxide dismutase (SOD) ……………………………125

7.10.9. Estimation of glutathione (GSH) ……………………………………….125

7.11. Results……………………………………………………………………..140

7.12. Discussion………………………………………………………………….142

CHAPTER # 8

8. GENERAL DISCUSSION……………………………………………....145-155

CONCLUSION……………………………………………………………..…...156

REFERENCES……………………………………………………………...157-184

LIST OF FIGURES

Title Page No.

Fig. 1.1. Causes of oxidative stress………………………………………….........14

Fig. 1.2. Showing free radical and oxidative stress……………………………….15

Fig. 1.3. Showing structure of Ibuprofen………………………………………....16

Fig. 1.4. Showing mechanism of inhibition by NSAID………………………….17

Fig. 1.5. Graphical representation of Lipid damage cycle………………………..19

Fig. 1.6. Showing antioxidant defense - enzymes………………………………..20

Fig. 2.1. Flow chart showing preparation of extract from Calotropis procera…..26

Fig. 2.2. Method for preparation of fractions of C. procera in different

solvents……….…………………………………………………………27

Fig. 2.3. Standard curve for total protein…………………………………………30

Fig. 2.4. Standard curve for total carbohydrates………………………………….33

Fig. 2.5. Standard curve for reducing sugar………………………………………36

Fig. 2.6. Standard curve for amino acids…………………………………………38

Fig. 2.7. Standard curve for phenol……………………………………………….42

Fig. 2.8. Standard curve for plasma urea………………………………………….46

Fig. 2.9. Standard curve for plasma creatinine……………………………………47

Fig. 2.10. Standard curve for MDA………………………………………………49

Fig. 2.11. Standard curve for 4-HNE……………………………………………..51

Fig. 2.12. Standard curve for catalase…………………………………………….53

Fig. 2.13. Standard curve for reducing sugar for amylase………………………..61

Fig. 4.1. Antimicrobial activity of flower extracts……………………………….78

Fig. 4.2. Antimicrobial activity of leaf extracts………….………………………80

Fig. 4.3. Showing zone of inhibition of C. procera flower extract……………...…81

Fig. 4.4. Showing zone of inhibition of C. procera leaf extract……………...……82

Fig. 5.1. Representing starch structure showing α-1,4-linkage

where α-amylase targets………………..…………………………………87

Fig. 5.2. Showing the effect of aqueous extract of C. procera

on glucoamylase activity………………………………………..………...93


on alpha amylase activity………………………………………..……….95


on urease activity………………………………………………...………97

Fig. 6.1. Effect of different (flower) extracts of C. procera

on Lipid peroxidation inhibition (%)………………………………...…108

Fig. 6.2. Effect of different (leaves) extracts of C. procera

on Lipid peroxidation inhibition (%)………………………………...…109

Fig. 6.3. DPPH radical scavenging activity of(flower) extracts of

C. procera…………………………………..…………………………..110

Fig. 6.4. DPPH radical scavanging activity of (leaves) extracts of

C. procera………………………………………………..……………..111

Fig. 6.5. Reducing power assay of(flower) extracts of C. procera……….…….112

Fig. 6.6. Reducing power assay of (leaves) extracts of C. procera……………113

Fig. 7.1. Effect on Body weight of rats in Control, Ibuprofen, Hexane

and Ibuprofen + Hexane treated groups…….………………………...130

Fig .7.2. Effect on Kidney weight of rats in Control, Ibuprofen, Hexane

and Ibuprofen + Hexane treated groups……………………………....131

Fig. 7.3. Effect on Plasma Urea level of rats in Control, Ibuprofen,

Hexane and Ibuprofen + Hexane treated groups…………………….132

Fig. 7.4. Effect on Plasma Creatinine level of rats in Control, Ibuprofen,

Hexane and Ibuprofen + Hexane treated groups……………………..133

Fig. 7.5. Effect on Tissue SOD level of rats in Control, Ibuprofen,

Hexane and Ibuprofen + Hexane treated groups……………………134

Fig. 7.6. Effect on Tissue Catalase level of rats in Control, Ibuprofen,


Fig. 7.7. Effect on Plasma MDA level of rats in Control, Ibuprofen,


Fig. 7.8. Effect on Tissue MDA level of rats in Control, Ibuprofen,


Fig. 7.9. Effect on Tissue 4HNE level of rats in Control, Ibuprofen,


Fig. 7.10. Effect on Tissue GSH level of rats in Control, Ibuprofen,


Fig. 8.1. Proposed inhibition of MDA formation and metabolism………..……...148

Fig. 8.2. Proposed inhibition of 4HNE production and metabolism…………..…149

Fig. 8.3. Proposed inhibition process of lipid peroxidation by C. procera extract

……………………………………………………………………….....150

LIST OF TABLES

Title Page No.

Table 3.1. Phytochemical Estimation of Calotropis procera……………………...67

Table 3.2. Detected Amino Acids in Calotropis procera…………………………67

Table 3.3. Phenolic constituents of Calotropis procera…..………………………68

Table 4.1. Antiicrobial activity of flower extracts against different

pathogenic strains…………………………………………………….....77

Table 4.2. Antimicrobial activity of leaf extracts against different

pathogenic strains…………………………………………………….....79

Table 5.1. Effect of aqueous extract of C. procera on glucoamylase activity……..90

Table 5.2. Effect of aqueous extract of C. procera on alpha amylase activity….....92

Table 5.3. Effect of aqueous extract of C. procera on urease activity……………..94

Table 6.1. Lipid peroxidation inhibition activity of C. procera flowers

extracts…………………………………………………………………102

Table 6.2. Lipid peroxidation inhibition activity (%) of C. procera leaves

extracts…………………………………………………………………103

Table 6.3. DPPH radical scavenging activity of C. procera flowers

extracts…………………………………………………………….…...104

Table 6.4. DPPH radical scavenging activity (%) of C. procera leaves

extracts………………………………………………………………....105

Table 6.5. Reducing power assay of C. procera flowers

extracts…………………………………………………………..……..106

Table 6.6. Reducing power assay of C. procera leaves

extracts…………………………………………………………………107

Table 7.1. Effect on Body and Kidney weight in control, ibuprofen, hexane,

and ibuprofen+hexane treated rats……………………………………..126

Table 7.2. Effect on renal function in control, Ibuprofen, hexane and

Ibu+hexane pretreated rats……………………………………………..127

Table 7.3. Tissue SOD and Catalase in Control, Ibuprofen, hexane and

Ibu+Hexane……………………………………………………………128

Table 7.4. Plasma MDA, Tissue MDA, 4HNE, and Glutathione levels…………129

1

ABSTRACT

Different soluble fractions viz., hexane, ethyl acetate, butanol and ethanol

of Calotropis procera (Ait.) R. Br. were screened for their antimicrobial

properties by using agar-well diffusion method against the human

pathogens viz., Escherichia coli and Salmonella typhi (Gram negative),

methcillin resistant Staphylococcus aureus and Micrococcus luteus (Gram

positive), in vitro antioxidant properties were analyzed by means of

DPPH free radical scavenging method, reducing ability assay and lipid

peroxidation inhibition method. Furthermore, in vivo protective efficacy

of C. procera extract against (NSAID) ibuprofen-induced nephrotoxicity

in rat model was also determined by evaluating renal function markers,

plasma measure of antioxidant enzymes superoxide dismutase (SOD) and

catalase (CAT) along with the determination of tissue lipid peroxidation

markers, i.e. aldehyde products malonyldialdehyde (MDA) and 4-

hydroxy-nonenal (4-HNE). Phytochemical analysis was also carried out

for the detection of phenolic constituents, amino acids, protein,

carbohydrates, reducing and non-reducing sugars in test plant.

2

In the present findings the hexane fraction of C. procera flower and leaf

have been proved very significant with maximum zones of inhibition i.e.,

flower (22mm) and leaf (23mm) against M. luteus. While, other tested

fractions of C. procera flower and leaf showed significant antimicrobial

activity against all pathogens. Whereas, in the present finding it was also

determined that the flower ethanol extract showed the highest DPPH free

radical scavenging activity (88.19% with 8 mg/ml) as compared to BHA

which showed 85% scavenging activity as standard. Similarly, C. procera

flower and leaf extracts were also analyzed for reducing capacity. The

highest absorbance (i.e., 1.827 with 10mg/ml) was recorded in C. procera

flower water extract as compared to standard which showed (0.238)

absorbance. In vitro lipid peroxidation inhibition, another model was used

to check the antioxidant capacity of C. procera. Flower water extract

exhibited a concentration dependent increase in lipid peroxidation

inhibition, the highest value is (89.58% with 10mg/ml) while the lipid

peroxidation value in C. procera leaf water extract (i.e., 75.11% with

10mg/ml) and leaf ethyl acetate extract showed (75.11% with 8mg/ml).

While, BHA (85%) and ascorbic acid (75.5%) showed lower values as

compared to tested plant.

3

However, body weight loss was successfully restored by the co-

administration of Ibuprofen with C. procera hexane extract. While,

increased level of renal function markers (urea, creatinine) was

normalized by the administration of C. procera hexane with ibuprofen

treatment. The imbalance in oxidative status was determined by

evaluating decreased level of catalase, superoxide dismutase and

glutathione along with increased levels of malonyldialdehyde and 4-

hydroxynonenal, which was counteracted by the co-administration of C.

procera hexane extract with ibuprofen which maintained cell

sustainability and indicated nephro-protective activity of C. procera.

Besides the above results C. procera leaf and flower aqueous extract were

also used to check enzymatic activities of glucoamylase, α-amylase and

urease enzymes. The flower extract is found proved to be a good enhancer

of glucoamylase, α-amylase and urease activity as compared to leaf

extract. A number of phytoconstituents were also detected. The presence

of phytochemicals in C. procera may indicate a good correlation with that

of antibacterial, antioxidant potential and protective role for in vivo model

which also proved as a good enhancer of enzyme activities. Thus due to

aforementioned activities, Calotropis procera may serve as a better and a

protective therapeutic agent than any other synthetic drug.

4

5

6

1.GENERAL INTRODUCTION

Plants are a major source of traditional medicine even modern medicine system

depends on pharmacologically active agents from plant to obtain useful drugs.

Calotropis procera (Aiton) R. Br. is one of the famous medicinal plant having

bioactive molecules which may serve against various ailments. C. procera is

commonly known as “Aak” and belongs to the family Asclepiadaceae, Plant is wide

spread in Arab, Africa and Asian countries (Mabberley, 2008). C. procera is

characterized by the presence of opposite and decussate leaves. Flowers in terminal or

axillary umbelloid cyme, consists of five deeply lobed and dirty white sepals with

purple tips and white base petals, corona of five fleshy laterally compressed lobes

surrounding the pentagonal stigma (Ali, 1983).

In the past C. procera was also used for epilepsy, mental stress, diarrhoea, earache,

sprain, toothache, anxiety, pain (Kew, 1985; Kareem et al., 2008; Ahmad et al., 2011).

Sometimes, leaves and stem are inhaled or smoked after burning to cure the fever,

swellings, paralysis and arthralgia. Similarly, leaves are also taken for the treatment of

various heart diseases and chest cold (Agharkar, 1991; Hemalatha et al., 2011). The

crude extracts, and dilutions in potencies of C. procera are being used in modern,

homeopathy, unani, as well as in veterinary practices (Dewan et al., 2000; Alencar et

al., 2004; Kareem et al., 2008; Johnson et al., 2011). Flowers decoction having

laxative and anthelmintic properties (Meena et al., 2011) and also cures jaundice

(Sharma et al., 2011). Plant is also used as antidiabetic (Roy et al., 2005; Jaiswal et

al., 2014; Shankar et al., 2014), and relief stomach pain and also acts as an

expectorant (Goyal and Mathur, 2011; Quazi et al., 2013), possesses analgesic,

7

antitumor, antioxidant, anticonvulsant, antidiarrhoeal, antimalarial (Oloumi, 2014) and

oestrogenic activity (Rahimi, 2015).

Biologically active compounds derived from plants are usually categorized in

secondary and primary metabolites. Whereas, last one is the part of metabolic

pathways and secondary metabolites are waste products or byproducts of metabolic

pathways. Regarding to the medicinal uses of Calotropis procera secondary

metabolites namely, phenolic compounds tannins, terpenoids and saponins have

received an immense attention (Vaya & others, 1997; Sengul & others, 2009; Patel &

others, 2010), such as Hassan & others (2006) evaluated roots, leaves and stem bark of

C. procera with aqueous, hexane, petroleum ether extracts for the detection of

phytochemicals where leaves and root extracts showed the presence of glycosides,

saponins, triterpenoids, steroids, alkaloids, tannins and flavonoids while stem bark

showed flavonoids, triterpenoids and saponins. Similarly, Qureshi & others (2007)

observed that the flower ethanol extract of C. procera having strong antioxidant

potential due to its Quercetin related flavonoids. Alam and Ali (2009) investigated

roots of C. procera and yield two phytochemical compounds, namely procerur acetate

and proceranol along with other known compounds. However, the studies of Kawo et

al. (2009) revealed that water extracts of leaf and milky sap of C. procera showed the

presence of flavonoids, saponins, steroids, and tannins. Leaf showed stronger

antibacterial potential while latex have no antibacterial activity. Similarly, methanolic

extract of C. procera leaf has been proved to contain alkaloids, flavonoids, phenolic

acids and tannins as significant constituents for drug development (Yadav et al.,

2010). Moustafa et al. (2010) evaluated that C. procera has cardenolides, flavonoids

8

and saponins. Mainasara et al. (2011) investigated the aqueous, methanol and ethanol

extracts of C. procera fruit and bark to evaluate their medicinal potentials, where a

number of phytochemicals including alkaloids, flavonoids, tannins, saponins and

cardiac glycosides were detected in water extract and revealed that aqueous extract of

C. procera may be used as a strong antibacterial agent. Similarly, Doshi & others

(2011) Ranjit & others (2012), Gajare & others (2012) screened ethanolic extract of

flower, leaves and stem of C. procera and detected alkaloids, glycosides, saponins,

triterpenoids, phenols and tannins in almost all parts of this plant. Prabha et al. (2012)

investigated phytochemicals in C. procera flower extracts of chloroform, acetone,

methanol and recorded the presence of alkaloids, tannins, asteroids, glycosides,

saponins, phenols and flavonoids. Srivastava et al. (2012) determined flavonoids by

the maximum quantity from C. procera leaves. Bouratoua et al. (2013) Isolated two

flavonoids isorhamnetin-3-o-rubinobioside and isorhamnetin-3-o-rutinoside from n-

butanol and ethyl acetate extracts of C. procera. Juca et al. (2013) investigated five

different latex fractions (hexane, dichloromethane, ethyl acetate, n-butanol and

aqueous) of C. procera and concluded that dichloromethane and ethyl acetate sample

showed anti-inflammatory properties. Chiranjeevi et al. (2013) and Quazi et al. (2013)

evaluated phytochemical profile of C. procera using various solvents. Verma et al.

(2013) also found out the cardiac glycosides, saponins, triterpenoids, alkaloids and

tannins and absence of flavonoids in ethanolic and chloroform extracts of C. procera.

According to Shrivastava et al. (2013) C. procera is the storehouse of secondary

metabolites such as flavonoids, terpenoids, alkaloids and steroids. Joshi and Kaur

(2013) analysed C.procera for the presence of bioactive compounds. While, Rajesh

9

et al. (2014) studied stem powder of C. procera with different extracts of hexane,

chloroform, methanol and sterile water for detection of saponins, flavonoids, sterols,

tannins and alkaloids. However, studies of Tiwari et al. (2014) evaluated the

phytochemicals of petroleum ether and methanol leaf extracts of C. procera and

determined the presence of glycosides, protein, triterpenoids, steroids and flavonoids

and suggested that these chemicals may be a significant indicator for the medicinal

importance of the plant. Moreover, Gholamshahi et al. (2014) also observed the leaf,

flower and fruit extracts of C. procera with the detection of phenolic constituents and

proved it as a strong antioxidant plant, which could be utilized in food and drug

industry. While, Al Snafi (2015) suggested that C. procera exhibited many

pharmacological aspects due to the presence of biologically active constituents.

Similarly, Shetty et al. (2015) studied the phytochemicals of C. procera leaves and

made a positive correlation between phytochemicals and antibacterial activity.

Beside the medicinal reports, C. procera also proved to be the antibacterial agent

against various human disease producing bacteria, which includes the bacteria with

violet stain and bacteria without violet stain. Akhtar et al. (1992) isolated a

cardenolide, proceragenin from C. procera and observed its strong antibacterial

activity against non-violet stain and violet stain bacteria. Ali et al. (2001) investigated

ethanolic extracts of 20 different plants, including C. procera against pathogenic

bacterial strains and concluded that C. procera affords antibacterial potential.

Similarly, acetone, methanol, ethanol, hexane, chloroform and ethyl acetate fractions

were used against S. aureus, S. epidermidis, Bacillus cereus, Pseudomonas

aeroginosa, Kleibsiella pneumonia, Serratia marcenes, Bacillus subtilis, and M.

10

luteus. (Parabia et al., 2008). It was reported that water, methanol, ethyl acetate flower

extracts are potent against fungal pathogens viz. Fusarium and T. vesiculatum (Devi et

al., 2008). Kareem et al. (2008) observed ethanol extract of the leaf and sap of C.

procera which showed widest zone of inhibition. Similarly, Yesmin et al. (2008) also

demonstrated the antibacterial activity through leaf watery & methanolic extracts of

C.procera and it was found that both extracts were active against bacterium with

violet stained and non-violet stained bacteria at low concentrations. Similarly, Kawo et

al. (2009) studied the antimicrobial potential of watery and ethanol leaf extracts and

sap of C. procera against the different bacterial strains where it was revealed that

aqueous extract did not show antibacterial activity while, leaf ethanol and latex have

significant antibacterial potential. Moreover, Mohanraj et al. (2010) observed that the

ethyl acetate extract of C.procera leaf and roots were effective against tested bacteria.

Antibacterial activities were performed from the flower extract with different organic

solvents viz., hexane, chloroform and methanol against Alternaria alternata,

Aspergillus flavus, Aspergillus niger, Bipolaris bicolour, Curvularia lunata, Penicillin

expansum, Pseudomonas marginalis and Rhizoctonia solani (Vadlapudi and Naido,

2010). While ethanol flower extract (Doshi et al., 2011) was used against the larvae of

A. stephansi. However, acetone and methanol flower extracts were further used

against Bacillus pumilis, E.coli, A. niger, Fusarium oxysporum, (David et al., 2011).

Amin and Khan (2011) proved antibacterial efficacy of methanolic fraction of leaf for

Enterobacter, Pseudomonas, S. aureus and Micrococcus. Similarly, Doshi et al.

(2011) utilized flower, young buds, mature leaves and stems of C. procera for

determination of antibacterial activity. While, mature leaves were found strong, potent

11

antimicrobial agent against all micro flora included during the test. Gajare et al. (2012)

evaluated ethanol root extract of C. procera, which was proved as potent antibacterial

agent. While, Vadlapudi et al. (2012) examined C. procera organic extract of aerial

parts against Pseudomonas marginalis and S. mutans. Prabha and Vasantha (2012)

screened C. procera flower extract of chloroform, acetone and ethanol against various

pathogens and maximum antibacterial activity was recorded against B. subtilis and S.

aureus. Velmurugan and his co-workers (2012) studied C. procera leaf extract of

hexane, ethyl acetate and methanol against aquatic micro pathogens from shrimp

fishes. It was noted that ethyl acetate extract effectively suppressed bacterial strains.

Mako et al. (2012) evaluated the antimicrobial activity of aqueous and ethanol root

and leaves extract of C. procera where ethanol extract showed more significant

potential than aqueous extract. Ranjit et al. (2012) observed ethanol flower extract of

C. procera and proved it as a strong inhibitory agent against human pathogenic

bacterias. Neenah (2013) studied the antimicrobial potential of solvent extract and

phenolic compounds of C. procera, using the agar well diffusion method. The crude

flavonoid fraction of methanolic extract was found to possess highest antimicrobial

activity and Gram positive bacteria were more vulnerable than the non-violet stained

bacteria and the yeast species were more vulnerable than filamentous fungi. Joshi and

Kaur (2013) determined the antimicrobial potential of ethanol, methanol and watery

extract of C. procera and found that ethanolic extract have strong antimicrobial

activity against Pseudomonas aeroginosa. Parabia et al. (2008) reported antibacterial

activity of aqueous elixir of twig and milky sap of C. procera. Both the samples

exhibited greater inhibition zone on S. aureus bacterial strain. Muzammal (2014)

12

determined antibacterial activity of aqueous, ethanol and methanol concentrate of C.

procera. While, ethanol concentrate of leaves and flower showed significant potential

against S. typhi and E. coli. Salem et al. (2014) demonstrated the antibacterial activity

through leaf and latex chloroform, ethanolic and methanolic concentrates of C.

procera where it was noted that watery and ethanolic leaf extracts showed maximum

potential against Gram negative and Gram-positive pathogenic bacteria. Kazemipour

et al. (2014) investigated antimicrobial activity of ethanol, chloroform and water

extracts of C. procera flowers and leaves. All extracts showed high potential against

Klebsiella pneumonia and S. epidermidis. Javadian et al. (2014) evaluated

antimicrobial activity of ethanol extract of C. procera and it found effective against E.

coli isolates. Ahmed et al. (2014) also proved the antibacterial activity of C. procera

latex against E.coli and Salmonella. Similarly, Ali et al. (2014) determined

antibacterial potential of different fractions including ethyl acetate, butanol and

aqueous flower extracts, where it was concluded that flower of C. procera have a

significant potential against many bacterial strains. Shetty et al. (2015) studied the

antibacterial effect of methanol, ethyl acetate, ethanol, acetone and aqueous extracts of

C. procera leaves against human pathogenic bacteria. It was revealed that leaves of C.

procera have significant antibacterial potential. Pandey et al. (2015) assessed

antibacterial activity of C. procera methanol, acetone, petroleum ether and ethyl

acetate extracts where methanol leaves extract and stem ethyl acetate extract showed

highest range of inhibition against E.coli and S. aureus.

However, toxicity is the degree to which a substance is able to damage an organism.

Toxicity can affect the whole organism or substructure of the organism and it may be

13

occur due to certain biological, physical or chemical effects. Drug induced toxicity can

damage any tissue depending on dosage such as acute dosage of a drug can produce

the toxicity for nervous system and its chronic exposure may cause the serious injuries

to the other organs. Toxicity can also be produced by the medicines which are

normally be used for curative purposes. Sometimes, the use of over the counter

medicines and long term use of overdoses of drugs may also cause toxicity to certain

specific organs. The Process of oxidation continuously takes place in all aerobic living

bodies, due to this ROS (reactive oxygen species) including O2 anion, H2O2 hydrogen

peroxide, -OH hydroxyl radical and nitric oxide/peroxinitrates (NO/NOO

-) are

constantly formed within the cells. The over production of these substances may cause

oxidative load in the cells. This oxidative stress produces deleterious effects to cells of

DNA, proteins and lipids. Lipid are specifically more damaged due to the formation of

lipid peroxidation products.

The toxicity of different metals, pollution, pesticides radiations, use of alcohols,

smoking, fast food, lack of good nutrition, stress, inadequate intake of fruits and

vegetables contaminants, excessive exercise and inadequate physical activity are the

reasons of free radical formation (Davies 1991; Halliwell and Aruoma 1993; Langseth

1995).

14

Figure 1.1. Causes of oxidative stress

(Adapted from http://currentscienceperspectives.com/)

15

Figure 1.2. Showing free radical and antioxidant in cell (Adapted from http://currentscienceperspectives.com/)

There are various reports available on toxicity producing substances like NSAIDS

(Derle & others, 2006; Fackovcova & others, 2000; Kocaoglu & coworkers, 1997),

Phenacetin (Murray and Brater, 1993), Mefenamic acid (Robertson & coworkers,

1980; Somchit & others, 2004), Caffeine (De Crespigny & coworkers, 1980),

Paracetamol (Younes & others, 1988), Acetaminophen (Tarloff & coworkers, 1990;

Trumper & others, 1992), Diclofenac (Hickey & coworkers, 2001; Yasmeen & others,

2007), Tenofovir (Morelle & others, 2009), Tacrolimus and NSAIDS (Soubhia &

others, 2005), Diclofenac (Kim & coworkers, 1999) and Metals including arsenic,

cadmium, lead and mercury (Nicholson, 1985; Fowler, 1992).

While, Cholestyramine was utilized against the Paracetamol induced toxicity in rats

and that was evident by a reduction in plasma enzyme activity and creatinine levels

(Siegers and Moller, 1989). On the other hand, Cadmium was used to prevent the

Acetaminophen induced toxicity in female rats (Bernard et al., 1988). Moreover,

16

Ibuprofen and Diclofenac were found useful protective drugs against Gentamicin

toxicity (Farag et al., 1996). While, Sharma et al. (2007) suggested that the algal

supplementation of Spirulina fusiformis can play a significant role against Mercuric

Chloride induced toxicity.

There are several substances which can initiate toxicity to the kidneys, called

nephrotoxic agents. These substances include antibiotics, anticancer drugs, heavy

metals, herbicides, pesticides, excess amount of uric acid and long term use and high

doses of analgesics may also cause nephrotoxicity these analgesics usually include

aspirin and ibuprofen.

Likewise, non-steroidal anti-inflammatory drugs or NSAID are the commonly used

over the counter drugs. They are pain relievers, help in reducing inflammation and

lower fever. They also prevent blood from clotting.

Ibuprofen is selected for present experimental studies. It is a derivative of propionic

acid its chemical name is Isobutylphenylpropionic acid, the structure containing a

benzene ring conjugated to a propionic acid. It was first derived during 1950-1960s at

the research laboratories of Boots group and discovered by the scientist Andrew RM

Dunlop with his co-researchers Stewart Adams, John Nicholson, Jeff Wilson and

Colin Burrows (Robertson, 2014).

Figure 1.3. showing structure of Ibuprofen

17

Mechanism of Ibuprofen

Ibuprofen is said to be an inhibitor of prostaglandin synthesis. The exact mechanism

of action is still unknown. Ibuprofen is an inhibitor of an enzyme (cyclooxygenase).

This enzyme converts arachidonic acid to prostaglandins. Prostaglandins are the

initiator of inflammation, fever and pain. There are two types of cyclooxygenase, one

is COX-1 which protects the lining of the stomach from digestive chemicals and also

maintains kidney function, whereas, COX-2 released when joints are injured or

inflamed (Robertson, 2014).

Figure 1.4. Showing mechanism of inhibition by NSAID

(Adapted from Balasubramaniam, 2001)

Similarly, antioxidants are the chemicals which prevent oxidative damage caused by

free radicals. Antioxidants are also strong protector against peroxidative damage

18

through their metal ion chelating and radical scavenging activities. A considerable

attention has been paid to the antioxidant activity of C. procera in respect to phenolic

constituents such as, Patel et al. (2014) studied the comparative antioxidant activity by

DPPH (1,1-Diphenyl-2-picryl hydrazyl) of C. procera and C. gigentia by using their

methanolic extract and it was reported that C. procera possesses high antioxidant

properties due to more phenols and flavonoids as compared to C. gigantea. Similarly,

Srividya et al. (2013) also proved antioxidant activity of ethanolic fruit extract of C.

procera by DPPH method.

Pooja et al. (2014) found out the antioxidant potential of ethyl acetate and acetone

fractions of C. procera by utilizing two different assays namely, Ferric reducing

antioxidant power (FRAP) and 2,2,Azino-bis-(3-ethyl) benzo thiazoline-6-sulfonic

acid (ABTS).

Lipid peroxidation is a process of oxidative degradation of lipids, in which a free

radical like hydroxyl group (OH) extract electrons from the unsaturated lipids present

in cell membranes. This result in the formation of a water molecule and lipid/fatty acid

radical. This radical again reacts with oxygen to form lipid peroxyl radical. Lipid

radical and lipid peroxyl radical both are unstable species, therefore, lipid radical

reacts with oxygen and convert into lipid peroxyl radical and lipid peroxyl radical

again react with other unsaturated lipid, this cycle continues and new lipid radical

reacts with same way and finally lipid peroxide is formed, that may cause cellular

damage. Cholesterol, Glycolipids, phospholipids, are also well-known targets of lipid

damaging and potentially cause fatal peroxidative change. Lipids also can be oxidized

19

by enzymes like cyclooxygenases, lipoxygenases, and cytochrome P450 (Ayala et al.,

2014).

There are two types of lipids found in cell polar and apolar. Triglycerides are the type

of (apolar) lipids, stored in various cells, but chiefly found in adipose (fat) tissue and

are usually the main form of energy storage in amphibians (Frayn 1998; Fruhbeck et

al., 2001). Polar lipids are structural components of cell membranes, where they take

part as a permeability barrier of cells and sub-cellular organelles in the form of a lipid

bi-layer, the main lipid of bi-layer is glycerol (Ayala et al., 2014).

Figure 1.5. Graphical representation of Lipid damage cycle

(Adapted from Clark, 2008)

20

The consequence of lipid peroxidation is the formation of reactive aldehyde

malonyldialdehyde (MDA) and 4 –hydroxy-nonenal (4-HNE). 4-HNE is a major

marker of lipid peroxidation.

Reducing power is another property of antioxidants, with this power an antioxidant

may reduce ferric Fe3+

ion into ferrous Fe2+

(Pohanka et al., 2009). So in this way

antioxidant may be beneficial for the living body.

Figure 1.6. Illustrates antioxidant defense - enzymes. Important intracellular enzymes

develop antioxidant defense; superoxide dismutase (SOD), catalase, and the GSH

peroxidase/GSSG reductase system. SOD catalyzes the dismutation of superoxide,

catalase the conversion of hydrogen peroxide to H2O and O2, while GSH peroxidase

transfers electrons from GSH to reduce peroxides to water. The oxidized glutathione

produced (GSSG) is re-reduced back to GSH by glutathione reductase utilizing

NADPH produced by the HMP shunt.acting as an enzyme cofactor (Adapted from

Proctor and Reynolds, 1984)

21

A considerable attention has been paid to the antioxidant, lipid peroxidation, and

reducing power of C. procera such as Roy et al. (2005) performed in vivo

experiments for determination of lipid peroxidation and antioxidant ability of dried

latex of C. procera which showed potential by increasing the levels of SOD,

catalase, and GSH. Similarly, Setty et al. (2007) evaluated lipid peroxidation and

antioxidant ability of C. procera ethanol flower extract which exhibited a marked

increase in tissue GSH level. Qureshi et al. (2007) determined the strong antioxidant

potential of flowers ethanol extract of C. procera. Yesmin et al. (2008) performed

DPPH method to determine the antioxidant activity of methanol leaves extract of C.

procera which shows strong antioxidant activity. Chavda et al. (2010) evaluated

hexane, ethyl acetate and chloroform root extract of C. procera, the fractions showed

significant lipid peroxidation inhibition activity and exhibited significant potential to

normalized the levels of tissue SOD, Catalase and GSH. Parihar et al. (2011)

elucidated a positive change in lipid peroxidation by increasing GSH contents in tissue

after administration of ethanol root extract of C. procera. Bouratoua et al. (2013)

determined a moderate antioxidant activity of butanol and ethyl acetate fractions of

aerial parts of C. procera. Srividya et al. (2013) also proved antioxidant activity of

ethanol fruit extracts of C. procera by DPPH method. Ahmed et al. (2014) determined

in vitro antioxidant activity of methanol extract of C. procera latex, which exhibited

positive activity to scavenge free radicals. Pooja et al. (2014) investigated the

antioxidant potential of ethyl acetate and acetone fractions of flowers of Alastonia

scholaris, Cassia auriculata, Catharanthus roseas and Calotropis procera. Amongst

all flowers, C. procera showed lower antioxidant activity.

22

While, the antioxidant capacity of extract may also be determined by reduction of the

ferricyanide Fe3+

complex in the ferrous Fe2+

form in the presence of plant extract

(Oyaizu, 1986; Kumar et al., 2013).

Enzymes are biological molecules that speed up the biological chemical reactions

(Brayer et al., 1995). The ability of an enzyme to catalyze a specific reaction could be

measured in terms of enzyme activity. There are various enzymes like, Glucoamylase,

also known as glucan 1,4-alpha-glucosidase, (EC 3.2. 1 .3). It is a type of digestive

enzyme which cleaves one glucose unit from a non reducing end of starch (amylose

and amylopectin). Most of the glucoamylases are also able to hydrolyze the 1,6-a

linkages in branch points of starch molecules.

Alpha amylase is a digestive enzyme. The formal title of alpha amylase is 1,4 α-D-

glucanohydrolase; EC 3.2.1.1. The enzyme alpha amylase aids in the hydrolysis of α–

1,4 glycosidic bond in the conversion of starch to maltose (Brayer et al., 1995). In

humans, it is found in both saliva and pancrease. Amylases are also used in various

industries like paper, food and textile industries (Windish et al., 1965; Gupta et al.,

2003).

The enzyme urease (EC 3.5.1.5) catalyzes the breakdown of urea into carbon

dioxide and ammonia. The reaction occurs as follows (Zimmer, 2000).

(NH2)2CO + H2O → CO2 + 2NH3

In view of the previous studies the present study was carried out to find out the in vitro

antioxidant potential of Calotropis procera which was further confirmed by in vivo

effects in rats against Ibuprofen induced nephrotoxicity.

https://en.wikipedia.org/wiki/Enzyme_Commission_number

http://enzyme.expasy.org/EC/3.5.1.5

https://en.wikipedia.org/wiki/Catalysis

https://en.wikipedia.org/wiki/Urea

https://en.wikipedia.org/wiki/Carbon_dioxide


https://en.wikipedia.org/wiki/Ammonia


https://en.wikipedia.org/wiki/Water



23

In vitro lipid peroxidation, reducing power and DPPH free radical scavenging activity

was also determined to ensure the degree of protective efficacy of naturally growing

Calotropis procera as a therapeutic agent.

24

2. GENERAL MATERIAL AND METHODS

2.1. Collection of Plant Material

Healthy and fresh flower and leaf of Calotropis procera were collected from different

population occurring in the Karachi. Collection were made in the year 2013-2014.

Sample specimen were deposited to the Herbarium, University of Karachi. General

herbarium = 86455.

2.2. Extraction methodology

After collection flower and leaf materials (c. 8-10 kg) were properly washed and air

dried for about 30 days. Then their powders were prepared by using grinder. The dried

powder matrial was soacked in 80% ethanol and left for one week. Then extract was

filtered with the help of filter paper. After filtration these extracts were concentrated

by rotary evaporator and kept for further use.

2.3. Fractioning of extract

Fractions of extract were prepared with the help of separating funnel. Various solvents

viz. butanol, hexane, ethyl acetate were used for fractioning. These fractions were

25

further concentrated. The obtained extracts were dried till converted into solid form.

This form can be used for further analyses.

26

Sorting of Flowers and

Leaves

Shade drying of

Flowers and Leaves

Grinding of

Flowers and Leaves

Collection of Plant

Calotropis procera

Extraction of Flowers and Leaves with

hexane, ethyl acetate, ethanol & butanol

Collection of

fractions from

each solvent

Storage of fractions

In vitro and In vivo experiments

Soaking of Flowers and Leaves

with Solvent (ethanol)

After 10 days soaked material was

filtered in a new bottle in form of

crude extract.

Crude Extract was concentrated

with the help of rotary evaporator

Figure 2.1. Flow Chart For Preparation of Extract From Calotropis procera

27

Ethanolic Crude Extract was concentrated with the help

of rotary evaporator

Concentrated Ethanolic Extract was mixed

with Hexane Solvent in Separating Funnel.

Let the mixture for few minutes to be separated

in two parts. A separation layer was observed.

Lower portion of the mixture contains

Aqueous Solution and Upper portion of the

mixture was taken as Hexane soluble part.

Hexane soluble Fraction was

collected Aqueous mixture was again treated with hexane

solvent until a clear upper part was obtained.

Aqueous portion was mixed with Ethylacetate

in separating funnel

Lower portion of the mixture contains

Aqueous Solution and Upper portion of the

mixture was taken as Ethylacetate soluble

part.

Ethylacetate soluble Fraction

was collected

Hexane soluble Fraction was

concentrated with the help of

rotary evaporator

Aqueous portion was mixed with Butanol in

separating funnel

Aqueous mixture was again treated with

Ethylacetate solvent until a clear upper part

was obtained.

Ethyacetate soluble Fraction

was concentrated with the

help of rotary evaporator

Lower portion of the mixture contains Aqueous

Solution and Upper portion of the mixture

was taken as Butanol soluble part.

Butanol soluble Fraction was

collected

Butanol soluble Fraction was

concentrated with the help of

rotary evaporator

Aqueous mixture was again treated with

Butanol until a clear upper part was obtained.

Figure 2.2. Method For Preparation of Fractions In Different Solvents

28

2.4. GENERAL CHEMICALS AND MATERIALS

Formalin, ethanol, ethyleacetate, methanol, hexane, Butanol, acetonitrile, pyridine,

sulphuric acid, eosin, EDTA, BHT, Na-tungstate from Fluka AG, phosphoric acid,

oxidized glutathione Amresco, disodium hydrogen phosphate from Merck, diacetyl

monoxime from Riedel de Haen, sodium hydroxide, potassium chloride, -NADPH,

formaldehyde, thiobarbituric acid, 2,4 dinitrophenyl hydrazine from Fisher Scientific,

disodium carbonate, nitro blue, paraffin, hydrogen peroxide, tetrazolium,

hydroxylamine hydrochloride, triton X-100, hematoxylin, 1,14,4 diethoxypropane,

sodium dihydrogen phosphate, picric acid and acetic acid.

PHYTOCHEMISTRY

2.5. Estimation of total protein (Bradford, 1976)

REAGENTS:

2.5.1. Coomassie Reagent

Coomassie stain (100gm) was mixed in 50ml of methanol and filtered. The solution

was added into 100ml of 85% phosphoric acid and volume was made up to 200ml.

The reagent was prepared by adding 1ml of dye stock with 4ml of water.

2.5.2. Tris HCl buffer (pH = 6.8)

Solution of 100mM Tris base was prepared and pH was maintained by the addition

of 0.2M HCl.

29

2.5.3 . Extraction

0.5 g fresh leaf and flowers were taken and macerated in 5ml of Tris HCl buffer and

centrifuged for 20 minutes at 2500rpm. Supernatant was collected in separate test

tubes for estimation.

2.5.4. Procedure for estimation of protein

0.04ml of leaf and flower extracts was added in test tubes. Then 2ml of assay reagent

was added in each tube. Test tubes were kept for incubation at room temperature for

30 minutes and finally the optical density was determined at 595nm.

2.5.5. Calibration of standard curve

Bovine serum albumin (BSA) was used to prepare standard curve.

2.5.6. Stock solution

Stock was processed by adding 0.1gram of BSA in 10ml of Tris HCl buffer.

2.5.7. Working standard solution and colour development

Dilutions of BSA stock solution were prepared as 200µg/ml, 400µg/ml, 600µg/ml, 800µg/ml

and 1000µg/ml. 0.04ml was taken from each dilution in separate test tubes. 2.0 ml of

reagent was also poured. All test tubes were kept at room temperature for 30 minutes.

Optical density was noted at 595nm.

30

Figure 2.3. Standard Curve for Total Proteins

31

2.6. Estimation of Carbohydrates (Yemm and Willis, 1954)

Reagents:

2.6.1. Anthrone Reagent

Anthrone (0.4ml) was added in 200 ml of concentrated sulpfuric acid with continuous

shaking. The acid solution was then cooled and 15 ml of 95% ethanol and 60ml of

distilled water were taken in a dark coloured flask which was placed in an ice bar.

Then acid solution was transferred drop by drop in a dark coloured flask with constant

shaking. Fresh Anthrone reagent was prepared each time.

2.6.2. Method for 100 mM Tris HCL

Solution A: 12.1gm of Tris base was added in 100ml of distilled water and the

volume was made up to 1000 ml.

Solution B: 1.21 ml of concentrated HCl was added in 100 ml distilled water and the

volume was made up to 1000 ml.

500ml of solution A added in to 200 ml of solution B. The pH of solution was

maintained up to 6.8. When pH was basic, solution B was added, and for acidic pH

solution A was added.

2.6.3. Extraction

1 gm fresh leaves and flowers were crushed in a mortar with 5 ml Tris HCl. Crushed

material was centrifuged at 2500 rpm for 20 minutes. The supernatant was separated

in test tubes.

32

2.6.4. Procedure for estimation of carbohydrates

To 1ml extract, 4ml distilled water was added in 10 ml of anthrone reagent and

shaked well. Content was kept for 16 minutes in a water bath. Test tubes were left for

cooling. Finally, optical density was observed at 660 nm.

2.6.5. Calibration of standard curve

To prepare standard curve sucrose was used.

Stock Solution

1000μgm/ml prepared by adding 0.1 gm of sucrose in 100 ml distilled water.

2.6.6. Working standard solution and colour development

An aliquot of 200 μgm/ml, 400 μgm/ml, 600 μgm/ml, 800 μgm/ml and 1000 μgm/ml

dilution of the sucrose stock solution was made 1 ml of each dilution in separate test

tubes. Distilled water for reagent blank was taken. 4.0 ml distilled water was added.

10 ml of anthrone reagent was poured drop by drop. Test tubes were kept in boiling

water bath for 16 minutes. Test tubes were then cooled and optical density was noted

at 660nm.

33

Figure 2.4. Standard Curve for Total Carbohydrates

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 200 400 600 800 1000 1200

Ab

sorb

an

ce a

t 6

60

nm

Concentration of Sucrose

34

2.7. Estimation of total reducing sugars (Miller, 1959)

Reagents

2.7.1. DNS Reagent 3, 5 di-nitro salicylic acid (100mg) was mixed in 20 ml of 2N

sodium hydroxide and dissolved in 50ml distilled water. 30 g of Sodium Potassium

tartrate was also added, then volume was made up to 100 ml and stored at 4oC in

refrigerator.

2.7.2. Preparation of 100 mM tris HCl

Solution A: Tris base (12.1gm) was mixed in 100ml of water and volume was made

up to 1000 ml.

Solution B: Concentrated HCl (1.21ml) was added in 100 ml water and made the

volume up to 1000 ml. Took 500 ml of solution A and added in to 200 ml of solution

B. PH of solution was maintained at 6.8. If the pH was basic then solution B was

added, if it was more acidic then solution A was added.

2.7.3. Extraction

1 gm fresh leaf and flower samples were crushed separately in a mortar with 5 ml tris

HCl. Crushed material were centrifuged at 4000 rpm for 15 minutes. Supernatent was

then collected in test tubes.

2.7.4. Procedure for estimation of reducing sugars

2 ml DNS was added in 1 ml extract. Then tubes were kept in boiling water bath for 2 to 3

minutes then cooled. Optical density was noted at 540 nm.

35

2.7.5. Preparation of standard curve

For the preparation of standard curve glucose was used.

2.7.6. Stock standard

Glucose (2.5 mg) was added in distilled water and volume was made up to 50 ml.

2.7.7. Working and colour standard

Different concentrations 0.2,0.4,0.6,0.8 and 1 ml of stock standard was taken into test

tubes. Each working standard was diluted up to 1ml with distilled water except last

one so that each test tube contains 50, 100, 150, 200 and 250 μgm/ml. However, for

the reagent blank only 1 ml distilled water was poured in test tube, then 1 ml of DNS

reagent was added, boiled it for 2-3 minutes. Then cooled and the optical density was

noted at 540 nm.

36

Figure 2.5. Standard Curve for Reducing Sugar

0

0.5

1

1.5

2

2.5

0 50 100 150 200 250 300

Ab

sorb

an

ce

Concentration of DNS

37

2.8. Estimation of total non-reducing sugars

Non reducing sugar was calculated by the following formula:

Total Non Reducing Sugars=Total Sugars-Total Reducing Sugars

2.9. Estimation of total amino acids (Spices, 1957).

2.9.1. Extraction

Leaf and flower were crushed in distilled water and these water extracts were

centrifuged twice at 3000rpm for 5 minutes.

2.9.2. Reagents and preparation of solution

Citrate buffer: For preparing citrate buffer citric acid monohydrate (21gm) mixed

with 200ml 1N sodium hydroxide and volume was made up to 50ml by adding water.

Dilution solvent: For making dilution solvent water and n-propanol were equally

added.

Acid ninhydrine mixture:

Ninhydrin 1.20gm was mixed in 200ml of methyl cellulose with pH 5. Then Tin

chloride 2.08gm was mixed in 500ml citrate buffer. Then a mixture was made by

adding ninhydrin and tin chloride and kept for further use.

38

2.9.3. Development of coloured complex

Took 1ml of flower and leaf extracts in test tubes and 1ml of ninhydrine mixture was

added to these test tubes, and covered with aluminum foil then these tubes were placed

in a water bath for 20 minutes at 50- 70oC. Diluted solvent (0.5ml) was mixed in test

tubes and kept these tubes at room temperature for 15 minutes. After the appearance of

purple coloration absorbance was noted at 570nm by using Schimadzu

spectrophotometer.

2.9.4. Preparation of standard calibration curve

Amino acid standards were prepared by adding 250µg amino acid in 1ml absolute

ethanol.

Figure 2.6. Standard Curve for Amino Acids

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 10 20 30 40 50 60 70 80

Ab

sorb

ance

nm

Lucine ug/ml

39

2.10. Estimation of amino acids by paper chromatography

2.10.1. Method of extraction

Leaf and flower material (1.0 gm) of Calotropis procera was kept in a flask containing 10ml

ethanol (80%). Then these extracts were boiled for 10 minutes and left for 24 hours. The

samples were centrifuged at 4000 rpm. About 1 ml of supernatant was collected and volume

was made up to 2 ml by adding 1 ml of 50 % ethanol.

2.10.2. Methodology for amino acids estimation

Extracts of leaf and flower were further applied on a chromatogram. These

chromatograms were allowed to run in ascending tank by using BAW as a solvent.

Then these chromatograms were kept for air drying. After drying chromatograms were

sprayed with 0.2% ninhydrin in acetone. Then these chromatograms were kept in a

heater at 80oC for about 10 minutes. Appearance of coloured spots indicated the

presence of amino acids. These chromatograms were observed under UV lamp for the

detection of amino acids (Harborne, 1984). Rf values were calculated with the help of

following formula:

Rf= Distance travelled by solute front/ Distance travelled by solvent front

The calculated Rf values were compared with known values for the identification of

amino acids.

40

2.11. Phenolic compounds

Phenolic contents were detected following the method of Swain and Hillis (1959).

2.11.1. Details of reagents

Reagent-I: 1N HCl (Conc. HCl 82.8ml (37%) was mixed with deionized water,

Allowed to cool and the volume was made up to 10ml with water.

Reagent-II: Pure ethanol

Reagent-III: Folin-ciocalteu reagent was prepared by mixing folin ciocalteu solution

with distilled water in the ratio of 1:9 in respective manner.

Reagent IV: Saturated NaHCO3 solution.

2.11.2. Extraction

1.0gm leaf and flower of C. procera were soaked in hot HCl (1N) for softening the

tissues. Further the tissues were crushed by adding HCl up to 10 ml. Material was

boiled for about 30 minutes and centrifuged for 5 minutes at 1000rpm. The

supernatant was extracted and dried by heating.

2.11.3. Methodology to estimate total phenolic contents

Dried extract was dissolved in 0.5ml ethanol and from this 0.1ml of mixture was

transferred separately in a test tube. Then 5ml distilled water and 0.2ml of folin

reagent were added to this tube. The tube was shaked well and then 1 ml sodium

bicarbonate was also added. Tube was again shaked and allowed to incubate for 30

41

minutes at 26oC. Absorbance was noted against reagent blank at 660nm. With the help

of standard curve total phenolic contents (µg/ml) were calculated.

2.11.4. Preparation of standard curve

To prepare the standard curve gallic acid was used.

2.11.5. Stock solution

For the preparation of stock solution 2 mg of gallic acid was dissolved in 1ml of pure

methanol.

2.11.6. Working standard

For working standard 1.0ml of stock solution was added into 10ml of pure methanol to

get 200µg/ml gallic acid.

2.11.7. Colour development

A series of 0.1–0.5ml of working standard were taken in test tubes each having 20, 40,

60, 80, 100µg of gallic acid in respective manner.Volume of tubes was maintained up

to 1.0ml by adding pure methanol. In these tubes 5 ml of reagent III was added and

shaked well. These tubes were left for 3 minutes. 1 ml of reagent IV was also added in

tubes. The tubes were allowed to incubate for 30 minutes at 26oC. The optical density

was observed at 660nm. The curve was plotted between micrograms of gallic acid and

optical density.

42

Figure 2.7. Standard Curve of Phenol

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 10 20 30 40 50 60

Ab

sorb

an

ce

nm

Gallic acid (ug/ml)

43

2.12. Detection of phenolic compounds by chromatography

2.12.1. Preparation of ethanolic extract

1 gm of C. procera leaves and flowers were extracted separately in 70% ethanol

overnight at room temperature. Extracts were filtered and concentrated.

2.12.2. Preparation of extract by hydrolysis method (Harborne, 1984)

Dried leaves and flowers (1 gm each) were dipped separately in 2M HCl and heated

for 30-40 minutes at 100oC. Then cooled extract was filtered and extracted with small

amount of ethyl acetate. The ethyl acetate layer was concentrated to dryness then it

was dissolved in a few drops of ethanol. The aqueous extract was further heated to

remove the last traces of ethyl acetate and re-extracted with small volume of amyl

alcohol. The amyl alcohol extract was concentrated to dryness then it was dissolved in

1% methanol HCl.

2.12.3. Chromatography

Whatman no. 1 paper was used to load the extracts. Quercetin was used as a marker,

and chromatographed 2-dimentionally using two different solvents i.e. (acetic acid:n-

butanol:water=1:4:5) and 15% acetic acid. Phenolic compounds were identified by

comparing the Rf values and colour in ultraviolet light before and after ammonia

fumigation.

44

IN-VIVO ANTIOXIDANT STUDIES

2.13. Method for protein free filtrate preparation

1.0 ml plasma was added in a test tube containing 3 ml deionized water and mixed

well. Then sodium tungstate 10% (0.3ml) and 2/3N H2SO4 (0.3ml) were also mixed in

test tube. The constituents were kept at room temperature for 5 minutes and samples

were centrifuged at 3000rpm for 5 minutes. The resulting supernatant was collected as

protein free filtrate.

2.13.1. Plasma urea estimation

Plasma urea was estimated by the standard procedure of Butler et al. (1981).

Reagent-I: (DAM) Diacetyl monoxime solution (2% glacial acetic acid was added in

2% diacetyl monoxime solution).

Reagent-II: Mixture of phosphoric and sulphuric acids (50ml Conc. H2SO4 + 150ml

85% phosphoric acid) and 140ml deionized water was also added to this mixture

Reagent-III: In blank test tube 2ml deionized water, 0.4ml DAM, 1.6ml of

Phosphoric acid-sulphuric acid mixture was also added.

Standard Curve: The standard curve was plotted by using a series of standards (0.01-

0.05mg) from calibration solution which was 0.025mg/ml (Fig. 2.8).

45

2.13.2. Methodology for plasma urea estimation

1 ml of protein free filtrate was poured in test tubes and 1ml of deionized water was

added into tubes. Then 0.4ml of DAM solution and 1.6 ml mixture of phosphoric and

sulfuric acid was also added in tubes. After shaking tubes were kept for half an hour in

boiling water bath. Stand for cooling, and optical density was noted at 480nm against

reagent blank. The contents of urea in plasma were estimated in mg/dl.

2.13.3. Plasma creatinine estimation

Plasma creatinine was estimated by following the procedure of Spierto et al. (1979).

Blank: 0.5ml of sodium hydroxide mixed with 1.5 ml of picric acid in 3ml of

deionized water.

Standard Curve: The standard curve was prepared by using a series of standard

values (0.005-0.035mg) from main standard solutions (0.015mg/ml) (Fig. 2.9).

2.13.3.1. Methodology for plasma creatinine

Took 3ml protein free filtrate in test tubes and 0.5 ml (4N) sodium hydroxide was

added. Then 1.5 ml (0.04M) picric acid was also mixed and the tubes were kept at

room temperature for 15 minutes. The optical density was noted at 530nm against

reagent blank. The quantity of creatinine in plasma was estimated in mg/dl.

46

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 0.01 0.02 0.03 0.04 0.05 0.06

Ab

sorb

an

ce

Concentration (mg)

Series1

47

48

2.13.4. Kidney homogenate

Kidney homogenate was prepared at 4oC by using Homogenizer model (Ultra

Taurax).

Tissues were divided into pieces. The homogenate 1:10w/v was made by adding

0.3mM EDTA and buffer of potassium chloride 100mM, at pH=7.0 was also added to

homogenate. Then centrifuged at 4oC for 60 minutes at 600nm and the supernatant

was collected for various analyses.

2.13.5. Malonyldialdehyde estimation

Standard Curve: Values of malonyldialdehyde were estimated by comparing with

the standard absorption values in nM/g of tissue. Whereas, the standard curve was

prepared in a range between 0.0002-0.00176mM from main mixture (0.02mM/L (Fig.

2.10).

Malonyldialdehyde was estimated by following the procedure of Okhawa et al. (1979).

The mixture for reaction contained 1.5ml of 20% acetic acid with 0.2ml of 8.1%

sodium dodecyl sulphate and pH was maintained at 3.5 by using NaOH. Then 1.5ml of

thiobarbituric acid diluted in water (0.8%) was also mixed to the homogenate. The

volume was made up to 4.0 ml by water and heated for 60 minutes at 95oC. The

49

mixture was stand for cooling. Then 5.0ml mixture of pyridine and n-butanol

(1:15v/v) with 1 ml water was added to homogenate. The mixture was centrifuged and

supernatant was collected then optical density was observed at 532nm and compared

with malonyldialdehyde standards.

50

2.13.6. 4-hydroxyl-2-nonenal estimation

The 4 hydroxyl-2-nonenal contents were calculated by following the procedure of

Kinter et al. (1996).

Standard curve: The calibration curve was obtained by using a series of standard

solutions (concentration range 0.0189-0.11378 mM) from main calibration solution

(0.006 mM/L) (Fig. 2.11.).

Methodology

In a clean glass tube, 2 ml of filtrate was taken and added 1 ml of 2,4 Dinitrophenyl

hydrazine and kept for 1 hour at room temperature. The Sample was then extracted

with hexane three times and the extract was evaporated at 40oC. The sample was then

cooled and reconstituted with 2 ml of methanol, the absorbance was then measured at

350 nm against methanol blank on Schimadzu-spectrophotometer UV 120- 01. The

concentration values were calculated from absorption measurement as standard

absorption in nM/g tissue.

51

52

2.13.7. Catalase estimation

For the determination of catalase level the methodology of Sinha et al. (1972) was

adopted.

Dichromate acetic acid Reagent: 150 ml of glacial acetic acid was added with 50ml

5% dichromic acid.

Phosphate buffer: 0.01M, pH=7.0

Hydrogen peroxide: 0.2M

Standard curve: The concentration values were calculated by measuring the optical

density of standards in mM/gram of tissue. The standard curve was obtained by using

a series of standard solutions (concentration range from 0.05-0.3mM) from main

calibrating solution (0.2mM/ml) (Fig. 2.12).

Methodology

1ml of hydrogen peroxide and 1.96ml of phosphate buffer was poured in a test tube

then 0.04 ml of 10% homogenate was also added. Then 2ml of dichromate acetic acid

reagent was mixed with 1 ml of test tube contents. The mixture was boiled for 10

minutes, stand for cooling and the optical density was noted at 570nm with the help of

Schimadzu spectrophotometer (UV 120-01).

53

54

2.13.8. Superoxide dismutase estimation

For the estimation of superoxide dismutase level the methodology of Kono (1978)

was adopted.

Reagents:

Reagent I: 0.1mM EDTA with 50mM sodium carbonate at pH=10.0.

Reagent II: 90µM nitro blue tetrazolium dye.

Reagent III: 0.6% Triton X-100 in reagent I.

Reagent IV: 20mM Hydroxylamine hydrochloride, pH=6.0.

Reference test tube: 0.1 ml of supernatant was added to the test and reference

cuvette.

Methodology

1.3ml of reagent I, 0.5ml of reagent II, 0.1ml of reagent III and 0.1 ml of reagent IV

were mixed with homogenate. The rate of nitro blue tetrazolium reduction was noted

for one minute at 560nm with the help of Schimadzu spectrophotometer (UV 120-01).

The activity was calculated by using the percent inhibition in gram of tissue and

expressed in U/gram of tissue.

Inhibition (%) = Abs of test – Abs of reference/ Abs of test – Abs of blank X 100

U/ml: % inhibition / gram of tissue

55

2.13.9. Glutathione estimation

For the determination of glutathione level the methodology of Carlberg and

Mannervik, (1985) was adopted.

Methodology

In a test tube 0.35 ml of 0.8 mM βNADPH was added then 0.3 ml of 10% BSA was

taken, after that 1.5 ml of 50 mM potassium phosphate buffer (pH = 7.6) was added

then 0.1 ml of 30 mM oxidized glutathione and 0.1 ml of homogenate was also

poured to test tube. After shaking, the absorbance was noted at 340nm for 5 minutes at

25oC temperature on Kinetic spectrophotometer PRIM 500 (Germany with automatic

aspiration and thermostat). The activity was calculated by using the molar coefficient

for NADPH of 6.22 µmol-1 x cm-1 and expressed in the Unit/g of tissue.

Activity of GSH

Activity U/L = µM / L = (340/min / 0.00622 x (Total Volume/Sample Volume in µl)

56

IN-VITRO ANTIOXIDANT STUDIES

2.14. Lipid peroxidation inhibition method (Halliwell and Gutteridge,

1999)

Effect of C. procera on inhibition of lipid peroxidation activity was studied in vitro,

according to the guidelines of Halliwell and Gutteridge (1999).

2.14.1. Preparation of tissue homogenate:

Fresh tissue of a normal albino rat was sliced into small pieces and transferred in a test

tube. Then phosphate buffer saline pH 7.4 was added. The homogenate was

centrifuged at 3000 rpm for 15 minutes, clear upper layer was collected for anti lipid

peroxidation assay.

2.14.2. Procedure for lipid peroxidation inhibition

C. procera leaf and flower extracts were taken with (1, 2, 4, 6, 8, 10 mg/ml) concentrations

from stock solution of (10mg/ml) i.e. 0.01, 0.02, 0.04, 0.06, 0.08 and 0.1 ml was added in test

tubes containing distilled water in 0.09, 0.08, 0.06, 0.04, 0.02, and 0.0 ml respectively. Further

test tubes were standing till dryness. To these dried test tubes 1 ml of 0.15M Potassium

chloride solution was added and then tissue homogenate (0.5ml) was added in each tube. To

start the lipid peroxidation process (0.1ml) of 0.2mM ferric chloride (FeCl2) was added. Then

all test tubes were incubated for 30 minutes at 37oC. The reaction was terminated by addition

of (2 ml) of Hydrochloric acid (0.125N) having 1.68 gms of 15% Tricarboxylic acid with

41.60mg Thiobarbituric acid (0.38%) and 0.5% BHT in ethanol was also added. Again

mixture was incubated at 80oC for 1 hour.

57

After cooling samples were centrifuged, after the appearance of pink layer, absorbance

was measured at 532nm. For comparison purpose BHA was used as a control. A

similar test was performed without the presence of the extract and standard to

determine the amount of lipid served as control. All tests were carried out in triplicate

and the results were expressed as mean ± SD. Lipid peroxidation percent of inhibition

was calculated by following formula:

LPOI (%) = [(A1-A

2)/A

s] x100

Where A1 is the absorbance of control and A

2 is the absorbance of the standard

/sample

2.14.3. 1,1-diphenyl -2-picrylhydrazyl radical scavenging method

The in vitro antioxidant power of C. procera extracts was determined by the method

of Kumar et al. (2013). Six different concentrations of test extracts were prepared from

(1-10mg/ml). 3.0 ml of 0.1mM DPPH solution was mixed to each test tube. The

contents were allowed to stand at room temperature for thirty minutes in dark. The

absorbance was determined at 517nm. Ascorbic acid was used as standard. The

percent inhibition was calculated by using following formula:

% I = [(AC-AS)/AC] x 100

58

Where I = inhibition, AC and AS = Absorbance values of the control and the sample

respectively. Each sample was used in triplicate and results were expressed as mean ±

SD.

2.14.4. Method for determining Reducing power

The reducing ability of C. procera flower and leaf extracts were evaluated by the

method of Kumar & others (2013), Oyaizu (1986) and Mishra & coworkers (2013).

1ml of each sample of four different solvents (water, ethanol, hexane and ethyl

acetate) extracts was taken in test tubes, each in different concentrations (1, 2, 4, 6, 8

and10mg/ml). To each test tube 2.5ml of 1% potassium hexacyanoferrate and 2.5ml of

phosphate buffer (0.2M, pH 6.6) were mixed. All tubes were incubated for 20 minutes

at 50OC temperature in a water bath. The reaction was terminated by mixing 2.5ml of

10% trichloroacetic acid and then centrifuged at 4000rpm for 10min. 1ml of the upper

layer was mixed with 0.5ml of ferric chloride solution (0.1%, w/v) and 1ml of distilled

water and tubes were stand for two minutes at room temperature. The optical density

was noted at 700 nm. The butylated hydroxyanisole was used as standard for

comparison. Higher reducing ability was noted in terms of higher optical density

reading. All the tests were run in triplicate. Results are reported as mean ± SD.

59

ENZYME ACTIVITY

2.15.1. Estimation of glucoamylase

Glucoamylase was estimated by the method described by Ghani et al. (2013).

2.15.1.1. Glucoamylase activity assay (Ghani et al., 2013)

The activity of Glucoamylase was determined by mixing 0.2 ml of enzyme with 1.0

ml soluble starch (1.0%) prepared in 0.05 M citrate buffer at pH 5.5. The reaction tube

was incubated for 30 and 60 minutes at 50oC. After incubation, the reaction was

stopped by dipping the tubes in a boiling water bath for 5 minutes and cooled

afterwards. The glucose released after the assimilation of starch in the reaction

solution was calculated by (GOD-PAP) glucose oxidase method using glucose as

control, and the optical density was noted at 546 nm.

2.15.1.2. (GOD-PAP) method

Glucose is oxidizes to gluconic acid and hydrogen peroxide in the presence of glucose

oxidase. Hydrogen peroxide reacts with phenol and aminophenazone to form a pink

coloured compound in the presence of peroxidase. The intensity of the pink coloured

formed is reciprocal to the glucose level in sample.

Reagents

Reagent I Enzyme,

Reagent II Standard 100mg/dl

Method

Three test tubes were taken and marked as sample, standard and blank respectively.

0.1 ml of sample and standard were added in respective tubes. Then 1.0 ml of Reagent

60

I was added in all three test tubes. After mixing, left them for 10 minutes at 37oC. The

absorbance of sample and standard was measured at 546 nm against reagent blank.

Glucose conc. mg/dl = A (sample) x 100

A(standard)

2.15.1.3. Reducing sugar estimation (DNS Method) for alpha amylase

activity

3’5’-Dinitrosalisylic acid (DNS) method was used to estimate the reducing sugar

(Miller, 1959) and maltose was used as standard.

Reagents I 3’ 5’-Dinitrosalisylic acid (II) Sodium potasium tartarate (III) Sodium

Hydroxide (2.0 N).

Preparation of DNS reagent

In 50 ml deionized water 1 gm of DNS was added with constant stirring.

1 gram of DNS was added in 50.0 ml deionized water under constant stirring. Sodium

potassium tartarate (30gm) was poured and stirred well. 20ml of NaOH (2N) was also

added, stirred and deionized water was added up to 100ml.

Preparation of standard curve

Maltose (0.4 gm) was used as standard in 100.0 ml of deionized water.

Methodology

Five tubes, with 0.2, 0.4, 0.6, 0.8 and 1.0 ml working standard were prepared

respectively. Deionized water was added for making volume upto 1 ml. 1.0 ml of

deionized water was also added in blank tube. DNS (1.0 ml) was added in all tubes.

61

Test tubes were placed in a boiling water bath for 5.0 minutes. After boiling, deionized

water (9.0 ml) was added in all test tubes including blank and mixed well. Absorbance

of all the standard tubes was measured against blank at 546 nm.

Figure 2.13. Standard Curve for Reducing Sugar for α-amylase

0

0.5

1

1.5

2

2.5

0 50 100 150 200 250 300

Ab

sorb

an

ce

Concentration of DNS

62

2.15.1.4. Alpha amylase enzyme activity assay

1. Buffer: 50mM Tris –HCl buffer (pH 7.00)

2. Substrate: Starch (2.00gm/dl) in Tris-HCl buffer was used as substrate.

3. Enzyme: Partially purified alpha amylase (0.10 ml)

Method

Two test tubes were marked as Control (C), and two test tubes were taken as Test (T).

In test tube (T) 0.1 ml enzyme having flower and leaf extract was mixed with 0.9 ml

substrate while pure enzyme 0.1 ml was added in control tube and mixed with 0.9 ml

of substrate. 1 ml deionized water was used in blank tube. Test tubes were incubated

at 60oC for 5 minutes and 1.0 ml DNS was added in all tubes (Test, Control and

Blank) to stop the enzyme–substrate reaction. All tubes were placed in boiling water

bath for 5.0 minutes and allowed to cool. After cooling 9 ml deionized water was

added in each tube. The absorbance of test and control was observed at 546 nm

against blank.

Calculation

mg of reducing sugar/ml/minute=

OD of test – OD of control x Conc. of standard x 1 x

1

OD of standard volume of sample

time (min)

1 µmol of maltose = 0.00036 mg/ml = 1 Unit

Therefore,

63

U/ml/minute = mg reducing sugar/ml/minute

0.00036 mg/ml

2.15.1.5. Estimation of urease activity

Urease activity was determined by kit method (Modified Berthelot’s Method, 1859)

(Fawcett and Scott (1960).

Reagents

Reagent I (Enzyme): Urease 5000 U/L,

Reagent II (Colour reagent): Phosphate Buffer pH 7.0 = 60.00 mmol/L,

Sodium Nitroprusside = 2.50 mmol/L, Sodium Salicylate = 31.25 mmol/L, EDTA

0.74 mmol/L

Standard: Urea = 50mg/dl

Preparation of mono-reagent

One ml of Reagent-I was mixed with 20 ml of Reagent-II

Method

Three test tubes were taken and marked as Sample, Standard and Blank. 0.1 ml of

sample (extract, Standard (Urea) and blank (deionized water were added in respective

tubes. Then 1.0 ml of mono-reagent was added in each of the tubes. After mixing,

tubes were incubated for 5 minutes at 37oC. Then 1.0 ml of reagent-III was added to

each of the tubes. After mixing, the tubes were again incubated for 5 minutes at 37oC.

Absorbance was recorded at 580 nm against reagent blank.

Urea = A(sample) x concentration of standard

A(standard)

64

2.16. Statistical analysis

Data were analyzed statistically by using mean values ± SE at P<0.05 through One-

way ANOVA and Duncan’s multiple comparison test (DMCT) with the help of a

computer software, statistical package for social sciences (SPSS) for windows version

14.0.0 (SPSS, 2005).

65

PHYTOCHEMICAL STUDIES OF CALOTROPIS

PROCERA

3.1. Introduction

Biologically active compounds derived from plants are usually classified as primary

and secondary metabolites. Whereas, primary metabolites are the part of metabolic

pathways and secondary metabolites are waste products or byproducts of metabolic

pathways. Regarding to the medicinal uses of Calotropis procera secondary

metabolites namely, phenolic compounds tannins, terpenoids and saponins have

received an immense attention (Vaya et al., 1997; Sengul et al., 2009; Patel et al.,

2010).

Therefore, C. procera was previously investigated for its phytochemicals in relation

to medicinal importance. Present studies are carried out to investigate phytochemical

properties of Calotropis procera and to substantiate the previous findings.

3.2. Material and Methods

3.2.0. Extract Methodology

Detailed method is described in Chapter 2 (2.2).

3.2.1. Total proteins estimation

Total protein in extract was evaluated by the procedure of Bradford (1976) as

described in Chapter 2 (2.5).

66

3.2.2. Carbohydrates estimation

Total carbohydrates were calculated by the guidlines of Yemm and Willis (1957) as

described in Chapter 2 (2.6).

3.2.3. Total reducing sugars

Total reducing sugars were calculated by the technique of Miller (1959) as explained

in Chapter 2 (2.7).

3.2.4. Total non-reducing sugars

Total non reducing sugars were calculated by the formula as mentioned in Chapter 2

(2.8).

3.2.5. Total amino acids estimation

Total amino acids were evaluated by the technique of Spices (1957) as mentioned in

Chapter 2 (2.9).

3.2.6. Amino acid detection by paper chromatography

Amino acid detection was performed by the technique described in Chapter 2 (2.10).

3.2.7. Phenolic compounds estimation

Total phenolic compounds were evaluated by the technique of Swain and Hillis (1959)

as mentioned in Chapter 2 (2.11).

3.2.8. Phenolic compounds detection by chromatography

Detection of phenolic compounds was performed by the technique of Harborne (1984)

as mentioned in Chapter 2 (2.12).

67

3.3. Results

Table 3.1. Phytochemical Estimation of Calotropis procera

Constituents Leaf µg/ml Flower µg/ml

Reducing Sugar 145.0±5.0 90.0±5.0

Non-reducing sugar 335.0±73.65 247.33±13.65

Proteins 373.33±25.16 473.33±23.09

Carbohydrates 480.0±72.4 337.33±16.16

Amino Acids 110.33±9.073 84.0±4.0

Phenols 17.66±0.57 22.0±1.0

Table 3.2. Detected Amino Acids in Calotropis procera

AminoAcids Leaf Flower

Alanine - +

Arginine + -

Aspartic acid - +

Cysteine - +

Glutamic Acid + +

Lysine - +

Proline + -

Serine - +

Threonine + -

Tryptophane + -

Tyrosine + -

Unknown Rf=0.92 + +

Unknown Rf=1.0 + +

68

Table 3.3. Phenolic constituents of Calotropis procera

Phenolic Compounds

Class of Ethanolic Extract Acid Hydrolysis

Phenolic

compounds

Leaf Flower Leaf Flower

Apigenin Flavones - + - -

Luteolin Flavones - + - +

Tricin Flavones + + - -

Orientin Glycosyl

flavones - + + +

Iso-orientin Glycosyl

flavones - + + +

Vitexin Gylcosyl

flavones + + - -

Isovitexin Glycosyl

flavones + + - +

Azaleatin Flavonols - + - -

Gossypetin Flavonols + + - -

Kaempferol Flavonols + - - -

Myricetin Flavonols + + - -

Quercetin Flavonols - + - +

3-

Glucoronide(quercetin

glycoside) Isoquercitrin Flavonols - + - -

Dihydrokaempferol Flavanol + - - -

Dihydromyricetin Flavanol + - - -

Naringenin Flavanones + + - -

Dihydroquercetin Flavanones - + - -

Mangiferin Xanthone + - - -

Isovanillin Phenolic

aldehyde - + - -

Unknown Rf=10 - + + - -

Unknown Rf=15 - + + + -

Unknown Rf=20 - - + - -

69

3.4. Discussion

In C. procera a large number of chemical constituents like phenolic compounds,

including phenolic acids and flavonoids, carbohydrates, proteins and amino acids are

detected (Table 3.1, 3.2, 3.3). Amongst the primary metabolites, value of amino acids

is higher in leaf (i.e. 110.33 µg/ml) than flower (84.0 µg/ml) these quantitative values

could also be supported by the qualitative detection as leaves having greater amino

acids in relation to flowers (Table 3.1). Regarding to the amount of carbohydrates,

reducing and non-reducing sugars, leaf showing greater value as compared to flower.

While, greater value of protein is recorded in flowers (460.7µg/ml) in comparison with

leaf (Table 3.1).

Flower extract showed 21 µg/ml total phenols and leaf exhibited 17.5 µg/ml phenols.

Similarly, in the qualitative determination of phenolic constituents more compounds

are observed in flower as compared to leaf (Table 3.3).

Amongst the detected phenolic compounds, especially flavonols and flavones of the

class flavonoids have been received considerable attention to prove their medicinal

importance (Ikken & others, 1999; Gao & others, 2000; Patel & coworker, 2010;

Gholamshahi & coworkers, 2014; Pooja & others, 2014; Shetty & coworkers, 2015),

such as quercetin, luteolin and kaempferol are most commonly consumable and

natural flavonoids and reported to have antibacterial and antioxidant properties (Bentz,

2009; Calderon-Montana et al., 2011; Majewska et al., 2011). Another flavonol

Gossypetin also showed antibacterial/antiseptic potential ( Mounnisamy et al., 2002).

Similarly Mangiferin is a natural phenolic compound exhibited antimicrobial and

70

antioxidant activities (Stoilova et al., 2005). Tricin is another O-methylated flavones

known to minimize the risk of intestinal cancer (Lutskii et al., 1971). The

dihydromyricetin belongs to flavonol and used as anti-inflammatory agent (Satoshi,

2007; Shihui et al., 2015). Isoorientin is a flavones, showing hypoglycaemic activity

(Lim et al., 2007). Besides these above information it has been observed that the

extract of C. procera flower exhibited comparatively large amount of phenolic

compounds as compared to leaf extract. While, on the other hand flower also showed

high scavenging potential by DPPH and more effective for lipid peroxidation

inhibition. So there seems a good correlation between phenolic compounds,

antioxidant and antibacterial potential.

71

EFFECT OF FLOWERS AND LEAVES EXTRACTS OF

CALOTROPIS PROCERA AS ANTIMICROBIAL AGENT.

4. Introduction

Infectious diseases are usually controlled by commercial antimicrobial drugs that

largely produce toxicity in the human body which intimated the attention of workers to

trace out any other alternate source for the prevention of infectious disease with less or

zero toxicity. In the last few decades, several new natural anti-microbial compounds

were discovered for the control of severe infections. A discovery of new antibacterial

agent against multidrug resistant organisms is still in full swing due to the

development of continuous resistance developed by microbes. The multidrug resistant

organisms have received great clinical attention because of increasing reported cases

around the globe. Along with this, there is an increase in consumer demand for those

drugs, which are isolated or derived from natural sources. The Threat posed to general

public health by various multidrug resistant organisms and pathogens can be resolved

by the discovery of natural antibacterial compounds having effective broad spectrum

inhibition against pathogens prevalent in the local community.

Calotropis procera is reported to have therapeutic properties such as flowers show

anti-inflammatory activity (Basu and Chaudhry, 1991; Neenah and Ahmed, 2011) for

curing cholera , wound, piles, asthma (Mohanraj et al., 2010) and also used as an

appetizer and tonic (Sharma et al., 2011). Sometimes, leaves and stem are inhaled or

smoked after burning to cure the fever, swellings, paralysis and arthralgia. Similarly,

72

leaves are also taken for the treatment of various heart diseases and chest cold

(Agharkar, 1991; Hemalatha et al., 2011). The leaves also have anthelmintic, laxative,

healing, and bitter properties. It acts as an expectorant, relieves stomach pain and

cures ulcers (Goyal and Mathur, 2011). Chopped leaves showed properties as a

nematicide (Kristova and Tissot, 1995; Kawo, 2009) and as a cure for jaundice

(Sharma et al., 2011). Besides this flower and leaves are also used as a source of anti-

bacterial agent against gram positive and gram negative bacteria (Moscolo et al., 1988;

Larhsini, et al., 1999; Dewan et al., 2000; Alencer et al., 2004; Parabia et al., 2008;

Vadlapudi and Naidu, 2009; Amin et al., 2011; David et al., 2011; Doshi et al.,

2011; Ahmad et al., 2011; Sharma et al., 2011; Johnson et al, 2011; Prabha and

Vasantha, 2012; Muzammal, 2014; Kazemipour et al., 2014) ).

It is also evident by various reports that various works gave attention only to the

flower extract to check its antibacterial activity such as Doshi et al. (2011) used

ethanolic flower and other parts extracts against larvae of A. stephansi, there was no

antibacterial activity observed in flower and stem extracts of C. procera against S.

aureus, Bacillus cereus, Bacillus subtilis, and Micrococcus luteus. Whereas, David et

al. (2011) acetone and methanol flower extracts were further used against Bacillus

pumilis, E.coli, A. niger, Fusarium oxysporum. Similarly, Prabha and Vasantha (2012)

screened C. procera flower extract of chloroform, acetone and ethanol against various

pathogens and maximum antibacterial activity was recorded against S. aureus and B.

subtilis. Ranjit et al., (2012) also used ethanol fraction against S. aureus, Bacillus

subtilis, Bacilis pumilis, Micrococcus luteus, E. coli, Pseudomonas Aeroginosa and

Proteus vulgaris. Observations revealed a significant inhibitory action against test

73

bacteria. Joshi and Kaur (2013) determined the antimicrobial potential of ethanol,

methanol and aqueous extract of C. procera and found that ethanol extract have strong

antimicrobial activity against P aeroginosa. While, Javadian et al. (2014) evaluated

the antimicrobial activity of ethanol extract of C. procera and found effective against

E. coli isolates.

The antibacterial efficacy of C. procera leaves against human disease producing

bacteria was already studies by various scientists. Yesmin et al. (2008) demonstrated

the antibacterial activity through the leaves aqueous and methanol extracts of C.

procera and it was revealed that both extracts were active against Gram positive and

Gram negative bacteria at low concentrations. Studies of Kareem et al. (2008)

revealed positive activity of ethanol and chloroform fractions against Candida,

Miocrosporum, Aspergillus niger, S. pneumonia, S. pyogens, E. coli and S. aureus.

Similarly, Kawo et al. (2009) studied the antimicrobial efficacy of water and ethanol

leaves extract of C. procera and it was revealed that the aqueous sample did not

showed any activity while, ethanol extract have significant antibacterial potential.

Whereas, Goyal and Mathur (2011) applied fractions of petroleum ether, ethanol and

butanol against Candida para, Candida albican, Enterococci, Pseudomonas

aeroginosa, Staphylococci and E. coli which exhibited significant antimicrobial

activity. In another study (Hemalatha et al., 2011) reported leaf extract of acetone,

ethyl acetate, methanol and water against Shigella, S. typhi, Vibrio cholera,

Pseudomonas aeroginosa, Lactobacillus, B. cireus, S. aureus and B. subtilis, all of the

solvent extracts showed inhibitory activity against bacterial samples. Similarly,

Johnson et al., (2011) determined that aqueous and alcoholic extract showed strong

74

antibacterial efficacy against Aspergillus, E.coli and S. aureus. While, Velmurugan et

al., (2012) studied C. procera leaf extract of hexane, ethyl acetate and methanol

against aquatic micro pathogens from shrimp fish and It was noted that ethyl acetate

extract effectively suppressed bacterial strains. On the other hand Mako et al. (2012)

evaluated the antimicrobial activity of aqueous and ethanol root and leaves extract of

C. procera where ethanol extract showed more significant potential than aqueous

extract. Salem et al. (2014) demonstrated the antibacterial activity through leaf and

latex chloroform, ethanol, methanol extracts of the leaf where it was observed that

aqueous and ethanol extracts of leaves showed maximum potential against Gram

negative and Gram-positive pathogenic bacteria. However, Shetty et al. (2015) studied

the antibacterial effect of methanol, ethyl acetate, ethanol, acetone and aqueous

extracts of C. procera leaves against human pathogenic bacteria and it was found that

leaves extract showed significant antibacterial activity against Micrococcus aureus in

all test solvents. Pandey et al. (2015) assessed the antibacterial activity of C. procera

methanol, acetone, petroleum ether and ethyl acetate extracts, amongst them

methanol leaves extract showed highest range of inhibition against E.coli and S.

aureus.

In view of the above mentioned studies the present studies were carried out to evaluate

the antimicrobial efficacy of C. procera from Karachi (Pakistan) using different

solvent fractions of flowers and leaves with butanol, hexane, ethyl acetate and aqueous

extracts against various disease producing bacteria to substantiate the earlier findings

for its significant use.

75


Detail of collection of plant samples already mentioned in Chapter 2 (2.1)

4.2. Preparation of Extract

Detailed method is mentioned in Chapter 2 (2.2).

4.3. Preparation of Fraction


4.4. Experimental Microbes

The antimicrobial activity of C. procera flowers and leaf extracts was evaluated

against pathogenic microorganism types viz. Gram negative organisms includes

Escherichia coli and Salmonella typhi were separated from a dirty water sample.

While, Gram positive organisms includes Micrococcus luteus (JQ 250612) and

methicillin resistant Staphylococcus aureus were isolated from soil sample and clinical

specimen respectively.

4.5. Culture Media

Nutrient broth was used to restore experimental strains for 24 hours at 37 C with the

shaking of 135 rpm. Strains were maintained on nutrient agar slants at 4 C for further

studies.

76

4.6. Procedure for Antimicrobial Activity

To evaluate the antimicrobial potential of leaf and flower extracts of Calotropis procera

against pathogenic bacterial species. Agar well diffusion method was adopted by Tagg and

McGiven (1971) and Ali (2014). For the revival of bacterial species sterilized petri plates were

filled with with nutrient agar and inoculated 100µl of bacterial species having 108 cfu/ml

(Iqbal, 1998; Ali et al., 2014) compared with the 0.5 McFarland turbidity index. Then 100µl of

concentrated leaf and flower extracts were introduced in the wells of petri plate and plates

were incubated for 24 hours at 37oC temperature. While, for control plates no extracts were

introduced. To determine the potential against pathogenic bacterial species, zone of inhibition

was calculated. All experiments were performed in triplicate.

77

Table 4.1. Antimicrobial activity of flower extracts against different

pathogenic strains.

Key: MRSA= Methicillin resistant Staphylococcus aureus, Significant zone= > 11 mm,

-ve = No activity detected.

S.No Extracts Zones of inhibition (mm)

S. typhi Control E. coli Control MRSA Control M. luteus Control

1 Butanol -ve -ve -ve -ve -ve -ve 30 -ve

2 Ethyl

acetate -ve -ve 15 -ve 18 -ve 25 -ve

3 Aqua -ve -ve -ve -ve -ve -ve 30 -ve

4 Hexane 13 -ve 12 -ve 15 -ve 22 -ve

78

Figure 4.1: Antimicrobial activity of flowers extracts (aqueous, butanol,

hexane, ethyl acetate) against A= Micrococcus luteus, B= Salmonella

typhi, C= Escherichia coli, D= (Methicillin resistant Staphylococcus

aureus ) MRSA

79

Table 4.2. Antimicrobial activity of leaves extracts against different pathogenic

strains.

Key: MRSA= Methicillin resistant Staphylococcus aureus, Significant zone = > 11 mm,

-ve = No activity detected.

S.# Extracts Zones of inhibition (mm)

S. typhi Control E. coli Control MRSA Control M. luteus Control

1 Butanol -ve -ve -ve -ve 7 -ve -ve -ve

2 Ethyl

acetate -ve -ve 12 -ve 15 -ve -ve -ve

3 Aqua -ve -ve -ve -ve -ve -ve 19 -ve

4 Hexane 15 -ve 18 -ve 12 -ve 23 -ve

80

Figure. 4. 2. Antimicrobial activity of leaves extracts (aqueous, butanol,

hexane, ethyl acetate) against A= Micrococcus luteus, B= (Methicillin

resistant Staphylococcus aureus ) MRSA, C= Escherichia coli and D=

Salmonella typhi.

81

Figure 4.3. Zone of inhibition of C. procera flowers extracts in different

solvents (butanol, ethyl acetate, aqueous and hexane).

0

5

10

15

20

25

30

35

S. typhi E. coli MRSA M. luteus

Zo

ne

of

inh

ibit

ion

(m

m)

Experimental Bacterial Strains

Butanol

Ethyl acetate

Aqua

Hexane

82

Figure 4.4. Zone of inhibition of C. procera leaves extracts in different

solvents (butanol, ethyl acetate, aqueous and hexane).

0

5

10

15

20

25

S. typhi E. coli MRSA M. luteus

Zo

ne

of

inh

ibit

ion

(m

m)

Experimental Bacterial Strains

Butanol

Ethyl acetate

Aqua

Hexane

83

4.7. RESULTS

To explore the antimicrobial potential of different fractions (hexane, ethyl acetate,

butanol and aqueous) of Calotropis procera flower and leaf were used.

Flowers: All solvents of flower extracts of C. procera showed dissimilar results of

inhibition against M. luteus, methicillin resistant Staphylococus aureus, E. coli, and S.

typhi (Table 4.1., Fig. 4.1. A-D & 4.3). Amongst four extracts, hexane fraction has

been proved very significant as an antimicrobial agent against all the examined

bacterial strains. A maximum zone of inhibition (22mm) was observed against M.

luteus. E. coli, Butanol and Aqueous fractions also exhibited inhibitory efficacy

against M. luteus, whereas, another bacterial strain were resistant to both fractions.

Fraction of ethyl acetate showed inhibitory activity not only against M. luteus (25mm)

but also against E. coli (15mm) and methicillin resistant Staphylococus aureus

(18mm).

Leaf: Different solvents of leaf extracts of C. procera exhibit variation in the

inhibition spectrum against E. coli, methicillin resistant Staphylococcus aureus,

Micrococcus luteus and Salmonella typhi (Table 4.2., Fig. 4.2. A-D). Amongst four

extracts, hexane fraction has been found highly significant antibacterial factor against

all bacterial strains. A maximum zone of inhibition (23mm) was observed against

Micrococcus luteus. Ethyl acetate also showed significant inhibitory activity against

MRSA (15mm) and E. coli (12mm). While, Butanol and hexane extract did not

produce any significant inhibitory activity against MRSA. Similarly, no activity was

observed in E. coli against butanol and aqua, M. luteus did not show any significant

activity in butanol and ethyl acetate. While, butanol and ethyl acetate samples were

proved insignificant against the activity of S. typhi.

84

4.8. DISCUSSION

Resistance to different broad-spectrum antibiotic has now become a global concern

due to emerging cases of drug resistance (Mohanraj et al., 2010). Due to these

emerging cases and also due to the increase consumer demand towards natural

antibacterial agents there is a need of screening of natural anti-microbial compounds

effective against different drug resistant pathogens. In the last few decades, several

new natural anti-microbial compounds were discovered for the control of severe

infections. Keeping this in view, the present study was designed to explore the anti-

bacterial potential of medicinally important flower of C. procera. Different soluble

flower extracts of C. procera showed dissimilar inhibition pattern against tested

disease producing microorganisms. Amongst four extracts, hexane fraction has been

proved very significant as an antibacterial agent against studied pathogens. A

maximum zone of inhibition was observed against Micrococcus luteus which can

cause infections in immune-compromised individuals (Seifert et al., 1995; Ali et al.,

2014). It is also noteworthy that present findings are in contrast to the earlier findings

of Parabia et al. (2008) where hexane fraction of stem of C. procera showed least

antibacterial activity (7mm) against Micrococcus luteus. Similarly, fraction of ethyl

acetate showed significant inhibitory activity not only against Micrococcus luteus but

also against E.coli and methicillin resistant Staphylococcus aureus (except S. typhi)

and these findings could be well supported by the studies of Patil and Saini (2012)

where they also found significant role of ethyl acetate against various pathogens. E.

coli is a toxin producing human pathogen. E. coli is an enteric hemorrhagic strain and

cause severe diarrhoea leads to kidney failure through food. However, Staphylococcus

85

aureus (methicillin resistant) is also a disease producing bacteria, commonly resistant

against β-lactam antibiotics (Iqbal, 1998; Que and Moreillon, 2009; Ali et al., 2014).

Presently ethyl acetate and hexane extracts of C. procera significantly inhibited the

growth of this multidrug resistant organism.

These findings are also in agreement with the previous reports of Kazemipour et al.

(2014) and Javadian et al. (2014). However, flower aqueous and butanol extracts

showed significant inhibitory activity only against M. luteus.

Similarly, leaf extracts of C. procera also showed various spectrums of inhibition

against E. coli, methicillin resistant S. aureus (MRSA), S. typhi and M. luteus (Table

4.2., Fig. 4.2. (A-D). Amongst all of the extracts, the C. procera hexane fraction has

been proved very significant as antibacterial agent against all of the studied pathogens.

A maximum significant zone of inhibition of hexane extract (23mm) was observed

against M. luteus as compared to butanol, and ethyl acetate fractions which did not

show any activity against M. luteus and S. typhi. However, present finding is in

contrast to that of the findings of (Doshi et al., 2011) where an inhibition zone of C.

procera leaf extract for M. luteus was observed at 9mm. Similar to the findings of

Joshi and Kaur (2013) hexane soluble leaf extract also shows significant activity

against E. coli. While, ethyl acetate fraction of leaf also showed significant zone of

inhibition (12mm) against E. coli and MRSA (15mm) . These significant readings are

in contrast with the observation of Doshi et al. (2011) where 7mm inhibition zone was

observed. Whereas, butanol soluble extract shows in significant (7mm) zone of

inhibition. Thus, flower and leaf extracts of C. procera found to be strong

antimicrobial agent.

86

EFFECT OF CALOTROPIS PROCERA AQUEOUS

EXTRACT ON GLUCOAMYLASE, ALPHA-

AMYLASE AND UREASE ACTIVITY.

5. Introduction

Enzymes are biological molecules that speed up the biological chemical reactions

(Brayer et al., 1995). Generally enzymes are classified based on the type of reaction

that they catalyze such as glucoamylase, also known as glucan 1,4-alpha-glucosidase,

(EC 3.2. 1 .3). It is a type of digestive enzyme which cleaves one glucose unit from a

non reducing end of starch (amylose and amylopectin). Most of the glucoamylases are

also able to hydrolyze the 1,6-a linkage in branch points of starch molecules.

Alpha amylase is a digestive enzyme. The formal title of alpha amylase is 1,4 α-D-

glucanohydrolase; EC 3.2.1.1. The enzyme alpha amylase aids in the hydrolysis of α–

1,4 glycosidic bond in the conversion of starch to maltose (Brayer et al., 1995). In

humans, it is found in both saliva and pancreas. Amylases are also used in various

industries like paper, food and textile industries (Windish et al., 1965; Gupta et al.,

2003).

The enzyme urease (EC 3.5.1.5) catalyzes the breakdown of urea into carbon

dioxide and ammonia. The reaction occurs as follows (Zimmer, 2000).

(NH2)2CO + H2O → CO2 + 2NH3

https://en.wikipedia.org/wiki/Enzyme_Commission_number

http://enzyme.expasy.org/EC/3.5.1.5

https://en.wikipedia.org/wiki/Catalysis






https://en.wikipedia.org/wiki/Water



87

Figure 5.1. Representing starch structure showing α-1,4-linkage where α-amylase

targets. (Adapted from El-Fallal et al., 2012).

88


5.1.1. Preparation of aqueous extract.

Leaves and flowers of Calotropis procera from two different collections, already

mentioned in Chapter 2(2.2) were powdered and mixed with deionized water to

extract its compounds. The extract was kept at 4oC for 24 hours and then it was

centrifuged at 12000 rpm for 10 minutes at 4oC.

5.1.2. Enzyme activity

Glucoamylase (100U/mg), Amylase (50U/mg) and Urease (5U/mg) were mixed

separately with leaf and flower extracts at a concentration of 50mg/ml for 30 and 60

minutes. Control was used for comparison, in which 50mM phosphate buffer (pH=7)

was added. After the incubation, enzyme activity was performed.

5.1.3. Estimation of glucoamylase

Glucoamylase was estimated by the method of Ghani et al. (2013) as described in

Chapter 2 (2.15.1).

5.1.4. Glucoamylase activity assay

Glucoamylase activity was determined by the method of Ghani et al. (2013) as

described in Chapter 2 (2.15.1.1).

5.1.5. Estimation of reducing sugar for alpha-amylase

3’5’-Dinitrosalisylic acid (DNS) method was used to estimate the reducing sugar

(Miller, 1959) as described in chapter 2 (2.15.1.3).

89

5.1.6. Estimation of urease activity

Urease activity was determined by modified Berthelot’s method (Fawcett and Scott,

1960) as described in Chapter 2 (2.15.1.5).

5.1.7. Alpha amylase enzyme activity assay

Detailed method is mentioned in Chapter 2 (2.15.1.4).

90

Table 5.1. Effect of aqueous extract of C. procera on glucoamylase activity

S. No. Samples

Glucoamylase activity

after 30 minutes

(%)


after 60 minutes

(%)

1 Control 100e±5.23 100

c±4.25

2 Leaf Extract 1 ***134d±3.35 100

c±3.62

3 Leaf Extract 2 ***148b±4.56 ***137

a±5.67

4 Flower Extract 1 ***168a±6.24 100

c±6.55

5 Flower Extract 2 ***141c±7.25 ***131

b±5.84

Extract concentration = 50mg/ml

n=6, mean values ± SE, ***= P<0.05, a-d= ranks of mean values, sharing of same letter do not

differ significantly.

91

Figure 5.2. Effect of aqueous extract of C. procera on glucoamylase activity.



***e

***d ***b

***a ***c

c c

***a

c

***b

0

50

100

150

200

Control Leaf Extract 1 Leaf Extract 2 Flower Extract 1

Flower Extract 2

Re

lati

ve

En

zy

me

Act

ivit

y (

%)

Extract conc. 50mg/ml


Incubation at 30 minutes Incubation at 60 minutes

92

Table 5.2. Effect of aqueous extract of C. procera on alpha amylase activity

S. No. Samples

Alpha amylase

activity after 30

minutes

(%)

Alpha amylase

activity after 60

minutes

(%)

1 Control 100a±3.5 100

c±4.0

2 Leaf Extract 1 100a±3.6 ***125

b±5.2

3 Leaf Extract 2 100a±4.5 100

c±4.0

4 Flower Extract 1 100a±5.6 ***146

a±6.0

5 Flower Extract 2 100a±5.0 100

c±4.5




93

Figure 5.3. Effect of aqueous extract of C. procera on alpha amylase activity.



a a a a a c ***b

c

***a

c

0

50

100

150

200


Flower Extract 2

Re

lati

ve

En

zy

me

Act

ivit

y (

%)


Alpha-amylase activity

Incubation at 30 minutes Incubation at 60 minutes

94

Table 5.3. Effect of aqueous extract of C. procera on urease activity.

S. No. Samples

Urease activity

after 30

minutes

(%)

Urease

activity after

60 minutes

(%)

1 Control 100d±5.2 100

a±4.25

2 Leaf Extract 1 ***111b±6.45 100

a±5.26

3 Leaf Extract 2 100d±7.58 100

a±4.32

4 Flower Extract 1 ***125a±4.25 100

a±3.59

5 Flower Extract 2 ***107c±5.12 100

a±5.26


n=3, mean values ± SE, ***= P<0.05, a-d= ranks of mean values, sharing of

same letter do not differ significantly.

95

Figure 5.4. Effect of aqueous extract of C. procera on urease activity.

n=3, mean values ± SE, ***= P<0.05, a-d= ranks of mean values, sharing of

same letter do not differ significantly.

d

***b

d

***a

***c a a a a a

0

20

40

60

80

100

120

140


Flower Extract 2

Re

lati

ve

En

zy

me

Act

ivit

y (

%)


Urease Activity Incubation at 30 minutes Incubation at 60 minutes

96

5.2. Results and Discussion

According to one way Anova values of glucoamylase activity in leaf and flower

extracts differ significantly (P<0.05) (Table 5.1, Fig. 5.2). While, Duncan’s multiple

comparison test showed an increase in glucoamylase activity in leaf and flower extract

after 30 minutes incubation period (Table 5.1, Fig. 5.2).

Flower extracts with concentration of 50mg/ml show comparatively greater

glucoamylase activity as compared to leaves extracts (P<0.05) and after increasing the

incubation time from 30-60 minutes, a marked decrease in glucoamylase activity was

observed (Table 5.1, Fig. 5.2).

One way Anova for alpha amylase activity showed insignificant difference (P>0.05)

after the 30 minutes incubation period in both leaf and flower extracts of C. procera

as compared to control (P>0.05) (Table 5.2, Fig. 5.3). However, a significant

difference was observed in alpha amylase activity with leaf and flower extract after the

60 minutes incubation period (P<0.05). On the other hand, Duncan’s multiple

comparison test showed that the leaves and flower extract after 30 minutes of

incubation did not cause any effect on α-amylase activity and results were remained

insignificant (P>0.05) (Table 5.2, Fig. 5.3). However, an increase in α-amylase

activity was observed (P<0.05) when the enzyme was mixed with leaf and flower

extract for 60 minutes (Table 5.2, Fig. 5.3).

97

According to one way Anova there was a significant difference for urease activity

after 30 minutes incubation period (P<0.05). While, after 60 minutes no significant

difference was found amongst samples and control (P>0.05) (Table 5.3, Fig. 5.4).

The Duncan’s multiple comparison test showed the highest urease activity in flower

extract 1 than leaf extract 1 and flower extract 2 respectively (P<0.05).

Thus, urease activity was increased when extracts were incubated for 30 minutes.

While, no effect on urease activity was observed after 60 minutes. Hence, flower

extract of C. procera is proven to be a good enhancer of urease as compared to

leaves of C. procera (Table 5.3, Fig. 5.4).

In general, it is concluded that flower of C. procera proves to be best suited to

enhance the enzymatic activities like α-amylase, glucoamylase and urease as

compared to leaves and the activities of these enzymes could be well correlated with

the change of incubation time and concentration of the extract.

98

IN VITRO ANTIOXIDANT PROPERTIES OF

CALOTROPIS PROCERA

6. Introduction

Antioxidants are the substance that reduces oxidative damage caused by free radicals.

While, free radical are part of metabolic pathways but over production of the free

radicals may injured the tissues which may lead to various diseases of heart by

damaging lipid, cancer by damaging DNA other age related diseases by damaging

proteins (Sen et al., 2010).

Antioxidant potential may be determined by various ways like reduction of

ferricyanide complex into ferrous or metal ions chelating, reducing ability, free radical

scavenging activity or lipid peroxidation.

Lipid peroxidation is a process of oxidative degradation of lipids, in which a free

radical like hydroxyl group (OH) extract electrons from the unsaturated lipids present

in cell membranes. This result in the formation of a water molecule and lipid/fatty

acid radical. This radical again reacts with oxygen to form lipid peroxyl radical.

Lipid radical and lipid peroxyl radical both are unstable species, therefore, lipid

radical reacts with oxygen and convert into lipid peroxyl radical and lipid peroxyl

radical again react with another unsaturated lipid, this cycle continues and new lipid

radical reacts with same way and finally lipid peroxide is formed, that may cause

cellular damage. Cholesterol, glycolipids, phospholipids, are also well-known targets

of lipid damaging and potentially cause fatal peroxidative change. Lipids also can be

99

oxidized by enzymes like cyclooxygenases, lipoxygenases, and cytochrome P450

(Ayala et al., 2014).

A number of plants are known to have antioxidant properties due to the presence of

various phytochemicals. Amongst them Calotropis procera is one of the most

popular plant which have a series of chemical constituents i.e., various favonoids and

phenolic acids are presently reported which may have antioxidant and other medicinal

uses.

Previously, Kumar et al. (2013) determined the antioxidant activity of C. procera by

using its root extract. Similarly, Krishnaveni et al. (2013) estimated free radical

scavenging activity of aqueous leaf extract of the plant and proved that antioxidant

potential of C. procera to chelate metal ions. Yesmin et al. (2008) determined the

antioxidant potential of methanol ectract of C. procera leaf by DPPH. While, Srividya

et al. (2013) evaluated antioxidant potential of C. procera fruit extract through DPPH.

Patel et al. (2014) studied the comparative antioxidant activity by DPPH (1,1-

Diphenyl-2-picryl hydrazyl) of C. procera and C. gigentia by using their methanol

extract and it was reported that C. procera possess high antioxidant properties due

to more phenols and flavonoids as compared to C. gigantea. Ahmed et al. (2014)

determined in vitro antioxidant activity of methanol extract of C. procera latex which

exhibited positive activity to scavenge free radicals. Pooja et al. (2014) investigated

the antioxidant potential of ethyl acetate and acetone fractions of four flowers

Alastonia scholaris, Cassia auriculata, Catharanthus roseas and Calotropis procera.

Amongst flowers of C. procera showed lower antioxidant activity.

100

Presently, leaf and flower extracts of C. procera are investigated to determine the

antioxidant properties of the plant through techniques like DPPH, Lipid peroxidation

and reducing power ability.


Detail of collection of plant samples already mentioned in Chapter 2 (2.1).

6.1.1. Preparation of extract


6.1.2. Preparation of fraction

Detailed method is explained in Chapter 2 (2.3).

6.1.3. Preparation of tissue homogenate

Fresh tissue of a normal albino rat was taken, the tissue was sliced into small pieces

and phosphate buffer saline pH 7.4 was added. The homogenate was centrifuged at

3000 rpm for 15 minutes, clear supernatant was collected for anti lipid peroxidation

assay.

101

6.2. Lipid peroxidation inhibition

Effect of C. procera on inhibition of lipid peroxidation activity was studied in vitro

according to the guidelines of Halliwell and Gutteridge (1999) as described in Chapter

2 (2.14).

6.3. 1,1-diphenyl -2-picrylhydrazyl (DPPH) radical scavenging assay

The in vitro antioxidant power of C. procera extracts were determined as described by

Kumar et al. (2013) in Chapter 2 ( 2.14.3).

6.4. Reducing power assay

The reducing power of C. procera flowers and leaves extracts were determined by

the methods of Oyaizu (1986) and Mishra et al. (2013) as described in Chapter 2

(2.14.4).

102

Table 6.1

Lipid peroxidation inhibition activity of C. procera flowers

extracts

Extract Conc. Water Hexane Ethanol Ethyl Acetate

mg/ml % % % %

1 21.36 27.48 56.87 28.43

2 26.07 38.62 75.11 42.83

4 43.13 43.83 78.54 46.6

6 55.69 46.68 88.38 56.87

8 70.86 53.79 88.62 65.23

10 89.58 54.99 89.33 63.12

BHA 85

Ascorbic Acid 75.5

103

Table 6.2

Lipid peroxidation inhibition activity of C. procera leaves extracts

Extract Conc. Water Hexane Ethanol Ethyl Acetate

mg/ml % % % %

1 13.98 27.25 29.14 27.98

2 20.14 28.43 38.63 28.43

4 31.99 38.61 42.93 56.87

6 38.62 43.93 56.94 44.31

8 51.18 46.68 39.43 75.11

10 75.11 54.98 20.04 54.99

BHA 85 85 85 85

Ascorbic Acid 75.5

104

Table 6.3

DPPH radical scavenging activity of C. procera flowers extracts

Extract

Conc.

mg/ml

Water Hexane Ethanol Ethyl Acetate

1 65.81 36.12 48.45 56.98

2 69.6 66.9 62.52 61.7

4 71.51 42.91 76.18 51.1

6 77.82 53.79 81.72 70.84

8 80.9 71.45 88.19 66.22

10 83.05 76.38 86.13 77.2

Ascorbic

Acid 75.5 75.5 75.5 75.5

BHA 85

105

Table 6.4

DPPH radical scavenging activity (%) of C. procera leaves

extracts

Extract

Conc.

mg/ml


1 70.1 27.25 29.14 27.98

2 75.97 28.43 38.63 28.43

4 55.8 38.61 42.93 56.87

6 41 43.93 56.94 44.31

8 33.6 46.68 39.43 75.11

10 7.9 54.98 20.04 54.99

Ascorbic

Acid 75.56 75.56 75.56 75.56

BHA 85

106

Table 6.5

Reducing power assay of C. procera flowers extracts

Extract

Conc.

mg/ml


1 0.673 0.296 0.484 0.049

2 1.287 0.427 0.403 0.132

4 1.167 0.864 0.287 0.137

6 1.403 0.613 0.236 0.193

8 1.746 0.849 0.487 0.033

10 1.827 1.469 0.572 0.145

Ascorbic

acid 0.238 0.238 0.238 0.238

107

Table 6.6

Reducing power assay of C. procera leaves extracts

Extract

Conc.

mg/ml


1 0.782 0.31 0.121 0.332

2 0.994 0.679 0.396 0.373

4 1.039 0.601 0.314 0.316

6 1.035 0.816 0.43 0.403

8 1.085 0.804 0.318 0.411

10 1.093 1.403 0.585 0.586

Ascorbic

Acid 0.238 0.238 0.238 0.238

108

Figure 6.1. Effect of different flower extracts of C. procera on Lipid

peroxidation inhibition (%).

0

10

20

30

40

50

60

70

80

90

100


(%)

Inh

ibit

ion

of

lip

id p

erox

idati

on

C. procera flower extracts in different solvents

1mg/ml

2mg/ml

4mg/ml

6mg/ml

8mg/ml

10mg/ml

BHA 2mg/ml

109

Figure 6.2. Effect of different leaves extracts of C. procera on Lipid

peroxidation inhibition (%).

0

10

20

30

40

50

60

70

80

90


(%)

Lip

id p

erox

idati

on

in

hib

itio

n

C. procera leaves extracts in different solvents

1mg/ml

2mg/ml

4mg/ml

6mg/ml

8mg/ml

10mg/ml

BHA 2mg/ml Linear (BHA 2mg/ml)

110

Figure 6.3. DPPH radical scavenging activity of flower extracts of C. procera

0

10

20

30

40

50

60

70

80

90

100


(%)

DP

PH

rad

ical

scaven

gin

g a

ctiv

ity


1mg/ml

2mg/ml

4mg/ml

6mg/ml

8mg/ml

111

Figure 6.4. DPPH radical scavanging activity of leaves extracts of C. procera

0

10

20

30

40

50

60

70

80


(%)

DP

PH

rad

ical

scaven

gin

g a

ctiv

ity


1mg/ml

2mg/ml

4mg/ml

6mg/ml

8mg/ml

10mg/ml

Ascorbic acid

112

Figure 6.5. Reducing power of flower extracts of C. procera

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2


Ab

sorb

an

ce a

t 7

00

nm


1mg/ml

2mg/ml

4mg/ml

6mg/ml

8mg/ml

10mg/ml

Ascorbic acid

113

Figure 6.6. Reducing power of leaves extracts of C. procera

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6


Ab

sorb

an

ce a

t 7

00

nm


1mg/ml

2mg/ml

4mg/ml

6mg/ml

8mg/ml

10mg/ml

114

6.5. Results

Results showed that highest lipid peroxidation inhibition activity was found in flower

water and ethanol extracts i.e. about 89.5%. While, leaf water and ethyl acetate

extracts exhibited highest LPOI activity i.e. about 75%. These values of LPOI were

more or less similar to the standard value of BHA and ascorbic acid (Table 6.1, Fig.

6.1).

Flower showed highest value of DPPH radical scavenging activity (88.19%) in

ethanol. While, leaf water extract showed highest value of DPPH radical scavenging

activity i.e. 75.95% and the value of standard are more or less similar to that of DPPH

scavenging values of plant extracts (Table 6.2, Fig. 6.2).

Highest reducing power value was about more than 1.0 in both flower and leaf water

extracts. Whereas, reducing power value of tested extracts are significantly higher than

the standard value of ascorbic acid that was 0.238.

115

6.6. Discussion

Plants are the main source of the natural antioxidants against free radicals and their

reactive derivatives (ROS), which are known to induce various diseases of heart by

damaging lipids, cancer by DNA damaging and ageing by the damage of protein

(Sen et al., 2014). In the present study to counter act this action C. procera leaf and

flower extracts are utilized in vitro as an antioxidant agent by analyzing DPPH

scavenging activity, reducing power and lipid peroxidation inhibition activities. While,

C. procera was earlier found to be beneficent to inhibit or reduce the lipid

peroxidation chain reaction (Kumar et al., 2015), free radical scavenging activity and

reducing power ( Yesmin et al., 2008).

Flower and leaf extracts of C. procera with various solvents possess effective lipid

peroxidation inhibition (LPOI) activity. Flower extract exhibited highest lipid

peroxidation inhibition value as compared to leaf extract. Lipid peroxidation

inhibition of flower extract showed concentration dependent increase in water, hexane

and ethanol extract except ethyl acetate extract. Highest LPOI activity is found in

water extract i.e. (89.58%), ethyl acetate (65.23%) and hexane (54.99%) respectively.

However, BHA and Ascorbic acid exhibited lower values (85 and 75%) as compared

to water extract value (Table 6.1, Fig. 6.1).

There is a concentration dependent LPOI activity in water and hexane. Whereas,

ethanol and ethyl acetate LPOI activity was independent to their concentrations. The

highest activity of LPOI in terms of percentage was observed in water (i.e., 75.11%

with 10gm/ml) and ethyl acetate ( 75.11% with 8mg/ml) as compared to ethanol

116

(56.94% with 6mg/ml) and hexane (54.98% with 10mg/ml) in respective manner.

Similarly BHA exhibited 85% activity as compared to ascorbic acid showing 75%

lipid peroxidation inhibition activity.

Similarly, leaf extracts of C. procera with various solvents possess effective DPPH

scavenging activity in all concentrations.

Flower extract exhibited highest scavenging activity as compared to leaf. The

antioxidant activity of flower extract usually increases with the increase of

concentration and highest activity is found in ethanol extract (i.e., 88.19%) followed

by water extract (83.05%), ethyl acetate (77.2%) and hexane (76.38) respectively.

However, BHA showed highest activity (85%) as compared to ascorbic acid (75.5%)

(Table 6.3, Fig. 6.3).

Leaf extract with various solvents showed different values of scavenging activity

irrespective of their concentration. Amongst all of the solvents water extract exhibited

highest activity (i.e., 75.95% with 2mg/ml) following ethyl acetate (75.11 with

8mg/ml), ethanol (56.94% with 6mg/ml) activity and 10mg/ml hexane showing

(54.98%) scavenging activity respectively. While BHA and ascorbic acid were used

as standard showing 85% and 75.56% activity as compared to test extracts (Table 6.4,

Fig. 6.4).

Different solvent extracts of C. procera flower and leaf were also analyzed to

determine the reducing power. Flower showed highest reducing power as compared

to leaf. Amongst all of the test extracts, flower extract showed different absorbance

pattern of reducing power irrespective of their concentrations. While, flower water

117

extract was found to have concentration dependent pattern of reducing power. Highest

reducing power was observed in water extract with 1.827 absorbance value followed

by hexane with 1.469 absorbance, ethanol with 0.572 absorbance and ethyl acetate

showed lowest absorbance respectively. However, ascorbic acid showed less reducing

power as compared to water, hexane and ethanol extract (Table 6.5, Fig. 6.5).

A concentration dependent absorbance of reducing power was found in leaf extract

with water. The highest value of reducing power was observed in water extract

(1.093). While, other three extracts showed improper values irrespective of their

concentrations. The absorbance value of ascorbic acid was found at lower side as

compared to test extracts (Table 6.6, Fig. 6.6).

Therefore, it is concluded that C. procera flowers and leaf extracts in different

solvents have a significant potential to inhibit free radical chain reaction. To a great

extent it was also found that the flower extract is more potent than leaf extract as

antioxidant.

118

EFFECT OF CALOTROPIS PROCERA LEAF

HEXANE SOLUBLE EXTRACT ON NSAID

(IBUPROFEN) TREATED RATS.

7. Introduction

The ability of a substance to damage any living body is called toxicity. Toxicity can

affect the whole organism or substructure of the organism and it may be occur due to

certain biological, physical or chemical effects. Drug induced toxicity can damage any

tissue depending on dosage such as acute dosage of a drug can produce the toxicity for

nervous system and its chronic exposure may cause the serious injuries to the other

organs. Toxicity can also be produced by the medicines which are normally be used

for curative purposes. Sometimes, the use of over the counter medicines and long term

use of overdoses of drugs may also cause toxicity to certain specific organs. The

Process of oxidation continuously takes place in all aerobic living bodies, due to this

ROS (reactive oxygen species) including O2 anion, H2O2 hydrogen peroxide -OH

hydroxyl radical and nitric oxide/peroxinitrates (NO/NOO-) are constantly formed

within the cells. The over production of these substances may cause oxidative load in

the cells. This oxidative stress produces deleterious effects to cells of DNA, proteins

and lipids. Lipid are specifically more damaged due to the formation of lipid

peroxidation products.

There are various reports available on toxicity producing substances like the NSAIDS.

Particularly, Paracetamol (Younes et al., 1988), Acetaminophen (Tarloff et al., 1990;

Trumper et al., 1992), Diclofenac (Hickey et al., 2001; Yasmeen et al., 2007),

Phenacetin (Murray and Brater, 1993; Kocaoglu et al., 1997; Fackovcova et al., 2000;

119

Soubhia et al., 2005; Derle et al., 2006), Mefenamic acid (Somchit et al., 2004),

Tenofovir (Morelle et al., 2009), Paracetamol (Somanawat et al., 2013) and metals

including arsenic, cadmium, lead and mercury (Nicholson et al., 1985; Fowler, 1992).

However, Cholestyramine was utilized against the Paracetamol induced toxicity in rats

and that was evident by a reduction in plasma enzyme activity and creatinine levels.

(Siegers and Moller, 1989). On the other hand Cadmium was used to prevent the

Acetaminophen induced toxicity in female rats (Bernard et al., 1988). Moreover,

Ibuprofen and Diclofenac were found useful protective drugs against Gentamicin

toxicity (Farag et al., 1996). While, Sharma et al. (2007) suggested that the

supplementation of Spirulina fusiformis can play a significant role against mercuric

chloride induced toxicity.

7.1. Nephrotoxic Agents

There are several substances which can initiate toxicity to the kidneys. These

substances include antibiotics, anticancer drugs, heavy metals, herbicides, pesticides,

excess amount of uric acid and long term use and high doses of analgesics may also

cause nephrotoxicity these analgesics usually include aspirin and ibuprofen

(Robertson, 2014).

7.2. NSAIDs

Nonsteroidal anti-inflammatory drugs or NSAID are the commonly used over the

counter drugs. They are pain relievers, help in reducing inflammation and lower

fever. They also prevent blood from clotting (Robertson, 2014).

120

7.3. Ibuprofen

Ibuprofen is selected for present experimental studies. It is a derivative of propionic

acid its chemical name is Isobutylphenylpropionic acid, the structure containing a

benzene ring conjugated to a propionic acid. It was first derived during 1950-1960s at

the research laboratories of Boots group and discovered by the scientist Andrew RM

Dunlop with his co-researchers Stewart Adams, John Nicholson, Jeff Wilson and

Colin Burrows (Robertson, 2014).

7.4. Mechanism of action of Ibuprofen

Ibuprofen is said to be an inhibitor of prostaglandin synthesis. The exact mechanism

of action is still unknown. Ibuprofen is an inhibitor of an enzyme (cyclooxygenase).

This enzyme converts arachidonic acid to prostaglandins. Prostaglandins are the

initiator of inflammation, fever and pain. There are two types of cyclooxygenase,

one is COX-1 which protects the lining of the stomach from digestive chemicals and

also maintains kidney function whereas, COX-2 released when joints are injured or

inflamed (Robertson, 2014).

A number of medicinal plants have been reported as antioxidants such as fruit extract

of Berberis vulgaris was used as an antioxidant (Motalleb et al., 2005; Hanachi et al.,

2008). Zingiber officinalis was reported to be used against ROS induced oxidative

stress (Ajith, 2010). Similarly the species of Gemelia, Kigelia, Hibiscus,Parthenium

(Patel et al., 2010) and Calotropis procera (Ahmed et al., 2014) have been reported

for their high radical scavenging activity.

121

In the present study Calotropis procera is selected as anti-oxidant against the NSAID

(Ibuprofen) induced toxicity. As the species has already been reported to have various

medicinal properties (Goyal and Mathur, 2011; Johnson et al., 2011; Doshi et al.,

2011; Prabha and Vasantha, 2012).


Detail of collection of plant samples already mentioned in Chapter 2 (2.1).

7.5.1. Preparation of extract


7.5.2. Preparation of fractions


7.5.3. Experimental animals and diet

Wistar white male rats (180-250g b.w.), bought from the animal house of Dow

University of Health Sciences, Karachi, Pakistan. Before starting experiments rats

were adjusted to the laboratory atmosphere and accommodated separately in a

artificially maintained temperature (22-26oC). Water and diet were provided to rats

Diet preparation is explained in the following table:

122

Rat’s Diet Preparation Table

INGREDIENTS QUANTITY

Wheat Flour 5 Kg

Barley Flour 2.5 Kg

Corn Flour 1.25 Kg

Cooking Oil 1.5Litre

7.6. Proper Recommendations

The scientific procedures were conducted in compliance with the ethical

recommendations of Institutional ERB (Ethical Review Board) and internationally

accepted ethics for laboratory use and care in animal research (Health Research

Extension Act, 1985).

7.7. Drug: Ibuprofen was purchased from market.

7.8. Study Protocol and Drug Administration Plan

Animals were divided into 3 different groups (n= 6)

Each group consists of six rats and treated as follows:

The Group I consists of healthy animals, untreated rats and termed as control.

Rat’s weight was recorded between 11:00 -12:00 hr for 10 alternate days.

Group II treated with prepared Ibuprofen suspension orally at a dose of 2ml /200gm

b.w. for 15 days. Termed as Ibuprofen Treated +ve Control. They were weighed

before administration of oral dose daily.

123

Group III treated with only hexane suspension orally at a dose of 2ml/200gm b.w.

Group IV treated with Ibuprofen + Hexane extract treated group, received hexane

extract orally according to the recommended doses i.e., they were weighed before

hexane extract treatment. Hexane extract was given 30 minutes prior to ibuprofen

administration.

7.9. Collection of Samples

7.9.1. Blood Samples

After 24 hours of last dose, rats were killed by chop off their heads and blood was

collected in the lithium heparin coated tubes. Then these tubes were shaked well.

After shaking, the tubes were centrifuged at 2000 rpm for 20 minutes. The plasma was

separated and collected in disposable eppendorff tubes and stored at -70oC for further

processing.

7.9.2. Kidney Sample

Kidneys were taken and blood was cleaned by passing through saline, Then dried

under filter paper and weighed. The kidneys were stored in the freezer at -70oC for

biochemical analysis.

124

7.10. Analytical methods

7.10.1. Preparation of protein free filtrate (PFF)

Detailed method of preparation for protein free filtrate is mentioned in Chapter 2

(2.13).

7.10.2. Estimation of plasma urea

Plasma urea was estimated by using Diacetyl monoxime method (Butler et al., 1981)

as mentioned in chapter 2 (2.13.1 and 2).

7.10.3. Estimation of plasma creatinine

Plasma creatinine was estimated by Modified Jeff’s method (Spierto et al., 1979) is

explained in Chapter 2 (2.13.3).

7.10.4. Preparation of kidney homogenate

Kidney homogenate was prepared by the procedure of Ricardo et al. (2005) as

mentioned in Chapter 2 (2.13.4).

7.10.5. Estimation of malonyldialdehyde (MDA)

MDA was estimated following the method of Okhawa et al. (1979) is explained in

Chapter 2 (2.13.5).

125

7.10.6. Estimation of 4-hydroxyl-2-nonenal (4-HNE)

4-HNE was measured by the method of Kinter et al. (1996) as mentioned in Chapter 2

(2.13.6).

7.10.7. Estimation of catalase

Method of Sinha (1972) was used to estimate catalase is explained in Chapter 2

(2.13.7).

7.10.8. Estimation of superoxide dismutase (SOD)

SOD was estimated by the method of Kono (1978) is explained in Chapter 2 (2.13.8).

7.10.9. Estimation of glutathione (GSH)

GSH was estimated by the method of Carlberg and Mannervik (1985) as mentioned in

Chapter 2 (2.13.9).

126

Table 7.1. Effect on body and kidney weight in control, Ibuprofen, hexane, and

Ibuprofen+hexane treated rats.

Parameters Control Ibuprofen

200mg/kg

Hexane

200mg/kg

Ibu+Hex.

200mg/kg

Mean Body

Weight (gms) ***261.66

a±12.88 ***238.1

d±12.35 ***245.49

c±10.66 ***259.2

b±11.61

Mean Kidney

Weight

(gms)

***0.75c±0.05 ***0.85

b±0.12 ***0.89

a±0.06 ***0.88

a±0.05

n=6, mean values ± SE, ***= P<0.05, a-d= ranks of mean values, sharing of same letter do not differ

significantly.

127

Table 7.2. Effect on renal function in control, Ibuprofen, hexane and

Ibuprofen+hexane pretreated rats.


200mg/kg

Hexane

200mg/kg

Ibu+Hex.

200mg/kg

Plasma Urea

(mg%) ***59.1

b±0.493 ***68.5

a±0.401 ***56.9

c±0.596 ***54.4

d±3.961

Plasma

Creatinine

(mg%)

***0.54c±0.046 ***1.8

a±0.692 ***0.67

b±0.448 ***0.43

d±0.443


significantly.

128

Table 7.3. Effect on tissue SOD and catalase in control, Ibuprofen, hexane and

Ibuprofen+hexane pretreated rats.


200mg/kg

Hexane

200mg/kg

Ibu+Hex.

200mg/kg

SOD

(u/g tissue) ***17.19

b±1.01 ***13.35

d±0.98 ***14.11

c±2.49 ***19.69

a±1.12

Catalase

(mM/g tissue)

***3.11b±0.008 ***1.99

d±0.63 ***2.99

c±0.68 ***3.50

a±0.17


significantly.

129

Table 7.4. Effect on plasma MDA, tissue MDA, 4HNE, and glutathione levels in

control, Ibuprofen, hexane and Ibuprofen+Hexane pretreated rats.


200mg/kg

Hexane

200mg/kg

Ibu+Hex.

200mg/kg

Plasma MDA

(nM/ml)

***3.31b±0.5

3 ***4.01

a±0.06 ***2.10

c±0.79 ***1.87

d±0.09

Tissue MDA

(nM/gm)

***0.55c±0.0

4 ***0.73

a±0.05 ***0.58

b±0.13 ***0.48

d±0.08

Tissue 4-

HNE (nM/g)

***159.61b±1

0.48

***210.14a±5.

23

***130.15c±11.

12

***143.15d±21.

25

Tissue GSH

(U/g tissue)

***3.56b±0.4

01 ***1.79

d±0.56 ***2.80

c±0.27 ***5.93

a±0.81


significantly.

130

Figure 7.1. Effect on Body weight of rats in Control, Ibuprofen, Hexane

and Ibuprofen + Hexane treated groups.

***a ***d ***c

***b

0

50

100

150

200

250

300

Control Ibuprofen Hexane Ibu+Hex

Me

an

Bo

dy

We

igh

t

(gm

)

Experimental Groups (200mg/kg body weight)

131

Figure 7.2. Effect on Kidney weight of rats in Control, Ibuprofen, Hexane

and Ibuprofen + Hexane treated groups.

***c

***b ***a ***a

0

0.2

0.4

0.6

0.8

1

1.2


Me

an

Kd

ne

y W

eig

ht

(gm

)


132

Figure 7.3. Effect on Plasma Urea level of rats in Control, Ibuprofen,

Hexane and Ibuprofen + Hexane treated groups.

***b

***a

***c ***d

0

10

20

30

40

50

60

70

80


Pla

sma

Ure

a

(mg

/dl)

Experimental Groups (200mg/kg. body weight)

133

Figure 7.4. Effect on Plasma Creatinine level of rats in Control, Ibuprofen,


***c

***a

***b

***d

-0.5

0

0.5

1

1.5

2

2.5

3


Pla

sma

Cre

ati

nin

e (

mg

/dl)


134

Figure 7.5. Effect on Tissue SOD level of rats in Control, Ibuprofen,


***b

***d ***c

***a

0

5

10

15

20

25


Tis

sue

SO

D l

ev

el

(U

/gm

tis

sue

)

Experimental Groups (200 mg/kg body weight)

135

Figure 7.6. Effect on Tissue Catalase level of rats in Control, Ibuprofen,


***b

***d

***c

***a

0

0.5

1

1.5

2

2.5

3

3.5

4


Tis

sue

Ca

tala

se l

ev

el

(m

mo

l/g

m

tiss

ue

)


136

Figure 7.7. Effect on Plasma MDA level of rats in Control, Ibuprofen,


***b

***a

***c ***d

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5


Pla

sma

MD

A l

ev

el

(n

mo

l/m

l)


137

Figure 7.8. Effect on Tissue MDA level of rats in Control, Ibuprofen,


***c

***a

***b

***d

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9


Tis

sue

MD

A l

ev

el

(n

mo

l/g

m t

issu

e)


138

Figure 7.9. Effect on Tissue 4HNE level of rats in Control, Ibuprofen,


***b

***a

***c ***d

0

50

100

150

200

250


Pla

sma

4H

NE

le

ve

l

(mg

/L)


139

Figure 7.10. Effect on Tissue GSH level of rats in Control, Ibuprofen,


***b

***d

***c

***a

0

1

2

3

4

5

6

7

8


Tis

sue

GS

H l

ev

el

(U

/gm

tis

sue

)


140

7.11. Results

According to one way Anova values of body and kidney weight differ significantly

(P<0.05) (Table 7.1, Fig. 7.1). Duncan’s multiple comparison test (DMCT) showed a

significant decrease in rat body weight in treated rats as compared to control (P<0.05).

Similarly marked decreased was observed in Ibu+hex and Ibuprofen respectively

(P<0.05) (Table 7.1, Fig. 7.1). According to DMCT kidney weight of treated rats was

significantly higher as compared to control (P<0.05). A marked increase in kidney

weight in hexane, Ibu+hex and Ibuprofen was observed respectively (P<0.05).

However, values of kidney weight were insignificant between hexane and Ibu+hex

(P<0.05) (Table 7.1, Fig. 7.2).

One way Anova showed, the values of plasma urea and plasma creatinine differ

significantly (P<0.05) (Table 7.2, Fig. 7.3). According to Duncan’s multiple

comparison test, Ibuprofen treated rats showed a marked increase in plasma urea level

as compared to control, hexane and Ibu+hex respectively (P<0.05). Similarly, plasma

creatinine level was significantly increased in Ibuprofen treated rats as compared to

hexane and control (P<0.05). While, a significant decrease was observed in Ibu+hex

than all the other treated and control rats (P<0.05) (Table 7.2, Fig. 7.4).

According to one way Anova values of SOD and catalase differ significantly (P<0.05)

(Table 7.3, Figs. 7.5, 7.6).While, Duncan’s multiple comparison test showed

significant increase in SOD level in Ibu+hex treated rats as compared to control,

hexane and Ibuprofen respectively (P<0.05) (Table 7.3, Fig. 7.5). However, increased

141

level of catalase was observed in Ibu+hex treated rats as compared to control, hexane

and Ibuprofen treated rats respectively (P<0.05) (Table 7.3, Fig. 7.6).

One way Anova showed a significant difference in plasma MDA, tissue MDA, tissue

4HNE and GSH levels (P<0.05). (Table 7.4, Figs. 7.7 – 10). Duncan’s multiple

comparison test showed a significantly increased level of plasma MDA in Ibuprofen

treated rats (P<0.05). Whereas, level of plasma MDA was significantly decreased in

control followed by hexane and Ibu+hex treated rats (P<0.05).

Similarly, tissue MDA level in Ibuprofen treated rats increased significantly as

compared to hexane, control and Ibu+hex respectively (P<0.05) (Table 7.4, Fig. 7.8).

The value of tissue 4HNE showed a significant increase in Ibuprofen treated rats

(P<0.05). While, the level of tissue 4HNe gradually decreased in control, hexane and

Ibu+hex treated rats respectively (P<0.05) (Table 7.4, Fig. 7.9).

On the other hand level of tissue GSH was significantly higher in Ibu+hex treated rats

as compared to control, hexane and Ibuprofen treated rats (P<0.05) (Table 7.4, Fig.

7.10).

142

7.12. Discussion

Nonsteroidal anti-inflammatory drugs (NSAIDS) are one of the sources for pre-renal

failure (Hoitsma et al., 1991). Ibuprofen (NSAID) is used to induce nephrotoxicity in

rats with pretreatment of C. procera leaf hexane extract. The antioxidant activity of

endogenous enzymes (SOD and Catalase) is evaluated. Presently increase in

antioxidant enzyme levels (SOD and Catalase) was observed after Ibuprofen+hexane

administration (P<0.05) (Table 7.3). While, SOD converts superoxide radicals into

H2O2 (Hydrogenperoxide) and serve as a first line of defense against ROS (Sen et al.,

2010).

Previously, it was concluded that accumulation of urea and creatinine is the indicator

of improper renal function (Javed et al., 2015). In the present study, serum urea and

creatinine levels (Table 5.2) were significantly increased (P<0.05) after the

administration of Ibuprofen and showing renal disorder, this result is also in

accordance with the previous findings of Mahalakshmi et al. (2010) where ibuprofen

was used as NSAID. The significant decrease in urea and creatinine values after

treatment of Ibuprofen with C. procera hexane extract also provide the evidence of

positive role to inhibit the toxicity in rats produced by Ibuprofen (P<0.05) (Table 7.2).

The reactive oxygen species (ROS) initiates the contraction of masangial cells which

change the filtration surface area and alter the ultrafiltration coefficient factor that

decreases the rate of glomerular filtration (Leena and Alaraman, 2005).

C. procera hexane extract prevented Ibuprofen induced decline in glutathione (GSH)

activity in the renal mitochondria of rats (P<0.05) (Table 7.4) which is also supported

143

by the report of Setty et al. (2007) where C. procera flower extract increased the

depleted concentration of GSH. Due to Ibuprofen administration different complexes

formed are taken up by renal cells and stabilized by intracellular GSH. GSH

Peroxidase present in the cytoplasm of the cells, removes H2O2 by coupling its

reduction to H2O with oxidation of GSH. In case of intracellular depletion the

complexes undergo the rapid transformation to receive metabolites, this depletion

seems to be the main factor that impairs antioxidant enzyme (Ozen et al., 2004; Ban et

al., 1994). It is also noteworthy that the presence of flavonoids in C. procera revealed

the correlation of antioxidant properties (Hesham et al., 2002; Javed et al., 2015)

which may ultimately useful for the treatment of kidney damage.

It is also shown that Ibuprofen administration is associated with increased formation

of free radicals, and with heavy oxidative stress (Chen et al., 1994; Setty et al., 2007;

Mahalakshmi et al., 2010; Javed et al., 2015).This will lead to oxidative damage of

cell components, like proteins and nucleic acids (Boya et al., 1999).

The high concentration of manonyldialdehyde (MDA) in kidney tissues may cause its

malfunction. Present findings (Table 7.4) showed decreased value of MDA in C.

procera treated tissue and plasma (P<0.05). These results are also supported with

previous findings of Roy et al. (2005) where, decrease in MDA level, treated with C.

procera latex was observed to correlate antioxidant acivity.

4-hydroxynonenal (4HNE) an unsaturated aldehyde is considerably more toxic for

cell in vivo, than MDA, it is very important to measure 4HNE levels (Ong et al.,

2000). In present study treatment by C. procera with Ibuprofen decreased the level

of 4HNE (P<0.05) (Table 7.4).

144

Thus, it is concluded that Ibuprofen induced nephrotoxicity in rats is the result of over

production of hydrogen peroxide and hydroxyl radical that may finally cause renal

oxidative stress. However, with the supplementation of C. procera oxidative stress

was significantly decreased by regularizing the levels of superoxide dismutase,

catalase, 4HNE and MDA.

145

8. GENERAL DISCUSSION

Phytochemically leaf and flower of C. procera are investigated and a large number of

chemical constituents are reported which exhibit diversity in quantitative and

qualitative values of carbohydrates, reducing and non reducing sugars, proteins, amino

acids and phenolic compounds.

At one side plant is considered as a tonic (Gholamshahi et al., 2014) due to the

presence of enormous amount of protein, amino acids, carbohydrates, reducing and

non reducing sugars. While on the other hand, plant usually serves against infectious

diseases caused by bacteria or fungi and other diseases related to free radical induced

oxidative stress (Patel et al., 2010; Moteriya et al., 2015). These properties of plant

may be correlated with the detected amount of phenolic acids and flavonoids, as

22ug/ml and 17.66ug/ml total phenol are detected from flower and leaf extracts

respectively. While, a large number of flavonoids (Table 3.3) were also detected from

flower and leaf.

Similarly, use of NSAIDs is very common in the treatment of rheumatism, pain, fever,

inflammation and cardiovascular diseases but over the counter (OTC) use for a long

period of time, is the beginning of the production of free radicals which may result in

the gastric or duodenal ulceration and severe complications such as perforation and

gastrointestinal hemorrhage as well as kidney failure (Hoitsma et al., 1991;Kamboj,

2000). A marked decrease was observed in body weight of Ibuprofen treated rats as

compared to control (P<0.05) (Table 7.1, Fig.7.1). This weight loss was restored by

146

the administration of pretreatment of C. procera leaf hexane with Ibuprofen (P<0.05)

(Table 7.1, Fig.7.1). Previously it was reported that Ibuprofen may cause

gastrointestinal disturbance due to damaging effect in gastrointestinal mucosa that

results in reduced ingestion of food (Mahalakshmi et al., 2010). This is the

consequence of inhibition of cyclooxygenase -1 enzyme system which protect

gastrointestinal lining from digestive chemicals. In the present study Ibuprofen treated

rats show increase in kidney weight as compared to control (P<0.05) (Table 7.1, Fig.

7.1). Similarly, a significant increase is also observed in plasma urea and creatinine

levels in Ibuprofen treated rats as compared to control (P<0.05) (Table 7.2, Fig. 7.2).

However, accumulation of urea and creatinine in plasma induce decrease in kidney

function which is an indication of decrease glomerular filtration rate due to

nephrotoxicity (Javed et al., 2015). However, Increased Ca+2

movement in the

masangial cells may also be a cause of reduced glomerular filtration rate (Stojiljkovic

et al., 2008; Javed et al., 2015). In the present study these increased levels are

neutralized by the administration of C. procera leaf hexane extract with Ibuprofen to

prevent the normal kidney function. While, Kaneko et al. (2008) found an opposite

relation between quantity of absorbed urea and rate of tubular urine flow.

There is an imbalance between oxidative stress occur in Ibuprofen treated rats,

which is responsible for the formation of reactive oxygen species (ROS). This was

determined by evaluating decreased level of catalase, super oxide dismutase (SOD),

Glutathione (GSH) and increased levels of malonyldialdehyde (MDA) and 4 hydroxy

nonenal (4HNE) (Table 7.3-7.4, Fig. 7.5-10). In order to counteract oxidative

imbalance due to administration of Ibuprofen, C. procera leaf hexane extract co-

147

administered with Ibuprofen to test rats which significantly restored decreased levels

of catalase and super oxide dismutase (SOD) (Table 7.3, 7.5-6). Whereas, catalase,

glutathione peroxidase and glutathione reductase belong to the endogenous type of

antioxidant defense system. While, SOD acts as a first line of defense against ROS

which traps superoxide radical and convert it into H2O2. However, increased amount

of hydrogen peroxide and hydroxyl radicals and decreased amount of glutathione are

responsible to initiate Ibuprofen nephrotoxicity (Parlakpinar et al., 2005). The higher

amount of glutathione peroxidase may reduces H2O2 to H2O with oxidation of

glutathione (GSH) and due to reducing nature, glutathione is one of the important

substance for maintaining cell sustainability (Sen et al., 2010).

Presently, a decrease in renal glutathione level is observed in Ibuprofen treated rats

(Table 7.4, 7.10). However, in some previous studies it was also observed that kidney

damage may cause due to increase in GSH levels (Antunes et al., 2000).

Similarly, in vivo lipid peroxidation can be evaluated by estimating lipid peroxidation

products malonyldialdehyde (MDA and 4 hydroxy nonenal 4HNE). However, Lipid

peroxidation is an important oxidative damage mechanism to cell structure which may

become the reason of cell disintegration. The presence of lipid peroxidation involves

generation and propagation of lipid radicals, collection of oxygen and shifting of

double bonds in unsaturated lipids, which leads to abolishtion of membrane lipids.

148

After this process a number of products are obtained which includes alkanes, ethers,

alcohols ketones and aldehydes (Dianzani and Barrera, 2008).

Figure 8.1. MDA inhibition, formation and metabolism: (black pathway) Proposed action of

C. procera extract which scavenges oxygen radical and convert lipid peroxide by reduction into

non toxic form of PUFA, PUFA peroxide radical is formed by the reaction of Ibuprofen with

PUFA, (blue pathway) MDA formed during the enzymatic biosynthesis of thromboxane

A2(TXA2) and 12-1-hydroxy-5,8,10-heptadecatrienoic acid (HHT) by in vivo decomposition of

arachidonic acid (AA) and longer PUFAs as a side products. Or by the non enzymatic production

of bicyclic endoperoxides during lipid peroxidation (red pathway). Formed MDA can be

enzymatically metabolized (green pathway). Enzymes responsible for the generation and

metabolism of MDA includes: cyclooxygenases (1), prostacyclin hydroperoxidase (2),

thromboxane synthase (3), aldehyde dehydrogenase (4), decarboxylase (5), acetyl CoA synthase

(6), and tricarboxylic acid cycle (7). (Modified from Ayala et al., 2014).

149

Figure 8.2. 4-HNE inhibition, production and metabolism. Proposed action of C. procera

extract on lipid acids to inhibit 4-HNE production, activation of 4-HNE production by Ibuprofen.

In plant enzymatic route to 4-HNE includes lipoxygenase (LOX),-hydroperoxide lyase (HPL),

alkenal oxygenase (AKO), and peroxygenases. 4-HNE metabolism may lead to the formation of

corresponding alcohol 1,4-dihydroxy-2-nonene (DHN), corresponding acid 4-hydroxy-2-nonenoic

acid (HNA), and HNE–glutathione conjugate products. 4-HNE conjugation with glutathione s-

transferase (GSH) produce glutathionyl-HNE (GS-HNE) followed by NADH-dependent alcohol

dehydrogenase (ADH-)catalysed reduction to glutathionyl-DNH (GS-DNH) and/or aldehyde

dehydrogenase (ALDH-)catalysed oxidation to glutathionyl-HNA (GS-HNA). 4-HNE is

metabolized by ALDH yielding HNA, which is metabolized by cytochrome P450 (CYP) to form 9-

hydroxy-HNA (9-OH-HNA). 4-HNE may be also metabolized by ADH to produce DNH.

(Modified from Ayala et al., 2014)

150

Figure 8.3. Lipid peroxidation process. During Initiation, Ibuprofen abstract the

hydrogen radical forming the carbon-centered lipid radical; the carbon radical tends to be

stabilized by a molecular rearrangement to form a conjugated diene (step 1). In the

propagation phase, lipid radical rapidly reacts with oxygen to form a lipid peroxy radical

(step 2) which abstracts a hydrogen from another lipid molecule generating a new lipid

radical and lipid hydroperoxide (step 3). In the termination reaction, C. procera extract

which contains flavonoids, donate a hydrogen atom to the lipid peroxy radical species

resulting in the formation of nonradical products (step 4) (Modified from Ayala et al., 2014)

151

RH + -- R + RH

R + O2 --- ROO

RH + ROO - RE _ ROOH

The end products of lipid peroxidation especially (HNE and MDA) cause protein

demage by addition reaction with lysine amino group, cystein sulfhydryl group and

histidine imidazole group (Esterbauer et al., 1991). In the present study MDA and 4

HNE levels found in higher levels in Ibuprofen treated rats (Table 7.4, 7.7-9), which

may cause damage to kidney tissues. While, co-administration of C. procera hexane

extract with Ibuprofen showed decreased value of MDA and 4 HNE (Table 7.4, 7.7-9)

which is the indication of nephro-protective activity of C. procera.

On the other hand in vitro lipid peroxidation inhibition activity also supports the in

vivo findings. The flower extract showed highest lipid peroxidation inhibition value

as compared to leaf extract. Lipid peroxidation of flower extract exhibited

concentration dependent increase in water, hexane and ethanol extracts (Table 6.1,

Fig. 6.1). However, a concentration dependent LPOI activity in leaf water and hexane

extract was observed (Table 6.2, Fig. 6.2). The highest inhibition activity in terms of

percentage was noted in leaf water (i.e., 75.11% with 10mg/ml) and leaf ethyl acetate

152

extract showed (75.11%, with 8mg/ml) as compared to ethanol and hexane. Similarly

BHA exhibited 85% inhibition activity as compared to ascorbic acid (75%) lipid

peroxidation inhibition activity. The results revealed that the test extracts have strong

potential to inhibit lipid peroxidation in vitro as well.

1,1-Diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging is a more sophisticated

method for in vitro determination of antioxidant ability. DPPH is a nitrogen

containing free radical which is largely being used to demonstrate antioxidant property

of any substance. It is also known that DPPH reacts quickly with substances which

have phenol group in their structure. It is a dark purple coloured solution which

contain an odd / unpaired electron which is responsible for transfer of electron from

antioxidant to DPPH radical, the colour of DPPH solution converts from purple to

yellow as the radical is scavenged by the antioxidant, due to this property DPPH is

being used for spectrophotometric analysis (Usmani, 2013; Kumar et al., 2013).

In the present study, flower extract showed highest scavenging activity as compared

to leaf extract (Table 6.3-4, Fig. 6.3-4). The highest activity is found in ethanol extract

(88.19%). While, leaf extract with water exhibited highest activity (75.95% with

2mg/ml). Whereas, BHA (85%) and ascorbic acid (75.5%) were used as standards

Table 6.3-4, Fig. 6.3-4).

Thus, C. procera found more effective as compared to standard DPPH values which

indicates that C. procera is more potent antioxidant for DPPH radical scavenging.

Besides this, in vitro antioxidant activity was also determined by the method of

reducing power capacity. The main principle of reducing power assay is that

153

compounds which have reduction potential may react with potassium ferricyanide

(Fe+3

) to form potassium ferrocyanide (Fe+2

) which then react with ferric chloride to

form ferrous complex. The colour intensity of the complex may be determined in

terms of absorbance at 532 nm. Higher absorbance indicates higher reduction

potential. The reducing ability (absorbance) of a given sample determines its

antioxidant potential with the ability to donate hydrogen atom to free radical chain

(Kumar et al., 2013; Patel et al., 2014).

In the present study different solvent extracts of C. procera flower and leaf were

analyzed for reducing power capacity. In general, flower shows highest reducing

power as compared to leaf extracts. However, C. procera flower and leaf water

extracts showed concentration dependent reducing power capacity (Table 6.5-6, Fig.

6.5-6).

Due to the global appearance of increasing drug-resistant cases there is a need to

search sources for production of natural antimicrobial compounds which may be

effective to destroy these drug resistant pathogens. During last few decades a lot of

natural antimicrobial compounds were discovered for the control of severe microbial

infections. Presently, the antimicrobial potential of the various fractions of C. procera

flower and leaf extracts against four human pathogenic strains viz., Salmonella typhi,

Escherecia coli, mithicillin resistant Staphylococcus aureus and Micrococcus luteus is

investigated. These pathogens are the major cause of infectious diseases such as M.

luteus is an opportunistic pathogen and cause infection in immune-compromised

individuals (Seifert et al., 1995). Similarly, E. coli is a toxin producing human

154

pathogen and cause severe diarrhoea leads to kidney failure through food. Whereas,

MRSA involved in various hospital acquired infections and found to be resistant to all

β-lactum antibiotics (Iqbal, 1998; Iqbal et al., 2005; Que and Moreillon, 2010).

However, S. typhi. mainly cause inflammatory bowel syndrome and typhoid fever.

Different soluble flower and leaf extracts of C. procera exhibited differential spectrum

of inhibition against all tested pathogenic strains. Similar to the previous findings of

Joshi and Kaur (2013), the hexane fractions of flower and leaf have been proved very

significant as an antimicrobial agent against all of the studied pathogens, as maximum

zone of inhibition was observed against M. luteus flower (22mm) and leaf ( 23mm).

While on the other hand, these findings are in contrast to the previous findings of

Parabia et al. (2008) and Doshi et al. (2011) where (7-9mm) maximum zone of

inhibition was observed.

Similarly, fractions of flower and leaf ethyl acetate showed significant inhibitory

activity against E. coli and MRSA (Table 4.1-2, Fig. 4.1-4). While, no inhibitory

activity was recorded against S. typhi. and only flower ethyl acetate fraction was found

to be significant inhibitor against M. luteus. Present inhibitory findings could be well

supported by the studies of Patil and Saini (2012) where they also found significant

role of ethyl acetate extract of C. procera against various pathogens. However,

aqueous leaf and flower extracts showed significant inhibitory activity only against M.

luteus. Whereas, butanol leaf extract showed insignificant inhibitory activities against

all of the studied pathogenic strains and flower butanol extract was found significant

only against M. luteus. Thus, amongst all of the tested extracts of C. procera flower

and leaf hexane, ethyl acetate, aqueous and butanol fractions were found significant

155

to inhibit the antibacterial activity against the studied pathogenic strains in respective

manner (Table 4.1-2, Fig. 4.1-4).

Besides the antibacterial and antioxidant properties C. procera is also effective for

enhancing various enzymatic activities such as, flower extract with concentration of

50mg/ml shows comparatively greater glucoamylase activity as compared to leaf

extract and after increasing the incubation time from 30-60 minutes, a marked

decrease in glucoamylase activity was observed (Table 5.1, Fig. 5.2). However, it was

found that the leaf and flower extracts did not cause any effect on α-amylase activity

after 30 minutes of incubation and results were remained same (P>0.05) (Table 5.2,

Fig. 5.3). An increase in α-amylase activity was noted when the enzyme was mixed

with leaf and flower extracts (25% and 46%) respectively for 60 minutes (P<0.05)

(Table 5.2, Fig. 5.3). While, urease activity was increased when leaf (11%) and flower

(25%) extracts were incubated for 30 minutes (P<0.05) (Table 5.3, Fig. 5.4). Whereas,

no effect on urease activity was reported at 60 minutes. Therefore, flower extract is

proven to be a good enhancer of glucoamylase, α amylase and urease as compared to

leaf.

156

CONCLUSION

It is concluded that the test extracts of C. procera leaf and flower have potential to

scavenge free radical and significant reducing power and also found effective to

inhibit lipid peroxidation in vitro. Results reveal that C. procera flower extracts have

more antioxidant and antibacterial potential than leaf extracts and this could be well

correlated with that of the presence of more phenolic compounds in flower extracts as

compared to leaf extracts. While, it is also established that C. procera could

completely protect nephrotoxicity induced by the long term use of nsaid (ibuprofen) in

a rat model.

Therefore, C. procera could be proposed as an antioxidant and antibacterial source of

natural origin, which may ultimately be much efficient than any synthetic drug.

157

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Pak. J. Pharm. Sci., Vol.27, No.5(Special), September 2014, pp.1565-1569 1565

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Antibacterial potential of Calotropis procera (flower) extract against various pathogens

Abid Ali1, Asma Ansari2, Shah Ali Ul Qader*2, Majid Mumtaz3, Sumayya Saied3 and Tabassum Mahboob1 1Department of Biochemistry, University of Karachi, Karachi, Pakistan 2The Karachi Institute of Biotechnology & Genetic Engineering (KIBGE), University of Karachi, Karachi, Pakistan 3Department of Chemistry, University of Karachi, Karachi, Pakistan

Abstract: Increased bacterial resistance towards commonly used antibiotics has become a debated issue all over the world in a last few decades. Due to this, consumer demand towards natural anti-microbial agents is increasing day by day. Natural anti-microbial agents have gained enormous attention as an alternative therapeutic agent in pharmaceutical industry. Current study is an effort to explore and identify a bactericidal potential of various solvent extracts of Calotropis procera flower. Flowers of C. procera were extracted with hexane, butanol, ethyl acetate and aqua to evaluate the antibacterial activity by agar well diffusion method against the various human pathogens. The microorganisms used in this study includes Salmonella typhi, Escherichia coli (O157:H7), Micrococcus luteus KIBGE-IB20 (Gen Bank accession: JQ250612) and methicillin resistant Staphylococcus aureus (MRSA) KIBGE-IB23 (Gen Bank accession: KC465400). Zones of inhibition were observed against all four pathogenic strains. Fraction soluble in hexane showed broad spectrum of inhibition against all the studied pathogens. However, fractions soluble in ethyl acetate inhibited the growth of E. coli, MRSA, and M. luteus. In case of butanol and aqueous extracts only growth of M. luteus was inhibited. Results revealed that the flower extracts of C. procera have a potential to be used as an antibacterial agent against these pathogenic organisms. Keywords: Calotropis procera, antibacterial potential, human pathogens, agar well diffusion method, hexane extract. INTRODUCTION In the last few decades, several new natural anti-microbial compounds were discovered for the control of severe infections. A discovery of new antibacterial agents against multidrug resistant organisms is still in full swing due to the development of continuous resistance developed by microbes. The multidrug resistant organisms have received great clinical attention because of increasingly reported cases around the globe. Along with this, there is an increase consumer demand for those drugs, which are isolated or derived from natural sources. Threat posed to general public health by various multidrug resistant organisms and pathogens can be resolved by the discovery of natural antibacterial compounds having effective broad spectrum inhibition against pathogens prevalent in the local community. The anti-microbial potential of Calotropis procera against human pathogens was previously investigated by several researchers. Calotropis procera belong to the family Asclepiadaceae and commonly known as “AAK”. The flower C. procera is widely distributed in Asia, Africa and Arab countries (Mohanraj et al., 2010). C. procera flowers (fig. 1) are arranged in terminal or axillary umbelloid cyme, consists

of five deeply lobed and dirty white sepals with purple tips and white base, corona of five fleshy laterally compressed lobes surrounding the pentagonal stigma (Ali 1983). C. procera is medicinally very important due to its anaesthetic properties (Kawo et al., 2009) and its crude extracts are commonly used in traditional medicines and also in veterinary practises (Dewan et al., 2000; Alencar et al., 2004; Kareen et al., 2008; Johnson et al., 2011). The milky sap of C. procera is also found to be very useful in alternative medicines (Goyal and Mathur, 2011). C. procera flowers are used as therapeutic agents to treat inflammation (Mascolo et al., 1988; Basu and Chaudhuri 1991; Neenah and Ahmed, 2011), cholera, wound, piles and asthma (Mohanraj et al., 2010). Sharma et al. (2001) and Mohanraj et al. (2010) also reported the use of C. procera as appetizer and tonic. Beside this the extracts of C. procera also used as an antibacterial agent against Gram’s positive and Gram’s negative bacteria (Mascolo et al., 1988; Sharma et al., 2001; Parabia et al., 2008; Devi et al., 2008; Varahalarao and Naido, 2010; Johnson et al., 2011; Ahmed et al., 2011; David et al., 2011; Doshi et al., 2011; Patil and Saini, 2012). The present study is an effort to evaluate the antibacterial potential of C. procera using different solvent fractions of flowers with butanol, hexane, ethyl acetate and aqueous against various human

*Corresponding author: e-mail: [email protected]



pathogens to substantiate the earlier findings for its significant use. MATERIALS AND METHODS

Plant materials The fresh flowers of Calotropis procera were collected from natural population growing around the vicinity of Karachi during 2010-2011. Voucher specimens were deposited in the Karachi University Herbarium (G.H. No. 86455).

Extract preparation About eight kilo-gram flowers of C. procera were collected and washed properly with tap water. The flowers were air dried at room temperature for one month. The dried flowers were then crushed into fine powder with the help of grinder. About 700gms of the dried flower was soaked in 80% ethanol for ten days. To obtain crude extract, the sample was filtered through a filter paper. The extract was concentrated by using Buchi Rotavapor R-200 (Buchi Labortechnik AG, Switzerland) rotary evaporator. The resulting residues were stored at 4°C until used for fractionation. Fraction preparation The ethanol concentrated extract was used for fractionation using separating funnel. A series of solvents were used to separate different fractions soluble in hexane, ethyl acetate and butanol. Aqueous fraction was collected during separating funnel fractionation. Fraction of hexane and ethyl acetate was concentrated on Buchi Rotavapor R-200 while butanol fraction was concentrated with the help of Eyela Rotary Vacuum Evaporator (Model No. N-10, Tokyo Rikakikai Co. Ltd. Japan). The resulting residues were then dried until it turns into solid form. The solid residue was stored at 4°C.

Indicator organisms The anti-bacterial activity of flower extracts was determined against four human pathogenic bacterial strains. Salmonella typhi and Escherichia coli (O157:H7) were Gram’s negative organisms isolated from contaminated water samples. Whereas, Micrococcus luteus KIBGE-IB20 (GenBank accession: JQ250612) and methicillin resistant Staphylococcus aureus (MRSA) KIBGE-IB23 (Gen Bank accession: KC465400) were Gram’s positive organisms isolated from soil sample and clinical specimen respectively. Culture conditions For the revival of the culture, all the strains were grown in nutrient broth at 37°C for 24 hours with the agitation of 135 rpm. For further studies strains were maintained on nutrient agar slants at 4°C. Anti-microbial activity assay To determine the anti-microbial potential of flower extracts fractionated in different solvents, agar well

diffusion method (Tagg and Mcgiven, 1971) was performed. Nutrient agar was poured in sterilized plates and was incubated at 37°C for 24 hours. Next day wells were punctured on nutrient agar plates previously spreaded with 100µl culture of each indicator strain containing 108cfu/ml compared with the 0.5 McFarland turbidity index. Concentrated fractions (100µl) were added in wells and plates were incubated at 37°C for 24 hours. Solvents without flower extracts were used as a negative control. Zones of inhibition were measured in millimeters to determine the anti-microbial activity. The values presented in table are means of three replicate experiments with the standard deviation of ±3. RESULTS The Current study was designed to explore the anti-bacterial potential of medicinally important flower C. procera against various pathogenic as well as drug resistant organisms of our community. Different soluble flower extracts of C. procera showed differential spectrum of inhibition against S. typhi, E. coli, methicillin resistant S. aureus (MRSA) and M. luteus (table 1). Amongst all the extracts, hexane fraction has been proved very significant as an antibacterial agent against all the studied pathogens. Maximum zone of inhibition (22mm) was observed against M. luteus (fig. 1). Butanol and Aqua fractions also exhibited inhibitory activity against M. luteus whereas; other indicator strains were resistant to both fractions. Fraction of ethyl acetate showed inhibitory activity not only against M. luteus (25mm) but also against MRSA (18mm) and E. coli (15mm). DISCUSSION Resistance to different broad-spectrum antibiotic has now become a global concern due to emerging cases of drug resistance (Mohanraj et al., 2010). Due to these emerging cases and also due to the increase consumer demand towards natural antibacterial agents there is a need of screening of natural anti-microbial compounds effective against different drug resistant pathogens. In the last few decades; several new natural anti-microbial compounds were discovered for the control of severe infections. Keeping this in view, the present study was designed to explore the anti-bacterial potential of medicinally important flower C. procera. Different soluble flower extracts of C. procera showed differential spectrum of inhibition against tested pathogenic organisms. Amongst all the extracts, hexane fraction has been proved very significant as an antibacterial agent against all the studied pathogens. Maximum zone of inhibition was observed against M. luteus which is an opportunistic pathogen and can cause infections in immune-compromised individuals (Seifert et

Abid Ali et al


al., 1995). It is also noteworthy that present findings are in contrast to the earlier findings (Parabia et al., 2008) where hexane fraction of apical twig showed least antibacterial activity (7mm) against M. luteus. Fraction of ethyl acetate showed inhibitory activity not only against M. luteus but also against MRSA and E. coli, which are complementary with the previous study (Patil and Saini, 2012). E. coli is a toxin producing human pathogen. E. coli (O157:H7) is an enteric hemorrhagic strain and cause severe diarrhea leads to kidney failure through food. However, MRSA is also a potent human

pathogen, involved in various hospital acquired infections and found to be resistant to all β-lactam antibiotics (Que and Moreillon, 2010; Iqbal et al., 2005) but in the current study ethyl acetate and hexane extracts of C. procera significantly inhibited the growth of this multidrug resistant organisms. Varahalarao and Naido (2010) demonstrated the antibacterial potential of extracts of C. procera extracted in hexane, chloroform and methanol against Alternaria alternate, Aspergillus flavus, Aspergillus niger, Bipolaris bicolor, Curvularia lunata, Penicillin expansum, Pseudomonas marginalis and Rhizoctonia solani. In another study ethanolic flower

Table 1: Antibacterial activity of flower extracts against different pathogenic strains.

Extracts Zones of inhibition (mm) Salmonella typhi control Escherichia coli control MRSA control Micrococcus control

Butanol -ve -ve -ve -ve -ve -ve 30 -ve Ethyl acetate

-ve -ve 15 -ve 18 -ve 25 -ve

Aqua -ve -ve -ve -ve -ve -ve 30 -ve Hexane 13 -ve 12 -ve 15 -ve 22 -ve

Key: MRSA: Methicillin resistant Staphylococcus aureus, Significant zone: > 11 mm, -ve: No activity detected.

A B

C D

Fig. 1: Zone of inhibitions of flower extracts of Calotropis procera against various pathogens using agar well diffusion assay. Micrococcus luteus (A), Salmonella typhi (B), E.coli (C), MRSA (D).



extract was used against the larvae of A. stephansi (Doshi et al., 2008). Parabia et al. (2008) used acetone, methanol, ethanol, hexane, chloroform and ethyl acetate fractions against Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Pseudomonas aeroginosa, Kleibsiella pneumonia, Serratia marcenes, Bacillus subtilis and Micrococcus luteus. Davis (2008) reported the anti-fungal potential of water, methanol and ethyl acetate flower extracts against Fusarium and T. vesiculatum. However, acetone and methanolic flower extracts were used against Bacillus pumilis, E.coli, A. niger, Fusarium oxysporum, (David et al., 2011) Salmonella para typhi A, Salmonella para typhi B, Bacillus subtilis, Bacillus thuringiensis, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeroginosa, S. aureus and E. coli (Prabha et al., 2012). After reviewing the antibacterial potential C. procera it is concluded that flower extracts of C. procera found to be highly effective not only against the common human pathogenic organisms of our community but also against multidrug resistant organism. In a nutshell, extracts of C. procera can be used to treat infections caused by aforementioned organisms after performing its characterization and clinical trials. ACKNOWLEDGEMENT Authors are indebted to the Dr. Iqbal Chaudhry, Director, HEJ Research Institute of Chemistry, University of Karachi, for providing facility of Eyela Rotary Vacuum Evaporator Model No. N-10, Tokyo Rikakikai Co. Ltd. Japan. This paper is a part of PhD thesis of first author. REFERENCES Ahmad N, Anwar F, Hameed S and Boyce MC (2011).

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