ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ...prr.hec.gov.pk/jspui/bitstream/123456789/2370/1/2828S.pdfMehmood, Aisha Ashraf for their assistance, good company, marvelous behavior

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ANTIOXIDANT AND ANTIMICROBIAL

ACTIVITIES OF SELECTED PLANTS OF

POTHOHAR PLATEAU

By

HUMA MUNIR

M. PHIL. (UAF)

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSPHY

IN

CHEMISTRY

DEPARTMENT OF CHEMISTRY & BIOCHEMISTRY

FACULTY OF SCIENCES

UNIVERSITY OF AGRICULTURE,

FAISALABAD, PAKISTAN

2014

ii

To

The Controller of Examination,

University of Agriculture,

Faisalabad.

“We, the Supervisory Committee, certify that the contents and form of the thesis

submitted by Miss Huma Munir, Reg. No. 2005-ag-187, have been found

satisfactory and recommend that it be processed for evaluation, by the External

Examiner(s) for the award of Ph.D degree”.

Supervisory Committee

1. Chairman ______________________

(Dr. Raja Adil Srafraz)

2. Co-supervisor ______________________

(Dr. Abdullah Ijaz Hussain)

3. Member ______________________

(Dr. Bushra Sultana)

4. Member ______________________

(Dr. Muhammad Shahid)

iii

Acknowledgement

I bow my head before Almighty Allah, The omnipotent, The omnipresent, The merciful, The

most gracious, The compassionate, The beneficent, who is the entire and only source of every

knowledge and wisdom endowed to mankind and who blessed me with the ability to do this

work. It is the blessing of Almighty Allah and His Prophet Hazrat Muhammad (Sallallaho

Alaihe Wasallam) which enabled me to achieve this goal.

I would like to take this opportunity to convey my cordial gratitude and appreciation to my

worthy, reverently and zealot supervisor Dr. Raja Adil Sarfraz, Assistant Professor,

Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan.

Without whose constant help, deep interest and vigilant guidance, the completion of this thesis

was not possible. I am really indebted to him for his accommodative attitude, thought

provoking guidance, immense intellectual input, patience and sympathetic behavior.

I would like to pay my deepest gratitude and appreciation to one of the member of my

supervisory committee Dr. Muhammad Shahid, Associate Professor, Department of

Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan, for his valuable

assistance, technical suggestions and kind help during my research work.

I am also very grateful to Dr. Bushra sultana and Dr. Abdullah Ijaz Hussain for their kind

cooperation during the research work and thesis write-up. I am also very thankful to the staff

members of Hi-Tech Lab, University of Agriculture, Faisalabad, for providing me facilities

during the whole study period. Special thanks to all of my teachers who taught me during my

academic career. I am also very much grateful to Higher Education Commission (HEC),

Pakistan, for awarding me the Indigenous PhD Fellowship.

I am also thankful to my friends and fellows especially, Faiza Nazir, Nadia Noor, Ayesha

Mehmood, Aisha Ashraf for their assistance, good company, marvelous behavior and friendly

attitude.

I really acknowledge and offer my heartiest gratitude to all members of my family especially,

my brother (Muhammad Iftkhar Ahmad), sisters (Hina, Sana, Rabia and Esha) and my

husband (Haider Ali Haider) for their huge sacrifice, moral support, cooperation,

encouragement, patience, tolerance and prayers for my health and success which enabled me

to achieve this excellence. I sincerely appreciate my husband Haider Ali Haider for his quick

help, whenever required.

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Lastly and most importantly I wish to say a big 'thank you' to my parents. They have always

supported my dreams and aspirations. I would like to thank them for all they are and all they

have done for me that has made my journey possible. Thank you my dear mother and father!

Huma Munir

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Dedicated To

My loving parents

vi

Abstract

Pothohar plateau has specific agro climatic and geographical environment which might affect

the antioxidant and antimicrobial attributes of native plants. In the present study, three

medicinal plants (Asphodilus tenifolius, Aerva javanica and Fagonia indica) were collected

from Pothohar plateau on the basis of ethno-botanical uses. Different solvent (absolute and

aqueous ethanol, absolute and aqueous methanol and absolute and aqueous acetone) extracts

of all the plants obtained through different extraction techniques (Stirring, orbital shaking,

sonication and reflux) were evaluated for their antioxidant activities (by 2, 2-diphenyl-1-

picrylhydrazyl and superoxide radical scavenging assays, reducing power and ß-carotene

linoleic acid system), antimicrobial activities (by Disc diffusion and Minimum Inhibitory

Concentration assays) and phytochemical studies (total phenolic and flavonoid contents).

All the plant extracts exhibited good antioxidant and antimicrobial activities. Aqueous solvent

extracts showed better activities as compared to pure solvent extracts. Out of four extraction

techniques, extracts obtained from sonication exhibited better activities and phytochemical

constituents. In case of Asphodilus tenifolius aqueous ethanol while for Aerva javanica and

Fagonia indica aqueous methanol proved to be best solvent for the extraction of bioactive

compounds. High Performance Liquid Chromatography analysis also revealed the presence of

phenolic acids in these plant extracts. The variation in yield, phytochemical constituents and

biological activities of most of the plant extracts, with respect to solvent systems and plant

species were statistically significant.

vii

Contents Introduction………………………………………………………………………………......1

Review of Literature…………………………………………………………………………7

2.1. Pothohar plateau…………………………………………………………………………..7

2.2. Medicinal plants…………………………………………………………………………..9

2.3. Asphodilus tenifolius…………………………………………………………………….10

2.3.1. Biological classification/ Taxonomy ............................................................................ 10

2.3.2. Description .................................................................................................................... 10

2.3.3. Ethnobotanical uses ...................................................................................................... 11

2.3.4. Chemical composition .................................................................................................. 11

2.3.5. Biological testing .......................................................................................................... 11

2.4. Aerva javanica ..................................................................................................................12


2.4.2. Description .................................................................................................................... 12

2.4.3. Ethnobotanical uses ...................................................................................................... 12



2.5. Fagonia indica ..................................................................................................................14


2.5.2. Description .................................................................................................................... 14

2.5.3. Ethnobotanical use ........................................................................................................ 14



2.7. Reactive oxygen species (ROS) ........................................................................................15

2.8. Antioxidant .......................................................................................................................17

2.9. Synthetic antioxidants .......................................................................................................17

2.10. Natural antioxidants ........................................................................................................18

2.11. Phenolic compounds .......................................................................................................18

2.11.1. Phenolic acids ..............................................................................................................19

2.11.2. Flavonoids ....................................................................................................................20

2.12. Antimicrobial activity .....................................................................................................23

2.13. Extraction ........................................................................................................................24

2.13.1. Extraction solvents ...................................................................................................... 25

2.13.2. Extraction techniques .................................................................................................. 26

viii

2.14. Antioxidant activity determination assays ......................................................................27

2.14.1. DPPH free radical scavenging assay ........................................................................... 27

2.14.2. Superoxide radical scavenging assay .......................................................................... 28

2.14.3. Reducing power assay (FRAP) ................................................................................... 29

2.14.4. Beta carotene linoleic acid assay ................................................................................ 29

2.14.5. Folin-Ciocalteu method .............................................................................................. 29

2.15. HPLC analysis ................................................................................................................30

2.15.1. Gallic acid ................................................................................................................... 30

2.15.2. P-coumaric acid .......................................................................................................... 31

2.15.3. Caffeic acid ................................................................................................................. 31

2.15.4. Ferulic acid.................................................................................................................. 32

2.15.5. Vanillic acid ................................................................................................................ 32

2.15.6. Chlorogenic acid ......................................................................................................... 32

2.15.7. Syringic acid ............................................................................................................... 32

Material and Methods .......................................................................................................... 34

3.1. Collection of Plants ...........................................................................................................34

3.2. Materials used in the whole research work .......................................................................34

3.3. Washing and Drying of plant samples ..............................................................................37

3.4. Extraction ..........................................................................................................................37

3.4.1. Stirring as extraction technique .................................................................................... 37

3.4.2. Orbital shaking as extraction technique ........................................................................ 37

3.4.3. Sonication as extraction technique................................................................................ 37

3.4.4. Reflux as extraction technique ...................................................................................... 38

3.5. Phytochemical Studies ......................................................................................................38

3.5.1. Determination of total phenolic contents (TPC) ........................................................... 38

3.5.2. Determination of total flavonoid contents (TFC) ......................................................... 39

3.6. Antioxidant activity ..........................................................................................................40

3.6.1 TLC based DPPH radical scavenging assays................................................................. 40

3.6.2. DPPH free radical scavenging activity (Spectrophotometric method) ......................... 40

3.6.3. Superoxide radical scavenging (SOR) activity ............................................................. 40

3.6.4. Determination of reducing power (FRAP contents) ..................................................... 41

3.6.5. Antioxidant activity determination in a ß-carotene linoleic acid system ...................... 42

3.7. Evaluation of Antimicrobial Activity ...............................................................................42

3.7.1. Antibacterial activity ......................................................................................................42

ix

3.7.1.1. Bacterial Strains ......................................................................................................... 42

3.7.1.2. Bacterial culture preparation ...................................................................................... 42

3.7.1.3. Disc diffusion assay for antibacterial activity ............................................................ 43

3.7.1.4. Spectrophotometric assay (Determination of MIC) ................................................... 43

3.7.2. Antifungal activity .........................................................................................................44

3.7.2.1. Fungal strains used ..................................................................................................... 44

3.7.2.2. Fungal inoculum preparation ..................................................................................... 44

3.7.2.3. Disc diffusion assay for antifungal activity ............................................................... 44

3.7.2.4. Spectrophotometric assay (Determination of MIC) ................................................... 44

3.8. High Performance Liquid Chromatography (HPLC) .......................................................45

3.8.1. Sample preparation for HPLC analysis ......................................................................... 45

3.8.2. Standard used ................................................................................................................ 45

3.9. Statistical Analysis ............................................................................................................47

Results and Discussion .......................................................................................................... 48

4.1. Extraction yield .................................................................................................................48

4.2. Phytochemical Studies ......................................................................................................53

4.2.1. Total phenolic contents ..................................................................................................53

4.2.2. Total Flavonoid contents................................................................................................59

4.3. Evaluation of Antioxidant activity ....................................................................................64

4.3.1. TLC based DPPH free radical scavenging assay .......................................................... 65

4.3.2. DPPH free radical scavenging activity (Spectrophotometric method) ......................... 66

4.3.3. Superoxide radical (SOR) scavenging assay .................................................................70

4.3.4. Reducing power assay (FRAP contents)........................................................................75

4.3.5. Antioxidant Activity Determination in a ß-Carotene Linoleic Acid System .................80

4.4. Evaluation of Antimicrobial activity ................................................................................85

4.4.1. Antibacterial activity ......................................................................................................85

4.4.2. Antifungal activity .......................................................................................................104

4.5. HPLC analysis of different solvent extracts of selected medicinal plants……………..126

Summary……………………………………………………………………………..........139

Literature cited..…………………………………………………………………………. 142

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List of Figures

2.1. Map of Punjab showing Pothohar region………………………………………..……….8

2.2. Asphodilus tenifolius……………………………………………………………..……...10

2.3. Aerva javanica……………………………………………………….…….….…...…….12

2.4. Fagonia indica…………………………………………………………………......…....14

2.5. Diseases caused by oxidative stress in humans….…………………………………........16

2.6. Structure of phenolic acids…………………………………………….……...................20

2.7. Basic skeleton of flavonoids………………………………………………………......…21

2.8. Basic structure and examples of different classes of flavonoids…………………………22

2.9. Chemical structure of gallic acid…………………………………...………………….…31

2.10. Chemical structure of p-coumaric acid………………………………….………......….31

2.11. Chemical structure of caffeic acid………………………………………………………31

2.12. Chemical structure of ferulic acid…………………………………………...….………32

2.13. Chemical structure of vanillic acid…………………………………………………......32

2.14. Chemical structure of syringic acid……………………………………………………..33

3.1. Standard curve of gallic acid for total phenolic contents……………………………......39

3.2. Standard curve of quercetin for total flavonoid contents…………….....………………..39

3.3. Standard curve of gallic acid for FRAP contents……………………..…………………41

3.4. HPLC chromatograms of standard phenolic acids used in the present research work (a)

gallic acid, (b) caffeic acid, (c) vanillic acid, (d) 4-hydroxy-3-metoxy benzoic acid, (e)

chlorogenic acid, (f) syringic acid, (g) p-coumaric acid, (h) m-coumric acid and (i) ferulic

acid………………………………………………………………………………...................46

4.1.1. Effect of different solvent systems on extraction yields (g/100g) of different plants using

stirring as extraction technique………………………………………………………….……49


orbital shaking as extraction technique……………………………………………...……….49


sonication as extraction technique…………………………………………………………...50


reflux as extraction technique…………………………………...……………………………51

4.2.1. Representative TLC plate showing TLC-DPPH free radical scavenging activity of plant

extracts ……………………………………………………….……………………………...65

4.3.1 DPPH Free radical scavenging activities (%inhibition) of different solvent extracts of

medicinal plants using stirring as extraction technique……………………………………….66


medicinal plants using orbital shaking as extraction technique………………..…………….67


medicinal plants using sonication as extraction technique…………………………..……….68

4.3.4. DPPH free radical scavenging activities (%inhibition) of different solvent extracts of

medicinal plants using reflux as extraction technique……………………………..…………69

xi

4.4.1 Superoxide radical scavenging activity (%inhibition) of different plant extracts using

stirring as extraction technique………………………………….…………………………...71

4.4.2. Superoxide radical scavenging activity (%inhibition) of different plant extracts using

orbital shaking as extraction technique……………………………………………………….72

4.4.3 Superoxide radical scavenging activity (%inhibition) of different plant extracts using

sonication as extraction technique………………………………….………………………..73

4.4.4. Superoxide radical scavenging activity (%inhibition) of different plant extracts using

reflux as extraction technique…………………………………………………………...……74

4.5.1. Representative HPLC chromatogram of absolute ethanolic extract of Asphodilus

tenifolius through sonication...……………………………………………..……………….136

4.5.2. Representative HPLC chromatogram of absolute methanolic extract of Asphodilus

tenifolius through sonication………………………………………………………………..136

4.5.3 Representative HPLC chromatogram of aqueous ethanolic extract of Aerva javanica

through stirring……………………………………………………………………………...137

4.5.4. Representative HPLC chromatogram of aqueous methanolic extract of Fagonia indica

through sonication…………………………………………………………………………..137

4.5.6. Representative HPLC chromatogram of aqueous acetone extract of Fagonia indica

through sonication ………………………………………………………………………….138

xii

List of Tables

3.1. Selected medicinal plants from Pothohar plateau, Pakistan…………………………….34

3.2.1. Materials used in the whole research work……………………………………………34

3.2.2. Standard compounds used in the research work……………………………………....36

3.2.3. Instruments used in the research work……………………………………………..….36

3.3.1. Specifications for HPLC analysis for phenolic compounds…………………………..45

4.1.1. Total phenolic contents (mg GAE/g of plant extract) of different solvent extracts using

stirring as extraction technique……………………………………………………………....54


orbital shaking as extraction technique…………………………………………………....….55


sonication as extraction technique……………………………………………………...…….56


reflux as extraction technique………………………………………………………………...57

4.2.1. Total flavonoid contents (mg QE/g of plant extract) of different solvent extracts using

stirring as extraction technique……………………………………………………………….60


orbital shaking as extraction technique……………………………………………………….61


sonication as extraction technique………………………………………………..……….….62


reflux as extraction technique…………………………………………………………..…….63

4.3.1. FRAP contents (mg GAE/g) of different solvent extracts using stirring as extraction

technique………………………………………………………..……………………………76

4.3.2. FRAP contents (mg GAE/g) of different solvent extracts using orbital shaking as

extraction technique………………………………………….………………………………77

4.3.3. FRAP contents (mg GAE/g) of different solvent extracts using sonication as extraction

technique…………………………………………………..…………………………………78

4.3.4. FRAP contents (mg GAE/g) of different solvent extracts using reflux as extraction

technique………………………………………………..……………………………………79

4.4.1. Effect of different solvent systems on the antioxidant activity coefficient (AAC) of

different plants using stirring as extraction technique………………………………………..81


different plants using orbital shaking as extraction technique…………………………….…82


different plants using sonication as extraction technique………………………………….…83


different plants using reflux as extraction technique………………………………………….84

4.5.1. Antibacterial activity of different plant extracts using stirring as extraction technique

against Escherichia coli……………………………………………………………………...86

4.5.2. Antibacterial activity of different plant extracts using orbital shaking as extraction

technique against Escherichia coli ………………………………………………………….87

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4.5.3. Antibacterial activity of different plant extracts using sonication as extraction technique

against Escherichia coli……………………………………………………………………..88

4.5.4. Antibacterial activity of different plant extracts using reflux as extraction technique

against Escherichia coli …………………………………………………..………………….89


against Pasteurella multocida…………………………………………..……………………90


technique against Pasteurella multocida……………………………….……………………91


against Pasteurella multocida………………………………………………………………..92


against Pasteurella multocida………………………………………………………………..93


against Bacillus subtillus……………………………………………………………………..94


technique against Bacillus subtillus…………………………………………………..………95






against Staphylococcus aureus……………………………………………………………….98


technique against Staphylococcus aureus…………………………………………………....99

4.8.3. Antibacterial activity of different plants using sonication as extraction technique against

Staphylococcus aureus……………………………………………………….………….….101


against Staphylococcus aureus……………………………………………………………...102

4.9.1. Antifungal activity of different plant extracts using stirring as extraction technique

against Aspergillus flavus…………………………………………………………………..106

4.9.2. Antifungal activity of different plant extracts using orbital shaking as extraction

technique against Aspergillus flavus……………………………………………………….107

4.9.3. Antifungal activity of different plant extracts using sonication as extraction technique

against Aspergillus flavus…………………………………………………………………..108

4.9.4. Antifungal activity of different plant extracts using reflux as extraction technique against

Aspergillus flavus…………………………………………………………………….……..109


against Aspergillus niger…………………………………………………………………....111


technique against Aspergillus niger…………………………………………………..…….112


against Aspergillus niger……………………………………………………………………113

4.10.4. Antifungal activity of different plant extracts using reflux as extraction technique

against Aspergillus niger……………………………………………………………………114


against Fusarium solani……...…………………………………………………………......116

xiv


technique against Fusarium solani………………………………………………………….117


against Fusarium solani………………………………………………………………....….118


against Fusarium solani……………………………………………………………..……119


against Helminthus spermium………………………………………………………………121


technique against Helminthus spermium……………………………………………………122


against Helminthus spermium………………………………………………………………123


against Helminthus spermium………………………………………………….………..….124

4.13.1. Phenolic compounds in different solvent extracts of Asphodilus tenifolius obtained

through sonication…………………………………………………………………………..128

4.13.2. Phenolic compounds in aqueous ethanolic extracts of Asphodilus tenifolius obtained

through different extraction techniques……………………………………………………..129

4.14.1. Phenolic compounds in different solvent extracts of Aerva javanica obtained through

sonication…………………………………………………………………………………...131

4.14.2. Phenolic compounds in aqueous ethanolic extracts of Aerva javanica obtained through

different extraction techniques…………………………………………………..………….132

4.15.1. Phenolic compounds in different solvent extracts of Fagonia indica obtained through

sonication……………………………………………………………………..…………….134

4.15.2. Phenolic compounds in aqueous ethanolic extracts of Fagonia indica obtained through

different extraction techniques………………………………………..…………………….135

1

Chapter 1

Introduction

Plants are used for curative purposes in both organized (Ayurveda, Unani) as well as

unorganized (folk, tribal, native) forms since ancient times (Girach et al., 2003). They play

very important role in the lives of humans and animals because of their use in the medicines

(Arshad et al., 2011). Medicinal plants are used for the treatment of many kinds of diseases all

over the world (Ghasemzadeh et al., 2010; Razaq et al., 2010; Mahmoud et al., 2011; Kamba

and Hassan, 2011). These are valuable natural resources considered as safe drugs playing an

important role in curing human diseases (Ahmad et al., 2007). Herbal drugs have proved to be

effective and have no side effects, having beneficial effects by the combination of bioactive

compounds with vitamins and minerals (Ahmad et al., 2008). The medicinal importance of

plants is due the phytochemicals particularly secondary metabolites produced by these plants

(Jayaraman et al., 2008; Mohammedi and Atik, 2011) and used as antimicrobials, pesticides

and as pharmaceutical agents in traditional medicine (Walteri et al., 2011).

Use of medicinal plants in the industrialized societies have directed towards the

extraction and development of drugs from these natural sources (Menghani et al., 2012). Plant

extracts have proved to be a potential source of natural products. They have been analyzed for

their medicinal activities, against many infections and also for their antioxidant activity. These

activities formed the basis of the use of plants in pharmaceuticals, and natural therapy

(Muthukumaran et al., 2011). Plants produce a variety of biochemical compounds, have

varying biological activities (Arshad et al., 2011) as a result of extreme environmental

conditions (Abutbul et al., 2005). Bioactive compounds are produced in all parts of plants such

as leaves, barks, stem, seeds ant roots. These compounds have much importance in the

treatment of various diseases of humans and animals (Khan et al., 2012).

Oxygen is very important for normal physiological and metabolic activities. About 5%

of it reduced to reactive oxygen species like hydrogen peroxide, superoxide and hydroxyl free

radicals. (Zia-ul-Haq et al. 2012; Sethi and Sharma, 2011). Free radicals are also formed due

to exposure to cigarette, automobile exhaust, smoke, air pollution and radiations (Praveen et

al., 2007). Excessive production of these free radicals or reactive oxygen species more than the

2

antioxidant capacity of biological system give rise to oxidative stress. This oxidative stress

causes oxidation of biomolecules like protein, carbohydrates and lipids leading to a number of

physiological disorders including cancer, diabetes, hepatotoxicity, inflammation, ageing,

cardiovascular and neurodegenerative diseases (Sethi and Sharma, 2011; Rakesh et al., 2010).

Antioxidant protect from these free radicals by neutralizing them (Bhanot et al., 2011; Zia-ul-

Haq et al. 2012). Antioxidants act by preventing of chain initiation, binding of pro-oxidant

metals, decomposing of peroxides, reducing ability and radical scavenging (Pracheta et al.,

2011). Synthetic antioxidants like propyl gallate (PG) butylated hydroxyanisole (BHA),

butylated hydroxyl toluene (BHT) are added in foods to prevent oxidation, however, these are

carcinogenic and have many other side effects (Jindal et al., 2012).

Infectious diseases are a major cause of unhealthiness and even deaths throughout the

world. Pathogenic microbes such as Escherichia coli (E. coli), Staphylococcus aureus (S.

aureus) and Bacillus subtilis (B. subtillus) are widely spread in environment responsible for

high mortality and morbidity rates in human beings. S. aureus causes several pus forming

infections and some serious infections such as meningitis, urinary tract infections, pneumonia

and mastitis. E. coli. and B. subtillus cause food poisoning (Sapkota et al., 2012). S. aureus, E.

coli. and Pasteurella multocida are also harmful for our livestock causing haemorrhagic

septicemia, diarrhea and dysentery problems in cattles (Hussain et al., 2011). Aspergillus

species cause many diseases such as allergic and nasal sinus diseases (Albrecht et al., 2011).

Fusarium species also cause a wide range of infections including allergic diseases and

mycotoxicosis in animals and humans (Kaplancikli et al., 2013).

Antibiotics and synthetic drugs have been used for the treatment of infections produced

by these microbial pathogens. However, number of resistant microbes and side effects of these

therapeutic agents have increased day by day (Qader et al., 2013). Resistance has also evolved

to newer and even more potent antimicrobial drugs like carbapenems (Patil et al., 2012).

Synthetic drugs are also expensive. This situation demands the need of more safe and effective

therapeutic agents for the treating these resistant microbes. Plant products have been used from

centuries for the treatment of infectious diseases (Vaghasiya and Chanda, 2007; Zahra et al.,

2011). Use of plant extracts and phytochemicals for their antimicrobial properties have gained

much significance in the treatment of various diseases. These plant products are preferred over

3

synthetic drugs (Manjamalai et al., 2010). These products are considered to be important

weapon against pathogenic microbes (Mahmood et al., 2011).

Phytochemicals are the chemicals produced by plants. These are further divided on the

basis of their role in metabolism into two groups: primary metabolites and secondary

metabolites. Primary metabolites such as sugars, proteins, amino acids and chlorophylls etc.

are involved in photosynthesis and normal growth of the plant. Secondary metabolites are

phenolics, terpenes, and alkaloids, saponins, involved in the defense system of plant

(Krishnaiah et al., 2007; Mazid et al., 2011; Hussain et al., 2011; Mohammedi and Atik, 2011).

These secondary metabolites produced by plants have important medicinal properties (Abutbul

et al., 2005) and produce therapeutic action on human body (Jayaraman et al., 2008).

Accumulation of secondary metabolites is different at different times of the year and is related

to temperature, day length and water availability (Abutbul et al., 2005). The production of

these secondary metabolites takes place in response to adverse environmental conditions or

particular developmental stages (Lin et al., 2007).

Antioxidative ability of plant origin components is mainly because of phenolic

compounds (Sultana et al., 2007). Phenolic compounds are ubiquitous in plants. Insoluble

phenolic compounds are present in cell walls and soluble phenolic compounds are present in

cell vacuoles. Outer layers of plants have more phenolics than inner layers. P-coumaric acid

and ferulic acid are major cell wall phenolic acids (Naczk and Shahidi, 2006; Stalikas, 2007).

Phenolic compounds from plants are also active as antimicrobial agents. Therefore, search for

such natural compounds has increased, not only for food preservation but also for stabilization

of fats/oils and for the treatment of human and plant diseases of microbial origin (Wong and

Kitts, 2006; Majhenic et al., 2007).

Phenolic compounds have aromatic ring possessing one or more hydroxyl groups along

with their functional group. The major and widely occurring classes of phenolic compounds

with antioxidant properties are phenolic acids and flavonoids (Cai et al., 2004; Komes et al.,

2010). Phenolic compounds also exhibit anti-inflammatory, anti-mutagenic and anti-

carcinogenic properties (Stalikas, 2007). Phenolic compounds increases the efficiency of

immune system to destroy cancer cells and to inhibit the angiogenesis (development of new

blood vessels) which is necessary for tumour growth. They also reduce the adhesiveness and

invasiveness of cancer cells thus decreasing their metastatic ability (Janarthanan et al., 2012).

4

Use of these natural products as antioxidants have replaced the consumption of synthetic

antioxidants (Jindal et al., 2012).

Extraction is the separation of biologically active compounds from plant tissues by

using appropriate solvents and extraction procedures (Tiwari et al., 2011). Antioxidant activity

and extraction yield are greatly affected by nature of plant matrix and solvent type used for

extraction, due to the presence of different phytochemicals (Sultana et al., 2008). A lot of

solvent systems such as water, ethanol, propanol, methanol, hexane, acetone, dimethyl

sulfoxide, ethyl acetate, dimethylformamide and mixtures of two or more solvents have been

used for the extraction of different classes of phenolics. Phenolic compounds have diverse

structure, having multiple hydroxyl groups conjugated to acid, alkyl or sugar groups. Thus the

polarities of phenolic compounds also vary considerably so it becomes very difficult to develop

a single method for maximum extraction of phenolics. Therefore, it is necessary to optimize

an extraction method for maximum recovery of phenolic compounds from plant materials

(Salas et al., 2010). Kratchanova et al., (2010) recommended to use more than one extraction

methods in order to study detailed antioxidant activity of plant extracts.

Pakistan has a unique position among developing countries due to its great resources

of medicinal plants. A large number of medicinal plants are present on its northern and

northwestern sides (Arshad et al., 2011). It has a variety of medicinal plants which are largely

used by local people (Ahmad et al., 2008). About 80% people living in rural areas depend on

these medicinal plants (Hameed et al., 2010). The local people are well known by the use of

these medicinal plants (Ahmad et al., 2008). Pakistan is rich in indigenous herbs and

provide a great scope for ethnobotanical studies. Traditional Unani medicine is part of

culture of Pakistan and is mostly used among the large number of its people (Qureshi

et al., 2009).

Pothohar plateau is one of the famous plateau of Pakistan. It falls in rainfed region

(Adnan et al., 2009). It is situated in the north of Punjab province of Pakistan comprising of

Jhelum, Attock, Rawalpindi, Islamabad and Chakwal districts. It is north central area ranging

from 1500-2000 feet in elevation. On its eastern side river Jhelum and on western side Indus

river are present. Khoshab district is located on its southern side and Hazara, Margalla and

5

Murree hills are located on its northern side (Rashid and Rasul, 2011; Kazmi and Rasul, 2012).

Due to its unique location it has very useful resources of medicinal plants.

Shinwari and Khan (2000) have done ethnobotanical studies in Margalla hills Nationa

park, Islamabad. They reported fifty species of herbs belonging to twenty seven families,

which were used for medicinal purposes by the local people of the studied area.

District Attock is a well-known historical place in the Punjab province, Pakistan. It has

useful resources of medicinal plants due to its unique location. A study was conducted to

investigate the indigenous plants used for the treatment of several diseases such as cough,

influenza, diabetes, diarrheoa, digestive disorders, ear and eye infections, asthma and

abdominal pain. Twenty five plant species belonging to twenty five genera were recorded to

use against these diseases (Ahmad et al., 2007). Qureshi and Ghufran (2007) conducted a

survey on the traditional medicinal plants of District Attock. This study was performed by

interviewing ten Hakims (Herbal doctors) and eighty inhabitants of the area. Attock consists

of semiarid area and is important for variety of important medicinal plants. About forty nine

medicinal plants were reported in this study. Hayat et al., (2008) also conducted an

ethnobotanical survey of Pindigheb tehsil of district Attock. They documented medicinal uses

of 100 plant species belonging to 44 families by the local people of the area. These plants are

locally used for the treatment of various diseases of humans and animals. Qureshi et al., (2009)

reported the traditional medicinal uses of twenty eight plants belonging to twenty five families

in Southern Himalayan regions of Pakistan. These plants were used medicinally as well as for

various purposes by local women.

Margallah Hills National Park consisting of an area of 15,883 hectares in North East of

Islamabad. It lies 33° 43’ N latitude and 72°55’ E longitude, elevated from 65 to 1600 m. The

average minimum and maximum temperature are 19.5 and 33.3°C, respectively. In this study,

245 plants belonging to 77 families were investigated from Margallah Hills National Park,

Islamabad. All the plant species had ethnobotanical uses in this area. Their common names

and traditional medicinal uses were also recorded (Jabeen et al., 2009). Ahmad et al., (2009)

also described traditional uses of indigenous medicinal plants of Margallah hills National Park,

Islamabad. Qureshi et al., (2009) recorded the medicinal uses of indigenous plants of Chakwal.

It is located in the south of Rawalpindi and lies between 32° 56' north and 72° 54' east. The

6

environment is cool with sub-humid climate. About 90% population lives in rural areas. They

found 29 plant species belonging to 25 genera and 18 families and recorded their botanical

name, local name, family name and ethobotanical uses. Qureshi et al., (2011) provided the

botanical survey of Koont farm located in the Rawalpindi at the borderline of Gujjar khan.

This area lies in the beginning of Pothohar plateu. It is rainfed area. In this survey 130 plant

species were identified from the studied area. These plant species belong to 105 genera and 35

families. They also reported the presence of Aerva javanica, Asphodilus tenifolius and Fagonia

indica in most of the regions of this area.

Although a lot of work was done on the ethnobotanical uses of the medicinal plants of

Pothohar region. But no scientific study was done on the biological activities of indigenous

medicinal plants of this region. That’s why this study was designed to study the antioxidant

and antimicrobial activities of selected medicinal plants indigenous to Pothohar plateau.

Fagonia indica, Asphodilus tenifolius and Aerva javanica are traditionally used to treat various

diseases in the studied area (Ahmad et al., 2007; Hayat et al., 2008; Qureshi et al., 2009).

Following are the major objectives of the proposed study:

Screening of selected Pothohar plateau plants as a potential source of antioxidant and

antimicrobial activities

Optimization of different methods of extraction

Characterization and quantification of selected bioactive constituents

7

Chapter 2

Review of Literature

Pakistan is located between 60° 55’ to 75° 30’ E longitude and 23° 45’ to 36° 50’ N

latitude. It covers an area of 80,943 sq. km. Out of which 46, 8000 sq. km area consists of

mountains lands and plateau in north and west, while the remaining 3, 28000 km is in form of

plains. Pakistan has varying environmental conditions from high snow fall areas of Himalaya

in the north to hot humid climate of shores of Arabian Sea in the South. The country is mostly

arid with 75% of its parts receiving an annual precipitation of less than 250 mm and 20% of

its less than 125mm. Only 10% of the area in the northern mountains ranges receives in

between 500 mm and 1500 mm rainfall.

Pakistan has large variation in its climate and is quite rich in medicinal plants, which

are scattered over a large area. Its soil is rich in naturally occurring medicinal plants and herbs

(Mahmood et al., 2011). These plants are used as natural health care in traditional system.

About 6,000 species of higher plants are present here. Out of which 600 to 700 species have

medicinal uses (Shinwari, 2010). About 350-400 species are sold in different drug markets and

are used to manufacture homeopathic and Unani medicines (Jabeen et al., 2009).

These medicinal plants have been mostly used by Hakims and the people in the rural areas

(Qureshi and Ghufran, 2007).

2.1. Pothohar plateau:

The area under study is known as “Pothohar plateau”, including Islamabad,

Rawalpindi, Chakwal, Attock and Jhelum districts (Chaudary et al., 2007; Arif and Malik,

2009; Mahmood et al., 2011). It is situated between latitude 30 and 34o N and longitude 70 and

74o E (Dasti et al., 2007; Rashid and Rasul, 2011). It is located between Jhelum River and the

Indus River and expanded from the salt range northward to the foot hills of Himalayas. It

covers an area of 1.5 million hectares and in altitude it rises from 350 to 575meters. The land

of Pothohar region is mainly regarded as fragmented land holding (Ashraf et al., 2007; Adnan

et al., 2009; Hussain et al., 2011). This rainfed area contributes significantly to agricultural

and livestock production. The soils of Pothohar plateau are low in natural fertility, deficient in

nitrogen and phosphorous, however, potassium level is adequate. Similarly, the soils are also

8

low in organic matter and having pH of 7.5 to 8.5 (Arif and Malik, 2009). Climate of Pothohar

plateu is of extreme nature, winter is bitterly cold and while summer is unbearably hot. June

and July are the hottest months (average maximum temperature 42oC) while December and

January are the coldest months (average temperature 1.7oC).

Fig. 2.1. Map of Punjab showing Pothohar region (Mufti et al., 2011)

2.2. Medicinal plants:

Plants have been a source of medicines throughout human history (Zereen and Khan,

2012). These are used as medicines in different drug systems like Ayurveda, Unani,

Homeopathy and allelopathy and many others. These medicinal plants are very popular

globally because of their safety, effectiveness and inexpensive nature (Anil et al., 2012).

According to WHO about 80% of populations of whole world depends on these traditional

medicines for health care (Kalim et al., 2010) and most of this therapy depends on the use of

plant extracts and their bioactive compouds. Drugs obtained from plant sources are generally

safer as compared to synthetic drugs, providing medicinal benefits and affordable treatment

(Panghal et al., 2011; Neelam and Khan, 2012).

9

Medicinal plants are potential source of pharmaceutical drugs that cause more

physiological effects and lesser side effects. They show antioxidant, anti-tumour, antidiabetic,

antihepatotoxicity, cytotoxic and antimicrobial activities (kalim et al., 2010; Gajalakshmi et

al., 2012). Plant derived medicines have gained much importance as these are based upon

natural products. These natural products can improve health and cure diseases. This curing

ability of plants and return towards natural products is the demand of our time (Sen et al.,

2010).

Medicinal plants produce a variety of compounds such as phenolics, flavonoids,

alkaloids, glycosides, saponins essential oils, mucilages and tannins in all of their parts (root,

stem, bark, leaves, seeds) which possess a healing physiological effect in the treatment of

various diseases of animals and humans (Adhikari et al., 2010). Medicinal herbs contain

significantly higher phenolic contents and also show strong antioxidant activity as compared

to fruits and vegetables taken as important natural sources of dietary antioxidants (Cai et al.,

2004). These bioactive compounds have varying mechanism of action depending on their

structure and environment (Matkowski et al., 2008).

Ethnobotany is the study of the relationship of a society and its environment especially

plants (Noor and Kalsoom, 2011). This field has become very important in the improvement

of health care system throughout the world. The aim of ethnobotanical studies is to record the

indigenous knowledge about plants (Ahmad and Husain, 2008). On the basis of ethnobotany

plant species are further analyzed for biological activities and phytochemical studies (Robards,

2003). Pakistan is rich in indigenous medicinal plants so it provides a great scope of

ethnobotanical studies (Rashid and Marwat, 2006).

10

2.3. Asphodilus tenifolius:

Fig 2.2. Asphodilus tenifolius

2.3.1. Biological classification/ Taxonomy:

Kingdom Plantae

Order Asparagales

Family Lilliaceae

Genus Asphodilus

Species Asphodilus tenifolius

Common name Piazi

2.3.2. Description:

Asphodilus tenifolius belongs to Lilliaceae family. This family occupies 15 genera and

780 species which are widespread all around the world especially south Africa. From these 3

genera and 11 species are indigenous to Pakistan. Asphodilus is one of these genera and

Asphodilus tenifolius belongs to it (Safder et al., 2012). A. tenifolius plant is erect, glabrous

annual herb with a perennial bulb.

http://en.wikipedia.org/wiki/Asparagales

http://en.wikipedia.org/wiki/Amaranthaceae

http://en.wikipedia.org/wiki/File:Asphodelus_tenuifolius.JPG

11

2.3.3. Ethnobotanical uses:

Seeds of the plants are traditionally used for the treatment of cold, hemorrhoids and

rheumatic pain. Seeds are applied externally to ulcers and inflamed parts by local people and

also used as diuretics (Vaghasiya and Chanda, 2007; Panghal et al., 2011). Leaf decoction of

A. tenifolius is used for the treatment of kidney stone and leaf paste is applied on swelling

(Mahmood et al., 2011). The plant is used as diuretic by local people (Ali, 2004; Safder et al.,

2012).

2.3.4. Chemical composition:

Safder et al., (2012) isolated two new triterpenes (asphorin A and B) in ethyl acetate

subfraction of methanolic extract of A. tenifolius. Vaghasiya et al., (2011) reported the

presence of alkaloids in A. tenifolius. Bulb and seeds of A. tenifolius contain flavonoids,

luteolin and its glycosides (Baker et al., 2000). Kalim et al., (2010) also recorded the presence

of phenolics and ascorbic acid. Menghani (2012) reported the presence of reducing sugars,

tannins and alkaloids in the methanolic extract of A. tenifolius. Safder et al., (2009) isolated

and elucidated the structures of ß-sitosterol, 3-hydroxybenzoic acid, 1- triacontanol, 1-

octacosanol, hexadecanoic acid, triacontanoic acid, tetracosanoic acid and ß-sitosterol 3-O-ß-

D-glucopyranoside through different spectroscopic techniques. Vaghasiya and Chanda (2007)

described the presence of cardiac glycosides, saponins, flavonoids and alkaloids in A.

tenifolius. Menghani et al., (2012) also mentioned the presence of these phytochemicals in A.

tenifolius.

2.3.5. Biological testing:

Asphodilus tenifolius is used for the treatment of atherosclerosis and also used as

diuretics (Safder et al., 2009). Panghal et al., (2011) examined the antimicrobial activities of

A. tenifolius against clinical isolates of oral cancer patients.

12

2.4. Aerva javanica:

Fig 2.3. Aerva javanica


Kingdom Plantae

Order Caryophyllales

Family Amaranthaceae

Genus Aerva

Species Aerva javanica

Common name Bui

2.4.2. Description:

Aerva javanica belongs to Amaranthacea family (Khan et al., 2012). It is a perennial

herb, native to Africa, Asia and extensively scattered in the far away areas of the world (Judd

et al., 2008). Amaranthaceae is important family for its medicinal uses having 169 genera and

2300 species (Anwar et al., 2011). Leaves of A. javanica are used as fodder to goats and whole

plant is used as a fuel (Qureshi and Bhatti, 2009).

2.4.3. Ethnobotanical uses:

Aerva javanica is locally used for the treatment of wounds, cough, diarrhoea, ulcer and

hyperglycaemia (Khan et al., 2012) and is also used as diabetic, demulcent and diuretic. Its

http://en.wikipedia.org/wiki/Caryophyllales


http://en.wikipedia.org/wiki/Aerva

13

decoction is used to remove swelling and to relieve toothache. Its powder is externally used to

ulcers in domestic animals and seeds for headache. Its flowers and roots are used for the

treatment of kidney problems and rheumatism. A paste prepared from its leaves and

inflorescence is applied externally to cure wounds and inflammation of joints. The whole plant

of A. javanica is used for the treatment of chest pain, diarrhea and ascaris (Samejo et al., 2012)

and also used as purgative and emetic for camels and horses. Its wooly seeds are used to relieve

headache (Chawla et al., 2012).


Srinivas and Reddy (2008) described that carbohydrates, flavonoids steroids and

triterpenoids are present in Aerva javanica. Samejo et al., (2012) were detected sixteen

compounds (terpenoids, ketones, hydrocarbons) in the essential oil obtained from the leaves

and stems of A. javanica through GCMS analysis. Chawla et al., (2012) described that Aerva

species have flavonol glycosides as a major constituent and minor constituents are sterols, ß-

cyanins and carbohydrates. Sharif et al., (2011) reported the isolation of a few natural bioactive

compounds from Aerva javanica which are Isoquercetrin, 5-methylmellein, Apigenin 7-O-

glucuronide and7-(1 ׳ hydroxyethyl)-2-(2″- hydroxyethyl)-3,4-dihydrobenzopyran.


Soliman (2006) has done cytogenetical studies on Aerva javanica. Samejo et al.,

(2012) described that Aerva javanica exhibit anti-inflammatory, anti-plasmodial, anti-

diarrhoeal and analgesic properties. Abutbul et al., (2005) investigated the activity of Aerva

javanica against bacterial pathogens in fish. Chawla et al., (2012) described significant

antidiarroheal activity of aqueous and ethanolic extractsof Aerva javanica and Aerva lanata at

a dose of 800mg/Kg in Inbred rats. Khan et al., (2012) reported that Aerva javanica showed

significant anti-ulcer activity. Rajesh et al., (2011) described the diuretic, hepatoprotective,

anti-inflammatory, nephroprotective ans antidiabetic activities of Aerva lanata.

14

2.5. Fagonia indica:

Fig 2.4. Fagonia indica


Kingdom Plantae

Order Fabales

Family Zygophyllaceae

Genus Fagonia

Species Fagonia indica

Common name Dhamian, Fagonia, Dhamasa

2.5.2. Description:

Fagonia indica belongs to Zygophyllaceae family and is distributed in deserts of Asia

and Africa. It is widely distributed in India and Pakistan It is a branched, pale green, annual or

perennial shrublets upto 60cm high (Alam, 2011; Sharma et al., 2010).

2.5.3. Ethnobotanical use:

Fagonia indica is a good source of safe drugs, used as blood purifier, antiseptic,

febrifuge, prophylactic against smallpox (Sharma et al., 2009). It is used therapeutically for

treating the fever, vomiting, urinary discharge, liver trouble, typhoid, asthma, toothache,


15

dysentery, stomach problems. The aqueous decoction of leaves and young twigs is used for the

treatment of skin lesions especially amongst children. Aqueoes decoction of aerial parts is also

used for the treatment of cancer and diseases of digestive system (Anil et al., 2012).


Stem and fruits of Fagonia indica have high amounts of saponins, its leaves and flowers

have large amounts of tannins and cardiac glycosides (Zafar et al., 2010). F. indica collected

from UAE have 3% flavonoids (Elhady, 2011). Sharma et al., (2009) also reported the presence

of flavonoids, terpenoids, glycosides, saponins and amino acids in the aqueous and ethanolic

extracts of F. indica. They also described that alkaloids, fats, tannins, gums and proteins were

absent in these extract.


Elhady (2011) described that alcoholic extract of whole plant showed analgesic

activity. Soomro and Jaferey (2003) studied antituour activity of F. indica in rats and

concluded that aqueous extract of this plant has tumourostatic effect on the experimentally

produced tumour of albino rats.

2.7. Reactive oxygen species (ROS):

Oxygen is an indispensable element for life. It is utilized by the cells to produce energy

by catabolism. However, in this process free radicals or reactive oxygen species are produced

as by products (Huy et al., 2008; Ragavendran et al., 2012). These byproducts are usually free

radicals or reactive oxygen species. In small quantities these free radicals acts as signal

transducers, growth regulator and also a part of the immune defense system (Jindal et al.,

2012). At lower level these reactive oxygen species are also neutralized by natural defense

system in body. Environmental factors such as cigarette smoke, UV-rays, radiation and toxic

chemicals are also initiate production of ROS. Excess production of these ROS is not

controlled by body defense system and leads to oxidative stress. This oxidative stress cause

damage to biomolecules (lipids, DNA and proteins) and involved in pathogenic diseases sucha

as aging diabetes mellitus, cardiovascular diseases, kidney damage, lung diseases,

inflammation, cataracts, neuronal diseases, carcinogenesis (Aliyu et al., 2011; Pracheta et al.,

16

2011; Battu et al., 2012; Raghavendra et al., 2013) and Alzheimer’s disease (Chang et al.,

2007).

Fig 2.5. Diseases caused by oxidative stress in humans

These free radicals or ROS are highly reactive species having one or more unpaired

electrons. ROS take or give their unpaired electrons to become stable molecules. ROS occur

in radical form like superoxide (O2•−), hydroperoxyl (HO2 •), lipid peroxyl (LOO•), alkoxyl

(RO•), peroxyl (ROO•) and hydroxyl (OH•); and nonradical form such as singlet oxygen (1O2),

ozone (O3), hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and lipid peroxide

(LOOH) (Panchawat et al., 2010; Sen et al., 2010; Pracheta et al., 2011).

These reactive oxygen species also react with fatty foods leading to lipid peroxidation. Lipid

peroxidation is one of the major problems in food industry as it is involved in the loss of quality

of fatty foods by the formation of such a products which having negative effect on taste,

colour, flavour, nutritional value and storage stability of food products (Rakesh et al., 2010).

Antioxidants are important in the prevention of oxidative damage (Battu et al., 2012).

17

2.8. Antioxidant:

Antioxidant are the compounds which prevent oxidation when present in small amounts

(Panchawat et al., 2010; Rakesh et al., 2010; Prabhune et al., 2013). Antioxidants prevent

oxidation in different ways (Krishnaiah et al., 2007):

by reducing the rate of free radical formation and called as preventive antioxidants.

They convert hydro-peroxides into such products which are not a potent source of free

radicals.

ROOH 2[H] ROH + H2 O

Chain breaking antioxidants: which scavenge and stabilize free radicals. They can also

trap peroxyl radicals. These are mostly phenols or aromatic amines.

L. + AH → LH + A. (1)

LO. + AH → LOH + A. (2)

LOO. + AH → LOOH + A. (3)

AH represents antioxidant molecule, L. is a lipid radical, LO. is an alkoxyl radical and LOO.

is a peroxyl radical. So radical initiation (1) or propagation (2 or 3) steps are inhibited by the

antioxidant (AH).

By chelating pro-oxidant metals (Pracheta et al., 2011)

Antioxidants are very important neutraceuticals on the basis of their health benefits and

wide use in the food industry to prevent from lipid peroxidation (Scherer and Godoy, 2009).

These are added in foods to prevent or delay oxidation of food, initiated by their exposure to air,

light and temperature (Kumar et al., 2008). Generally there are two main types of antioxidants:

synthetic and natural (Zheng and Wang, 2001; Alam et al., 2012).

2.9. Synthetic antioxidants:

Antioxidants which are manufactured synthetically called synthetic antioxidant. For

example propyl gallate, Butylated hydroxyl toluene (BHT), tertiary butylated hydroquinone

(TBHQ) Butylated hydroxyl anisole (BHA) etc. These are added in food as preservative.

However, these synthetic antioxidant cause liver damage, carcinogenesis (Krishnaiah et al.,

2010; Pracheta et al., 2011; Alam et al., 2012; Priyanka et al., 2013) and mutagenesis

(Umamaheswari and Chatterjee, 2008). BHA cause carcinogenic as well as genotoxic effects

and BHT is proved to exhibit hemorrhaging effect (Stankovic et al., 2011). Therefore, plant

18

derived antioxidants have received a special attention (Mahmoud et al., 2011; Raghavendra et

al., 2013).

2.10. Natural antioxidants:

Plants such as fruits, vegetables, herbs, spices and legumes provide a potential source

for natural antioxidants (Iqbal and Bhanger, 2006). These natural antioxidant do not cause side

effects (Alam et al., 2012) and possess antiviral, antibacterial, antiallergic, anti-inflammatory,

vasodilatory and antithrombotic activities (Oliveira et al., 2012). Natural antioxidant

compounds are ascorbic acid, phenolics, carotenoids, tocopherols, flavonoids, tocotrienols and

folic acid to prevent oxidation (Krishnaiah et al., 2007). These natural products are involved

in decreasing the risk of cancer, diabetes, cardiovascular diseases and aging (Mahmoud et al.,

2011). Bioactive compounds mainly responsible for the health benefits are phenolic

compounds (Atoui et al., 2005; Chang et al., 2007). Among all the secondary metabolites

produced by the plants, phenolic compounds are major group having antioxidant potential

(Rafat et al., 2010). The antioxidant attributes of phenolic compounds may be due to their

potential to scavenge free radicals and singlet oxygen (Sethi and Sharma, 2011), chelate metals

and inhibit lipoxygenase (Chang et al., 2007). Rehman and Moon (2007) also reported that

radical scavenging activities of plant extract are related to phenolic compounds.

2.11. Phenolic compounds:

Plants produce a large and heterogenous group of compounds called phenolic

compounds (Mazid et al., 2011). According to structure, over 8000 phenolics have been

identified and are widely distributed in plant kingdom (Crozier et al., 2009). These are

synthesized from carbohydrates through shikimate pathway in response to ecological and

physiological factors like pathogens attack and UV radiations etc. Quantities of phenolic

compounds are different in different tissues and also vary within different populations of the

same plant. Their quantities depend upon plant species, growing conditions, soil conditions,

harvest time, etc. For example, simple phenolic acid quantities are greater in younger tissues,

but later on these phenolic acids condense to form polyphenolic constituents such as flavonoids

(Robards, 2003; Sultana et al., 2007; Khoddami et al., 2013). Phenolic compounds are also

19

necessary to plant life, e.g., by protecting from pathogen attacks and by making food

unpalatable to herbivorous predators (Apak et al., 2007).

Phenolic compounds include simple phenolic acids (low molecular weight) such as

gallic acid, caffeic acid, chlorogenic acid, coumaric acid, ferulic acid and large, complex

polyphenols such as flavonoids, quinones, coumarins, stilbenes, curcuminoids and lignans etc.

(Crozier et al., 2009). All plant based foods have phenolic compounds (Pohjala and Tammela,

2012). Blue, red and purple colours of vegetables, fruits, teas and herbs are due to the presesnce

of polyphenolic compounds (Rakesh et al., 2010). Phenolics possess great potential of

antioxidant activity and also have anticancer, antimutagenic, antidaibetic and antimicrobial

activities (Cai et al., 2004; Umamaheswari and Chatterjee, 2008).

The antioxidant potential of phenolic compounds is because of their reducing ability

which is responsible of neutralizing of free radicals. This activity of phenolic compounds is

related to their structure. Generally, antioxidant and radical scavenging activities of phenolic

compounds are due to position and number of hydroxyl groups on their aromatic ring. Their

activities are also affected by the glycosylation of aglycones and other H-donating groups (-

NH, -SH), etc. The glycosylation of flavonoids decrease their activity (Cai et al., 2004).

Phenolic acids and flavonoids are the typical phenolics which exhibit antioxidant activities

(Zheng and Wang, 2001; Raghavendra et al., 2013; Rahman et al., 2012).

2.11.1. Phenolic acids:

Phenolic acids are the chemicals having an aromatic ring, one or more hydroxyl groups

on the aromatic ring along with their functional derivatives. Simple phenolic acids are major

group of phenolics including benzoic acid derivatives (vanillic acid, p- hydroxyl benzoic acid,

protocatechuic acid and gallic acid) and cinnamic acid derivatives (p- coumaric acid, caffeic

acid and ferulic acid) (Cai et al., 2004). Benzoic acid derivatives possess analgesic, antipyretic,

anti-inflammatory, and antiseptic properties and cinnamic acid derivatives possess cholagogic,

choleritic, and antibiotic properties (Hajnos et al., 2007). Cinnamic acid derivatives are cell wall

phenolic acids play an important role in plant growth by providing protection against

pathogens, wounds and UV radiations (Khoddami et al., 2013).

20

Fig 2.6. Structure of phenolic acids

Phenolic acids are formed from shikimic acid in plants (Hajnos et al., 2007). Phenolic

acids play an important role in antioxidant activity (Cai et al., 2004) such as ferulic acid, caffeic

acid and vanillic acid are widely spread in plants (Zheng and Wang, 2001). They work as

antioxidant and convert to univalent oxidized form (phenoxyl radicals) (Sakihama et al., 2002).

Phenolic acids possess redox properties, by which they can adsorb, neutralize free radicals,

decompode peroxides and scavange singlet and triplet oxygen (Hasan et al., 2008; Hasan et

al., 2009; Mahmoud et al., 2011).

These important compounds also show antimicrobial, anti-infective and antiseptic

activities. These phenolic acids also combine with polyphenols thus forming complexes with

enhanced activities. For example, gallic acid combine with catechin (polyphenol) to yield

catechin gallate complex which is an antioxidant with enhanced antitumour and anticancer

properties (Rakesh et al., 2010).

2.11.2. Flavonoids:

Flavonoids are the largest group of polyphenolic compounds, having 15 carbons central

pyran ring (C6–C3–C6) (Cai et al., 2004) and are important natural phenolics (Pracheta et al.,

2011). Flavonoids are widely spread in plant tissues and are responsible for their blue, yellow,

red, orange and purple colours alongwith the chlorophylls and carotenoids (Khoddami et al.,

2013) and provide protection from UV radiations and other environmental stresses (Saleem et

al., 2010). In excess of 4000 flavonoids structures have been reported (Huay et al., 2008) and

are widely spread in the plants especially in their leaves and skin of fruits (Crozier et al., 2009).

21

Fruits, vegetables, herbs, spices, nuts, seeds, stems, flowers, tea and red wine are potential

sources of flavonoids (Huay et al., 2008). Flavonoids are further divided into several classes

such as flavanols, flavanones, flavones, isoflavones, catechins, anthocyanins,

proanthocyanidins (Huay et al., 2008; Lotito and Feri, 2006; Khoddami et al., 2013).

Fig 2.7. Basic skeleton of Flavonoids (Pyran ring)

In plants flavonoids are responsible for the colour of seeds and flowers, also involved

in defence system of the plant to protect them from abiotic stress like UV light and biotic stress

like pathogen and predators attack. These compounds are found in plants as glycosides or

aglycones and are present in the vacuoles of plant cells in the form of soluble fraction. That’s

why they can be easily extracted with polar solvents (Rispail et al., 2005; Leela et al., 2013).

Benefits of flavonoids for human body are mainly due to their potential as antioxidants (Huay

et al., 2008). Flavonoids are the important secondary metabolites having antioxidant,

anticancer, anticoagulative, antihistaminic, anti-allergic, antimicrobial, anti-inflammatory

(Agoreyo et al., 2012; Hossain et al., 2013), antiulcer and antiviral activities (Umamaheswari

and Chatterjee, 2008). An important sub-group of flavonoids is known as flavan-3ols includes

catechin, anthcyanidins, proanthocyanidins and tannins. These possess very good antioxidant,

anticancer and cardioprotective properties (Rakesh et al., 2010). Flavonoids show antioxidant

activity through a number of mechanisms such as free radical scavenging by donation of

hydrogen atoms, chelating metals atoms such as iron and copper, inhibiting lipid peroxidation

thus protect us from several diseases such as heart attacks, strokes and even cancer (Kalim et

al., 2010). Quercetin, one of the flavones, having antitumor, antiallergic and

immunomodulatory activities (Leela et al., 2013).

22

Fig 2.8. Basic structure and Examples of different classes of flavonoids

23

2.12. Antimicrobial activity:

Anti-microbial agents are definitely one of the most important discovery of the 20th

century in the field of medicines (Kamaraj et al., 2012). Microorganisms such as bacteria,

viruses, fungi and nematodes are the major cause for serious infections in human beings

throughout the world. Now a days these pathogenic microbes have become resistant to most

of the antibiotics, because of careless use of these antimicrobial agents (Sharma et al., 2009).

Thus it causes a great problem in treating the infectious diseases. Antibiotics also have a

number of side effects. Staphylococcus aureus, a gram positive bacteria (Joshi et al., 2011) is

said to be responsible for skin diseases such as pimples, boils and infection in wounds.

Although S. aureus is still susceptible to some common antibiotics, there is still the need to

find alternative drugs before it develop resistance to the current ones (Faruq et al., 2010).

Due to this resistance of microbes, there is a need to explore antimicrobial agents from

nature. Crude extracts from plants have been used traditionally since ancient times which can

be useful resources of these new antimicrobial drugs. Some plant families showed antibacterial

as well as antifungal activities. Plants produce compounds such as secondary metabolites

which are active against pathogenic microbes (Fatimi et al., 2007; Walteri et al., 2011;

Huwaitat et al., 2013) having less side effects, low cost and better patient tolerance (Joshi et

al., 2011). These compounds are of low molecular weight synthesized in the plants in stressed

conditions such as polyphenolics, flavonoids, terpenoids and glycosteroids found in various

plant parts such as leaves, bark, stems, roots, seeds, flowers and fruits. These phytochemicals

are synthesised by plants in response to microbial infection. Therefore, it is logically proved

that they can be used against a broad range of micro-organisms (Borchardt et al., 2008; Panghal

et al., 2011). Flavonoids and phenolic acids show good activity against fungi and bacteria

(Narayana et al., 2001). The important characteristic of these plant products is their

hydrophobicity, due to which these plant extracts can disturb the lipid portion of bacterial cell

membrane thus destroying the cell structure and transforming them into more permeable form

(Joshi et al., 2011). Doughari et al., (2008) also reported that the study of plants for their

antimicrobial properties gives useful results and show that in some plants there are many

substances such as peptides, alkaloidal constituents, unsaturated long chain aldehydes,

essential oils, phenols and some other compounds which exhibit activities against viruses,

fungi and bacteria.

24

Plant phenolics have a lot of defensive functions such as antibacterial and antifungal

potential. Phenolic compounds present on the surface of plants or in the cytoplasm of the

epidermal cells, they protect from pathogens. Phenolic acids such as pyrogallol and gallic acid,

flavonoids such as rutin, myricetin and daidzein are significantly effective as antibacterial

agents (Ferrazzano et al., 2011).

The exact mechanisms of antimicrobial activity of secondary metabolites are not fully

known. Phenolic compounds exert antimicrobial activity primarily by their potential to act as

non-ionic surface-active agent, therefore, disturbing the lipid-protein interface or denaturing

the proteins and inactivating of enzymes in the pathogens. Secondly, phenols alter the

permeability of the membrane which results in inhibition of active transport and coupling of

oxidative phosphorylation, and also loss of metabolites due to membrane damage (Manoj and

Murugan, 2012). Flavonoids may show their activity by inhibiting the cytoplasmic membrane

function, DNA gyrase and β-hydroxyacyl-acyl carrier protein dehydratase activities (Paiva et

al., 2010). Biological activities of phenolic compounds are related to their molecular structure;

hydroxyl groups or phenolic ring (Nitiema et al., 2012). Disc diffusion assay is a simple and

rapid method for anlyzing the antimicrobial activity of large number of samples (Abutbul et

al., 2005).

2.13. Extraction:

Extraction is first important step for the characterization and isolation of bioactive

compounds (Gupta et al., 2012). Extraction method and choice of suitable solvent are very

important for extraction purposes. (Robards, 2003; Mussatto et al., 2011). Aim of extraction

is to gain the medicinally active compounds and to remove the unwanted materials by using

suitable solvents through standard extraction methods (Tiwari et al., 2011). Plants contain a

variety of secondary metabolites as many as 200,000 compounds. Not every compound is

found in every plant species. There are different classes of metabolites such as phenolic,

flavonoids etc., having different physico-chemical properties (Krishnaiah et al., 2007).

Extraction of phytochemicals from plant matrices is greatly effected by various factors such as

extraction procedures, solvent polarity, particle size and other extraction conditions

(temperature, time etc.). Extraction solvent and technique are basic factors for optimization of

extraction yield (Victorio et al., 2009). Therefore, suitable extraction methods have to be

25

chosen for such a variety of compounds. The products obtained after extraction are mixtures

of phytochemicals in semisolid, liquid or in dry powder form. During extraction, solvents

penetrate into the plant matrice and dissolve the compounds of similar polarity (Tiwari et al.,

2011).

2.13.1. Extraction solvents:

Maximum recovery of bioactive compounds from plant material depends upon the type

of solvent used for extraction. A good solvent for extraction purposes must be; less toxic, easily

evaporated at low heat, provide rapid physiological absorption of the extract, acting as

preservative, unable to cause dissociation of extracts. Choice of solvent is effected by different

factors such as variety and quantity of different bioactive compounds to be extracted, rate of

extraction, easy of handling of the extracts and variety of inhibitory compounds extracted.

For the extraction of bioactive compounds from plants a lot of solvents such as water,

ethanol, chloroform, ethyl acetate, methanol, acetone, hexane, etc. are commonly used either

singly or mixture of solvents to get better extraction efficiency(Gupta et al., 2012). Methanol,

ethanol and water are considered for the extraction of polar compounds and for non-polar

compounds, chloroform is mostly used (Krishnaiah et al., 2007). Water is used as a solvent

by traditional people, however, different studies showed that plant extracts obtained from

organic solvents show good antibacterial activities as compared to water extracts (Vaghasiya

and Chanda, 2007).

No single solvent can provide optimized recovery of phenolics because phenolics

exhibit diversity in polarity, from hydrophobic to hydrophilic character. DMSO and

ethylacetate can also be used for extraction but aqueous mixtures of ethanol, methanol or

acetone are best suited for the extraction of a wide range of phenolics in oats, soy, spices, fruits

and vegetables. Solvent of low polarity and ethyl acetate can only solublize the compounds

from dried plant material, while alcoholic solvents enhance extraction by rupturing of cell

membranes so that intracellular materials also come out (Robards, 2003).

Phenolics are not evenly spread at tissue, cellular and subcellular levels in the plants.

They can be found in free, conjugated and polymeric forms or may exist by forming complexes

with sugars. All these factors result in difficulty to optimize a single solvent for all the

compounds (Luthria et al., 2006; Salas et al., 2010). The solubility of polyphenols also depends

26

upon the hydroxyl groups, molecular size and length of hydrocarbon. Addition of water in

alcohols increases the polarity of the medium thus enhancing the extraction of polyphenols.

Water is not good for the extraction of polyphenols (Mohammedi and Atik, 2011). Chirinos et

al., (2007) also reported that water proportion in these binary solvent should not be more than

50%. Jakopic et al., (2009) also reported that extraction of plant phenolics greatly depends on

the type and concentrations of the solvents.

Alcohols such as ethanol and methanol are efficient solvents for the extraction of

phenolic compounds from plant materials as compared to water. They degrade cell walls and

seeds which have non polar character thus releasing phenolic compounds from cells (Lapornik

et al., 2005; Tiwari et al., 2011). Low Dog, (2009) described that ethanol is mostly used organic

solvent by herbal drugs manufacturer because the finished products can be used safely by the

consumers. Acetone has the ability to solubilize hydrophilic and lipophilic compounds from

plants. It is volatile and less toxic and very useful for the extraction of antimicrobial compounds

(Das et al., 2010).

Lu et al., (2012) also studied the effect of different solvent systems on the antioxidant

and cytotoxic activities of Actinidia macrosperma. They used methanol, hexane, water,

chloroform and ethyl acetate as extracting solvents and found that highest TPC, antioxidant

and cytotoxic activities were given by the methanolic extract. These activities depend upon

phenolic contents. Methanol extracted more TPC so exhibited highest activities.

2.13.2. Extraction techniques:

Extraction techniques have been widely used to obtain valuable natural compounds

from plants for commercialization. The conventional methods of extraction are mostly based

on the solvent choice, temperature or mixing to improve the solubility of matrices and thus rate

of extraction. Sonication extraction is a potentially useful technology used on small as well as

large scale in phyto-pharmaceutical extraction industry (Gupta et al., 2012). Sonication is the

production of ultrasound waves with frequencies 20-2000 kHz which enhances the

permeability of cell walls. In this extraction method, the grounded material is mixed with the

appropriate solvent and kept into an ultrasonic bath. As the intensity of ultrasound is increased

in the solvent, then it reaches at a point at which the intra-molecular forces cannot hold the

molecular structure intact, so it breaks down and bubbles are produced. Break down of these

27

bubbles can cause chemical, physical and mechanical effects which resulted in the destruction

of biological membranes thus releasing more extractable compounds and also increasing the

diffusion of solvent into cellular materials and improving the recovery of desired compounds.

The main advantages of sonication are shorter extraction time, need of small amounts of

material, minimum expenditure on solvents and the increase in extraction yield (Lou et al.,

2011; Khoddami et al., 2013).

Extraction through reflux is one of the most applicable conventional methods in

extraction studies. In this method, first the container is heated and then heat is transferred

molecule by molecule into solvent. Conventional extraction has an advantage of being cheap

in terms of equipment. This method is not suitable for heat sensitive compounds as heating

may cause decomposition of bioactive compounds. (Nikhal et al., 2010). This method is

mostly used for the extraction of flavonoids (Stalikas, 2007).

2.14. Antioxidant activity determination assays:

Antioxidant activity is very important property for human life as many other biological

activities may also orginate from it such as antiaging, anticarcinogenicity and antimutagenicity

properties (sim et al., 2010). For the evaluation of antioxidant activity, a large number of

number of methods have been developed. Oxidative stress process involves many active

species and different reaction mechanism. That’s why, there is no simple and universal method

which can be used for accurate and quantitative measurement of antioxidant activity.

Generally, these methods involve the generation of a radical and the antioxidant ability of a

sample against this radical is measured (Jindal et al., 2012). Lu et al., (2012) suggested to use

more than one assay for the evaluation of antioxidant activity because a single assay does not

give full description of antioxidant activity.

2.14.1. DPPH free radical scavenging assay:

It is direct and reliable method for evaluation of free radical scavenging activity. DPPH

(1, 1-diphenyl-2-picrylhydrazyl) molecule is a stable free radical because its free electron

delocalized over the whole molecule and do not dimerise like other free radicals. The

delocalization also gives rise to the deep violet colour, characterised by an absorption band at

about 520 nm. When DPPH solution is added to sample solution (hydrogen atom donar), then

28

violet colour of DPPH changes to yellow because of reduction of 1,1-diphenyl-2-

picrylhydrazyl radical into 1,1-diphenyl-2-picrylhydrazine (reduced form). The yellow colour

left is due to picryl group.

Z• + AH = ZH + A•

Where Z• is DPPH radical and AH is donar molecule. ZH represents as the reduced form of

DPPH and A• as a free radical produced in this reaction and further reacts with another DPPH

radical (Molyneux, 2004; Daud et al., 2011). The reduction in the number of DPPH molecules

can be related with the number of available hydroxyl groups on phenolic compounds

(Umamaheswari et al., 2008).

DPPH + H-A → DPPH-H + A

(Purple) (Yellow)

DPPH assay is widely used because of its simplicity, feasibility, sensitivity and has a

good stability. DPPH can be used for the measurement of antioxidant activity of natural

products obtained from plant as well as microbial sources (Marinova and Batchvarov, 2011;

Prabhune et al., 2013). This assay applied for both plant extracts as well as pure compounds

(Aliyu et al., 2012).

2.14.2. Superoxide radical scavenging assay:

Superoxide anions are one of the most damaging free radicals that attack on

biomolecules directly or indirectly by forming OH, H2O2, peroxynitrite or singlet oxygen

during pathological diseases (Pracheta et al., 2011). In the oxidation-reduction reactions of

29

cells, superoxide radicals are formed normally, later on their effects can be magnified as they

produce many types of cell destroying free radicals other oxidizing agents (Subramanian and

Vedanarayanan, 2012). SOR scavenging activity was measured using NBT (Nitro blue

tetrazolium reagent). In this assay, superoxide radicals are generated in the reaction mixture.

These radicals react with NBT, which is reduced to nitrite. Nitrites in the presence of EDTA

gives a colour that can be measured at 560 nm (Veena and Mishra, 2011).

2.14.3. Reducing power assay (FRAP):

FRAP assay is a simple, inexpensive and used to measure the ability of antioxidants to

donate electron to ferric ion (Fe+3) (Majer et al., 2010). It is also used to compare antioxidant

activity of plants and mammals (Niemeyer and Metzler, 2003). This assay is based on the

reduction of ferric ions to ferrous ions (Lotito and Frei, 2006; Jindal et al., 2012) and is a

simple and inexpensive method. In this assay antioxidants from test samples reduce Fe+3 in the

reaction mixture to Fe+2 (blue colour), which is measured by spectrophotometer (Sultana et al.,

2007: Baig et al., 2011; Prabhune et al., 2013; Priyanka et al., 2013). Reducing power of an

extract significantly indicates the antioxidant activity of that extract (Chang et al., 2007;

Premanath and Lakshmidevi, 2010). Reducing power of different compounds shows that they

can donate electron and are also involved in the reduction of intermediates of lipid oxidation

reactions, thus acting as primary and secondary anti-oxidants (Umamaheswari and Chatterjee

et al., 2008).

2.14.4. Beta carotene linoleic acid assay:

In this assay linoleic acid, an unsaturated fatty acid undergoes oxidation by reactive

oxygen species (hydroperoxides) produced from halogenated water (Baig et al., 2011). These

hydroperoxides in the absence of antioxidants oxidize ß-carotene molecules, which loss their

double bonds and characteristic orange colour. This change in colour is measured by a

spectrophotometer. Antioxidants prevent the oxidation of ß-carotene molecules by neutralizing

hydroperoxides and other free radicals produced in the reaction mixture (Sim et al., 2010).

2.14.5. Folin-Ciocalteu method:

Total phenolic content of an extract can be measured with spectrophotometer method

using Folin-Ciocalteu reagent (Naczk and Shahidi, 2006). It is an electron transfer based assay

30

(Komes et al., 2011). Folin–Ciocalteu reagent is a yellow mixture of phosphotungstic and

phosphomolybdic heteropoly acids (Ozkan et al., 2011). This method is based on the principle

of reduction ability of phenol functional group at basic condition. The reduction of Folin-

Ciocalteu reagent by phenolate ion will change its color to be blue (Stalikas, 2007). The

reduction of the reagent increases when the extract contain more phenolic compounds. Thus,

the color will be darker and the absorbance will be higher. Due to the diverse nature of plant

phenolic compounds and interference of some readily oxidized compounds in the plant

extracts, different methods have been used for measurement of total phenolic contents such as

Folin-Ciocalteu method (FC), Folin-Denis method (FD), ultraviolet absorbance, permanganate

titration, and colorimetry with iron salts. However, FC method is preferred over other methods

because it is a simple, reproducible and most widely used for the determination of phenolic

compounds from plant extracts (Dai et al., 2010; Oliveira et al., 2012).

2.15. HPLC analysis:

HPLC is a preferred technique for identifying and quantifying phenolic compounds.

Phenolic compounds are non-volatile so their analysis on GC (Gas chromatography) is not

generally recommended. But with suitable derivatization, phenolic compounds can be

analyzed through GC or GCMS. However, HPLC is currently most popular and reliable

technique for phenolic compounds (Robards, 2003; Khoddami et al., 2013) with a wide range

of commercially available columns (Stalikas, 2007). For the analysis of phenolic compounds

on HPLC, C18 columns are mostly used. Gradient elution is recommended because of

complexity of phenolic compounds. Mostly binary solvents are used as mobile phase, one

component is water and other is less polar organic solvent such as acetonitrile or methanol.

Reverse phase LC is typically used. Polar compounds (phenolic acids) elute first then

compounds with decreasing polarity like cinnamic acid derivatives followed by flavonoids

(Bernal et al., 2011).

2.15.1. Gallic acid:

It belongs to the benzoic acid derivatives group of phenolic acids and is a polar

compound (Hanjos et al., 2007). It is a 3, 4, 5- trihydroxy benzoic acid, having astringent

(Leela et al., 2013) and strong antioxidant activity (Raghavendra et al., 2013). It act as a free

radical scavenger and can be used for the treatment of lung cancer (Ghasemzadeh et al., 2010)

31

and brain tumour by suppressing the proliferation, cell viability and angiogenesis (Lu et al.,

2010). Gallic acid easily absorbed in human body and show activity against cancer cells

(Shabir et al., 2011).

Fig 2.9. Chemical structure of gallic acid

2.15.2. p-coumaric acid:

p-coumaric acid is a cinnamic acid derivative. It has antioxidant attributes and reduces

the risks of stomach cancer (Nagavani et al., 2010) by reducing the formation of carcinogenic

nitrosamines (Shabir et al., 2011).

Fig 2.10. Chemical structure of p-coumaric acid

2.15.3. Caffeic acid:

It is found naturally in vegetables, fruits, olive oil, tea, coffee beans and wine, possess

anti-inflammatory, anti-carcinogenic, antioxidant and anti-mutagenic activities (Yumrutas et

al., 2011). Caffeic acid possess good antioxidant activity as compared to quercetin (flavonoid)

and is a cinnamic acid derivative (Zheng and Wang, 2001).

Fig 2.11. Chemical structure of caffeic acid

32

2.15.4. Ferulic acid:

It is a cinnamic acid derivative, abundantly found in vegetables and maize bran

(Archivio et al., 2007). This phenolic acid prevents the photo-oxidation of linoleic acid at its

high concentration (Zheng and Wang, 2001).

Fig 2.12. Chemical structure of ferulic acid

2.15.5. Vanillic acid:

Vanillic acid is benzoic acid derivative (Hanjos et al., 2007) and exhibit antioxidant

activity but less than caffeic acid (Zheng and Wang, 2001; Simic et al., 2007), hepato-

protective activity (Kim et al., 2010) antimicrobial, antimalarial activities (Erdem et al., 2012)

and also act as an inhibitor of snake venom (Khadem and Marles, 2010).

Fig 2.13. Chemical structure of vanillic acid

2.15.6. Chlorogenic acid:

Chlorogenic acid have antioxidant, antitumour, anti-allergic, hepatoprotective,

hypoglycemic, anti-inflammatory, antiviral and antibacterial activities (Mussatto et al., 2011).

2.15.7. Syringic acid:

Syringic acid is found in many natural sources such as soybean, thyme, rosemary, date,

clove, medicinal shrub, propolis and cereal grains like barley, maize, millet, oat, rice, rye,

33

sorghum, and wheat (Dykes and Rooney, 2007). It exhibits antioxidant, antibacterial (Kong et

al., 2008) and hepatoprotective (Itoh et al., 2009; Itoh et al., 2010) activities.

Fig 2.14. Chemical structure of syringic acid

34

Chapter 3

Material and Methods

The research work presented in this dissertation was conducted in the Hi-Tech

Laboratory and Protein and molecular biology laboratory (PMBL) of Department of Chemistry

and Biochemistry, University of Agriculture, Faisalabad.

3.1. Collection of Plants:

Selected medicinal plants were collected from different areas of Pothohar Plateau in

the months of March and April, 2011. Authentic identification plants was carried out by Dr.

Mansoor Ahmad, Associate Professor, Department of Botany, University of Agriculture,

Faisalabad. The investigated plants are given in the Table 3.1.

Table 3.1. Selected medicinal plants from Pothohar plateau, Pakistan

Sr. No. Biological name Family name Local name

1 Asphodilus tenifolius Liliaceae Piazi

2 Aerva javanica Amaranthaceae Bui

3 Fagonia indica Zygophyllaceae Dhamasa

3.2. Materials used in the whole research work:

Materials, standard compounds and instruments used in research work along with their

company names are given in the tables 3.2.1, 3.2.2 and 3.2.3, respectively:

Table 3.2.1. Materials, chemicals and reagents used in the present research work

Name Company name

2, 2-diphenyl-1-picrylhydrazyl (DPPH) Sigma-Aldrich, Germany

Folin-Ciocalteu reagent Applichem, Germany

Nitro blue tetrazolium (NBT) MP Biomedicals LLC, France

Ethylenediamine tetraacetic acid (EDTA) Sigma-Aldrich, Germany

ß-carotene Fluka, Sigma Aldrich

Linoleic acid Sigma-Aldrich, Germany

35

Chloroform Merck, Germany

Aluminium chloride Fluka chemic, Switzerland

Sodium acetate Sigma-Aldrich, Germany

Potassium ferricyanide Daejung, Korea

Trichloroacetic acid Sigma-Aldrich, Germany

Ferric chloride BDH Laboratory supplies, England

Sodium dihydrogen phosphate Applichem, Germany

Disodium hydrogen phosphate Applichem, Germany

Hydroxylamine hydrochloride Daejung, Korea

Sodium carbonate Applichem, Germany

Sodium nitrite Applichem, Germany

Sodium hydroxide Merck, Germany

Acetone Merck, Germany

Nutrient agar Oxoid, Hamsphire, UK

Nutrient broth LAB M, Limited, UK

Potato dextrose agar Applichem, Dermastadt, Germany

Sabarose dextrose broth Applichem, Dermastadt, Germany

Resazurin WRH Int. Limited, Lutterworth, UK

Methanol Merck, Germany

Ethanol Sigma-Aldrich, Germany

Dimethyl sulfoxide Merck, Germany

Hydrochloric acid Labscan, Thailand

Sulphuric acid BDH Laboratory supplies, England

Whattman No. 1 filter paper Sigma-Aldrich, Germany

0.45µm filter paper Biotech, Germany

TLC silica gel 60 F254 Aluminium sheets Merck, Germany

36

Table 3.2.2. Standard compounds used in the research work

Name Company name

Gallic acid BDH Laboratory supplies, England

Ascorbic acid Sigma Aldrich, Germany

Butylated hydroxyl toluene(BHT) Merck, Germany

Quercetin Sigma Aldrich, Germany

Rifampicin Sigma-Aldrich, Germany

Terbinafine Saffron pharmaceuticals, Faisalabad, Pakistan

Table 3.2.3. Instruments used in the research work

Name Company name

Magnetic stirrer Yellowline, IKA, USA

Orbital shaker Yellowline, OS10 Basic

Sonicator Transsonic T 460/H Elma, Germany

Centrifuge Sigma

Waterbath Memmert, Germany

Vortex mixer Heidolph Reax Top D-91, Schwabach

Laminar air flow Memmert, Germany

ELISA microplate reader Bio-Tek-USA

Autoclave JICA, Japan

Double beam spectrophotometer Lambda 25, Perkin Elmer, USA

Incubator Memmert, Germany

Rotary evaporator EYELA, SB-651, Rikakikai Co. Ltd.

Tokyo, Japan

pH meter WTW Inolab multi, 720, Germany

Analytical balance AUY 220, SHIMADZU, Japan

High performance liquid chromatography

(HPLC) LC-10A, SHIMADZU, Japan

37

3.3. Washing and Drying of plant samples:

Whole plant materials were collected. After washing with distilled water, these were

dried under room temperature. Dried plant materials were ground by a mechanical grinder and

stored for further analysis.

3.4. Extraction:

Following solvent systems were used for the extraction of plant material:

Absolute (100%) and aqueous ethanol (80%)

Absolute (100%) and aqueous methanol (80%)

Absolute (100%) and aqueous acetone (80%)

Extraction was performed by using each of above solvent system through four extraction

techniques:

3.4.1. Stirring as extraction technique:

20 g powdered plant material was extracted with 200 mL of each solvent system in a

magnetic stirrer for 4h at room temperature according to the method of Chandrappa et al.

(2011). After 4h, the above mixture was centrifuged at 10,000 rpm for 10 min at 40oC. After

centrifugation, the supernatant was separated and residue was re-extracted with same fresh

solvent. The supernatents were mixed and the solvent was removed by using rotary evaporator.

3.4.2. Orbital shaking as extraction technique:

Plant material (20 g) was mixed with 200 mL of each solvent system in conical flasks.

Extraction was carried out in an orbital shaker for 6h at room temperature (Sultana et al., 2009).

The extract was filtered and residue was extracted twice. The combined supernatents were

dried in a rotary evaporator. The dried extract was stored in a cool dry place for further analysis.

3.4.3. Sonication as extraction technique:

20 g plant material was extracted in 200 mL of above solvent systems. Extraction was

done in a sonication bath for 1h at room temperature (Anwar et al., 2006). After sonication it

was subjected to centrifugation for 10 min at 3800 × g. Residue was again extracted with fresh

solvent system. Supernatents were mixed and evaporated to dryness by a rotay evaporator.

38

3.4.4. Reflux as extraction technique:

20 g plant powder was extracted for 6 h in each solvent system (200 mL) as described

by Khan et al. (2012). Extraction was carried out in a soxhlet apparatus under reflux. The raw

extracts were filtered and evaporated to dryness in a vacuum oven.

After drying of each extract, extractive value of each method was calculated by using this

formula (Mandal et al., 2009):

Extractive value (% yield) = Weight of extract × 100

Weight of plant material

Dried extracts were stored in a refrigerator (-4oC) in air tight bottles until used for further

analysis.

3.5. Phytochemical Studies:

3.5.1. Determination of total phenolic contents (TPC):

Total phenolic contents of plant extracts were determined by the method of Aiyegoro

and Okoh (2010) using Folin-Ciocalteu reagent. 2.5 mL of 10% Folin-Ciocalteu reagent was

added to 0.5 mL of plant extract solution (250 µg/mL). Then 2 mL of 2% (w/v) sodium

carbonate solution was added and mixed thoroughly. After 30 minutes of incubation at 45oC,

absorbance was taken at 765 nm using UV/Vis spectrophotometer. All the determinations were

carried out in triplicates. A standard curve was also obtained using various concentrations of

gallic acid (0-30µg/mL) (Fig 3.1). Solution of gallic acid was made in water and the above

procedure was followed. Results were expressed as milligrams of gallic acid (GAE) per g of

plant extract.

39

Fig 3.1. Standard curve of gallic acid for total phenolic contents

3.5.2. Determination of total flavonoid contents (TFC):

Aluminium chloride colorimetric method was used for determination of total flavonoid

contents (Kumar and Kumar, 2009). One mL of plant extract was diluted with 4 mL distilled

water in a volumetric flask. Initially 0.3 mL of 5% NaNO2 was added in the mixture. At 5th

minute 10% AlCl3 (0.3 mL) and at 6th minute 2 mL of NaOH (1M) were added. Total volume

was made up to 10 mL by distilled water and mixed well. Absorbance of the reaction mixture

was measured at 510 nm using a spectrophotometer. Standard curve was obtained by using

different concentration of quercetin (0-20 µg/mL) (Fig 3.2). TFC were determined as quercetin

equivalents (mg/g of plant extract). Three readings were taken for each sample and the results

reported as average.

Fig 3.2. Standard curve of quercetin for total flavonoid contents

y = 0.0089x + 0.1187R² = 0.9955

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 5 10 15 20 25 30 35

Ab

sorb

ance

at

76

5 n

m

Concentration(µg/mL)

y = 0.0011x + 0.0688R² = 0.9558

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

0 5 10 15 20 25

Ab

sorb

ance

at

51

0 n

m

Concentration (µg/mL)

40

3.6. Antioxidant activity:

3.6.1 TLC based DPPH radical scavenging assays:

The antioxidant activity was analyzed qualitatively using thin layer chromatography

(TLC) followed by DPPH (2, 2-Diphenyl-1-picrylhydrazyl) technique according to the method

of Patnibul et al.,(2008) and Bhatnagar et al.,(2012) with slight modification. About 10μL of

extract solution was loaded on TLC plates. The plates were developed in acetone: methanol

(1:1). After development of plate, the plate was air-dried. To evaluate antioxidant activity,

plates were sprayed with 0.02% methanolic solution of DPPH. The presence of antioxidant

compounds was detected by yellow spots against a purple background on TLC plate.

3.6.2. DPPH free radical scavenging activity (Spectrophotometric method):

Free radical scavenging activity was determined by using 2, 2-diphenyl-1-

picrylhydrazyl (DPPH) radical as described by Kumar and Kumar (2009). DPPH solution (33

mg/L) was prepared in methanol. Absorbance of this solution was taken at 0 min. Then extract

solution (250 µg/mL) were prepared. Then 5mL of methanolic solution of DPPH was added

in 1mL of extract solution. The mixture was left in the dark for 30 minutes. Then absorbance

was measured at 517 nm using a spectrophotometer with methanol as blank. Free radical

scavenging activity was expressed as percentage inhibition and calculated by using the

following formula:

Inhibition (%) = Control Absorbance - Sample Absorbance × 100

Control Absorbance

Where control absorbance is absorbance of methanolic solution of DPPH taken at zero minute

3.6.3. Superoxide radical scavenging (SOR) activity:

Superoxide radical scavenging activity was evaluated by using NBT (Nitro blue

tetrazolium reagent) according to the method of Veena and Mishra (2011). Here extract

solution was taken at a concentration of 250 μg/mL. In 1mL of extract solution, 1mL of 50

mM sodium carbonate and 0.4mL of 24mM NBT were added. To this reaction mixture, 0.2mL

of 0.1 mM EDTA solution was added. Now the reaction was started by adding 0.4 mL of 1

mM hydroxylamine hydrochloride. Reaction mixture was left for 15 minutes at 25oC. After 15

41

minutes, absorbance was measured at 560nm using a spectrophotometer. %inhibition was

calculated by following formula:

% inhibition = [(Ao-At) / Ao x 100]

Ao is absorbance of the control (blank, without extract) and At is absorbance in the presence

of the extract.

3.6.4. Determination of reducing power (FRAP contents):

Reducing power of plant extracts was determined by ferric-reducing antioxidant power

(FRAP) assay (Chan et al., 2007). In this method, 0.2M phosphate buffer (2.5 mL, pH=6.6)

was added in 1mL plant extract solution. Then 1% (w/v) potassium ferricyanide (2.5 mL) was

added into mixture and incubate for 20 minutes at 50oC. After incubation, 10% (w/v)

trichloroacetic acid solution (2.5 mL) was added to stop the reaction. Then 2.5 mL of the

mixture was separated in a test tube and diluted with 2.5 mL distilled water. 500 µL of ferric

chloride solution (0.1% w/v) was added into diluted mixture and left it for 30 minutes. Then

absorbance was taken at 700 nm using spectrophotometer.

Different concentrations of gallic acid were used to draw standard curve (Fig 3.3).

Ferric reducing power of extract was calculated as mg gallic acid equivalent/ g of dry plant

extract (mg GAE/g).

Fig 3.3. Standard curve of gallic acid for FRAP contents

y = 0.0333x + 0.2012R² = 0.9918

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 10 20 30 40 50 60

Ab

sorb

ance

at

70

0 n

m

Concentration (µg/mL)

42

3.6.5. Antioxidant activity determination in a ß-carotene linoleic acid system:

In this assay antioxidant activity of plant extracts was determined in terms of bleaching

of ß-carotene according to the method of Chatha et al., (2011). 40 milligram of linoleic acid

and 400 mg of Tween 40 were mixed with 2 mL of ß-carotene solution. ß-carotene solution

was prepared in chloroform at a concentration of 1 mg/mL. After mixing, chloroform was

removed on a water bath at 45oC for few minutes. 100 mL distelled water was added in this

semisolid material with continuous agitation to form an emulsion. 5 mL of this emulsion was

added to 0.2 mL of extract solution (500 mg/L). Absorbance of the mixture was measured at

470 nm using a spectrophotometer immediately. Emulsion without ß-carotene was taken as

blank in spectrophotometer. Then mixture was kept in a water bath at 50oC for 120 minutes.

Absorbance was measured again after 120 minutes at 470 nm. All determinations were

performed in triplicate. Antioxidant activity coefficient of extracts was calculated as follows

AAC= (AA(120)-AC(120))/(AC(0)-AC(120))×1000

Where AA(120) is the absorbance of the sample after 120 minutes, AC(120) is the absorbance of

the control after 120 minutes and AC(0) is the absorbance of the control at 0 minute.

3.7. Evaluation of Antimicrobial Activity:

3.7.1. Antibacterial activity:

3.7.1.1. Bacterial Strains:

Four bacterial strains were used in this study. Two gram positive (Bacillus subtillus

and Staphylococcus aureus) and two gram negative (Escherichia coli and Pasteurella

moltocida) bacterial species were used.

3.7.1.2. Bacterial culture preparation:

Cultures of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pasterella

multocida were prepared in nutrient broth medium and kept incubation at 37°C for 24 hours at

120 rpm in a shaker. After growth was observed, the cultures were stored in the refrigerator at

2-8°C (Dhale and Markandeya, 2011).

43

3.7.1.3. Disc diffusion assay for antibacterial activity:

Disk diffusion assay was used for the determination of antibacterial activity of plant

extracts (Bulbul et al., 2011). The required number of Petri plates were washed and autoclaved.

And they were allowed to cool under laminar air flow. Solutions of plant extracts (10 mg/mL)

were prepared in 10% dimethyl sulfoxide (DMSO). Nutrient agar solution was prepared and

autoclaved. Aseptically transfer about 20 mL of nutrient agar medium (containing bacterial

culture, 50 µL) into each sterile petri plate and allowed to solidify. Dried and sterilized discs

(10 mm) of wicks sheet were prepared. Then discs containing 100 µL plant extracts solution

(10mg/mL) were placed on nutrient agar medium. Discs containing standard antibiotic

(Rifampicin, 100 µg/mL) and 10% dimethyl sulfoxide were also placed in each petri plate as

positive and negative control. The plates were then incubated at 37oC for 24h. After incubation,

the antibacterial activity was recorded by measuring the diameter of zone of inhibition around

the disc using zone reader (mm). The experiment was carried out in three replicates and average

was calculated.

3.7.1.4. Spectrophotometric assay (Determination of MIC):

Minimum inhibitory concentration (MIC) was determined spectrophotometrically for

each plant extract showing antimicrobial activity against test pathogens. Broth micro-dilution

method (Mehmood et al., 2012) was followed for determination of MIC values. Plant extracts

were used at a final concentration of 10 mg/mL. Plant extract solutions (100 µL) were added

in the wells of first row of microtiter plate and in the remaining wells 50 µL nutrient broth

medium was added. Then two fold serial dilutions were done using a micropipette so that each

well had 50 μL plant extract solution in a serially decreasing concentration. Thereafter 10 μl

inoculum was added to each well. Dimethyl sulfoxide in broth medium was used as negative

control, while broth containing standard drug (Rifampicin) was used as positive control. The

microtiter plates were incubated at 37°C for 24 h. Each extract was assayed in triplicate. After

24 h of incubation, 10µL of resazurin solution (an indicator) was added in each well. In the

presence of bacterial growth, blue colour of resazurin solution was changed into pink.

Absorbance was recorded at 620 nm by keeping microtiter plate in an ELISA reader. The

minimum inhibitory concentration was defined as the lowest concentration able to inhibit any

visible bacterial growth.

44

3.7.2. Antifungal activity:

3.7.2.1. Fungal strains used:

Four fungal strains were used for antifungal activity of plant extracts. Helminthus

spermium, Fusarium solani, Aspergillus flavus and Aspergillus niger were used.

3.7.2.2. Fungal inoculum preparation:

Fungus were grown in sabouraud dextrose broth at 28oC for 72 h. The turbidity in

broth medium showed the growth of fungus. The fungus culture was stored in refrigerator at

2-8°C for further analysis.

3.7.2.3. Disc diffusion assay for antifungal activity:

Antifungal activity of plant extracts were determined according to the method of Saha

and Paul (2012) with some modifications. Potato dextrose agar (PDA) solution was prepared

and autoclaved. About 20 mL PDA solution was poured in sterilized petri plates under laminar

air flow. Sterilized discs (10 mm) of wicks sheet impregnated with 100 µL of plant extract

solution (10 mg/mL) were placed on the agar plates. DMSO was used as negative control and

Terbinafine (100 µg/mL) was used as positive control. The activity was determined after 48 h

of incubation at 28oC. The diameter of the inhibition zones were measured in mm. All

determinations were carried out in triplicates.

3.7.2.4. Spectrophotometric assay (Determination of MIC):

The minimum inhibitory concentration (MIC) of the extract and fractions was evaluated

by the method reported by Sharma and Kumar (2009). Briefly, 100 μL of plant extract solution

(10 mg/mL) was transferred into the first row of the 96 well microtiter plates. To all other

wells, 50 μL of Sabouraud dextrose broth was added. Two-fold serial dilutions were performed

using a micropipette such that each well had 50 μL of the test material in serially descending

concentrations. Finally, 10 μL of fungal suspension was added to each well. Each plate had a

row of control (DMSO), a row of positive control (Terbinafine). The plates were prepared in

45

triplicate, and incubated at 28°C for 48 h. The absorbance was measured at 520 nm by ELISA

reader. The lowest concentration at which there was no growth was taken as the MIC value.

3.8. High Performance Liquid Chromatography (HPLC):

HPLC was used as an analytical tool for the qualitative and quantitative analysis of

phenolic compounds. Analysis was done on reverse phase high performance liquid

chromatography (RP-HPLC) equipped with UV-Vis detector (Table 3.5).

Table 3.3.1. Specifications for HPLC analysis for phenolic compounds

Column(Stationary phase) Shim-pack CLC ODS(C-18), 25cm×4.6mm, 5µm

Eluent (Mobile phase) A(H2O:AA-94:63, pH=2.27),

B (ACN 100%)

Gradient elution Program 0-15min= 15%B, 15-30=45%B, 30-45min=100%B

Flow rate 1mL/min

Column thermostate Room temperature

Detector UV-Vis detector, 280 nm

3.8.1. Sample preparation for HPLC analysis:

For HPLC analysis sample preparation was done according to the method of Dek et al.,

2011. Briefly, 50 mg of extracts was dissolved in 24 mL methanol and homogenized. Sixteen

mL distilled water was then added followed by 10 mL of 6 M HCl. The mixture was then kept

in an oven for 2 hrs at 95ºC. The final solution was filtered using a 0.45μm filter paper prior

to high performance liquid chromatography (HPLC) analysis.

3.8.2. Standard used:

Gallic acid, caffeic acid, vanillic acid, 4-hydroxy-3-metoxy benzoic acid, chlorogenic acid,

syringic acid, p-coumaric acid, m-coumaric acid and ferulic acid were used as standard

compounds for qualitative and quantitative analysis.

46

(a) (b)

(c) (d)

(e) (f)

(g) (h)

(i)

Fig 3.4. HPLC chromatograms of standard phenolic acids used in the present research

work (a) gallic acid, (b) caffeic acid, (c) vanillic acid, (d) 4-hydroxy-3-metoxy benzoic

acid, (e) chlorogenic acid, (f) syringic acid, (g) p-coumaric acid, (h) m-coumric acid and

(i) ferulic acid

47

3.9. Statistical Analysis: Each sample was analyzed individually in triplicate for its phytochemical studies, antioxidant

and antimicrobial activities and data was reported as mean ±SD. Data was further analyzed by

analysis of variance ANOVA using Minitab 2000 Version 13.2 statistical software (Minitab

Inc. Pennysalvania, USA) at 5% significance level (Shahat et al., 2011).

48

Chapter 4

Results and Discussion

In this study three plants (Asphodilus tenifolius, Aerva javanica and Fagonia indica)

belonging to different plant families were collected from different areas of Pothohar plateau.

These plants were analyzed for antioxidant and antimicrobial properties and phytochemical

studies. To optimize the suitable extraction method these plants were extracted with six solvent

systems such as absolute methanol (100%), aqueous methanol (80%), absolute ethanol (100%),

aqueous ethanol (80%), absolute acetone (100%) and aqueous acetone (80%). Four different

extraction techniques (stirring, orbital shaking, sonication and reflux) were used with each

solvent system.

4.1. Extraction yield:

Extraction is the first important step in the preparation of plant formulations. Extraction

means recovery of bioactive compounds from plant material using appropriate solvent and

extraction technique. The extracts obtained consist of mixture of plant metabolites in semisolid

form (Gupta et al., 2012).

Variation in extraction yields revealed that solvents and extraction techniques have

significant effect on the extraction of bioactive compounds from plants. The effect of different

solvent systems on extraction yield (g/100g of dry plant powder) of different plants with

stirring as extraction technique is given in Fig 4.1.1. In case of Asphodilus tenifolius, maximum

extraction yield was given by aqueous ethanol (13.85 g/100g). While in case of Aerva javanica

and Fagonia indica, maximum extraction yields were obtained with aqueous methanol, 11.04

and 12.32 g/100g, respectively.

Fig 4.1.2 shows the effect of different solvent systems on extraction yield of different

plants using orbital shaking as extraction technique in g/100g of dry plant powder. Here in case

of

49

Fig 4.1.1. Effect of different solvent systems on extraction yields (g/100g) of different

plants using stirring as extraction technique


plants using orbital shaking as extraction technique

7.99

13.855

11.93 12.05

9.47

11.84

5.18

10.076

7.114

11.036

2.3

10.364

4.89

10.17

7.68

12.32

3.11

11.392

0

2

4

6

8

10

12

14

16

Absoluteethanol

Aqueousethanol

Absolutemethanol

Aqueousmethanol

Absoluteacetone

Aqueousacetone

yiel

d (

g/1

00

g)

Solvent systems

Asphodilus tenifolius Aerva javanica Fagonia indica

7.94

13.42

9.52

11.255

8.69

11.3

4.628

8.47

6.486

9.93

2.9843.96

4.06

8.227.212

12.13

3.076

6.8

0

2

4

6

8

10

12

14

16

Absolute ethanol Aqueous ethanol Absolutemethanol

Aqueousmethanol

Absoluteacetone

Aqueousacetone

Yiel

d (

g/1

00

g)

Solvent systems


50

Asphodilus tenifolius, maximum yield was obtained with aq. ethanol (13.4 g/100g). Aqueous

methanol showed maximum extraction yield in case of A.javanica and F. indica, 9.93and 12.13

g/100g, respectively.

The effect of different solvent systems on extraction yields of different plants using

sonication as extraction technique is presented in Fig 4.1.3. Aqueous methanol gave maximum

extraction yield in all the three plants using sonication, Asphodilus tenifolius (15.39 g/100g),

Aerva javanica (12.95 g/100g) and Fagonia indica (12.71 g/100g).

Fig 4.1.3 Effect of different solvent systems on extraction yields (g/100g) of different

plants using sonication as extraction technique

8.59

14.475 14.815.39

13.235

14.5

5.552

10.29

7.5

12.95

3.62

10.658

6.73

11.66

8

12.71

5.224

11.82

0

2

4

6

8

10

12

14

16

18

Absoluteethanol

Aqueousethanol

Absolutemethanol

Aqueousmethanol

Absoluteacetone

Aqueousacetone

Yiel

d (

g/1

00

g)

Solvent systems


51


plants using reflux as extraction technique

Extraction yields of different solvent systems from plant materials through reflux

extraction is given in Fig 4.1.4. Maximum extraction yield of Asphodilus tenifolius was

obtained with aqueous ethanol and aqueous acetone 18.86 and 18.74 g/100g, respectively. In

case of Aerva javanica, aqueous methanol and aqueous ethanol gave maximum extraction

yield, 14.57 and 14.53 g/100g, respectively. Among all the extracts of Fagonia indica,

maximum extraction yield (14.44 g/100g) was obtained by using aqueous methanol as

extracting solvent. The variations in the yield of the all the plants with respect to different

solvent systems and plant species were significant (p < 0.05).

Overall results showed that in case of Ashodilus tenifolius, extraction efficiency of

different solvents was ethanol>methanol>acetone. While extraction efficiency of different

solvents in Aerva javanica was methanol>acetone>ethanol and in Fagonia indica was

methanol>ethanol>acetone. All the solvents in combination with water showed better

extraction yield than pure solvent. The presence of water in a solvent mixture is important as

it increases the extraction efficiency by swelling the plant material and allows the diffusion of

extraction solvent into the plant material. Thus increasing the extractability. Sultana et al.,

17.4218.86

16.1017.22

16.38

18.74

6.79

14.53

8.708

14.57

6.93

11.326

12.12

13.88

8.97

14.44

6.27

13.78

0

5

10

15

20

25

Absoluteethanol

Aqueousethanol

Absolutemethanol

Aqueousmethanol

Absoluteacetone

Aqueousacetone

Yiel

d (

g/1

00

g)

Solvent systems


52

(2007) also reported that methanol and ethanol are effective solvents for the extraction of

phenolic compounds.

From all the plants maximum extraction yield was obtained from Asphodilus tenifolius

(7.94-18.86 g/100g) followed by Fagonia indica (3.08-14.44 g/100g) and minimum from

Aerva javanica (2.30-14.57 g/100g). By comparing extraction techniques, reflux technique

showed maximum extraction yield from all the three plants followed by sonication, stirring

and orbital shaking. This is because, heating raised the efficiency of solvent to solubilize most

of the compounds (Victorio et al., 2009). Arnnok et al., (2012) studied the effect of shaking

and magnetic bar stirring on extraction yield and found that efficiency of extraction yield was

much better with magnetic bar stirring.

Vaghasiya and Chanda (2007) also studied Asphodilus tenifolius, obtained 3.6% and

11.4% extraction yield by using absolute acetone and methanol as extracting solvent,

respectively through rotary shaker. Yield with methanol in our study is comparable with their

results while yield with acetone in our results is much much higher than reported. Fatimi et al.,

(2007) studied the Aerva javanica and obtained 10.5% extraction yield with pure methanol as

solvent. These results are comparable to our findings. Srinivas and Reddy (2012) studied

different solvents extracts of Aerva javanica and found that maximum extraction yield was

obtained with pure methanol using shaking as extraction technique.

Sethi and Sharma (2011) reported that pure methanol gave maximum extraction yield

(2.30%) through soxhlet extraction of Aerva tomentosa as compared to petroleum ether

(0.35%), dichloromethane (0.65%), ethalyacetate (0.50%) and water (2.09%). Bukhari et al.,

(2008) studied Fenugreek seeds by extracting with different solvents and showed that methanol

and ethanol gave maximum extraction yield. Panghal et al., (2011) reported that maximum

extraction yield was obtained with methanol as extracting solvent. Mahmoud et al., (2011)

explained that extraction efficiency depends upon the polarity of solvent system. Hayouni et

al., (2007) also reported that aqueous solvents are mostly used for the extraction of

phytochemicals. Mohammedi and Atik (2011) reported that aqueous solvents are better for the

extraction of phenolic compounds from Tamarix aphylla leaves. Anwar et al., (2010) also

described that aqueous methanol gave better extraction yield of barley seeds.

53

4.2. Phytochemical Studies:

4.2.1. Total phenolic contents:

Plants phenolic extracts are mixtures of different types of phenolic acids having varied

solubility in different solvents (Mohammedi and Atik, 2011). Folin ciocalteu reagent is

generally used for the determination of phenolic contents. Blue colour formed after 15-60

minutes is measure spectrophotometrically at 715 nm and results are calculated in terms of

molar equivalent of a standard compound sucah as gallic acid (Robards, 2003). Phenolic

compounds are hydrogen donating so they possess good antioxidant activity (Kalim et al.,

2010).

TPC of plant extracts obtained through six extracting solvent systems and four

extraction techniques are given in table 4.1.1, 4.1.2, 4.1.3 and 4.1.4. TPC are calculated by

taking gallic acid a standard and expressed as mg GAE/g of plant extract. Table 4.1.1 shows

the effect of different solvent systems on the TPC of different plants using stirring as extraction

technique. Highest TPC of Asphodilus tenifolius was obtained in the aqueous ethanolic extract

(70.39 mg GAE/g) and least TPC in absolute acetone (40.68 mg GAE/g). In case of Aerva

javanica, maximum and minimum TPC were given by aqueous methanolic (124.00 mg

GAE/g) and absolute ethanolic (84.41 mg GAE/g) extracts. Aqueous methanolic and absolute

acetone extracts showed maximum (139.60 mg GAE/g) and minimum (40.40 mg GAE/g) TPC

among all the extracts of Fagonia indica.

54

Table 4.1.1. Total phenolic contents (mg GAE/g of plant extract) of different solvent

extracts using stirring as extraction technique

Solvent systems

Selected medicinal plants


Absolute ethanol 51.83±2.59cdc 84.41±4.22d

a 69.93±3.50cb

Aqueous ethanol 70.39±3.52ac 112.40±5.62ab

a 99.47±4.97bb

Absolute methanol 45.59±2.28deb 88.98±4.45cd

a 81.98±4.10ca

Aqueous methanol 56.26±2.81bcc 124.00±6.20a

b 139.60±6.98aa

Absolute acetone 40.68±2.03eb 96.53±4.83cd

a 40.40±2.02db

Aqueous acetone 59.54±2.98bc 102.30±5.12bc

a 76.48±3.82cb

Values (mean ± SD) are average of three replicates; Superscript letters within the same

column indicate significant (P< 0.05) differences of means within the extracting solvent

systems; Subscript letters within the same row indicate significant (P< 0.05) differences of

means within the medicinal plants.

TPC of different plants using orbital shaking as extraction technique are given in Table

4.1.2. Aqueous ethanolic and absolute acetone extracts of Asphodilus tenifolius showed

maximum (58.46 mg GAE/g) and minimum (37.20 mg GAE/g) antioxidant activities,

respectively. Among Aerva javanica extracts, aqueous methanolic extract gave maximum TPC

(121.85 mg GAE/g) and absolute methanol gave minimum TPC (74.06 mg GAE/g). In case

of Fagonia indica, aqueous methanolic and absolute acetone extracts showed maximum

(124.90 mg GAE/g) and minimum (57.58 mg GAE/g) antioxidant activities, respectively.

55


extracts using orbital shaking as extraction technique

Solvent systems



Absolute ethanol 53.63±2.68abb 80.18±4.01bc

a 72.75±3.64ca

Aqueous ethanol 58.46±2.92ac 109.36±5.47a

a 97.44±4.87bb

Absolute methanol 38.11±1.91cc 74.06±3.70c

b 99.07±4.95ba

Aqueous methanol 48.09±2.40bb 121.85±6.09a

a 124.90±6.25aa

Absolute acetone 37.20±1.86cc 82.31±4.12bc

a 57.58±2.88db

Aqueous acetone 39.35±1.97cb 92.17±4.61b

a 91.37±4.57ba





Table 4.1.3 shows the effect of different solvent systems on the TPC of different plants

using sonication as extraction technique. Aqueous ethanolic and absolute methanolic extracts

gave maximum and minimum TPC (76.23 and 53.40 mg GAE/g, respectively) among all the

extracts of Asphodilus tenifolius.In case of Aerva javanica and Fagonia indica, aqueous

methanolic extracts gave maximum TPC, 130.19 and 161.00, respectively.

TPC of different plants as affected by different solvent systems using reflux as

extraction technique are shown in Table 4.1.4. In case of Asphodilus tenifolius and Aerva

javanica, aqueous methanolic extracts gave maximum TPC (48.72 and 90.76 mg GAE/g,

respectively) and minimum TPC were obtained by absolute acetone extracts (24.78 and 56.67

mg GAE/g, repectively). While in case of Fagonia indica, aqueous methanolic extract gave

maximum TPC (109.70 mg GAE/ g) and absolute acetone extract showed least TPC (35.78

mg GAE/g).

56


extracts using sonication as extraction technique

Solvent systems



Absolute ethanol 58.73±2.93cdb 104.78±5.23b

a 62.64±3.13db

Aqueous ethanol 76.23±3.81ac 105.45±5.27b

b 122.11±6.11ba

Absolute methanol 53.4±2.67dc 87.73±4.39c

b 101.75±5.09ca

Aqueous methanol 68.45±3.42abc 130.19±6.51a

b 161.00±8.05aa

Absolute acetone 66.71±3.34bcb 85.85±4.29c

a 65.75±3.29db

Aqueous acetone 70.56±3.53abb 110.06±5.50b

a 113.12±5.66bca





57


extracts using reflux as extraction technique

Solvent systems



Absolute ethanol 44.13±2.21ac 66.57±3.33cd

a 53.69±2.69db

Aqueous ethanol 48.72±2.44ab 90.76±4.54a

a 90.49±4.52ba

Absolute methanol 31.41±1.57bc 75.24±3.76bc

a 67.10±3.36cb

Aqueous methanol 46.27±2.31ac 80.45±4.02ab

b 109.70±5.48aa

Absolute acetone 24.78±1.24cc 56.67±2.83d

a 35.78±1.80eb

Aqueous acetone 25.56±1.28cc 85.01±4.25ab

a 66.34±3.32cb





Overall results showed that aqueous solvents gave more TPC than pure solvents.

Maximum TPC of Asphodilus tenifolius were obtained by extracting it with aqueous ethanol.

While TPC of Aerva javanica and Fagonia indica were obtained by using aqueous methanol

as extracting solvent. So, aqueous methanol and aqueous ethanol are best for extracting more

total phenolic contents from plant materials. This is because, addition of water in these solvents

increases the polarity of the solvents thus increasing their efficiency to extract phenolic

compounds (Anwar et al., 2010; Mohammedi and Atik, 2011). Absolute acetone gave least

TPC in all the three plants under investigation. By comparing the plants, highest TPC were

obtained from Fagonia indica followed by Aerva javanica and then Asphodilus tenifolius.

Extraction techniques also affect total phenolic contents. Out of four extraction techniques,

trend obtained was sonication>stirring>orbital shaking>reflux. Ultrasound-assisted extraction

is a useful method for the extraction of phytochemicals from different materials in shorter times

as compared to other extraction methods because energy of ultrasound waves speed up the

extraction (Salas et al., 2010). From reflux technique, least total phenolic contents were

58

obtained because at high temperature phenolic compounds are decomposed (Khoddami et al.,

2013). Sultana et al., (2009) also reported that TPC decreased when using reflux as extraction

technique, without regarding the nature of solvent used for extraction.

Herrera and Castro (2005) also found that sonication extraction is best for phenolic

compound extraction, much faster method and causes less phenolics degradation as compared

to Solid–liquid method, Subcritical water extraction and Microwave-assisted extraction.

Kalim et al., (2010) reported 15.74 mg GAE/g TPC in aqueous methanolic (50%) extract of

Asphodilus tenifolius using stirring as extraction technique, while working on Unani medicinal

plants, Which is less than our results. This may be due to different geographical and agro-

climatic conditions of both the areas under study. Plants contain a variety of compounds and

also differing in their quantities of these compounds according to age, harvesting process and

geographical conditions (Vazirian et al., 2011).

Our findings are in agreement with Vaghasiyaand Chanda (2011) who reported that

more total phenolic contents were obtained from methanolic extract (23.88mg GAE/g) than

acetone extract (17.23mg GAE/g) of Asphodilus tenifolius collected from western region of

India. However, the TPC in this study are less than our results. By comparing with previous

studied, it was found that plants native to Pothohar plateau provide great resources for phenolic

compounds. This may be due to the fact that Pothohar plateau bear extreme climatic conditions

and because of this severity in climate, plants produced more phenolic compounds.

Environmental factors such as temperature (both high and low) affect the activity of

phenylalanine ammonia-lyase thus changes the quantities of phenolics produced by plants

(Anttonen, 2007).

Sethi and Sharma (2011) reported 34.5mg GAE/g total phenolic contents in Aerva

tomentosa by using methanol as extraction solvent through soxhlet apparatus. Methanol is

polar and is an efficient solvent for the extraction of large number of phenolic compounds

(Mussatto et al., 2011; Srinivas and Reddy, 2012). Anwar et al., (2010) also described that

highest TPC were obtained from 80%methanolic extract, while working on barley seeds. Liu

and Yao (2007) described that higher amounts of TPC from barely seeds were obtained with

aqueous (70%) ethanol and aqueous (70%) methanol. Jindal et al., (2012) also reported that

methanol extract showed the maximum TPC in Ardisia crispa. Chirinos et al., (2007) described

that mixtures of solvents with water enhances the extraction efficiency by extracting more

59

phenolic glycosides, which are more water soluble. Yu et al., (2005) reported that methanol

and ethanol are more efficient extracting solvents than water for the extraction of phenolic

compounds from peanut skin. Neelam and Khan (2012) evaluated TPC of different solvent

extracts of Galium aparine L. and found that methanol is best solvent for the extraction of

phenolic compounds. Jakopic et al., (2009) described that methanolic extract of walnut fruits

exhibited more TPC while working on its methanolic and ethanolic extracts.

4.2.2. Total Flavonoid contents:

Flavonoids are important and widely distributed group of secondary metabolites,

having a lot of medicinal activities such as antioxidant, antimicrobial, antithrombotic and

anticancer activities (Raghavendra et al., 2013). Flavonoids are also involved in wound healing

either by wound contraction or by increasing the epithelialization (Varadharajan et al.,

2012).TFC in plant extracts depend on polarity of extracting solvent. Here polarity is

considered to be very important. Flavonoids of low polarity such as flavanones, flavonols,

isoflavones and methylated flavones are extracted with dichloromethane, diethyl ether,

chloroform or ethylacetate while more polar flavonoid aglycones and flavonoid glycosides are

extrcated with alcohols or alcohol-water mixtures. Glycosylation increases polarity and

alcohol-water mixtures are suitable for extraction (Stankovic et al., 2011).

Flavonoids are not easily detected that’s why in the extract AlCl3 was used as

complexing reagent (Bukhari et al., 2008). Some flavan-3-ols (catechin) are unstable in neutral

and alkaline medium. However, aluminium chloride reduces the pH and stabilizes the extract

so that these flavan-3-ol are precipitated with AlCl3 (Robards, 2003). A quercetin standard

curve was used for the calculation of flavonoids content.

Table 4.2.1, 4.2.2, 4.2.3 and 4.2.4 show the total flavonoid contents of plant extracts

using different solvent systems and extraction procedures. All the solvents had significant

effect on the flavonoid extraction from plants. Table 4.2.1 shows the TFC of all the plant

extracts using stirring as extraction technique. Results showed that aqueous ethanolic extract

among all the extracts of Asphodilus tenifolius gave highest TFC, 272 mg QE/g of plant extract

and least TFC were obtained from absolute acetone extract (116.73mg QE/g). From all the

extracts of Aerva javanica, aqueous methanolic and absolute ethanol extracts exhibited

maximum and minimum TFC, 229.26 and 57.26 mg QE/g, respectively. By comparing TFC

60

of all the solvent extracts of Fagonia indica, it was observed that aqueous methanolic extract

gave maximum TFC (176.57 mg QE/g) and absolute acetone gave minimum TFC (36.90 mg

QE/g).

Table 4.2.1. Total flavonoid contents (mg QE/g of plant extract) of different solvent


Solvent systems



Absolute ethanol 216.18±10.81ba 57.27±2.86c

b 54.35±2.72db

Aqueous ethanol 272.36±13.62aa 107.21±5.36b

b 111.48±5.57bb

Absolute methanol 165.82±8.29ca 100.94±5.05b

b 56.38±2.82dc


a 176.57±8.83ab

Absolute acetone 116.73±5.84da 62.97±3.15c

b 36.90±1.85ec

Aqueous acetone 138.91±6.95da 70.39±.3.52c

b 70.02±3.50cb





Total flavonoid contents of all the plant extracts as affected by different solvent systems

obtained through shaking are given in Table 4.2.2. Results show that aqueous ethanolic extract

exhibited maximum TFC from all the extracts of Asphodilus tenifolius. Among all the extracts

of Aerva javaniva and Fagonia indica, highest TFC were obtained from aqueous methanolic

extracts, 229.91 and 151.55mg QE/g, respectively. Absolute extracts of all the plants showed

least total phenolic contents, 125.23 (Asphodilus tenifolius), 57.27mg QE/g (Aerva javaniva)

and 42.50 mg QE/g (Fagonia indica).

61



Solvent systems



Absolute ethanol 217.15±10.86ba 70.29±3.51de

b 54.79±2.74db


b 91.83±4.59bc

Absolute methanol 161.45±8.07ca 90.69±4.53c

b 56.23±2.81dc

Aqueous methanol 181.27±9.06cb 229.91±11.49a

a 151.55±7.58ac

Absolute acetone 125.23±6.26da 57.27±2.86e

b 42.50±2.13ec

Aqueous acetone 162.36±8.12ca 80.05±4.00cd

b 70.93±3.55cb





Total flavonoid contents of all the plants extracts using sonication as extraction

technique are presented in Table 4.2.3. Among all the extracts of Asphodilus tenifolius

maximum and minimum total flavonoid contents were given by aqueous ethanolic (312.12 mg

QE/g) and absolute acetone extracts (96.55mg QE/g). From all the extracts of Aerva javaniva

and Fagonia indica, highest total flavonoid contents were obtained from aqueous methanolic

extracts (345.06 and 212.24 mg QE/g, respectively) and least contents were obtained from

absolute acetone extracts (68.34 and 46.59 mg QE/g, respectively).

62



Solvent systems



Absolute ethanol 213.21±10.67ba 74.64±3.73de

b 53.40±2.67dc


b 135.02±6.75bb

Absolute methanol 165.82±8.29ca 114.93±5.75c

b 59.12±2.96cdc


a 212.24±10.61ab

Absolute acetone 96.55±4.83da 68.34±3.42e

b 46.59±2.33dc

Aqueous acetone 165.09±8.25ca 93.26±4.66cd

b 74.08±3.70cc





Total flavonoid contents of all the plant extracts as affected by different solvent systems

are shown in the Table 4.2.4. Aqueous ethanolic extract from all the extracts of Asphodilus

tenifolius exhibited highest total flavonoid contents (245.94 mg QE/g) and absolute ethanolic

extract gave least total flavonoid contents (100.45 mg QE/g). By comparing all the extracts of

Aerva javanica, aqueous methanolic and absolute ethanolic extracts gave maximum and

minimum total flavonoid contents (202.50 and 40.31 mg QE/g of plant extract, respectively).

In case of Fagonia indica, maximum and minimum total flavonoid contents were given by

aqueous methanolic (162.30 mg QE/g) and absolute acetone extract (41.64 mg QE/g).

63



Solvent systems



Absolute ethanol 207.45±10.37aa 40.31±2.01d

b 51.84±2.59cdb

Aqueous ethanol 245.94±12.30ba 85.05±4.25b

b 80.02±4.00bb

Absolute methanol 109.27±5.46da 95.18±4.76b

b 54.77±2.74cc

Aqueous methanol 150.73±7.54cb 202.50±10.13a

a 162.30±8.12ab

Absolute acetone 100.45±5.02ca 52.81±2.64d

b 41.64±2.08dc

Aqueous acetone 145.00±7.25da 68.12±3.41c

b 62.87±3.14cb





The variation in the TFC of different solvent extracts was statistically significant (P <

0.05). Overall results showed that aqueous ethanol proved to be better solvent for the extraction

of flavonoid contents from the Asphodilus tenifolius. While aqueous methanol was most

suitable solvent for the extraction of flavonoid contents from Aerva javanica and Fagonia

indica. So, it is concluded that aqueous methanol and aqueous ethanol are efficient for the

maximum recovery of flavonoid compounds from plant extracts. Absolute ethanol and acetone

extracted least flavonoid contents. Aqueous solvents gave more flavonoid content as compared

to their pure solvents. The use of water in combination with organic solvents increases the

polarity of the medium thus ensures the extraction of more flavonoid contents. Out of the four

extraction techniques, maximum flavonoid extraction was carried out by using sonication

extraction technique (46.59-345.06 mg QE/g), followed by stirring (36.90-272.66 mg QE/g)

and shaking (42.50-254.73 mg QE/g) and least contents were obtained from reflux extraction

(40.31-245.94 mg QE/g). By comparing plant species for the total flavonoid contents the

decreasing trend obtained was Aerva javanica>Fagonia indica>Asphodilus tenifolius.

64

Our results are in accordance with Dek et al., (2011) who described that highest TFC

were obtained by using sonication extracting technique as compared to shaking extracting

technique. Xia et al., (2006) also reported that sonication extraction proved to be most effective

technique for the extraction of phytochemicals from tea at low temperature as compared to

other conventional techniques. Sun et al., (2011) reported that sonication provide highest

extraction yield of some flavonoids in lesser time as compared to soxhlet and maceration

extraction. In soxhelt extraction, due to high temperature plant phenolic compounds undergo

degradation and some other enzymatic oxidation reactions due to high temperature and

prolonged extraction time (Khoddami et al., 2013).

Kalim et al., (2010) reported 11.98 mg QE/g TFC in aqueous methanolic (50%) extract

of Asphodilus tenifolius using stirring as extraction technique, while working on Unani

medicinal plants, Which is lower than our reported results. This possibly may be due to

different geographical and agroclimatic conditions of both the areas under study. Vaghasiya

and Chanda (2011) reported 34.02 and 8.79 mg QE/g TFC in the acetone and methanolic

extracts of Asphodilus tenifolius through shaking, working on the medicinal plants of western

region of India. Arya et al., (2010) described that phyto-chemistry also vary depending upon

geographical regions, season and time of collection of time and different climatic conditions.

TFC are strongly affected by the solvent type used for extraction and on the different

concentrations of solvent (Ghasemzadeh et al., 2011). Jindal et al., (2012) also reported that

methanol as an extracting solvent gave high TFC in Ardisia crispa. All the Plant extracts

showed high contents of polyphenols thus exhibit good antioxidant and antimicrobial activities

(Baravalia et al., 2009). Shabir et al., (2011) reported that 80% methanol is an efficient and

mostly used solvent for the extraction of phenolics and flavonoids from plant matrix, due to

high polarity on comination of methanol and water, thus exhibiting higher efficacy to extract

of polar phytochemicals.

4.3. Evaluation of Antioxidant activity:

The antioxidant activity of the plant extracts basically depends on the composition of

the extracts, hydrophobic or hydrophilic nature of the antioxidants, type of extracting solvent,

method of extraction, temperature and conditions of the test systems. Therefore, it is necessary

65

to use more than one method for evaluation of antioxidant activity of plant extracts defining

various mechanisms of antioxidant actions (Zargar et al., 2011). Antioxidant activity was

determined by the following different methods:

4.3.1. TLC based DPPH free radical scavenging assay:

It is a qualitative type of analysis. In this assay extracts of all plants were spotted on

the TLC plates. Then plates were developed in a solvent system having acetone and methanol

(1:1). After developing, plates were air dried and then sprayed with methanolic solution of

DPPH. Yellow spots appeared on purple background on TLC plates which showed the DPPH

radical scavenging activity of those spots (Fig 4.2.1). All the plant extracts showed antioxidant

activity by scavenging DPPH free radical in a higher or lesser extent. Free radical scavenging

is very important due to the harmful effects of radicals in food industry as well as biological

systems (Mandade et al., 2011).

Fig 4.2.1. Representative TLC plate showing TLC-DPPH free radical scavenging

activity of plant extracts

66

4.3.2. DPPH free radical scavenging activity (Spectrophotometric method):

It is a type of colorimetric assay used to evaluate free radical scavenging activity of

extracts in short time (Sim et al., 2010). DPPH is a nitrogen centered free radical stable at room

temperature. It is used for the evaluation of free radical scavenging activity of plant extracts.

It gives purple colour in its free radical form. During the reaction, its purple colour changes to

yellow on receiving proton from the plant bio-actives especially phenolics (Sultana et al.,

2007). This change in colour is measured as decrease in absorbance, which indicates free

radical scavenging (antioxidant) power of the plant extract (Daud et al., 2011).

DPPH radical scavenging activity (% inhibition) of the tested plant extracts prepared

by using different solvent systems and extraction techniques are shown in Fig 4.3.1-4.3.4.

Considerable variations in antioxidant profile were recorded among different solvent extracts.

The effect of different solvent systems on DPPH radical scavenging activities (% inhibition)

of different plants using stirring as extraction technique is given in Fig 4.3.1. The maximum

DPPH radical scavenging

Fig 4.3.1 DPPH Free radical scavenging activities (%inhibition) of different solvent

extracts of medicinal plants using stirring as extraction technique

44.41

57.6749.33

55.61

35.53 33.85

53.76

69.0466.04

78.86

38.08

60.81

56.81

72.58

62.54

75.08

40.86

65.11

0

10

20

30

40

50

60

70

80

90

Absoluteethanol

Aqueousethanol

Absolutemethanol

Aqueousmethanol

Absoluteacetone

Aqueousacetone

% in

hib

itio

n

Solvent systems

Asphodilus tenifoliu Aerva javanica Fagonia indica

67

activity (%inhibition) of Asphodilus tenifolius was shown by aqueous ethanolic extract

(57.67%) and minimum was observed by aqueous acetone extracts (33.85%). In case of Aerva

javanica and Fagonia indica, maximum DPPH radical scavenging activity was shown by

aqueous methanolic extracts, 78.86 and 75.08%, respectively. While minimum DPPH radical

scavenging was shown by pure acetone extracts 38.08 and 40.86% of Aerva javanica and

Fagonia indica, respectively.


extracts of medicinal plants using orbital shaking as extraction technique

DPPH Free radical scavenging activities (%inhibition) of different plants using orbital

shaking as effected by different solvent systems are shown in Fig 4.3.2. Maximum and

minimum DPPH free radical scavenging activities of Asphodilus tenifolius were given by

aqueous methanolic extract (66.24%) and pure ethanolic extract (46.23%), respectively. In case

of Aerva javanica, maximum DPPH radical scavenging activity was shown by aqueous

ethanolic (75.62%) and methanolic (75.58%) extracts. Maximum free radical scavenging

46.23

59.96 57.7566.24

55.98 57.64

59.05

75.63

64.45

75.58

58.99

71.7659.70

72.99 71.87

79.90

59.48

73.95

0

10

20

30

40

50

60

70

80

90

Absoluteethanol

Aqueousethanol

Absolutemethanol

Aqueousmethanol

Absoluteacetone

Aqueousacetone

% in

hib

itio

n

Solvent systems


68

activity of Fagonia indica was demonstrated by aqueous methanolic extract (79.90%) and

minimum by pure ethanolic and acetone extracts (59.70 and 59.47%, respectively).


extracts of medicinal plants using sonication as extraction technique

DPPH Free radical scavenging activities (%inhibition) of different plant extracts using

sonication as extraction technique are given in Fig 4.3.3. Maximum free radical scavenging

activity of Asphodilus tenifolius was recorded by aqueous ethanolic extract (58.06%) and

minimum by pure acetone extract (34.69%). Maximum and minimum DPPH radical

scavenging activity of Aerva javanica were exhibited by aqueous methanolic extract (87.04%)

and absolute ethanolic extract (58.56%). In case of Fagonia indica, aqueous methanolic and

ethanolic extracts exhibited maximum radical scavenging activities, 76.94 and 76.89%,

respectively.

DPPH Free radical scavenging activities (%inhibition) of different plants are affected

by different solvent systems using reflux as extraction technique (Fig 4.3.4.). Here greater

DPPH free radical scavenging activity of Asphodilus tenifolius was observed in aqueous

56.32 58.06 54.96 54.12

34.69

57.14

58.56

76.44

64.06

87.04

61.83

73.8959.00

76.89

67.57

76.94

57.13

65.81

0

10

20

30

40

50

60

70

80

90

100

Absoluteethanol

Aqueousethanol

Absolutemethanol

Aqueousmethanol

Absoluteacetone

Aqueousacetone

% In

hib

itio

n

Solvent systems


69

acetone extract (42.85%). Aqueous ethanolic extracts of Aerva javanica (42.44%) and Fagonia

indica (57.69%) showed maximum DPPH radical scavenging activities.

Fig 4.3.4. DPPH free radical scavenging activities (%inhibition) of different solvent

extracts of medicinal plants using reflux as extraction technique

Variations in the DPPH radical scavenging activities (%inhibition) with respect to

different solvent systems and plant species were significant (p < 0.05).Overall results showed

that aqueous methanol and aqueous ethanol are better extracting solvents for the extraction of

such bioactive compounds which can scavenge DPPH radical free radicals, from all the studied

plants. By comparing plant species, Aerva javanica showed DPPH radical scavenging activity

ranging from 31.87- 87.04%, followed by Fagonia indica (41.33-79.90%) and then Asphodilus

tenifolius (28-66.24%). Out of four extraction methods, extracts obtained from sonication

exhibited highest DPPH radical scavenging potential. Dek et al., (2011) also reported that

highest DPPH radical scavenging activity was shown by extract obtained from sonication than

extract obtained from shaking.

32.68 30.39 28.63 28.69

41.57 42.86

37.32

42.44

32.5835.34

31.87

41.41

51.79

57.69

41.3343.70

49.0952.68

0

10

20

30

40

50

60

70

Absoluteethanol

Aqueousethanol

Absolutemethanol

Aqueousmethanol

Absoluteacetone

Aqueousacetone

% in

hib

itio

n

Solvent systems


70

Kalim et al., (2010) reported aqueous methanolic (50%) extract of Asphodilus tenifolius

using stirring as extraction technique showed 50% DPPH free radical scavenging activity at a

concentration of 2.01mg/g while working on Unani medicinal plants. Which is lower than our

findings. Variations from our results may be due to different geographical and agro-climatic

conditions of both the areas under study.

Sethi and Sharma (2011) reported 94.65% DPPH free radical scavenging activity of

methanolic extract of Aerva tomentosa at a concentration of 80 µg/mL. Fatimi et al., (2007)

reported 91.6% DPPH radical scavenging activities of methanolic extract of Aerva javanica at

a concentration of 500 µg/mL, working on medicinal plants from Yemen. While our results

showed 71.87% DPPH radical scavenging activities of methanolic extract through shaking at

a concentration of 250µg/mL. Majhenic et al., (2007) reported that extracts prepared from

aqueous organic solvents showed greater DPPH free radical scavenging activity than pure

water extract.

Ghasemzadeh et al., (2011) reported that extracts obtained from high polarity solvents

like methanol are more effective free radical scavengers as compared to the less polar solvents

such as acetone. Neelam and Khan (2012) analyzed different solvent extracts of Galium

aparine L. and concluded that methanolic extract showed highest DPPH radical scavenging

activity.

4.3.3. Superoxide radical (SOR) scavenging assay:

Superoxide anion radicals are highly reactive oxygen species among free radicals

(Aiyegoro and Okoh, 2010) and are involved in the production of other reactive oxygen species

which cause oxidation of lipids, proteins and DNA. That’s why scavenging of superoxide

radicals is very important to analyze antioxidant activity of plant extracts (Battu and Kumar,

2012).

Superoxide radical scavenging activities of different solvent extracts of plants using

different extraction techniques are presented in Fig 4.4.1-4.4.4. Plants were extracted by six

solvent systems using four extraction techniques. Fig 4.4.1 shows the effect of different solvent

systems on the superoxide radical scavenging activity (%inhibition) of different plant extracts

using stirring as extraction technique. Maximum SOR scavenging activity of Asphodilus

71

tenifolius was given by its aqueous methanolic extract (69.28%) and minimum activity by

absolute acetone extract (47.05%). SOR scavenging activity of Aerva javanica ranges from

56.83-70.35% and Fagonia indica ranges from 60.91-74.19%.

Fig 4.4.1 Superoxide radical scavenging activity (%inhibition) of different plant


57.4264.96 63.69

69.28

47.0556.00

69.44 70.15 70.36 70.12

56.83

68.61

66.01

73.93 72.43 74.20

60.9165.40

0

10

20

30

40

50

60

70

80

90


Aqueousmethanol

Absoluteacetone

Aqueousacetone

%in

hib

itio

n

Solvent systems


72

Fig 4.4.2. Superoxide radical scavenging activity (%inhibition) of different plant


Different solvent systems effect the superoxide radical scavenging activity

(%inhibition) of different plant extracts using orbital shaking as extraction technique (Fig

4.4.2). Here, aqueous methanolic extracts of all the plants showed maximum radical

scavenging activities, Asphodilus tenifolius (69.76%), Aerva javanica (72.01%) and Fagonia

indica (73.45%). Lowest activity was observed by absolute acetone extracts of all the plants:

Asphodilus tenifolius (34.64%), Aerva javanica (55.15%) and Fagonia indica (67.77%).

53.4463.84

52.50

69.76

34.65

53.62

67.2371.49

65.22

72.01

55.15

67.18

69.6172.99 72.49 73.45

67.77 69.46

0

10

20

30

40

50

60

70

80

90


Aqueousmethanol

Absoluteacetone

Aqueousacetone

% in

hib

itio

n

Solvent systems


73

Fig 4.4.3 Superoxide radical scavenging activity (%inhibition) of different plant


Superoxide radical scavenging activity (%inhibition) of different plant extracts using

sonication as extraction technique are presented in Fig 4.4.3. In case of Asphodilus tenifolius,

aqueous ethanolic extract showed maximum SOR scavenging activity (78.01%) and absolute

methanolic extract exhibited lowest activity (56.60%). In case of Aerva javanica and Fagonia

indica, aqueous methanolic extracts gave maximum SOR scavenging activity, 72.47 and

75.11%, respectively. While extracts of both of these plants showed least activities, 56.04 and

68.80%, respectively.

74.21 78.01

56.60

70.26 70.3175.95

68.87 70.71 71.47 72.48

56.04

65.94

71.59 73.72 73.99 75.11

68.8173.78

0

10

20

30

40

50

60

70

80

90


Aqueousmethanol

Absoluteacetone

Aqueousacetone

% in

hib

itio

n

Solvent systems


74

Fig 4.4.4. Superoxide radical scavenging activity (%inhibition) of different plant


The influence of different solvent systems on the superoxide radical scavenging activity

(%inhibition) of different plant extracts using reflux as extraction technique are given in Fig

4.4.4. Aqueous ethanolic extracts of Asphodilus tenifolius and aqueous methanolic extracts of

Aerva javanica and Fagonia indica exhibited maximum SOR scavenging activities as

compared to other solvent extracts of the respective plant. While absolute ethanolic extracts of

all of these plants showed minimum SOR activities.

Significant Variations (p ˂ 0.05) were observed in the superoxide radical scavenging

activities with respect to different plant species and different solvent systems. Overall results

showed that aqueous methanol is better solvent for the extraction of bioactive components

responsible for SOR scavenging activity followed by aqueous ethanol and then remaining

solvent systems. By comparing the SOR scavenging of plant species, the trend formed was

Fagonia indica> Aerva javanica> Asphodilus tenifolius. It was also observed that aqueous

solvents showed more SOR activity as compared to absolute solvents. The possible reason

behind this is that by combining organic solvents with water, polarity of solvent increased

which result in extraction of more phenolic compounds thus increasing SOR activity. Results

31.87

50.66

34.89 37.5543.04

49.9028.99

64.91

55.37

67.71

43.48

61.2450.71

59.01 60.96

65.98

53.67

63.44

0

10

20

30

40

50

60

70

80


Aqueousmethanol

Absoluteacetone

Aqueousacetone

% in

hib

itio

n

Solvent systems


75

reflected that extracts with the higher phenolic contents also exhibit greater antioxidant

activity. Phenolics are important plant components showing radical scavenging activities

because of their hydroxyl groups. So, phenolic compounds of plant extracts directly contribute

to their antioxidant activity (Stankovic et al., 2011).

From the four extraction procedures, maximum SOR scavenging activity was achieved

by the extracts prepared from sonication followed by orbital shaking, stirring and reflux.

Ultrasound waves travel into the plant material followed by the breakdown of cell walls thus

promoting the release of bioactive compounds into the solvent at low temperature. Sonication

extraction also carried out in shorter time as compared to other techniques (Dek et al., 2011).

Kalim et al., (2010) found 50% superoxide radical scavenging activity of 50%

methanolic extract of Asphodilus tenifolius using stirring as extraction technique at

concentration of 425.92 µg/mL. While our results showed 69.28% SOR scavenging activity of

aqueous methanolic extract of Asphodilus tenifolius (using stirring) at a concentration of 250

µg/mL. These variations may be due to different geographical conditions and also due to the

difference in polarity of solvent system used for extraction (Hossain et al., 2013). Premanath

and Lakshmidevi (2010) evaluated different solvent systems for the extraction of bioactive

components from Tinospora cordifolia (Miers.) leaves and obtained maximum SOR activity

of methanolic and ethanolic extracts as compared to aqueous, chloroform and hexane extracts.

4.3.4. Reducing power assay (FRAP contents):

Reducing power is a direct measure of antioxidant activity of plant extracts (Komes et

al., 2011). The reducing power of plant extracts are generally due to the presence of reductones

which show antioxidant activity by breaking the free radical chain by donating hydrogen atom

(Rahman et al., 2012). Active compounds in the extracts reduce ferric ions into ferrous ions

and the gallic acid was used as standard. Ferric reducing power of extract was calculated as

ferric reducing antioxidant contents (FRAP contents) in mg gallic acid equivalent/ g of dry

plant extract (mg GAE/g).

The FRAP contents of plant extracts using different solvent systems and extraction

techniques are shown in Table 4.3.1, 4.3.2, 4.3.3 and 4.3.4. Table 4.3.1 shows the effect of

76

different solvent systems on FRAP contents of plant extracts using stirring as extraction

technique. Among different extracts of Asphodilus tenifolius, aqueous ethanolic extract

showed maximum FRAP contents (13.07mg GAE/g) and least FRAP contents were shown by

absolute ethanol (5.35 mg GAE/g). In case of Aerva javanica, maximum FRAP contents were

given by aqueous ethanol extract (30.55mg GAE/ g) and minimum by absolute ethanol (12.30

mg GAE/g). Among Fagonia indica extracts, maximum FRAP contents were given by

aqueous methanolic extract (29.43mg GAE/g) and minimum by absolute acetone extract

(10.07mg GAE/g).

Table 4.3.1. FRAP contents (mg GAE/g) of different solvent extracts using stirring as

extraction technique

Solvent systems



Absolute ethanol 5.35±0.27ec 12.30±0.62e

b 16.22±0.81da


a 25.48±1.27bcb

Absolute methanol 5.86±0.29dec 20.16±1.01c

b 22.44±1.12ca

Aqueous methanol 6.96±0.35db 28.89±1.45a

a 29.43±1.47aa

Absolute acetone 8.56±0.43cc 16.07±0.80d

a 10.07±0.50eb

Aqueous acetone 10.70±0.54bb 23.71±1.19b

a 25.74±1.29ba





The FRAP contents (mg GAE/g of plant extract) of different plant extracts using orbital

shaking as extraction technique are presented in Table 4.3.2. From different extracts of A.

tenifolius, maximum and minimum FRAP contents were obtained from aqueous ethanol

(13.95mg GAE/ g) and absolute ethanol extracts (8.35mg GAE/g), respectively. In case of

Aerva javanica and Fagonia indica, maximum FRAP contents were given by aqueous

77

methanolic extracts (31.17 and 33.06 mg GAE/g, respectively) and minimum contents by

acetone extract (18.42 and 11.05 mg GAE/g, respectively).

Table 4.3.2. FRAP contents (mg GAE/g) of different solvent extracts using orbital

shaking as extraction technique

Solvent systems



Absolute ethanol 12.24±0.61bcb 21.42±1.07b

a 20.34±1.02ca

Aqueous ethanol 13.95±0.70ab 27.98±1.40a

a 26.29±1.31ba

Absolute methanol 11.45±0.57bc 21.09±1.05b 22.30±1.12c

Aqueous methanol 13.00±0.65abb 31.17±1.56a

a 33.06±1.65aa

Absolute acetone 8.35±0.42dc 18.42±0.92b

a 11.05±0.55db

Aqueous acetone 11.04±0.55cb 29.50±1.48a

a 26.61±1.33ba





Reducing power (mg GAE/g of plant extract) of plant extracts is affected by different

extracting solvents using sonication as extraction technique (Table 4.3.3). Among different

extracts of A. tenifolius, aqueous ethanolic (18.48 mg GAE/g) and absolute methanolic (9.06

mg GAE/g) extracts showed maximum and minimum FRAP contents, respectively. In case of

Aerva javanica and Fagonia indica, maximum FRAP contents were given by aqueous

methanolic extracts (42.29 and 42.79 mg GAE/g, respectively) and minimum FRAP contents

of Aerva javanica was showed by absolute methanol (17.93 mg GAE/g) and of Fagonia indica

by absolute acetone (17.06 mg GAE/g).

78

Table 4.3.3. FRAP contents (mg GAE/g) of different solvent extracts using sonication as


Solvent systems



Absolute ethanol 9.10±0.46cc 30.51±1.53b

a 19.16±0.96eb


a 35.58±1.78bb

Absolute methanol 9.06±0.45cc 17.93±1.00c

b 28.82±1.44ca

Aqueous methanol 11.66±0.58bc 42.29±2.12a

a 42.79±2.14ab

Absolute acetone 9.46±0.47cc 20.73±1.04c

a 17.06±0.85eb

Aqueous acetone 11.99±0.60bc 37.89±1.89a

a 23.29±1.16db





All the plant extracts obtained through reflux also exhibited reducing power (Table

4.3.4). Aqueous acetone extract gave maximum FRAP contents (10.70 mg GAE/g) among all

the extracts of Asphodilus tenifolius and absolute methanolic extract (4.60 mg GAE/g) showed

least FRAP contents. In case of Aerva javanica and Fagonia indica, maximum FRAP contents

were documented by aqueous methanolic extracts (15.96 and 31.92 mg GAE/g, respectively)

and minimum contents were obtained from absolute acetone extracts (5.03 and 10.15%,

respectively).

79

Table 4.3.4. FRAP contents (mg GAE/g) of different solvent extracts using reflux as


Solvent systems



Absolute ethanol 5.28±0.26cdc 10.32±0.52b

b 17.23±0.86ca

Aqueous ethanol 6.13±0.31bcc 15.81±0.79a

b 26.99±1.35ba

Absolute methanol 4.60±0.23dc 8.74±0.44b

b 26.05±1.30ba

Aqueous methanol 4.64±0.23dc 15.96±1.00a

b 31.92±1.60aa

Absolute acetone 6.63±0.33bb 5.033±0.25c

c 10.15±0.51da

Aqueous acetone 10.70±0.54ac 14.74±0.74a

b 18.45±0.92ca





The differences in the yield of the all the plants with respect to different solvent systems

and plant species were significant (p < 0.05). Overall results showed aqueous methanol and

aqueous ethanol are best solvents for the extraction of ferric reducing antioxidant contents and

absolute acetone gives least FRAP contents. Results showed significant effect of extracting

solvents on the FRAP contents. Among plants Fagonia indica exhibited maximum FRAP

contents (10.07-42.79 mg GAE/g) followed by Aerva javanica (5.03-42.29 mg GAE/g) and

least contents by Asphodilus tenifolius (4.60-18.48 mg GAE/g). By comparing extraction

techniques, the order obtained was sonication>orbital shaking>stirring>reflux. In reflux,

extraction at high temperature cause a loss in natural antioxidants because heat may induce

their own oxidation and degenerative reactions (Sultana et al., 2009). Dek et al., (2011)

reported that highest FRAP contents were exhibited by the extracts obtained from sonication

as compared to the extracts obtained from shaking.

Premanath and Lakshmidevi (2010) reported that methanolic and ethanolic extracts of

Tinospora cordifolia (Miers.) exhibited highest reducing power while working on the different

80

solvent extracts. Generally the plant extracts obtained from aqueous solvents, showing high

TPC also exhibited highest FRAP contents. Reducing power of plant extracts is mainly due to

presence of phenolic compounds. So extracts with high TPC also exhibit high reducing power

(Sultana et al., 2009).

4.3.5. Antioxidant Activity Determination in a ß-Carotene Linoleic Acid

System: The β-carotene linoleic acid assay is mostly used to evaluate the antioxidant activity of

plant products because β-carotene is highly sensitive to free radical mediated oxidation of

linoleic acid (Umamaheswari and Chatterjee, 2008). This test is based on the de-colorization

of ß- carotene by reacting with free radicals produced from the oxidation of linoleic acid

(Marxen et al., 2007). Antioxidant activity of plant extracts is determined by measuring the

inhibition of hydroperoxides, conjugated dienes and other volatile organic compounds by the

action of these extracts (Sengul et al., 2009; Yumrutas et al., 2011). In this assay, the

antioxidant compounds donate hydrogen atoms to the peroxyl (R1R2CHOO-) radicals

produced as a result of oxidation of linoleic acid and reduce them to hydroperoxides

(R1R2CHOOH) leaving behind β-carotene molecules intact (Liu et al., 2010). Antioxidant

activity was calculated as antioxidant activity coefficient (AAC).

Antioxidant activity of different plant extracts are shown in Table 4.4.1, 4.4.2, 4.4.3

and 4.4.4. Table 4.4.1 shows the effect of different solvent systems on the antioxidant activity

of different plants using stirring as extraction technique. Aqueous ethanolic and absolute

acetone extracts showed maximum and minimum antioxidant activities (157.70 and 68.46

AAC, respectively) among all the extracts of Asphodilus tenifolius. In case of Aerva javanica

and Fagonia indica, aqueous methanolic extract showed maximum antioxidant activity

(378.77 and 341.40 AAC, respectively) and minimum antioxidant activity was given by

absolute acetone extracts (259.83 and 220.03 AAC).

81

Table 4.4.1. Effect of different solvent systems on the antioxidant activity coefficient

(AAC) of different plants using stirring as extraction technique

Solvent systems



Absolute ethanol 127.30±6.36bb 263.37±12.17c

a 245.24±12.26cda

Aqueous ethanol 157.70±7.89ab 314.46±15.72b

a 284.97±12.25ba

Absolute methanol 82.66±4.13cc 352.93±17.65ab

a 246.72±12.34cdb


a 341.40±17.07aa

Absolute acetone 68.46±3.42cc 259.83±13.00c

a 220.03±11.00db

Aqueous acetone 119.90±6.00bc 324.54±16.23b

a 271.92±13.59bcb





82

Table 4.4.2 Effect of different solvent systems on the antioxidant activity coefficient

(AAC) of different plants using orbital shaking as extraction technique

Solvent systems



Absolute ethanol 133.21±6.66bc 250.47±12.52c

a 222.92±11.15cb

Aqueous ethanol 175.18±8.76ac 335.57±16.78b

a 282.08±14.10bb

Absolute methanol 98.05±4.90dc 323.12±16.16b

a 233.66±11.68cb

Aqueous methanol 113.77±5.70cdc 385.48±19.27a

a 336.06±16.80ab

Absolute acetone 63.129±3.16eb 243.05±12.15c

a 203.83±10.19ca

Aqueous acetone 117.77±6.00bcc 342.65±17.13ab

a 235.97±11.80cb





The effect of different solvent systems on the antioxidant activity of different plants

using orbital shaking is given in Table 4.4.2. Here aqueous methanolic extracts of three plants

showed maximum antioxidant activities, Asphodilus tenifolius (175.18 AAC), Aerva javanic

(385.48 AAC), Fagonia indica (336.06 AAC) and absolute acetone extracts showed minimum

antioxidant activities, Asphodilus tenifolius (63.13 AAC), Aerva javanic (243.05 AAC),

Fagonia indica (203.83 AAC).

Different extraction solvents effect the antioxidant activity of plants with sonication as

extraction technique (Table 4.4.3). Here like orbital shaking technique aqueous methanolic

extracts of three plants showed maximum antioxidant activities i.e. Asphodilus tenifolius,

225.69 AAC; Aerva javanic, 391.02 AAC; Fagonia indica, 380.40 AAC, whereas absolute

acetone extracts showed minimum antioxidant activities i.e Asphodilus tenifolius , 82.45 AAC;

Aerva javanica, 278.56 AAC; Fagonia indica, 224.24 AAC.

83


(AAC) of different plants using sonication as extraction technique

Solvent systems



Absolute ethanol 139.05±6.95cc 315.25±15.76bc

a 261.17±13.06deb

Aqueous ethanol 225.69±11.28ab 355.87±17.79ab

a 344.62±17.23aba

Absolute methanol 104.56±5.23dc 352.93±17.65ab

a 299.21±14.96cdb


a 380.40±19.02aa

Absolute acetone 82.45±4.12ec 278.56±13.93c

a 224.24±11.21eb

Aqueous acetone 120.93±6.05cdc 382.32±19.12a

a 317.19±15.86bcb





The influence of different solvent systems on the antioxidant activity of different plants

using reflux as extraction technique is presented in Table 4.4.4. Among all the extracts of

Asphodilus tenifolius and Aerva javanic, maximum antioxidant activity was recorded by

aqueous actone extracts (124.47 and 186.37 AAC, respectively). In case of Fagonia indica

maximum antioxidant activities was obtained by aqueous ethanolic extract (270.39 AAC) and

minimum activity by absolute ethanolic extract (202.54 AAC).

84


(AAC) of different plants using reflux as extraction technique

Solvent systems



Absolute ethanol 122.59±6.13abc 146.36±7.32b

b 202.54±10.13ca

Aqueous ethanol 112.65±5.63abc 166.48±8.32ab

b 270.30±13.52aa

Absolute methanol 56.421±2.82dc 176.74±8.84a

b 206.06±10.30ca

Aqueous methanol 109.68±5.48bc 178.19±8.66a

b 240.02±12.00aba

Absolute acetone 79.13±3.96cc 173.93±8.95a

b 217.22±10.86bca

Aqueous acetone 124.47±6.22ac 186.37±9.32a

b 229.20±11.46bca





Overall results showed that aqueous methanolic extracts showed highest antioxidant

activities followed by aqueous ethanolic and then remaining extracts which is in accordance

with their concentration of TPC and TFC. By comparing antioxidant activity of plant species,

the trend obtained was Aerva javanica>Fagonia indica>Asphodilus tenifolius. From all the

extraction techniques, highest antioxidant activity was shown by the extracts obtained through

sonication (82.45-391.02AAC) followed by orbital shaking (63.13-385.48AAC), stirring

(68.46-378.77AAC) and reflux (56.42-270.30AAC). Antioxidant activity of all the plants was

adversely affected by the reflux extraction, regardless of the solvents used. Smelcerovic et al.,

(2006) reported that sonication proved to be the powerful extraction method for many

phytochemicals when compared to maceration, soxhlet and accelerated solvent extraction

methods. Our results are also in agreement with Sultana et al., (2009) who reported that extracts

obtained from shaking showed good antioxidant activity in ß-carotene linoleic acid system as

compared to extracts obtained from reflux extraction technique. The activities showed by the

85

different types of extracts may be due to the uneven distribution and variety of chemical

compounds in the extracts (Mohamed et al., 2010).

4.4. Evaluation of Antimicrobial activity:

Medicinal plants produce nutritive and non-nutritive compounds which show

antimicrobial activities and protect from pathogens. Thus it is important to characterize

medicinal plants for their antimicrobial activities (Sengul et al., 2009). Use of plant extracts

for their antimicrobial activity has become very important (Gislene et al., 2000). Disc diffusion

and MIC assays were performed to determine antibacterial and antifungal activities of plant

extracts. The extracts which had not shown activity in disc diffusion assay, were not analyzed

for MIC.

4.4.1. Antibacterial activity:

The antibacterial activity of plant extracts as affected by different solvent systems and

extraction techniques is shown in Table 4.5.1, 4.5.2 4.5.3, 4.5.4, 4.6.1, 4.6.2, 4.6.3, 4.6.4, 4.7.1,

4.7.2, 4.7.3, 4.7.4, 4.8.1, 4.8.2, 4.8.3, 4.8.4. Antibacterial activities of plant extracts were

evaluated against food borne and pathogenic bacteria. All the plant extracts showed

antibacterial activity against Gram negative and Gram positive bacteria. As given in Table

4.5.1, 4.5.2 4.5.3, 4.5.4, all the plant extracts exhibited good antibacterial activity against

Escherichia coli, a Gram negative bacterium. Aqueous ethanolic extract of Fagonia indica

showed excellent antibacterial activity with inhibition zone (34 mm) followed by MIC value

(31 µg/mL) which is equivalent to the standard Rifampicin inhibition zone (34mm) and MIC

value (31 µg/mL). From all the extracts of Asphodilus tenifolius, aqueous ethanolic extracts

showed larger inhibition zones (18-26 mm) and smaller MIC values (56-215 µg/mL) through

all the extraction techniques. Aqueous methanolic extracts of Aerva javanica exhibited larger

inhibition zones (24-30 mm) and smaller MIC values (56-104 µg/mL) among all the extracts

obtained through different extraction methods.

The antibacterial activity of different plant extracts obtained through different

extraction techniques against Pasteurella multocida is shown in Table 4.6.1, 4.6.2, 4.6.3, 4.6.4.

All the plant extracts were active against P. multocida, a Gram negative bacterium.

86

Table 4.5.1. Antibacterial activity of different plant extracts using stirring as extraction

technique against Escherichia coli

Solvent systems



Inhibition zones (mm)

Absolute ethanol 24±1.2 14±0.7 20±1.0

Aqueous ethanol 26±1.3 24±1.2 22±1.1

Absolute methanol 14±0.7 24±1.2 15±0.8

Aqueous methanol 18±0.9 25±1.3 18±0.9

Absolute acetone 15±0.8 22±1.1 14±0.7

Aqueous acetone 16±0.8 24±1.2 22±1.1

Rifampicin 34±1.9

Minimum inhibitory concentration (MIC), µg/mL


Aqueous ethanol 104±5.2 119±7.2 150±6.3




Aqueous acetone 250±11.5 119±5.7 150±6.9

Rifampicin 15.6±0.8

Values are mean ± standard deviation of the plant extracts analyzed individually in triplicate.

87

Table 4.5.2. Antibacterial activity of different plant extracts using orbital shaking as

extraction technique against Escherichia coli

Solvent systems





Aqueous ethanol 22±1.1 20±1.0 26±1.3




Aqueous acetone 14±0.7 23±1.2 20±1.0

Rifampicin 34±1.9



Aqueous ethanol 150±7.3 170±7.3 105±5.4




Aqueous acetone 303±14.7 119±6.3 170±7.2



88

Table 4.5.3. Antibacterial activity of different plant extracts using sonication as

extraction technique against Escherichia coli

Solvent systems





Aqueous ethanol 30±1.5 22±1.1 34±1.7




Aqueous acetone 18±0.9 26±1.3 32±1.6

Rifampicin 34±1.9



Aqueous ethanol 56±3.2 150±8.5 31±3.1




Aqueous acetone 215±10.0 103±5.2 56±4.6



89

Table 4.5.4. Antibacterial activity of different plant extracts using reflux as extraction

technique against Escherichia coli

Solvent systems





Aqueous ethanol 18±0.9 18±0.9 18±0.9




Aqueous acetone 14±0.7 24±1.2 16±0.8

Rifampicin 34±1.9



Aqueous ethanol 215±11.2 215±11.7 215±10.4




Aqueous acetone 303±14.2 119±5.9 250±11.5



90


technique against Pasteurella multocida

Solvent systems





Aqueous ethanol 22±1.1 13±0.6 22±1.1




Aqueous acetone 14±0.7 14±0.7 19±0.9

Rifampicin 28±1.6



Aqueous ethanol 150±7.9 325±16.5 148±7.0




Aqueous acetone 303±15.0 303±14.9 170±9.0



91


extraction technique against Pasteurella multocida

Solvent systems





Aqueous ethanol 20±1.0 14±0.6 22±1.1




Aqueous acetone 14±0.7 14±0.7 20±1.0

Rifampicin 28±1.6



Aqueous ethanol 160±7.6 310±14.3 148±7.0




Aqueous acetone 303±15.2 306±20.7 160±9.0



92


extraction technique against Pasteurella multocida

Solvent systems





Aqueous ethanol 22±1.1 17±0.9 24±1.2




Aqueous acetone 16±0.8 22±1.1 25±1.3

Rifampicin 28±1.6



Aqueous ethanol 148±7.9 235±10.9 119±6.2




Aqueous acetone 260±13.3 150±7.0 104±7.4



93


technique against Pasteurella multocida

Solvent systems





Aqueous ethanol 18±0.9 13±0.8 22±1.1




Aqueous acetone 12±0.6 14±0.7 17±0.9

Rifampicin 28±1.6



Aqueous ethanol 215±11.7 327±16.0 148±8.4




Aqueous acetone 342±19.1 306±17.0 220±12.9



94


technique against Bacillus subtillus

Solvent systems





Aqueous ethanol 28±1.4 22±1.1 22±1.1




Aqueous acetone 24±1.2 24±1.2 30±1.5

Rifampicin 38±2.00



Aqueous ethanol 83±6.2 150±9.2 153±8.7


Aqueous methanol 170±10.5 82±6.2 31±.3.5


Aqueous acetone 119±7.0 119±6.5 62±5.2



95


extraction technique against Bacillus subtillus

Solvent systems





Aqueous ethanol 18±0.9 24±1.2 20±1.2




Aqueous acetone 18±0.9 24±1.2 31±1.6

Rifampicin 38±2.0



Aqueous ethanol 215±11.6 24±4.4 170±7.9


Aqueous methanol 145±8.6 20.2±3.3 32±2.9


Aqueous acetone 215±11.9 119±6.6 62±5.2



96


extraction technique against Bacillus subtillus

Solvent systems





Aqueous ethanol 30±1.5 38±0.9 18±0.9




Aqueous acetone 26±1.3 26±1.3 36±1.8

Rifampicin 38±2.0



Aqueous ethanol 62±5.2 17±1.3 215±9.9




Aqueous acetone 104±7.2 106±6.6 150±9.2



97


technique against Bacillus subtillus

Solvent systems





Aqueous ethanol 16±0.8 22±1.1 18±0.9




Aqueous acetone 18±0.9 24±1.2 27±1.4

Rifampicin 38±2.00



Aqueous ethanol 250±13.5 153±8.2 215±11.4

Absolute methanol 303±14.9 250±11.9 156.2±8.2



Aqueous acetone 215±11.0 119±6.9 84±5.7



98


technique against Staphylococcus aureus

Solvent systems





Aqueous ethanol 30±1.5 20±1.0 18±0.9




Aqueous acetone 25±1.3 14±0.7 29±1.5

Rifampicin 34±1.9



Aqueous ethanol 82±7.1 174±10.3 217±11.7




Aqueous acetone 103±6.5 303±17.2 82±7.1

Rifampicin 31±2.2


99


extraction technique against Staphylococcus aureus

Solvent systems





Aqueous ethanol 27±1.4 19±1.0 18±0.9




Aqueous acetone 24±1.2 14±0.7 29±1.5

Rifampicin 34±1.9



Aqueous ethanol 80±5.0 166±7.7 217±11.8




Aqueous acetone 120±8.0 303±14.7 84±5.2

Rifampicin 31±2.2


100

Table 4.8.3. Antibacterial activity of different plants using sonication as extraction


Solvent systems





Aqueous ethanol 30±1.5 20±1.0 20±1.0




Aqueous acetone 24±1.2 18±0.9 34±1.7

Rifampicin 34±1.9



Aqueous ethanol 82±4.0 170±9.0 172±9.6




Aqueous acetone 120±6.0 215±12.2 40±3.6

Rifampicin 31±2.2


101



Solvent systems





Aqueous ethanol 22±1.1 17±0.9 18±0.9




Aqueous acetone 21±1.1 18±0.9 25±1.3

Rifampicin 34±1.9



Aqueous ethanol 150±8.5 210±11.2 215±10.7


Aqueous methanol 148±8.8 166.1±9.5 104±7.2


Aqueous acetone 149±7.9 217±10.0 101±7.7

Rifampicin 31±2.2


102

Aqueous ethanolic extracts from all the extracts of Asphodilus tenifolius obtained through

different extraction processes gave appreciable antibacterial activity with inhibition zones (18-

22 mm) and MIC values (148-215 µg/mL). While among all the extracts of Aerva javanica and

Fagonia indica, maximum activity against P. multocida was obtained from aqueous

methanolic extracts with inhibitions zones 18-26 mm and 23-26 mm, respectively. Whereas

their MIC values were 103-215 µg/mL and 103-119 µg/mL, respectively.

Antibacterial activity of plant extracts against Bacillus subtillus, a Gram positive

bacteria, is given in Table 4.7.1, 4.7.2, 4.7.3, 4.7.4. All the plants extracts exhibited

antibacterial activities. Among all the extracts of Asphodilus tenifolius, aqueous ethanolic

extract obtained through sonication exhibited larger inhibition zone (30 mm) and smaller MIC

value (62 µg/mL). Aqueous methanolic and queous ethanolic extracts of Aerva javanica and

aqueous methanolic extract of Fagonia indica obtained through sonication show activity

against B. subtillus equivalent to standard used with inhibition zone (38 mm) followed by MIC

values (16-17µg/mL).

The impact of different solvent systems and different extraction techniques on the

antibacterial activity of plant extracts against Staphylococcus aureus is shown in Table 4.8.1,

4.8.2, 4.8.3, 4.8.4. Results from the disc diffusion followed by the MIC indicated that aqueous

methanolic and aqueous acetone extracts of Fagonia indica prepared through sonication

showed appreciable antibacterial activity with inhibition zone (34 mm) and MIC value (37-40

µg/mL) almost equivalent to standard (Rifampicin) used. From all the extracts of Asphodilus

tenifolius, aqueous ethanolic extracts obtained through all the extraction techniques gave larger

inhibition zones (22-30 mm) followed by smaller MIC values (80-150 µg/mL). In case of

Aerva javanica, aqueous methanolic extracts from all the extraction techniques exhibited

maximum antibacterial activity with inhibition zones ranged 19-22 mm and MIC values 152-

172 µg/mL.

Overall results showed that all the plant extracts showed antibacterial activity against

all four the bacterial strains, two Gram negative (E.coli and Pasteurella multocida) and two

Gram positive (Bacillus subtillus and Staphylococcus aureus) bacteria. The activity of plant

extracts against both Gram positive and Gram negative bacteria indicate that these plants have

broad spectrum antibiotic compounds. Plants extracts showed more activity towards Gram

positive bacteria as compared to Gram negative bacteria. This is because that Gram-negative

103

bacteria possess an outer phospholipidic membrane carrying the structural lipopolysaccharide

components which makes the cell membrane of Gram negative bacteria impermeable to

antimicrobial agents. Gram-positive bacteria are more sensitive because they have only an

outer peptidoglycan layer which do not act as effective permeability barrier. Therefore, the

complex nature of cell walls of Gram-negative organisms make them less sensitive to

antimicrobial compounds as compared to Gram-positive bacteria (Negi et al., 2005; Joshi et

al., 2011; Qader et al., 2013). Instead of this permeability differences, some of the extracts

have still showed some degree of inhibition against Gram-negative organisms as well.

The results from disc diffusion and MIC indicate that Fagonia indica showed higher

antibacterial activities followed by Aerva javanica and then Asphodilus tenifolius against three

bacterial stratins, E.coli, P. multocida and B. subtillus. While in case of S. aureus the trend

obtained is Fagonia indica> Asphodilus tenifolius>Aerva javanica. Among all the solvent

systems aqueous solvent systems (aq. methanol, aq. ethanol and aq. acetone) exhibited more

activity against all the bacterial strains as compared to pure (absolute) solvents. Because the

addition of water in the organic solvents increases their polarity thus improving their efficiency

of extracting phenolic compounds. From all the extraction techniques, extracts of all the plants

obtained through sonication showed maximum antibacterial activity followed by stirring and

orbital shaking and least was shown by extracts obtained from reflux. In reflux high

temperature decompose the active antimicrobial compounds. Borchardt et al., (2008) also

reported that antimicrobial and antioxidant attributes of plant extracts vary with extraction

methods.

Panghal et al., (2011) isolated the microbes from oral cancer patients and reported that

acetone extract of Asphodilus tenifolius showed antibacterial activity against E.coli and

Staphylococcus aureus with inhibition zones, 13.33 and 10.33 mm, followed by MIC values

125 and 250 µg/mL, respectively. Its methanolic extract had shown activity against E. coli

(12.33 mm inhibition zone and 250 µg/mL MIC value), but no activity against Staphylococcus

aureus. Menghani et al., (2012) reported inhibition zones of methanolic extract of Asphodilus

tenifolius against E. coli. (7.3 mm), B. subtillus (9.0 mm) and S. aureus (7 mm), which are less

than our findings. These variations may be due to different geographical conditions. Mahmoud

et al., (2011) reported that chemical contents of plants are affected with weather and

geographical factors such as temperature, humidity and soil type, etc. Mohanasundari et al.,

104

(2007) described that antimicrobial agents are either polar or non-polar and they are extracted

through the organic solvents. Our studies are in agreement with Vaghasiya and Chanda (2007),

who reported that methanol extract of plants show more antibacterial activity as compared to

acetone extracts. So, methanol has better tendency to extract antimicrobial compounds from

medicinal plants. Wendakoon et al., (2012) also described that plants produce a variety of

chemicals which exhibit antibacterial activity. Determination of these bioactive compounds

from plant materials mainly depends upon the type of solvent used for extraction.

Srinivas and Reddy (2012) described the activity of methanolic extract of Aerva

javanica leaf against E.coli, B. subtillus and S. aureus, obtained inhibition zones 14, 12 and 12

mm, respectively at an extract concentration of 100 mg/mL. They also reported MIC values

2.5, 5 and 2.5 mg/dL against E.coli, B. subtillus and S. aureus, respectively. S. aureus is a

Gram positive pyrogenic bacterium significantly cause invasive skin diseases such as

superficial and deep follicular lesion and food poisoning (Joshi et al., 2011; Mahmoud et al.,

2011). All the plant extracts showed activity against S. aureus, thus these can be used for the

treatment of skin disorders. It was found from the above study that antibacterial activity was

affected by a lot of factors such as plant extract, the solvent and technique used for extraction

and the organism tested.

4.4.2. Antifungal activity:

Fungi are pathogenic microorganisms causing a number of infections of skin, nail or

hair, minor infections of mucous membranes or systemic infections causing progressive often

fatal disease. The antifungal of different plant extracts against four fungal strains are shown in

Table 4.9.1, 4.9.2, 4.9.3, 4.9.4, 4.10.1, 4.10.2, 4.10.3, 4.10.4, 4.11.1, 4.11.2, 4.11.3, 4.11.4,

4.12.1, 4.12.2, 4.12.3, 4.12.4. Antifungal activity of plant extracts were assessed against

Aspergillus flavus (A. flavus), Aspergillus niger (A. niger), Fusarium solani (F. solani) and

Helmithus spermium (H. spermium). Antifungal activity of plant extracts obtained from

different solvents and extraction techniques against A. flavus is shown in Table 4.9.1, 4.9.2,

4.9.3, 4.9.4. Effect of different extracting solvents on the antifungal activity of plant extracts

is given in Table 4.9.1 Results of disc diffusion followed by MIC indicated that among all the

extracts of Asphodilus tenifolius, aqueous ethanolic extract exhibited maximum inhibition

zones (14 mm) followed by smaller MIC values (303 µg/mL). From all the extracts of Aerva

105

javanica, highest antifungal activity was shown by aqueous ethanolic and aqueous methanolic

extracts with large inhibition zone (15 mm) and small MIC value (303 µg/mL). In case of

Fagonia indica, aqueous methanolic extract showed larger inhibition zone (24 mm) and

smaller MIC value (119 µg/mL) as compared to its other extracts.

The antifungal activity of different solvent extracts of all plants using shaking as

extraction technique is given in Table 4.9.2. Here, aqueous methanolic extracts of Asphodilus

tenifolius and Fagonia indica in comparison to their remaining extracts exhibited more activity

with larger inhibition zones (14 and 22 mm) and smaller MIC values (303 and 146 µg/mL).

Among all the extracts of Fagonia indica, aqueous methanolic and aqueous acetone extracts

showed maximum inhibition zones (14 mm) and lowest MIC values (303 µg/mL).

Plant extracts obtained through sonication were good inhibitor to the growth of A.

flavus (Table 4.9.3). Maximum activity of Asphodilus tenifolius and Fagonia indica were

obtained from their aqueous methanolic extracts with inhibition zones (16 and 25mm,

respectively) and MIC values (303 and 103µg/mL, respectively). Out of the six solvent extracts

of Aerva javanica, extracts obtained from aqueous ethanol and aqueous methanol exhibited

lager inhibition zone (15mm) and smaller MIC value (275µg/mL).

The influence of different solvent on the antifungal activity of plant extracts using

reflux as extraction method is presented in Table 4.9.4. Results obtained from disc diffusion

assay and MIC indicated that aqueous ethanolic and aqueous methanolic extracts of Asphodilus

tenifolius and Fagonia indica indicated larger inhibition zones (14 and 19 mm, respectively)

and smaller MIC values (303 and 201 µg/mL) than their remaining extracts. While in case of

Aerva javanica, maximum activity was achieved from aqueous ethanolic and aqueous acetone

extracts with inhibition zone (14 mm) and MIC value (303 µg/mL).

106

Table 4.9.1. Antifungal activity of different plant extracts using stirring as extraction

technique against Aspergillus flavus

Solvent systems





Aqueous ethanol 14±0.7 15±0.8 12±0.6




Aqueous acetone 11±0.6 13±0.7 16±0.8

Terbinafine 45±4.5



Aqueous ethanol 303±15.2 275±13.7 345±17.6




Aqueous acetone 350±15.7 320±16.5 250±11.9

Terbinafine 13±0.7


107

Table 4.9.2. Antifungal activity of different plant extracts using orbital shaking as

extraction technique against Aspergillus flavus

Solvent systems





Aqueous ethanol 12±0.6 13±0.6 13±0.7




Aqueous acetone 12±0.6 14±0.9 19±0.9

Terbinafine 45±4.5



Aqueous ethanol 345±17.0 320±15.3 320±14.0




Aqueous acetone 345±17.2 303±15.3 201±10.5

Terbinafine 13±0.7


108

Table 4.9.3. Antifungal activity of different plant extracts using sonication as extraction


Solvent systems





Aqueous ethanol 14±0.6 15±0.8 14±0.7




Aqueous acetone 13±0.7 14±0.7 21±1.1

Terbinafine 45±4.5



Aqueous ethanol 303±15.5 275±13.0 303±15.1




Aqueous acetone 320±16.5 303±14.3 150±6.0

Terbinafine 13±0.7


109

Table 4.9.4. Antifungal activity of different plant extracts using reflux as extraction


Solvent systems





Aqueous ethanol 14±0.8 14±0.7 13±0.7




Aqueous acetone 11±0.5 14±0.7 17±0.9

Terbinafine 45±4.5



Aqueous ethanol 303±14.2 303±14.4 320±15.0




Aqueous acetone 350±18.0 303±15.1 215±10.7

Terbinafine 13±0.7


110

Antifungal activity of plant extracts prepared by using different solvent systems and

extraction techniques against Aspergillus niger are given in Tables 4.10.1, 4.10.2, 4.10.3, and

4.10.4. Effect of different extracting solvent systems on the antifungal activity of plants using

stirring as extraction method is given in Table 4.10.1. As seen in Table 4.10.1 that aqueous

ethanolic and aqueous methanolic extracts of Asphodilus tenifolius and Aerva javanica

exhibited more activity as compared to their remaining extracts with 15 and 23 mm inhibition

zones and 272 and 119 µg/mL MIC values. Among all the extracts of Fagonia indica, larger

inhibition zone (22 mm) and smaller MIC value (151 µg/mL) were given by aqueous

methanolic and aqueous acetone extracts.

Antifungal activity of different plant extracts obtained through shaking is presented in

Table 4.10.2. Results showed that aqueous ethanolic extract of Asphodilus tenifolius and

aqueous methanolic extracts of Aerva javanica and Fagonia indica were found to be

comparatively more active with larger inhibition zones (15, 22 and 23 mm, respectively) and

smaller MIC values (275, 151 and 119 µg/mL, respectively).

The plant extracts prepared by using different solvent systems through sonication

extraction technique also showed inhibitory activity against A. niger (Table 4.10.3). Aqueous

ethanolic and absolute ethanolic extracts among all the extracts of Asphodilus tenifolius

showed larger inhibition zones (16 mm) and smaller MIC values (247 and 250 µg/mL).

Aqueous methanolic extracts of Aerva javanica and Fagonia indica exhibited good antifungal

activity with larger inhibition zones (25 and 26 mm) and smaller MIC values (104 µg/mL).

Effect of different solvent systems on the antifungal activity of plants when reflux was used as

extraction technique is shown in Table 4.10.4. From all the extracts of Asphodilus tenifolius,

aqueous ethanolic and aqueous acetone extracts gave larger inhibition zones (15 mm) and small

MIC values (270 and 272 µg/mL, respectively). Aqueous acetone extracts among all the

111


technique against Aspergillus niger

Solvent systems





Aqueous ethanol 15±0.8 13±0.7 20±1.0




Aqueous acetone 13±0.6 13±0.6 22±1.1

Terbinafine 50±4.2



Aqueous ethanol 272±12.6 325±14.5 170±7.0




Aqueous acetone 325±16.0 346±17.2 151±8.4

Terbinafine 9.3±1.0


112


extraction technique against Aspergillus niger

Solvent systems

Medicinal plants




Aqueous ethanol 15±0.8 15±08 19±1.0




Aqueous acetone 12±0.6 15±0.8 22±1.1

Terbinafine 50±4.2



Aqueous ethanol 275±13.8 270±12.8 183±10.2




Aqueous acetone 345±16.2 272±14.2 151±8.8



113

Table 4.10.3. Antifungal activity of different plant extracts using sonication as

extraction technique against Aspergillus niger

Solvent systems





Aqueous ethanol 16±0.9 15±0.8 25±1.3




Aqueous acetone 13±0.7 20±1.0 25±1.3

Terbinafine 50±4.2



Aqueous ethanol 247±12.3 270±15.1 104±6.3




Aqueous acetone 325±15.7 170±9.5 104±7.3



114


technique against Aspergillus niger

Solvent systems





Aqueous ethanol 15±0.8 13±0.7 17±0.9

Absolute methanol 11±00 14±0.6 14±0.7

Aqueous methanol 12±00 14±0.7 18±0.9


Aqueous acetone 15±0.8 18±0.8 19±0.9

Terbinafine 50±4.2



Aqueous ethanol 270±13.5 325±15.5 225±14.5




Aqueous acetone 272±13.8 220±12.3 183±9.2



115

extracts of Aerva javanica and Fagonia indica exhibited more activity with inhibition zones

(18 and 19 mm, respectively) and MIC values (220 and 183 µg/mL, respectively).

Antifungal activity of plant extracts obtained through different solvent systems and

extraction methods against Fusarium solani is shown in Table 4.11.1, 4.11.2, 4.11.3 and

4.11.4. Table 4.11.1 shows the effect of different solvent systems on the antifungal activity of

plants when stirring was used as extraction technique. Out of six solvent extracts of Asphodilus

tenifolius, aqueous methanolic and aqueous acetone extracts gave large inhibition zones (20

mm) and small MIC value (170 µg/mL). In case of Aerva javanica, maximum activity was

shown by aqueous methanolic and aqueous ethanolic extracts (20 mm inhibition zone, 170

µg/mL MIC value). Aqueous methanolic and aqueous acetone extracts of Fagonia indica

exhibited maximum activity against F. solani (22 mm, 150 µg/mL).

Different solvent extracts of all the plants obtained through shaking were also

possessed activity against F. solani (Table 4.11.2). Aqueous methanolic extracts of Asphodilus

tenifolius, Aerva javanica and Fagonia indica among all the extracts of respective plants were

given maximum antifungal activity with inhibition zones ranged 15-21 mm and MIC values

161-280 µg/mL. However, aqueous ethanolic extract of Asphodilus tenifolius also showed 15

mm inhibition zone and 280 µg/mL.

Antifungal activity of plant extracts as affected by different solvent systems through

sonication is given in Table 4.11.3. Again in this case maximum activity was shown by

aqueous methanolic extracts of Asphodilus tenifolius, Aerva javanica and Fagonia indica from

all the remaining extracts with inhibition zones (21-23 mm, respectively) and MIC values (150-

161 µg/mL).

Antifungal activity of different plant extracts obtained through reflux is presented in

Table 4.11.4. From disc diffusion assay and MIC results, it was observed that the aqueous

ethanolic extract from all the extracts of Asphodilus tenifolius, exhibited maximum inhibitory

activity (17 mm inhibition zone and 223 µg/mL MIC value). Among all the extracts of Aerva

javanica and Fagonia indica, larger inhibition zones (20 and 17 mm, respectively) and MIC

values (170 and 220 µg/mL) were achieved from their aqueous acetone and aqueous

methanolic extracts.

116


technique against Fusarium solani

Solvent systems





Aqueous ethanol 13±0.7 20±1.0 17±0.9




Aqueous acetone 20±1.1 18±0.9 22±1.2

Terbinafine 48±4.0



Aqueous ethanol 327±15.4 170±8.1 223±11.0




Aqueous acetone 170±7.8 215±11.1 150±8.3

Terbinafine 11±0.6


117


extraction technique against Fusarium solani

Solvent systems





Aqueous ethanol 15±0.7 17±0.8 18±0.9




Aqueous acetone 14±0.7 18±0.9 19±1.0

Terbinafine 48±4.0



Aqueous ethanol 280±13.4 223±10.5 215±11.5




Aqueous acetone 303±14.2 215±9.4 177±9.4

Terbinafine 11±0.6


118


extraction technique against Fusarium solani

Solvent systems





Aqueous ethanol 20±1.0 23±1.2 20±1.0




Aqueous acetone 18±0.9 20±1.0 21±1.1

Terbinafine 48±4.0



Aqueous ethanol 170±9.0 127±6.4 170±8.5




Aqueous acetone 215±10.4 170±9.0 161±8.1

Terbinafine 11±0.6


119


technique against Fusarium solani

Solvent systems





Aqueous ethanol 17±0.9 15±0.7 14±0.6




Aqueous acetone 15±0.7 20±1.0 14±0.7

Terbinafine 48±4.0



Aqueous ethanol 223±11.2 280±12.0 301±12.7




Aqueous acetone 280±14.0 170±7.5 303±17.3

Terbinafine 11±0.6


120

Effect of different extraction solvents and extraction techniques on the antifungal

activity of plants is presented in Table 4.12.1, 4.12.2 .4.12.3 and 4.12.4. Antifungal activity of

plant extracts obtained through stirring extraction technique against Helminthus spermium is

given in Table 4.12.1. Results showed that different solvent extracts of Asphodilus tenifolius

exhibited moderate activity, inhibition zone ranged 11-14mm and MIC values 301-370µg/mL.

Its absolute methanolic and aqueous methanolic extracts had not shown any activity against H.

spermium. From all the extracts Aerva javanica, maximum activity was given by aqueous

methanolic extract (24mm inhibition zone and 119µg/mL MIC value). However, no activity

was obtained from its absolute and aqueous acetone extracts. In case of Fagonia indica, all the

extracts showed activity ranging 12-15mm inhibition zones and 268-345µg/mL MIC values.

Table 4.12.2 depicts the antifungal activity of different plant extracts using different

extracting solvents through shaking. Again methanolic (absolute and aqueous) extracts of

Asphodilus tenifolius and acetone extracts (absolute and aqueous) of Aerva javanica were

remained inactive against H. spermium. Activities of the remaining extracts ranged, for

Asphodilus tenifolius (11-14 mm inhibition zones, 301-370 µg/mL), for Aerva javanica (13-

27 mm inhibition zones, 103-334 µg/mL) and for Fagonia indica (12-16 mm inhibition zones,

250-345 µg/mL).

Effect of different extracting solvents on the antifungal activity of plant extracts using

sonication extraction against H. spermium is given in Table 4.12.3. Activity of different

extracts of Asphodilus tenifolius were ranged 12-14 mm inhibition zones and 301-345 µg/mL

MIC values, with no activity of methanolic (absolute and aqueous) extracts. Activity of

different extracts of Aerva javanica were ranged 12-28mm inhibition zones and 83-345 µg/mL

MIC values, with no activity of its acetone (absolute and aqueous) extracts. While in case of

Fagonia indica, inhibition zones of all the extracts were in the range of 12-18 mm inhibition

zones and 229-345 µg/mL MIC values. Different solvent extracts of the plants showed activity

against H. spermium to a varying degree while using reflux as extraction technique (Table

4.12.4). None of the extract of Asphodilus tenifolius inhibited the growth of H. spermium.

Aerva javanica extracts exhibited activity with inhibition zones 12-21 mm and MIC values

170-345 µg/mL, with no activity of its methanolic (absolute and aqueous) extracts. In case of

Fagonia indica, all the extracts showed activity (12-14 mm inhibition zones and 301-345 MIC

values).

121


technique against Helminthus spermium

Solvent systems





Aqueous ethanol 12±0.6 14±0.7 12±0.6

Absolute methanol 0 18±0.9 14±0.7

Aqueous methanol 0 24±1.2 15±0.7

Absolute acetone 11±0.6 0 12±0.6

Aqueous acetone 14±0.7 0 13±0.7

Terbinafine 44±3.0



Aqueous ethanol 345±16.2 301±16.5 345±18.0

Absolute methanol --- 215±11.0 301±15.4

Aqueous methanol --- 119±6.7 268±17.3

Absolute acetone 370±19.0 --- 345±18.4

Aqueous acetone 301±15.0 --- 334±15.0

Terbinafine 13±0.8


122


extraction technique against Helminthus spermium

Solvent systems





Aqueous ethanol 12±0.6 13±0.7 14±0.7



Absolute acetone 12±0.6 0 12±0.6


Terbinafine 44±3.0



Aqueous ethanol 345±16.6 334±16.4 301±14.3




Aqueous acetone 301±15.1 --- 334±15.4

Terbinafine 13±0.8


123


extraction technique against Helminthus spermium

Solvent systems





Aqueous ethanol 14±0.6 14±0.7 18±0.9



Absolute acetone 12±0.7 0 ±0.6


Terbinafine 44±3.0



Aqueous ethanol 301±15.5 301±14.2 215±10.8




Aqueous acetone 301±15.1 --- 301±15.0

Terbinafine 13±0.8


124


technique against Helminthus spermium

Solvent systems




Absolute ethanol 0 12±0.6 12±0.6

Aqueous ethanol 0 13±0.7 12±0.7



Absolute acetone 0 0 12±0.7

Aqueous acetone 0 0 13±0.6

Terbinafine 44±3.0


Absolute ethanol --- 345±17.2 345±14.4

Aqueous ethanol --- 334±15.8 345±16.2



Absolute acetone --- --- 345±17.4

Aqueous acetone --- --- 334±16.0

Terbinafine 13±0.8


125

Overall results showed that all the plant extracts showed antifungal activity but less

than standard Terbinafine. All the plant extracts gave activity against A. flavus, A. niger and F.

solani. But a few extracts had not shown activity against H.spermium. From the results of disc

diffusion assay and MIC, it was observed that aqueous mixtures of all the solvents (ethanol,

methanol and acetone) exhibited better inhibitory activities against all the four fungal strains

in comparison to their pure state. Tiwai et al., (2011) described that more flavonoid compounds

were extracted with 70% ethanol as compared to pure ethanol due to higher polarity of the

solvent by the addition of water. Ethanol can easily penetrate through cell membrane to extract

phyto-constituents from plant material. They also reported that all the antimicrobial

compounds of plants are saturated or aromatic organic compounds and can be easily extracted

through methanol or ethanol. Ganora (2008) also reported that biological activities of plant

extracts dependens on the alcohol and water concentration in the extraction method.

By comparing plant species, Fagonia indica and Aerva javanica exhibited higher

activities and Asphodilus tenifolius showed least activities. Out of four extraction technique,

sonication proved to be suitable extraction technique for antifungal compounds while extracts

obtained from reflux showed least activities. Phenolic compounds are responsible for the

antifungal activity of plant extracts. This decrease in activity of the extracts, prepared by the

reflux technique are because of thermal decomposition of phenolics.

Menghani et al., (2012) reported that methanolic extract of Asphodilus tenifolius

showed no activity against A. flavus and A. niger. While in our experiments methanolic extracts

of A. tenifolius showed activity against both of these microbes. This may be due to different

agro-climatic conditions. The type and extent of biological activity shown by any plant

material is dependent on many factors, including the plant part, geographical source, soil

conditions, harvest time, moisture content, drying method, storage conditions, and post-harvest

processing (Wendakoon et al., 2012). Shabir et al., (2011) also reported that aqueous

methanolic extracts of flowers and leaves of Gold Mohar exhibited most effective antifungal

activity against all the tested microbes than its ethanolic, acetone and water extracts.

Flavonoids contents in the ethyl acetate fraction of alcoholic and aqueous extracts of

Aerva javanica demonstrated good antimicrobial activities against fungi, yeast and Gram

negative bacteria (Chawla et al., 2012). The antimicrobial activity of plant extracts against the

tested microbes is associated with the presence of phenolic acids such as protocatechuic,

126

chlorogenic, 3- hydroxybenzoic, gallic acids and flavonoids. Some earlier reports

demonstrated that the changes in chemical composition of an extract directly affected their

biological activities (Shabir et al., 2011). Igbinosa et al., (2009) also reported that methanolic

extract of Jatropha curcas was found to be effective than its ethanolic and aqueous extracts

against all the microorganisms. Das et al., (2010) described that acetone is very useful solvent

for the extraction of antimicrobial compounds and it can be used in bioassays due to its low

toxic and volatile nature.

4.5. HPLC analysis of different solvent extracts of selected medicinal

plants:

Reversed phase high-performance liquid chromatography (RP-HPLC) with UV-Vis

detector was used for qualitative and quantitative analysis of phenolic compounds. Nine

medicinally important phenolic acids were used as standard compounds, such as gallic acid,

caffeic acid, vanillic acid, 4-hydroxy-3-methoxy benzoic acid, chlorogenic acid, syringic acid,

p- and m-coumaric acid and ferulic acid. Extracts of all the three plants obtained through

sonication were further analyzed through HPLC for analyzing the effect of different extracting

solvents as these showed better antioxidant and antimicrobial activities in the previous tests.

Table 4.13.1 shows the qualitative and quantitative determination of phenolic

compounds in different solvent extracts of Asphodilus tenifolius and also evaluating the effect

of different extracting solvents on the phenolic compounds of plants with sonication as

extraction technique. Absolute ethanolic extracts showed the presence of vanillic acid (1.09

mg/g), 4-hydroxy-3-methoxy benzoic acid (1.21 mg/g), p-coumaric acid (2.89 mg/g) and

ferulic acid (0.77 mg/g). Aqueous ethanolic extract showed the presence of caffeic acid (3.94

mg/g), p-coumaric acid (4.68 mg/g) and ferulic acid (2.36 mg/g). From the other solvent

extracts it was observed that vanillic acid (1.52 mg/g), p-coumaric acid (2.92 mg/g) and m-

coumaric acid (5.25 mg/g) were present in absolute methanolic extract, gallic acid (0.34mg/g),

p-coumaric acid (3.95 mg/g) in aqueous methanolic extract, syringic acid (1.33 mg/g) and

ferulic acid (2.85 mg/g) in absolute acetone extract and 4-hydroxy-3-benzoic acid (2.42 mg/g),

p-coumaric acid (2.43 mg/g) and m-coumaric acid (2.16 mg/g) in aqueous acetone extracts.

From all the extracts of Asphodilus tenifolius, aqueous ethanolic extracts exhibited the

appreciable quantities of phenolic acids.

127

The phenolic compounds of aqueous ethanolic extracts of Asphodilus tenifolius

obtained through different extraction methods is depicted in Table 4.13.2. Aqueous ethanolic

extracts prepared from sonication showed highest quantities of phenolic compounds than other

methods. So, it showed that sonication had improved the extraction of phenolic compounds

from plant material.

128

Table 4.13.1. Phenolic compounds in different solvent extracts of Asphodilus tenifolius obtained through sonication

Values are average of the plant extracts analyzed individually in triplicate.

Plant extracts Phenolic acids (mg/g of plant extract)

Gallic

acid

Caffeic

acid

Vanillic

acid

4-hydroxy-3-

methoxy

benzoic acid

Chlorogenic

acid

Syringic

acid

p-coumaric

acid

m-coumaric

acid

Ferulic

acid

Absolute ethanol --- --- 1.09 1.21 --- --- 2.89 --- 0.77

Aqueous ethanol --- 3.94 --- --- --- --- 4.68 --- 2.36

Absolute methanol --- --- 1.52 --- --- --- 2.92 5.25 ---

Aqueous methanol 0.34 --- --- --- --- --- 3.95 --- ---

Absolute acetone --- --- --- --- --- 1.33 --- --- 2.85

Aqueous acetone --- --- --- 2.42 --- --- 2.43 2.16 ---

129

Table 4.13.2. Phenolic compounds in aqueous ethanolic extracts of Asphodilus tenifolius obtained through different extraction

techniques



Gallic

acid

Caffeic

acid

Vanillic

acid

4-hydroxy-3-

methoxy

benzoic acid

Chlorogenic

acid

Syringic

acid

p-coumaric

acid

m-coumaric

acid

Ferulic

acid

Absolute ethanol

(Stirring)

0.04 --- 0.10 --- --- 0.07 --- --- ---

Aqueous ethanol

(Orbital shaking)

--- --- 0.08 --- 0.10 --- --- 0.02 ---

Aqueous ethanol

(Sonication)

--- 3.94 --- --- --- --- 4.68 --- 2.36

Aqueous ethanol

(Reflux)

--- --- --- --- --- --- --- 0.02 ---

130

The qualitative and quantitative analysis of phenolic compounds in different solvent

extracts of Aerva javanica obtained through sonication is presented in Table 4.14.1. Results of

HPLC analysis showed that gallic acid (0.67 mg/g), caffeic acid (1.63 mg/g), 4-hydroxy-3-

benzoic acid (2.10 mg/g), p-coumaric acid (5.41 mg/g) and ferulic acid (1.27 mg/g) were

present in absolute ethanolic extract, gallic acid (0.95 mg/g), caffeic acid (1.10 mg/g), vanillic

acid (1.29 mg/g), 4-hydroxy-3-benzoic acid (2.10 mg/g), p-coumaric acid (5.01 mg/g) and m-

coumaric acid (3.01 mg/g) in aqueous ethanolic extract, caffeic acid (1.29 mg/g), vanillic acid

(2.41 mg/g), syringic acid (1.63 mg/g) and m-coumaric acid (4.70 mg/g) in absolute methanolic

extract, gallic acid (1.34 mg/g), caffeic acid (1.94 mg/g), syringic acid (1.30 mg/g), p-coumaric

acid (6.11 mg/g) and ferulic acid (1.86 mg/g) in aqueous methanolic extract. Absolute acetone

extract showed the presence of caffeic acid (1.33 mg/g), 4-hydroxy-3-benzoic acid (2.46 mg/g)

and m-coumaric acid (2.56 mg/g) while aqueous acetone extract exhibited 4-hydroxy-3-

benzoic acid (1.23 mg/g), m-coumaric acid (0.83 mg/g) and ferulic acid (1.70 mg/g).

Qualitative and quantitative distribution of phenolic compounds in aqueous ethanolic

extracts of Aerva javanica as effected by different extraction techniques is documented in

Table 4.14.2. Aqueous ethanolic extract obtained through sonication and orbital shaking

showed highest quantities of phenolic compounds followed by reflux and stirring.

131

Table 4.14.1. Phenolic compounds in different solvent extracts of Aerva javanica obtained through sonication



Gallic

acid

Caffeic

acid

Vanillic

acid

4-hydroxy-

3-methoxy

benzoic

acid

Chlorogenic

acid

Syringic

acid

p-

coumaric

acid

m-

coumaric

acid

Ferulic

acid

Absolute ethanol 0.67 1.63 --- 2.10 --- --- 5.41 --- 1.27

Aqueous ethanol 0.95 1.10 1.29 2.10 --- --- 5.01 3.01 ---

Absolute methanol --- 1.29 2.41 --- --- 1.63 --- 4.70 ---

Aqueous methanol 1.34 1.94 --- --- --- 1.30 6.11 --- 1.86

Absolute acetone --- 1.33 --- 2.46 --- --- --- 2.56

Aqueous acetone --- --- --- 1.23 --- --- --- 0.83 1.70

132

Table 4.14.2. Phenolic compounds in aqueous ethanolic extracts of Aerva javanica obtained through different extraction

techniques



Gallic

acid

Caffeic

acid

Vanillic

acid

4-hydroxy-

3-methoxy

benzoic

acid

Chlorogenic

acid

Syringic

acid

p-

coumaric

acid

m-

coumaric

acid

Ferulic

acid

Aqueous ethanol

(Stirring)

0.92 1.19 --- --- --- 0.23 --- --- ---

Aqueous ethanol

(Orbital shaking)

1.13 2.91 --- --- --- 1.43 --- 7.11 2.17

Aqueous ethanol

(Sonication)

0.95 1.10 1.29 2.10 --- --- 5.01 3.01 ---

Aqueous ethanol

(Reflux)

--- 1.97 1.90 --- 3.10 --- --- --- 2.87

133

Effect of different extracting solvents on phenolic compounds of Fagonia indica using

sonication as extraction technique is shown in Table 4.15.1. Results showed that gallic acid

was present in all the extracts with varying quantities. HPLC chromatograms showed the

presence of gallic acid (0.56 mg/g), chlorogenic acid (1.33 mg/g) and m-coumaric acid (6.54

mg/g) in absolute ethanolic extract, gallic acid (1.18 mg/g), caffeic acid (1.91 mg/g), 4-

hydroxy-3-methoxy benzoic acid (2.23 mg/g) and p-coumaric acid (8.67 mg/g) in aqueous

ethanolic extract, gallic acid (0.94 mg/g), 4-hydroxy-3-methoxy benzoic acid (2.93 mg/g),

syringic acid (3.73 mg/g) and m-coumaric acid (1.93 mg/g) in absolute methanolic extract and

gallic acid (1.58 mg/g), -hydroxy-3-methoxy benzoic acid (5.79 mg/g), p-coumaric acid (3.06

mg/g) and m-coumaric acid (7.48 mg/g) in aqueous methanolic extract. Absolute acetone

extract exhibited gallic acid (1.02 mg/g), caffeic acid (2.74 mg/g), 4-hydroxy-3-methoxy

benzoic acid (1.83 mg/g), syringic acid (2.24 mg/g) and m-coumaric acid (3.83 mg/g). Results

also depicted the presence of gallic acid (1.06 mg/g), vanillic acid (2.86 mg/g), syringic acid

(1.99 mg/g) and ferulic acid (7.08 mg/g).

Qualitative and quantitative distribution of phenolic compounds in different solvent

extracts of Fagonia indica for is shown in Table 4.15.2. Aqueous ethanolic extracts prepared

from different extraction techniques were analyzed through HPLC. Chromatograms obtained

from HPLC showed the presence of gallic acid and vanillic acid in all the aqueous ethanolic

extracts. However, aqueous ethanolic extract obtained through sonication showed the presence

of more phenolic compounds followed by stirring, orbital shaking and reflux.

Overall results showed that extracts obtained through aqueous solvents exhibited more

phenolic compounds with higher quantities as compared to pure solvents. Because the presence

of water increases the polarity of solvent and enhancing solubility of phenolic compounds. Out

of four extraction techniques, extracts obtained through sonication had more phenolic

compounds. A few HPLC chromatograms of three medicinal plants are given in Fig 4.5.1,

4.5.2, 4.5.3, 4.5.4, 4.5.5 and 4.5.6.

134

Table 4.15.1. Phenolic compounds in different solvent extracts of Fagonia indica obtained through sonication



Gallic

acid

Caffeic

acid

Vanillic

acid

4-hydroxy-3-

methoxy

benzoic acid

Chlorogenic

acid

Syringic

acid

p-coumaric

acid

m-coumaric

acid

Ferulic

acid

Absolute ethanol

(Stirring)

0.56 --- --- --- 1.33 --- --- 6.54 ---

Aqueous ethanol

(Sonication)

1.18 1.47 1.91 2.23 --- --- 8.67 --- ---

Absolute methanol

(Sonication)

0.94 --- --- 2.93 --- 3.73 --- 7.48 ---

Aqueous methanol

(Orbital shaking)

1.58 --- --- 5.79 --- --- 3.06 1.93 ---

Absolute acetone

(Reflux)

1.02 2.74 --- 1.83 --- 2.24 --- 3.83 ---

Aqueous acetone

(Sonication)

1.06 --- 2.86 --- --- 1.99 --- --- 7.08

135

Table 4.15.2. Phenolic compounds in aqueous ethanolic extracts of Fagonia indica obtained through different extraction

techniques



Gallic

acid

Caffeic

acid

Vanillic

acid

4-hydroxy-3-

methoxy

benzoic acid

Chlorogenic

acid

Syringic

acid

p-coumaric

acid

m-coumaric

acid

Ferulic

acid

Aqueous ethanol

(Stirring)

1.05 --- 4.48 --- --- --- --- --- 4.09

Aqueous ethanol

(Orbital shaking)

0.99 --- 1.29 --- 1.17 --- --- --- ---

Aqueous ethanol

(Sonication)

1.18 1.47 1.91 2.23 --- --- 8.67 --- ---

Aqueous ethanol

(Reflux)

0.59 --- 3.71 --- 1.70 --- 11.70 --- ---

136

Fig 4.5.1. Representative HPLC chromatogram of absolute ethanolic extract of

Asphodilus tenifolius through sonication

Fig 4.5.2. Representative HPLC chromatogram of absolute methanolic extract of

Asphodilus tenifolius through sonication

137

Fig 4.5.3 Representative HPLC chromatogram of aqueous ethanolic extract of

Aerva javanica through stirring

Fig 4.5.4. Representative HPLC chromatogram of aqueous methanolic extract of

Fagonia indica through sonication

138

Fig 4.5.6. Representative HPLC chromatogram of aqueous acetone extract of

Fagonia indica through sonication

139

Chapter 5

Summary

The research work presented in this dissertation was conducted in the central Hi-Tech

laboratory and laboratories of the Department of Chemistry and Biochemistry, University of

Agriculture, Faisalabad. The study was accomplished on medicinal plants of Pothohar plateau,

which is one of the famous plateau of Pakistan. Pothohar plateau provides great resources of

medicinal plants because of its unique geographical location and varied environment. Large

number of ethnobotanical studies in this area showed its importance for medicinal plants. Three

medicinal plants; Asphodilus tenifolius (Family; Liliaceae), Aerva javanica (Family;

Amaranthaceae) and Fagonia indica (Family; Zygophyllaceae) locally used in this area for

different ailments were collected from Pothohar plateu. Experiments were conducted to investigate

the yield, phenolic and flavonoid contents, antioxidant and antimicrobial activities of the extracts

of the studied plants. Efforts were also made to appraise the effect of extracting solvent and

extraction techniques on the yield, phenolic compounds and biological activities of the medicinal

plants.

Methanol (Absolute and aqueous), ethanol (Absolute and aqueous) and acetone (Absolute

and aqueous) were used as extraction solvents and stirring, orbital shaking, sonication and reflux

as extraction techniques. Antioxidant activity was evaluated by chromatographic as well as

spectroscopic methods; TLC based DPPH radical scavenging assays, DPPH radical scavenging

assay, superoxide radical scavenging assay, reducing power determination and Antioxidant

activity determination in a ß-Carotene Linoleic Acid System. For antimicrobial activity, disc

diffusion and MIC determination assays were performed. Phenolic compounds were analyzed by

spectroscopic methods and High performance liquid chromatography (HPLC).

Results showed that among all the solvent systems maximum extraction yield of

Asphodilus tenifolius was obtained from aqueous ethanolic extracts (14.48-18.86 g/100g of plant

powder) and that of Aerva javanica and Fagonia indica from aqueous methanolic extracts; 9.93-

14.57 g/100g of plant powder and 12.13-14.44 g/100g of plant powder, respectively, of all the four

extraction techniques. Out of four extraction techniques, reflux technique showed maximum

extraction yield plants followed by sonication, stirring and orbital shaking. From all the plants

140

maximum extraction yield was obtained from Asphodilus tenifolius (7.94-18.86 g/100g) followed

by Fagonia indica (3.08-14.44 g/100g) and minimum from Aerva javanica (2.30-14.57 g/100g).

Phytochemical studies revealed that highest phenolic and flavonoid recovery were

achieved by using aqueous solvents as compared to pure solvents. Maximum TPC and TFC of

Asphodilus tenifolius were obtained by extracting it with aqueous ethanol (76.23 mg GAE/g of

plant extract and 312 mg QE/g of plant extract, respectively). While TPC and TFC of Aerva

javanica were obtained by using aqueous methanol (130.19 mg GAE/g of plant extract and 345.06

mg QE/g of plant extract, respectively)as extracting solvent and that of Fagonia indica also from

aqueous methanolic extracts (161.00 mg GAE/g of plant extract and 212 mg QE/g of plant extract,

respectively). By comparing the plants, highest TPC and TFC were shown by Fagonia indica

followed by Aerva javanica and then Asphodilus tenifolius. Extraction techniques also affected

total phenolic and flavonoid contents; trend obtained was sonication>stirring>orbital

shaking>reflux.

All the plant extracts showed good antioxidant and radical scavenging activities. TLC

based DPPH assay was conducted for qualitative determination of antioxidant activity which

showed that all the plant extracts exhibited DPPH free radical scavenging potential with varying

degrees. Higher DPPH radical scavenging activity in the spectrophotometric assay was shown by

Aerva javanica ranging from 31.87- 87.04%, followed by Fagonia indica (41.33-79.90%) and then

Asphodilus tenifolius (28.00-66.24%). Plant extracts also showed appreciable superoxide radical

scavenging activities; Asphodilus tenifolius (31.87-78.01%), Aerva javanica (43.48-72.48%) and

Fagonia indica (50.71-75.11%). In the reducing power assay, Fagonia indica exhibited maximum

FRAP contents (10.07-42.79 mg GAE/g) followed by Aerva javanica (5.03-42.29 mg GAE/g) and

least contents by Asphodilus tenifolius (4.60-18.48 mg GAE/g). The greater antioxidant potential

in the ß-carotene linoleic acid system was shown by Aerva javanica (146.36-391.00AAC) then

Fagonia indica (202-380AAC) and Asphodilus tenifolius (63-225AAC). From all the other four

antioxidant activity determination assays, it was observed that aqueous ethnaol and aqueous

methanol were best solvents for the extraction of such bioactive compounsds which possess

antioxidant activities. Among four extraction techniques, extracts obtained through sonication

proved to be best for antioxidant activity. Extracts obtained through reflux exhibited least

antioxidant activities.

141

Antimicrobial (antibacterial and antifungal) activity of all the plant extracts was evaluated

against four bacterial (Escherichia coli, Pasteurella multocida, Bacillus subtillus and

Staphylococcus aureus) ans four fungal strains (Aspergillus flavus, Aspergillus niger, Fusarium

solani and Helminthus spermium). All the plant extracts showed activity against all the four

bacterial strains. Extracts of Fagonia indica showed highest antibacterial activities followed by

Aerva javanica and then Asphodilus tenifolius against three bacterial stratins, E.coli, P. multocida

and B. subtillus. While in case of S. aureus the trend obtained is Fagonia indica> Asphodilus

tenifolius>Aerva javanica. All the plant extracts also showed activity against four fungal strains

except a few extracts of Asphodilus tenifolius and Aerva javanica which were inactive against

Helminthus spermium. From all the solvent systems, extracts of aqueous solvent systems (aq.

methanol, aq. ethanol and aq. acetone) exhibited more activity against all the microbial strains as

compared to pure (absolute) solvents. By the comparison of extraction techniques, extracts of all

the plants obtained through sonication showed maximum antimicrobial activity followed by

stirring and orbital shaking and least was shown by extracts obtained from reflux.

HPLC analysis was performed only for the extracts obtained through sonication which

showed better antioxidant and antimicrobial activities. All the plant extracts showed appreciable

quantities of phenolic acids. Extracts obtained from aqueous solvents showed more phenolic acids

as compared to pure solvents. For comparison of extraction techniques, aqueous ethanolic extracts

of all the plants obtained from four techniques were analyzed through HPLC. Aqueous ethanolic

extracts obtained through sonication gave more phenolic contents.

This study provides useful information about the phytochemicals, antioxidant and antimicrobial

activities of the selected medicinal plants native to Pothohar plateau. The utilization of these

indigenous plants as potential source of natural antioxidants and antimicrobial agents is

encouraged. However, future in-vivo studies are recommended to further evaluate the

antimicrobial and antioxidant actions of these plants for therapeutic applications. Moreover, the

isolation and characterization of bioactive compounds is also suggested.

142

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ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ...prr.hec.gov.pk/jspui/bitstream/123456789/2370/1/2828S.pdfMehmood, Aisha Ashraf for their assistance, good company, marvelous behavior