i

Investigation of traditional Australian Aboriginal and

Indian Ayurvedic medicinal plants for their role in

management of type 2 diabetes

A Thesis submitted for the degree of Doctor of Philosophy

By

Vandana Gulati (M. Pharmacy)

Faculty of Life and Social Sciences

Swinburne University of Technology

Melbourne

Australia

January 2013

ii

I dedicate this thesis to my dear parents and

daughter for being a source of inspiration and for

their unconditional love to me.

iii

Abstract

Diabetes mellitus is not a single disease but is a group of metabolic diseases affecting a

large number of people globally. It is mainly characterized by hyperglycemia due to in-

sufficient production of insulin. Plants have been the main source of medicines since

ancient times and are the richest source of natural compounds. Plants continue to pro-

vide new chemical entities for the development of drugs against various diseases like

cancer, diabetes, inflammation, hypertension and neurodegeneration. In this project, the

bioactivity of extracts of seven Australian Aboriginal and nineteen Indian Ayurvedic

plants were studied. The main aim of the project was to evaluate these plant extracts for

their potential to help in the management of type 2 diabetes and related complications.

Antimicrobial screening of the plant extracts revealed that the Australian Aboriginal

extracts were active against only Gram-positive bacteria whereas Indian Ayurvedic ex-

tracts showed a broad spectrum of activity against Gram-positive and Gram-negative

bacteria. The antioxidant activity of Acacia ligulata was found to be strongest with an

IC50 of 6.98 µg/ml against DPPH. Acacia kempeana showed strongest activity against

ABTS with IC50 of 8.86 µg/ml. Among the Indian plant extracts, Bacopa moneirrei and

Andrographis paniculata showed good free radical scavenging activity with IC50 of

14.95 µg/ml each in the DPPH assay and 23.56 µg/ml and 41.65 µg/ml, respectively, in

the ABTS assay. Cytotoxicity studies revealed that Acacia kempeana and Acacia

tetragonophylla were found to have good activity against HeLa cancer cells, suggesting

their potential use as anti-cancer agents.

The main focus of the study was to investigate the ability of the plant extracts to inhibit

enzymes responsible for carbohydrate digestion (thus controlling hyperglycemia), spe-

cifically -amylase and -glucosidase. Extracts of Santalum spicatum (IC50 5.43

µg/ml), Chlorophytum borivilianum (IC50 4.17 µg/ml), Plumbago zeylenica (IC50 4.36

µg/ml), Solanum nigrum (IC50 4.87 µg/ml) and Pterocarpus marsupium (IC50 6.98

µg/ml) were particularly active against -amylase enzyme, while Beyeria leshnaultii

(IC50 0.48 µg/ml), Chlorophytum borivilianum (IC50 0.58 µg/ml) and Mucuna pruriens

(IC50 0.80 µg/ml) showed potent activity against -glucosidase enzyme. The IC50 value

was compared with positive control acarbose. The same plant extracts were also

screened against angiotensin converting enzyme (ACE-I) and the results were compared

iv

with the positive control, captopril, however none of the extracts were found be compa-

rable with captopril against this enzyme.

Twelve plant extracts were selected and further evaluated for their antidiabetic activity

against murine adipocytes to investigate glucose uptake and adipogenesis in cultured

cells i.e. 3T3-L1 adipocytes. At 100 µg/ml, Acacia kempeana and Curculigo orchioides

enhanced basal glucose uptake into differentiated 3T3-L1 cells by 19 % and enhanced

insulin-stimulated uptake by 45 % and 48 %, respectively. The plants were compared to

the positive control, rosiglitazone. Among the plant extracts examined, Acacia

tetragonophylla reduced lipid accumulation by 82 % whereas Beyeria leshnaultii, Eu-

phorbia drummondii and Curculigo orchioides were found to significantly reduce lipid

accumulation by 73 %, 65 % and 31 %, respectively, in 3T3-L1 adipocytes, thus found

to be helpful in reducing adipogenesis. The Australian Aboriginal plants have been tra-

ditionally used for general illnesses but have no documented use for diabetes, thus this

is the first study to report their potential use in the management of type 2 diabetes. The

plants showing potent activity may be developed into novel and safe treatments for type

2 diabetes and related complications.

v

Acknowledgements

On this gracious occasion, first of all, I bow to the Almighty without whose blessings

this work could have never been blossomed and completed.

I would like to express my deepest gratitude to my supervisor Associate Professor Enzo

Palombo for his valuable guidance, enthusiastic and dynamic approach, encouragement,

and above all his immense faith on me which had always motivated me in this endeav-

our. I cannot thank him enough for the care and friendship which made my life easy and

comfortable in a new country. I am also extremely thankful to Professor Ian Harding for

his advice and support for me to achieve my best in this project.

Many thanks are also given to the staff at Swinburne University, Mr. Ngan Nguyen,

Mrs. Soula Mougos, Mr. Christopher Key, Mrs. Savitri Galappathie and Mrs. Angela

Mckellar who were always there to help and shared their knowledge with me through-

out this project.

This thesis would have never been a success without Dr. Sateesh Chauhan (Promed Re-

search Centre, India) and Dr. Susan Semple (University of South Australia, Adelaide)

for providing Indian and Australian plant samples. I greatly appreciate Dr. Greog Ramm

and Dr. Ming Je Hsieh of Monash University, Australia for providing the cell lines.

I greatly appreciate the help and support I received from my dear friends Anjula and

Shakuntala who were always present in hours of need whether it was day or night.

I am extremely thankful to my friends Swarna, Raji, Melissa and Gaurav who left fun-

filled memories in my heart. I also extend my thanks to my departmental colleagues Su-

chetana, Dhivya, Babu, Kaylass, Shanthi, Bita, Sarah, Elisa, Abirami, Jafar, Rashida,

Jun, Jiawey, Natalia, Runyar, Rohan, Snehal and Avinash for their support and under-

standing.

I extend my gratitude to Dr. Mandvi, Dr. Usha, Dr. Anita, Dr. Vishal and Dr. Kiran for

their moral support and encouraging words. My cousines Vivek and Kanika also de-

serve special thanks for their constant support during this journey.

vi

Most of all I would like to express my profound love to my parents whose blessings and

faith made me strong. I really want to dedicate this PhD to you mom as you did not give

a single thought to your health and other priorities and the day you came to know that I

was going low with emotions, you just came here to help me. Big thanks to my parents

who supported me emotionally, physically and financially during this tenure.

I solicit my thanks to my In-laws, my brother, sister and their families and my ever

smiling cheerful nieces Srishti, Dhairya and Nikki.

Any amount of vocabulary cannot express my acknowledgement and indebtedness to

my lovely baby Ryka who was a greatest gift from God last year. I am really thankful

for your sacrifices. Your lovely smiles, cuddles and kisses added cheer in my life and

inspired me to finish this task.

Last but not least I would like to thank my dear husband Pankaj Gulati whose help and

support was there with me at each and every step of this entire journey, who encouraged

me to continue my passion towards research in plants. Words are not enough to express

my thanks to you my dear hubby.

vii

Declaration

This thesis contains no material which has been accepted for the award of any other de-

gree or diploma, in any University, college or any other educational institute. To the

best of my knowledge, this thesis contains no material previously published or written

by another person except where due reference is made in the text of the thesis.

Signature of Candidate: .....................................................................

Date: ........................................................................

viii

List of Publications

Following is a list of publications and conference presentations arising from the work

contained in this thesis.

Book Chapter

Gulati, V., Harding, I.H. and Palombo, E.A. Management of diabetes with diet

and plant-derived drugs. In Medicinal Plants: Classification, Biosynthesis and

Pharmacology. Edited by A. Varela and J. Ibañez. Nova Science Publish-

ers, New York. pp. 167-187, 2009.

Refereed journal publications

Gulati, V., Harding, I.H. and Palombo, E.A. Enzyme inhibitory and antioxidant

activities of traditional medicinal plants: potential application in the manage-

ment of hyperglycemia. BMC Complementary and Alternative Medicine 2012,

12:77.

Shah, R., Gulati, V. and Palombo, E.A. Pharmacological properties of guggul-

sterones, the major active components of gum guggul. Phytotherapy Research

2012, 26:1594-1605.

Conference presentations

Inhibition of alpha-amylase and alpha-glucosidase enzymes by medicinal plant

extracts: potential application in the management of Type 2 diabetes at ADS-

ADEA (Australian Diabetes Society- Australian Diabetes Educators Associ-

ation), Adelaide, Australia, Aug. – 2009 (Poster).

Enzymatic inhibition, antioxidant and antibacterial activity of medicinal plant

extracts: potential application in the management of Type 2 Diabetes.

ix

WorldPharma 2010, 16th World Congress of Basic and Clinical Pharma-

cology, Copenhagen, Denmark, July 2010 (Poster).

Enzymatic inhibition, antioxidant and antibacterial activity of medicinal plant

extracts: potential application in the management of Type 2 Diabetes at The

Royal Australian Chemical Institute (RACI). Melbourne, Australia. July –

2011 (Poster).

x

Table of Contents

Acknowledgements ........................................................................................................... v

Declaration ...................................................................................................................... vii

List of Publications ........................................................................................................ viii

Table of Contents .............................................................................................................. x

Index of Figures ............................................................................................................. xiv

Index of Tables ............................................................................................................... xvi

Abbreviations ................................................................................................................ xvii

Chapter 1. Literature Review ........................................................................................ 1

1.1. Abstract ............................................................................................................. 2

1.2. General introduction.......................................................................................... 3

1.2.1. Symptoms and related complications ......................................................... 3

1.3. Types of diabetes............................................................................................... 4

1.3.1. Current therapies for diabetes ..................................................................... 5

1.3.2. Management of diabetes with diet .............................................................. 7

1.3.3. Management of diabetes with plants ......................................................... 13

1.4. Phytochemicals with Anti-Diabetic Activities ................................................ 18

1.4.1. Flavonoids and Polyphenolic compounds ................................................. 18

1.4.2. Glycosides ................................................................................................. 20

1.4.3. Alkaloids ................................................................................................... 21

1.4.4. Essential oils ............................................................................................. 22

1.5. Brief overview of plants used in this study ..................................................... 24

1.6. The specific aims............................................................................................. 29

Chapter 2. Antimicrobial activity of plants ................................................................ 30

2.1. Abstract ........................................................................................................... 31

xi

2.2. Introduction ..................................................................................................... 32

2.2.1. Chapter Aims ............................................................................................ 33

2.3. Materials and methods .................................................................................... 34

2.3.1. Preparation of extracts............................................................................... 36

2.3.2. Media used ................................................................................................ 36

2.3.3. Anti-bacterial assay ................................................................................... 36

2.3.4. Anti-fungal assay ...................................................................................... 37

2.4. Results and Discussion .................................................................................... 38

2.5. Conclusions ..................................................................................................... 48

Chapter 3. Antioxidant activity of plants .................................................................... 50

3.1. Abstract ........................................................................................................... 51

3.2. Introduction ..................................................................................................... 52

3.2.1. Chapter Aims ............................................................................................ 54

3.3. Materials and methods .................................................................................... 54

3.3.1. Determination of total phenolic ................................................................ 55

3.3.2. Determination of total flavonoids ............................................................. 55

3.3.3. Antioxidant activity determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH)

radical inhibition ..................................................................................................... 55

3.3.4. Antioxidant activity determined by 2, 2′-azinobis-3-ethylbenzothiazoline-

6-sulfonate (ABTS) radical inhibition .................................................................... 57

3.3.5. Ferric reducing antioxidant power assay................................................... 57

3.3.6. Statistical analysis ..................................................................................... 58

3.4. Results ............................................................................................................. 58

3.4.1. Determination of total flavonoids and phenolic content ........................... 58

3.4.2. 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical inhibition ....................... 61

xii

3.4.3. 2, 2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS) radical

inhibition ................................................................................................................. 65

3.4.4. Ferric-reducing antioxidant power assay (FRAP)..................................... 68

3.5. Discussion ....................................................................................................... 73

3.6. Conclusions ..................................................................................................... 76

Chapter 4. Inhibition of enzymes relevant to the control of hyperglycemia and other

diabetes-related conditions .............................................................................................. 77

4.1. Abstract ........................................................................................................... 78

4.2. Introduction ..................................................................................................... 79

4.2.1. Chapter Aims ............................................................................................ 80

4.3. Materials and methods: ................................................................................... 80

4.3.1. Alpha-amylase enzyme inhibition............................................................. 81

4.3.2. Alpha-amylase enzyme inhibition by quantitative starch hydrolysis ....... 81

4.3.3. Alpha-glucosidase enzyme inhibition ....................................................... 82

4.3.4. Angiotensin converting enzyme inhibition ............................................... 83

4.3.5. Statistical analysis ..................................................................................... 84

4.4. Results ............................................................................................................. 85

4.4.1. Alpha-amylase enzyme inhibition by disc diffusion ................................. 85

4.4.2. Alpha-amylase activity determined by spectrophotometric assay ............ 88

4.4.3. Glucosidase inhibition assay ..................................................................... 91

4.4.4. ACE inhibition assay ................................................................................ 94

4.5. Discussion ....................................................................................................... 97

4.6. Conclusions ................................................................................................... 102

Chapter 5. Assessing the Anti-Diabetic and Anti-Cancer Potential of Plant Extracts

using Cell-Based Assays ............................................................................................... 103

5.1. Abstract ......................................................................................................... 104

xiii

5.2. Introduction ................................................................................................... 105

5.2.1. Chapter Aims .......................................................................................... 107

5.3. Materials and Methods .................................................................................. 107

5.3.1. Cell lines ................................................................................................. 107

5.3.2. Passaging of cell lines ............................................................................. 108

5.3.3. Cryopreservation of cells ........................................................................ 108

5.3.4. MTT cytotoxicity studies ........................................................................ 108

5.3.5. Adipocyte differentiation of 3T3-L1 cells .............................................. 109

5.3.6. Glucose uptake measurements ................................................................ 110

5.3.7. Lipid accumulation inhibition assay and Oil Red O staining of

intracellular triglycerides ...................................................................................... 110

5.3.8. Statistical analysis ................................................................................... 111

5.4. Results ........................................................................................................... 112

5.4.1. Cytotoxicity studies ................................................................................. 112

5.4.2. Glucose uptake assay .............................................................................. 115

5.4.3. Inhibition of lipid accumulation in 3T3-L1 cells .................................... 120

5.5. Discussion ..................................................................................................... 124

5.6. Conclusions ................................................................................................... 127

Chapter 6. General Discussion.................................................................................. 128

6.1. Discussion ..................................................................................................... 129

6.2. Future directions............................................................................................ 140

References ..................................................................................................................... 142

xiv

Index of Figures

Figure 1 - Digestion of food and mechanisms of currently available therapies, and their

side effects, for diabetes. ................................................................................................... 6

Figure 2 - Structures of six groups of flavonoids ............................................................ 21

Figure 3 - DPPH radical scavenging IC50 of Australian Aboriginal plant extracts. ...... 62

Figure 4 - DPPH radical scavenging IC50 of Indian Ayurvedic plant extracts. ............. 64

Figure 5 - ABTS radical scavenging IC50 of ethanolic extracts of Australian Aboriginal

plants. .............................................................................................................................. 66

Figure 6 - ABTS radical scavenging IC50 of ethanolic extracts of Indian Ayurvedic

plants. .............................................................................................................................. 68

Figure 7 - Ferric ion-reducing power effects of Australian Aboriginal plant extracts... 70

Figure 8 - Ferric ion-reducing power effects of Indian Ayurvedic plant extracts. ........ 73

Figure 9 - Disc diffusion assay of α-amylase for starch hydrolysis by plant extracts. .. 85

Figure 10 - Inhibitory activity of Australian Aboriginal plants against α- amylase. ..... 89

Figure 11 - Inhibitory activity of Indian Ayurvedic plants against α-amylase. ............. 91

Figure 12 - Inhibitory activity of Australian Aboriginal plants against α-glucosidase. . 92

Figure 13 - Inhibitory activity of Indian Ayurvedic plants against α- glucosidase. ....... 94

Figure 14 - Inhibitory activity of Australian Aboriginal plants against ACE. ............... 95

Figure 15 - Inhibitory activity of Indian Ayurvedic plants against ACE. ...................... 97

Figure 16 - Effects of Australian Aboriginal plant extracts at 10 µg/ml on basal and

insulin-stimulated glucose uptake in 3T3-L1 adipocytes. ............................................. 116

Figure 17 - Effects of Australian Aboriginal plant extracts at 100 µg/ml on basal and

insulin-stimulated glucose uptake in 3T3-L1 adipocytes. ............................................. 117

Figure 18 - Effects of Indian Ayurvedic plant extracts at 10 µg/ml on basal and insulin-

stimulated glucose uptake in 3T3-L1 adipocytes. ......................................................... 118

Figure 19 - Effects of Indian Ayurvedic plant extracts at 100 µg/ml on basal and

insulin-stimulated glucose uptake in 3T3-L1 adipocytes: ............................................ 119

xv

Figure 20 - Micrographs showing effects of plant extracts on lipid accumualtion ...... 120

Figure 21 - Effect of Australian Aboriginal plant extracts on Oil Red O staining in

cultured 3T3-L1 adipocytes. ......................................................................................... 122

Figure 22 - Effect of Indian Ayurvedic plant extracts on Oil Red O staining in cultured

3T3-L1 adipocytes. ....................................................................................................... 123

Figure 23 - Physiological pathway representing the generation of free radicals and their

contribution to various disorders which can be moderated or prevented by antioxidants

found in plant species. ................................................................................................... 131

Figure 24 - The proportion of active Australian Aboriginal and Indian Ayurvedic plants

investigated by various assays. ..................................................................................... 140

xvi

Index of Tables

Table 1 - Ethnobotanical uses of Australian Aboriginal plants ..................................... 24

Table 2 - Ethnobotanical uses of Indian Ayurvedic plants ............................................ 27

Table 3 - Australian Aboriginal plants used in this study ............................................... 34

Table 4 - Indian Ayurvedic plants used in this study ...................................................... 35

Table 5 - Zones of inhibition by Australian Aboriginal plant extracts .......................... 39

Table 6 - Zone of inhibition by Indian Ayurvedic plant extracts ................................... 41

Table 7 - Minimum inhibitory concentrations (MIC) and minimum bactericidal

concentrations (MBC) of plant extracts .......................................................................... 43

Table 8 - Total phenolic and flavonoid contents of Australian Aboriginal plant extracts

......................................................................................................................................... 59

Table 9 - Total phenolic and flavonoid contenst of Indian Ayurvedic plant extracts .... 60

Table 10 - Inhibition of α-amylase by Australian Aboriginal plant extracts. ................ 86

Table 11 - Inhibition of α-amylase by Indian Ayurvedic plant extracts. ....................... 87

Table 12 - IC50 values (μg/ml) of Australian Aboriginal plant extracts on two cancer

cell lines and the non-cancerous MDCK and 3T3-L1 cell lines. .................................. 113

Table 13 - IC50 values (μg/ml) of Indian Ayurvedic plant extracts on two cancer cell

lines, MDCK and 3T3-L1 cell line. .............................................................................. 114

Table 14 - Results summary of Australian Aboriginal plant extracts ........................... 136

Table 15 - Results summary of Indian Ayurvedic plant extracts .................................. 139

xvii

Abbreviations

ABTS 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate

ACN Acetonitrile

ACE-I Angiotensin converting enzyme

ANOVA Analysis of variance

AMPK Adenosine mono phosphate kinase

ATCC American Type Culture Collection

A549 Lung adenocarcinoma

2-NBDG 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose

3T3-L1 Fibroblast cell line

BC Before Christ

BDA Broth dilution assays

BHA Butylated hydroxyanisole

BHT Butylated hydroxyl toluene

CAM Complementary and Alternative Medicine

dH2O Distilled water

DMEM Dulbecco’s Modified Eagle’s Media

DMEM/F12 Dulbecco’s modified Eagle medium/Ham’s nutrient mixture F12

DNA Deoxyribonucleic acid

DNJ Deoxynojirimycin

DPPH 1, 1-diphenyl-2-picrylhydrazyl

EDTA Ethylenediaminetetraacetic acid

EGCG Epigallocatechin-3-gallate

FBS Foetal bovine serum

FRAP Ferric reducing antioxidant power

GAE Gallic acid equivalent

GPx Glutathione peroxidase

HCl Hydrochloric acid

HeLa Human ovarian carcinoma cells

HPLC High performance liquid chromatography

IAA Indoleacetic acid

xviii

IBMX 3-isobutyl-1-methylxanthine

IC50 50% inhibitory concentration

IDDM Insulin dependent diabetes mellitus

IPA Isopropyl alcohol

KCl Potassium chloride

KH2PO4 Potassium dihydrogenphosphate

LPO Lipid peroxidation

MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

MDCK Madin-darby canine kidney epithelial cells

MIC Minimum inhibitory concentration

MBC Minimum bactericidal concentration

NA Nutrient agar

NaCl Sodium chloride

Na2CO3 Sodium bicarbonate

NaH2PO4 Sodium dihydrogen phosphate

Na2HPO4 Disodium orthophosphate

NaOH Sodiun hydroxide

NaNO2 Sodium Nitrite

NB Nutrient broth

NCI National Cancer Institute

NIDDM Non-insulin dependent diabetes mellitus

NIH National Institute of Health

OD Optical density

OST Oral saccharinity tolerance

PBS Phosphate-buffered saline

PDA Potato dextrose agar

PDB Potato dextrose broth

RAAS Renin angiotensin aldosterone system

ROS Reactive oxygen species

RNS Reactive nitrogen species

rpm Revolutions per minute

RT Room temperature

xix

RTZ Roziglitazone

SD model Spring Dawley rat model

SEM Standard error mean

STZ Streptozotocin

SOD Superoxide dismutase

TFC Total flavonoid content

TPC Total phenolic content

TZD Thiazolidinedione

UV Ultra violet

QE Quercetin equivalent

WHO World Health Organization

Chapter 1 1

Chapter 1. Literature Review

Chapter 1 2

1.1. Abstract

Diabetes mellitus is a metabolic syndrome resulting from low levels of insulin. The in-

creasing worldwide incidence of diabetes mellitus in adults constitutes a major global

public health burden. The World Health Organization (W.H.O.) estimates that currently

more than 180 million people worldwide have diabetes.

Plants have been the main source of medicines since ancient times. Despite tremendous

advances in medicinal chemistry, synthetic drugs have not provided cures to many dis-

eases due to their adverse side effects or diminution in response after prolonged use.

Recently, there has been a renewed interest in traditional medicines with the belief that

plant-derived drugs may be less toxic and safer than synthetic drugs. With respect to

diabetes, numerous studies have indicated that plant-derived chemicals can be useful in

the therapeutic treatment of diabetes. However, before the development of therapeutic

insulin, diet was (and still is) the main method of treatment and modern treatment fo-

cuses on a combination of drugs and diet. Dietary measures included the use of tradi-

tional medicines mainly derived from plants. While drugs will continue to be an im-

portant part of diabetes therapy, the mass of evidence available in the literature regard-

ing the medicinal properties of vegetables, fruits and other herbs, suggests that diet (in-

cluding herbal medicines) should not be ignored or neglected.

This review will focus on recent examples of traditional medicines and foods that have

been validated by scientific evaluation as having promising activity for the prevention

and/or treatment of diabetes. Intriguing questions that await further elucidation include

how plants, plant-derived molecules and diet can be used in the future to complement

current treatment strategies for diabetes.

Chapter 1 3

1.2. General introduction

Diabetes mellitus is a metabolic syndrome which results from low levels of insulin

when β cells of the pancreas are not able to secrete sufficient insulin. The symptoms of

diabetes are hyperglycemia (high blood glucose), polyuria (increase in urine produc-

tion), polydipsia (increased thirst), blurred vision, lethargy and weight loss. The increas-

ing worldwide incidence of diabetes mellitus in adults constitutes a global public health

burden. It is predicted that by 2030, India, China and United States will have largest

number of people with diabetes (Wild et al., 2004, Frode and Medeiros, 2008). The

World Health Organization (W.H.O.) estimates that more than 180 million people

worldwide currently have diabetes and this figure is likely to be more than double by

2030. In 2005, an estimated 1.1 million people died from diabetes related illnesses and

almost 80% of diabetes deaths occurred in low and middle-income countries. Almost

half of diabetes deaths occur in people under the age of 70 years; 55% of diabetes

deaths are in women and the W.H.O. projects that diabetes deaths will increase by more

than 50% in the next 10 years without urgent action. Most notably, diabetes deaths are

projected to increase by over 80% in upper-middle income countries between 2006 and

2015 (WHO, 2012).

1.2.1. Symptoms and related complications

Over time, diabetes can damage the heart, blood vessels, eyes, kidneys, and nerves. The

major complications related to diabetes are diabetic retinopathy, a cause of blindness

which results from long-term accumulated damage to the small blood vessels in the ret-

ina, and diabetic neuropathy, the destruction of nerves as a result of diabetes with

common symptoms of tingling, pain, numbness, or weakness in the feet and hands.

Combined with reduced blood flow, neuropathy in the feet increases the chance of foot

ulcers and eventual limb amputation. Diabetes is among the leading causes of kidney

failure and increases the risk of heart disease and stroke. Ten to twenty percent of peo-

ple with diabetes die of kidney failure and fifty percent of people with diabetes die of

cardiovascular disease (primarily heart disease and stroke). The overall risk of dying

from these conditions among people with diabetes is at least double the risk of their

peers without diabetes (Organisation, 2008).

Chapter 1 4

1.3. Types of diabetes

Diabetes is categorized into; Type 1 – Insulin Dependent Diabetes Mellitus (IDDM) –

which is an autoimmune destruction of pancreatic β cells; Type 2 – Non-Insulin De-

pendent Diabetes Mellitus (NIDDM) – which is characterized by insulin resistance in

target tissues; and gestational diabetes which occurs during pregnancy (Borch-Johnsen,

2007).

In Type 1 diabetes, there is a loss of insulin secreting β cells of the Islets of Langerhans

(in the pancreas) which causes deficiency of insulin. The main cause of this β-cell loss

is autoimmune attack by T-cells. The principal treatment is replacement of insulin

(Garg, 2008).

Type 2 diabetes is caused by reduced insulin sensitivity due to increased glucose levels

in the blood. Hyperglycaemia can be rectified by medications that improve insulin sen-

sitivity or decrease glucose production by the liver. Hyperglycaemia can be treated by

increasing physical activity, decreasing carbohydrate intake, selection of proper diet,

modification of life-style and losing weight (Clemens et al., 2004).

Gestational diabetes, although temporary, increases the risk of developing Type 2 diabe-

tes later in life. Although insulin injections are sometimes necessary, this type of diabe-

tes is also commonly treated by life-style changes such as moderate physical activity

and diet (Kim et al., 2002).

The principal clinical features of diabetes mellitus were described by Hindu scholars as

long ago as about 1500 BC as a condition featuring polydipsia, polyuria and the produc-

tion of urine which was sweet enough to attract flies and ants (JDRF, 2004). The current

focus of drug discovery research in diabetes includes the exploration of alternative med-

icines, discovery of new synthetic antidiabetic agents as well as isolation of active com-

pounds from plants which have been the source of traditional herbal medicines and have

been documented and described for their antidiabetic properties in ancient texts such as

Ayurveda. The W.H.O. has recommended that alternative medicines should be investi-

gated and explored for discovery of new drugs for the treatment of diabetes mellitus

(Saxena and Vikram, 2004).

Chapter 1 5

1.3.1. Current therapies for diabetes

Currently available therapies for diabetes include insulin and various oral antidiabetic

agents such as sulfonylureas, biguanides, α-glucosidase inhibitors, α-amylase inhibitors

and glinides, all of which can be used as monotherapies or in combination to achieve

better glycemic regulation (Nathan et al., 2006). The medications available in the mar-

ket, and their side effects, are as follows and are summarized in Figure 1:

Metformin, is the only biguanide available to most of the world and its major effect is

to decrease hepatic glucose output and lower fasting glycemia. It is generally well toler-

ated, with the most common adverse effects being gastrointestinal symptoms.

Sulfonylureas lower glycemia by increasing insulin secretion. The major adverse effect

is hypoglycemia, while weight gain is also a common concern.

Glinides, like the sulfonylureas, also stimulate insulin secretion but bind to a different

site within the sulfonylurea receptor and have a shorter half-life than the sulfonylureas

and therefore must be administered more frequently. The glinides have a similar risk for

weight gain as the sulfonylureas, but hypoglycemia may be less frequent (nateglinide)

than with some sulfonylureas.

Enzyme inhibitors lower the rate of digestion of polysaccharides in the proximal small

intestine, primarily lowering postprandial glucose levels without causing hypoglycemia.

Since carbohydrates are absorbed more distally, malabsorption and weight loss are ame-

liorated, however, increased delivery of carbohydrate to the colon commonly results in

gas production and gastrointestinal symptoms.

Thiazolidinediones (TZD) or glitazones are peroxisome proliferator–activated receptor

γ modulators which increase the sensitivity of muscle, fat, and liver to endogenous and

exogenous insulin (“insulin sensitizers”). The most common adverse effects with TZDs

are weight gain and fluid retention.

Chapter 1 6

Figure 1 - Digestion of food and mechanisms of currently available therapies, and their side effects, for diabetes.

Chapter 1 7

The alternative to the above oral hypoglycemic agents is injectable Insulin which is the

oldest among the currently available medications, initially developed to treat Type 1

diabetic patients for whom it is lifesaving. Insulin is the most effective in lowering gly-

cemia. It has beneficial effects on triglyceride and cholesterol levels but is also associat-

ed with weight gain (Nathan et al., 2006).

Although oral hypoglycemic agents and insulin play important roles in the treatment of

diabetes by controlling hyperglycemia, these have serious side effects which may cause

other diabetic complications and most of the medicines available in the market are asso-

ciated with the adverse consequences of hypoglycemia or weight gain (Grover et al.,

2002). Thus, treatment of diabetes without any side effects is still a challenge (Jung et

al., 2006b).

It is clear from the Figure 1 that enzyme inhibitors such as diet and herbs rich in poly-

phenolic compounds have bearable side effects as compared to other oral hypoglycemic

agents.

When selecting an appropriate therapy for Type 2 diabetes, factors such as other co-

existing medical conditions (high blood pressure and elevated cholesterol), adverse ef-

fects of that therapy, contraindications to therapy (Grundy et al., 2005), issues which

may affect compliance (timing of medication, frequency of dosing, over-dosing etc.)

and cost to the patient and the healthcare system should be considered alongside the

magnitude of change in blood sugar control that each medication will provide. Moreo-

ver, the relatively complication-free option of diet and life-style change should be con-

sidered (Garg, 2008).

1.3.2. Management of diabetes with diet

An important research area is the discovery and development of more effective and saf-

er antidiabetic agents. In this context, medicinal plants and diet continue to play an im-

portant role in the treatment of diabetes, particularly in developing countries where most

people have limited resources and do not have access to modern treatment (Saxena and

Vikram, 2004). A recent survey of the frequency of use of complementary and alterna-

tive medicine (CAM) in diabetes patients found that most of the patients using CAM are

Chapter 1 8

better educated, born in cities, live in large families and were suffering from diabetes for

longer duration. The survey’s definition of CAM included herbal preparations (garden

thyme, pomegranate syrup, stinging nettle, dog-rose, chervil, cinnamon and bitter al-

mond), acupuncture and meditation. Further, it was reported that more than half of the

subjects who were using CAM experienced beneficial effects (Suleyman Ceylan, 2009).

Many plants and their active chemical compounds have demonstrated activity in the

treatment of Type 2 diabetes and various other disorders. Diet has long been the key-

stone in the treatment of diabetes and various other diseases. The Ebers Papyrus pre-

scribed in 1550 BC that a diet rich in wheatgerm and ochra has glucose-lowering effica-

cy (Day, 2005). Diet and lifestyle play an important role in the management of several

diseases, including diabetes. Before the introduction of the therapeutic use of insulin,

diet was the main form of treatment and dietary measures included the use of traditional

medicines mainly derived from plants (Swanston-Flatt et al., 1991).

The ancient Indian medical system of Ayurveda, which is based on scientific principles,

has also described diabetes under the name madhumeha, stating it to be mainly influ-

enced by dietary factors such as excessive eating of sugary, acidic or salty food, certain

non-vegetarian foodstuffs, and life-style factors such as lack of exercise, overindulgence

in sleep, sedentary habits, lack of cleanliness and “suppression of natural urges”. Cur-

rent studies have confirmed that there is increased risk of developing Type 2 diabetes

from lack of exercise and sedentary life-style (Manyam, 2004). Many studies have con-

firmed the benefits of medicinal plants with hypoglycemic effects in the management of

diabetes mellitus. The effects of these plants may delay the development of diabetic

complications and correct metabolic abnormalities. Moreover, during the past few

years, some of the new bioactive drugs isolated from plants showed antidiabetic activity

with more efficacy than oral hypoglycemic agents used in clinical therapy (Bnouham et

al., 2006).

Dietary management of diabetes includes consumption of food, spices, fruits, vegeta-

bles, traditional medicines and herbs. The diet should provide adequate amounts of vit-

amins, minerals, carbohydrates, fats and proteins. Diet which enhance glycemic control

are high in fibre, low to moderate in fats and moderate in biological value proteins such

as legumes, beans, vegetables, soy and other plant-based proteins which our body can

Chapter 1 9

digest, absorb and easily utilize. A decrease of calorie intake in diabetic patients helps in

weight loss. Diets rich in fibre and containing 60% carbohydrates improve blood sugar

and lipid levels. Thus, dietary modification is the first line of therapy for diabetic pa-

tients. Dietary strategies normalize blood glucose and lipoprotein levels to reduce mor-

bidity and mortality caused by the disturbance of carbohydrate and lipoprotein metabo-

lism in diabetes mellitus. These goals can be achieved by considering the quantity and

quality of diets according to the clinical conditions of an individual (Garg, 2008). Some

examples of dietary management of diabetes which have been evaluated scientifically

are described below:

A considerable number of human and animal experiments have been carried out to

evaluate the efficacy of common spices and natural food adjuncts for several physiolog-

ical effects such as antidiabetic, digestive stimulant, cholesterol lowering, anti-

carcinogenic, anti-inflammatory, antioxidant and anti-lithogenic potential. Several

common spices such as fenugreek (Trigonella foenumgraecum) were studied on diabet-

ic and normal rats, mice, rabbits and dogs. Subsequent human clinical trials confirmed

that fenugreek possess beneficial hypoglycemic potential. Garlic (Allium sativum), on-

ion (Allium cepa), cumin (Cuminum cyminum) and turmeric (Curcuma longa) are some

other spices with beneficial anti-diabetic properties (based on animal studies). Experi-

mental data with above spices indicated that dosages of 25-50 grams of fenugreek seeds,

5-6 garlic cloves, 1 onion bulb, and 1 gram of turmeric powder incorporated into the

daily diet of diabetics were effective as a support therapy in the prevention and man-

agement of diabetes and related complications such as hypertension and obesity. The

mechanisms of action are recognized as stimulation of the pancreas to secrete insulin,

interference with dietary glucose absorption and insulin sparing action of bioactive

compounds. Ginger (Zingiber officinale), curry leaf (Murraya koenigii), mustard (Bras-

sica nigra) and coriander (Coriandrum sativum) also improved glucose tolerance in ex-

perimental diabetic animals (Srinivasan, 2005).

Apart from serving as flavouring agents, spices can also be used in the management of

certain metabolic disorders such as diabetes. Rhus coriaria L., also called sumac, and

Bunium persicum Boiss, also known as black Persian cumin, are two spices used as

condiments, particularly in Iran and Afghanistan. The methanolic, ethyl acetate and n-

Chapter 1 10

hexane extracts of both spices have been studied for their ability to inhibit the enzyme

α-amylase. The ethyl acetate extract of Rhus coriaria fruits showed significant α-

amylase inhibitory activity and thus has the potential to be used in the management of

diabetes (Giancarlo et al., 2006). Various samples of fruit-enriched yoghurts have been

tested for diabetes and hypertension management. Dairy and soy yoghurt enriched with

strawberry, blueberry and peach were screened, in vitro, for total phenolic content, anti-

oxidant activity, α-glucosidase inhibition, α-amylase inhibition and the angiotensin con-

verting enzyme-I (ACE-I) inhibition. Soy yoghurt enriched with blueberry showed the

highest antioxidant activity, phenolic content, α-glucosidase inhibition and α-amylase

inhibition. The results indicated that enrichment of yoghurts with fruit phytochemicals,

e.g. blueberries showed high health functional value in terms of Type II diabetes man-

agement. Soy yoghurt, enriched with blueberries, appeared to be the best food system in

the management of diabetes and its long term complications (Apostolidis et al., 2006).

Different types of cheese – cheddar, feta and Roquefort have been shown to be benefi-

cial in the treatment of Type 2 diabetes, and inhibited α-glucosidase, α-amylase and

ACE-I. All samples of cheese showed very high ACE-I inhibition. They were also en-

riched with cranberries and showed highest activity against α–glucosidase and α-

amylase. Therefore, cheeses enriched with cranberries have promising anti-diabetic po-

tential such that enrichment with herbs and fruit phytochemicals enhanced functional

value of cheese in relation to Type 2 diabetes management (Apostolidis et al., 2007).

The aqueous extracts of some American foods (fresh green pepper, string beans, baby

spinach, broccoli sprouts, red pepper, fresh carrot, romaine lettuce, red grape, tomato

and basil leaves, Graham crackers, Chips Ahoy, cookies and Wheat Thins crackers) and

Asian foods (powdered Asian spices fenugreek, mustard, ginger, cinnamon, turmeric,

fennel powder, cardamom powder, fresh eggplant, coccinia, bittergourd, small brinjal,

ginger, mustard and fresh carrot) have been screened using in vitro enzymatic assays.

Overall, Asian foods were found to be more active than the American foods and it was

suggested that antioxidant activity was associated with amylase inhibition and phenol-

ic–phenolic synergies may be involved in the food extract enzyme-inhibition mecha-

nism. The results from experiments showed that common vegetables and spices con-

tained significant antidiabetic activity in vitro, as well as anti-ACE activity, and sug-

Chapter 1 11

gested that dietary modification to include these types of foods along with balancing

carbohydrate intake throughout the day may represent a promising strategy to help con-

trol postprandial hyperglycemia through modulation of carbohydrate absorption. Die-

tary α-amylase and α-glucosidase inhibitors from common foods are potentially safer,

therefore, may be a preferred alternative for the reduction of carbohydrate absorption

and control of blood glucose (McCue et al., 2005).

The inhibitory effects of polyphenol components of berries on various digestive en-

zymes have been studied and it was found that anthocyanins inhibit α-glucosidase and

reduce blood glucose levels after ingestion of meals rich in starch, and they may there-

fore control hyperglycemia. Ellagitannins, present in berries inhibit α-amylase activity.

Raspberries and strawberries contain high amounts of ellagitannins and anthocyanins.

Berry polyphenols such as flavonols, anthocyanidins, ellagitannins and proanthocya-

nidins can inhibit protease enzyme which could, in turn, affect protein digestion in the

gastrointestinal tract. Proanthocyanidins can inhibit gastrointestinal lipase activity

which helps in the control of obesity by reducing fat digestion. Polyphenol components

in berries, fruits and other vegetables provide health benefits by inhibition of these di-

gestive enzymes thus providing an alternative to pharmaceutical and nutraceutical

treatment for non-insulin dependent diabetes and obesity (McDougall and Stewart,

2005).

The National Diabetes Education Program of the National Institutes of Health (NIH)

recommends that eggplant should be included in the diet for the management of Type II

diabetes. The phenolic-enriched antioxidant activity and α-glucosidase inhibitory poten-

tial might help to reduce hyperglycemia-induced pathogenesis. This was tested experi-

mentally in vitro by extracting four varieties (Purple, White, Graffiti, Italian) of fresh

and well-ripened eggplant (Solanum melongena), with water and screened for activity

using α-amylase, α-glucosidase and ACE-I inhibition, DPPH and total phenolic assays.

The results indicated that phenolic-enriched extracts had high α-glucosidase inhibitory

activity, moderate antioxidant activity and moderate to high ACE-I inhibitory activity.

Eggplant may control glucose absorption and decrease the risk of related hypertension

because of its high fibre, phenolic compounds and low soluble carbohydrate content.

Inhibition of these enzymes provides a strong biochemical basis for management of

Chapter 1 12

Type 2 diabetes by controlling glucose absorption and associated hypertension. The

phenolic antioxidant-enriched dietary strategy also has the potential to reduce cellular

oxidation stress which is also related to diabetes (Kwon et al., 2008a).

In an in vitro epididymal fat cell assay, tea has been shown to increase insulin activity.

Black, green, oolong and herbal teas all increased insulin activity with the insulin poten-

tiating activity of green and oolong teas considered to be due to epigallocatechin gallate.

The other compounds responsible for enhancing insulin activities are epicatechin gal-

late, tannins and theaflavins (Anderson and Polansky, 2002). These data were supported

by in vitro enzyme inhibition analysis of other phenolic phytochemicals including the

four types of tea (green tea, oolong tea, black tea and white tea) and several varieties of

red and white wine. The aqueous extract of black tea showed the highest α-glucosidase

inhibition followed by white and oolong tea. Red wine had high α-glucosidase inhibi-

tion compared to white wine and the inhibitory activity was correlated to phenolic con-

tent, antioxidant activity and phenolic profile of the extracts. These extracts showed less

α-amylase inhibition which indicates the potential to overcome the side effects of undi-

gested starch, and thus may have benefits for the management of hyperglycemia (Kwon

et al., 2008b). Routine consumption of green tea has been reported as showing benefi-

cial effects on various metabolic disorders such as Type 2 diabetes, obesity and cardio-

vascular risks because of its catechin (specifically EGCG (-)-epigallocatechin-3-gallate)

content in various in vitro and animal studies (Thielecke and Boschmann, 2009).

Varieties of pumpkin (Cucurbita pepo), maize (Zea mays) and beans (Glycine max,

Vigna angularis, Canavalia spp., Cicer arietinum, and Canavalia ensiformis) have been

screened using in vitro enzyme (α-glucosidase, α-amylase and ACE-I) inhibition assays.

Round orange and spotted orange green pumpkin extracts had the highest potential for

glucosidase and ACE-I inhibition and help in reducing hyperglycemia and associated

complications linked to cellular oxidation stress and hypertension (Kwon et al., 2007a).

Aqueous extracts of nine types of pepper, Capsicum annum, (green, red, orange, yellow,

cubanelle, red sweet, yellow sweet, long hot and jalapeno) were investigated for inhibi-

tory activities against α-glucosidase, α-amylase and ACE-I. Green, red sweet, long hot

and yellow sweet had high inhibitory activity against α-glucosidase from both rat intes-

tine and yeast; red sweet possessed highest α-amylase activity and yellow pepper had

Chapter 1 13

the highest ACE-I inhibitory activity followed by cubanelle, red and red sweet. Some

peppers showed high α-glucosidase with low α-amylase activity which could be a good

dietary strategy to control glucose absorption without the side effects of undigested

starch. This study indicated that peppers are rich in phenolic phytochemicals and have

high free radical scavenging-linked antioxidant activity. These foods have the potential

to reduce hyperglycemia-induced vascular complications and tissue damage resulting

from oxidation and help reduce hyperglycemia and related long term complications of

diabetes (e.g. hypertension) (Kwon et al., 2007c).

Legumes, including soybeans, chickpeas, lentils, kidney beans, cannellini beans, soy-

beans, berlotti beans, baked beans and peanuts all reduced the risk of developing Type

II diabetes as they are low in fat, high in fibre, are a good source of protein and have

low glycemic index. Animal studies of obesity and diabetes showed soybeans reduced

serum insulin and insulin resistance, while a study of middle aged Chinese women has

also shown that consumption of legumes, in particular soybeans, was inversely associat-

ed with the risk of Type 2 diabetes (Villegas et al., 2008). Alpha-amylase inhibitor (α-

AI) has been isolated and purified from kidney beans (Phaseolus vulgaris L. cv Tender-

green). Two isoforms, α-AI1 and α-AI1', of 43 kDa have been isolated showing a dif-

ference in their isoelectric point and neutral sugar content. The major isoform, α-AI1,

inhibited human and porcine pancreatic α-amylase (PPA) but not bacterial or fungal α-

amylase enzymes (Le Berre-Anton et al., 1997). Douchi, a fermented soybean Chinese

food, has been screened for α-glucosidase inhibition and found to possess significant α-

glucosidase inhibitory activity. Douchi fermented with A. oryzae had strong inhibition

as compared to the same food fermented with other fungi such as A. elegans and R.

arrhizus (Chen et al., 2007). Genistein, an isoflavone isolated from soybeans, is a potent

α-glucosidase inhibitor (Lee and Lee, 2001).

1.3.3. Management of diabetes with plants

According to ethnobotanical information, more than 800 plants are used as traditional

remedies in one or other form for the treatment of diabetes (Alarcon-Aguilara et al.,

1998). Many different moieties, chemical groups and chemical constituents with thera-

peutic efficacy have been isolated and purified from plants which were traditionally

Chapter 1 14

used to treat disease. One should note that metformin, the single most prescribed agent

for the treatment of diabetes, originated from herbal medicine (Bailey and Day, 2004,

Day, 2005) and was derived from galegine. Experimental and clinical evaluations of

galegine, isolated from Galega officinalis, provided the pharmacological and chemical

basis for the subsequent discovery of metformin (Howlett and Bailey, 2007, Bailey and

Day, 2004). 1- Deoxynojirimycin (DNJ), a potent α-glucosidase inhibitor which helps in

prevention of diabetes, was isolated from the water extract of leaves of mulberry trees

(Morus alba L.) (Asano et al., 1994).

The folk medicines used for the treatment and prevention of diabetes include garlic, on-

ion, ginseng, bitter melon, fenugreek, Gymnema sylvestre, Pterocarpus marsupium and

other plants containing flavonoid compounds, bilberry, Aloe vera, and holly. The active

ingredients derived from plants used for antidiabetic preparations have been identified,

and potentially beneficial effects on the rate of food ingestion, glucose transport, poten-

tiation of insulin release, inhibition of insulin clearance, insulin-mimetic effects, re-

duced gluconeogenesis, and β-cell protection have been attributed to these agents (Dey

et al., 2002). Some plants, such as G. sylvestre, M. charantia and P. marsupium, may

also help in regeneration of β-cells in the pancreas, which is an important discovery be-

cause none of the conventional oral hypoglycemic agents shows this action (Saxena and

Vikram, 2004).

The anti-diabetic potential of ten plants, agrimony (Agrimony eupatoria), coriander

(Coriandrum sativum), eucalyptus (Eucalyptus globulus), juniper (Juniperus com-

munis), Lucerne (Medicago sativa), avocado (Persea americana), elder (Sambucus

nigra), nettle (Urtica diocia), mushroom (Agaricus campestris) and mistletoe (Viscum

album) were evaluated by an in vitro dialysis model of glucose movement. The glucose

movement was decreased by more than 50% by agrimony and avocado. It was found

that agrimony and avocado have the ability to inhibit glucose diffusion and represent

potential dietary supplements that could be useful for allowing flexibility in meal plan-

ning for management of Type 2 diabetes (Gallagher et al., 2003). In an in vivo study, the

aqueous and methanolic leaf extracts of avocado resulted in a reduction in plasma glu-

cose level, total cholesterol and LDL-cholesterol levels in albino rats (Brai et al., 2007).

The methanolic extract of the flowering part of pomegranate (Punica granatum Linn.)

Chapter 1 15

was evaluated by in vivo and in vitro diabetes assays. The extract was shown to decrease

plasma glucose levels and possess potent inhibitory activity against α-glucosidase. It

was suggested that it could improve postprandial hyperglycemia during treatment of

Type 2 diabetes and obesity (Li et al., 2005). The inhibitory activity of anthocyanins

and tannins was proved by removing the anthocyanin and tannin fractions from the ber-

ries and it was shown that tannins were related to amylase inhibition while anthocyanins

were responsible for glucosidase inhibition (McDougall et al., 2005).

In another study, a new natural α-glucosidase inhibitor from red wine vinegar (made by

the fermentation of storage root paste of purple fleshed sweet potato, Ipomea batata)

was identified as caffeoylsophorose. The compound was tested against α-glucosidase

and studied in Sprague Dawley rats; the experiments demonstrated that

caffeoylsophorose suppressed the increased postprandial blood glucose level achieved

by inhibition of maltase (Matsui et al., 2004).

Clonal herbs of family Lamiaceae were evaluated for the management of diabetes and

hypertension. Water extracts of clonal lines of rosemary Rosmarinus officinalis, lemon

balm (Melissa officinalis), sage (Salvia officinalis), chocolate mint (Mentha piperata)

and oregano (Origanum vulgare) were screened using enzymatic inhibition assays and

oregano showed the greatest α-glucosidase inhibition activity, followed by chocolate

mint and lemon balm. Clonal lines of rosemary also showed significant α-glucosidase

inhibition. ACE-I inhibition activity was greatest in rosemary followed by lemon balm

and oregano (Kwon et al., 2006).

Other nutraceutical compounds which reduce the risk of diabetes are found in diets rich

in fibres, legumes, coffee (chlorogenic acid), barley malt, biotin, magnesium, chromium

picolinate, calcium/vitamin D, bitter melon and cinnamon extracts (McCarty, 2005).

Hot water extracts of coffee seeds showed significant inhibition against both the en-

zymes α-glucosidase and α-amylase and reduced postprandial hyperglycemia (results

assessed by in vivo assays on Wistar rats using the Oral Saccharinity tolerance test

(OST) (Zheng et al., 2007). Consumption of foodstuffs which are digested at slow rates

is a good strategy to manage diabetes and its related complications of obesity and hy-

pertension. Grains which are rich in β-glucans, such as Prowashonupana (a cultivar of

barley that is less digestible than regular barley) are good for diabetic patients. Both bar-

Chapter 1 16

ley varieties have been studied for their digestion and absorption and it was found that

absorption of Prowashonupana was lower compared to barley (Lifschitz et al., 2002).

Whole wheat seeds, partially decorticated wheat (belila), fenugreek seed powder, fenu-

greek germinated seeds, lupine, chickpeas and composite biscuits of whole

wheat/fenugreek and whole wheat/chickpea have also shown beneficial effect in diabe-

tes patients. It has been reported that daily consumption of whole grain foods and leg-

umes in many forms improves glucose tolerance and serum insulin levels in diabetic

patients (Ghallas et al., 2008). The phenolic compounds of finger millet or ragi (Eleu-

sine coracana L.) from the seed coat have been screened against α-glucosidase and pan-

creatic amylase and found to exhibit strong inhibition against both enzymes (Shobana et

al., 2009).

Bitter gourd (Momordica charantia L.) is consumed as a vegetable and herbal medicine

in various parts of the world is believed to prevent and help in the management of dia-

betes and its related complications. It has been proven (by cell culture and glucose up-

take assay) that the hypoglycemic potential of bitter gourd was due to activation of

AMP-activated protein kinase (Cheng et al., 2008). Leaves of Tamarindus indicus

showed 90% inhibition of α-amylase (Funke and Melzig, 2006). The by-products of

pineapple (Ananas cosmosus) processing (i.e. remaining pulp, peels and skin) are rich in

phenolic compounds, soluble sugars and high in fibre. After being dried, ground and

mixed with organic soy bean flour in a ratio of 1:1 and 9:1 and bio-processed with Rhi-

zopus oligosporus for 12 days, the 9:1 mixture showed the highest level of α-amylase

inhibition after 2 days of R. oligosporous growth (Correia et al., 2004b).

Other plants which have been reported as helping to decrease hyperglycemia are the

rind of bitter cucumber (Citrullus colocynthis Schard), roots of Anthocleista voglii,

fruits of Eugenia jambolana, seeds of Malabar kola (Garcinia kola), leaf extract of

Mangifera indica, flowers and fruits of Musa sapientum Kuntze, leaves of olive (Olea

europea L.), seeds of Cajanus cajan Millsp., leaves of mulberry (Morus alba L.), Eri-

obotrya japonica Lindl., leaves of Atrocarpus heterophyllus Lam., leaves of Camellia

sinensis L., husk of isphagula (Plantago ovate), bitter gourd (Momordica Charantia),

Ivy gourd (Coccinia indica), leaves of mustard (Brassica juncea), cinnamon (Cinnam-

omi cassia), Beta vulgaris var. Cicla L., Aegle marmelose (Bnouham et al., 2006).

Chapter 1 17

In addition, other well-known plants with this activity are Aloe barbedensis, Ocimum

album, Achyranthes aspera, Withania somnifera, Equisetum myriochaetum, Salacia ob-

longa Wall, Swertia chiraita, Swertia japonica, Aralia cachemirica Decne., Cryptolepis

Sanguinolenta, Ocimum sanctum Linn, Stevia rebaudiana Bertoni, Cantharanthus

roseus, Azadirachta indica, Mucuna pruriens, Eruka sativa, Opuntia steptacantha, Lan-

tana camara, Agrimony eupatoria L., Eucalyptus globules Labill, Semecarpus anacar-

dium Linn., Chamaemelum nobile, Salvia officinalis, Coscinium fenestratum, Pterocar-

pus marsupium, Asparagus adscendens, Selaginella tamariscina Beauv, Nelumbo nucif-

era, Phyllanthus amarus, Tinospora cardifolia, Acanthopanax senticosus, Silybum

marianum, Panax ginseng, Aesculus hippocastanum L., Kochia scoparia, Salvia la-

vandifolia Vahl., Butea monosperma, Gymnema sylvestre, Acrocomia mexicana, Pan-

danus odorus and Salicornia herbacea L. (Garg, 2008).

An extract of pine bark and needle showed inhibition against salivary α-amylase and

yeast α-glucosidase enzymes and significantly reduced postprandial glucose level (Kim

et al., 2005). The astringent extract of chestnut skin (Tsujita et al., 2008), extract of

Pycnanthus angolensis fruits (Tchinda et al., 2008), ethanolic extract of Butea mono-

sperma (Somani et al., 2006), leaf extract of kiwi fruit (Shirosaki et al., 2008), seed ker-

nel of Syzigium cumini (Shinde et al., 2008), leaves of guava (Psidium guajava Linn.)

(Shen et al., 2008) have all shown good hypoglycemic potential.

Chapter 1 18

1.4. Phytochemicals with Anti-Diabetic Activities

A number of bioactive compounds have been isolated from plants which are potent α-

glucosidase and/or α-amylase inhibitors and show good antidiabetic properties. The

main phytochemicals with reported antidiabetic activities are flavonoids, polyphenolic

compounds, tannins, glycosides, alkaloids and terpenoids. The active phytochemicals

were isolated, purified and scientifically validated for antidiabetic action by in vitro or

in vivo experiments.

1.4.1. Flavonoids and Polyphenolic compounds

Luteolin isolated from Lonicera japonica, amentoflavone isolated from the leaves of

Ginkgo biloba, luteolin-7-O-glucoside isolated from Salix gracilistyla and daidzein iso-

lated from soybeans are all natural flavanoids which show strong inhibitory activity

against α-glucosidase and α-amylase; with luteolin exhibiting greater activity than acar-

bose (Kim et al., 2000). Similarly, hydnocarpin, luteolin and isohydnocarpin isolated

from acetone extracts of seed hulls of Hydnocarpus wightiana Blume were screened

against yeast α-glucosidase and it was found that luteolin showed the strongest inhibito-

ry activity; isohydnocarpin was also a potent inhibitor while hydnocarpin was a mild

inhibitor (Reddy et al., 2005). Quercetin 3-O-β-D-xylopyranosyl (1″ -β-D-(׳2″ → ׳

galactopyranoside and (-)-lyoniresinol 3-O-β-D-glucopyranoside isolated from the

leaves of Alstonia scholaris, also known as Devil tree, is a traditional Thai medicinal

plant. Quercetin 3-O-β-D-xylopyranosyl (1″ β-D-galactopyranoside was found-(׳2″ → ׳

to possess maltase inhibitory activity and (-)-lyoniresinol 3-O-β-D-glucopyranoside

showed significant inhibition against both the sucrase and maltase activities of α-

glucosidase (Jong-Anurakkun et al., 2007).

Crude 50% methanolic extracts of rhizomes of Berginia ciliata, a Nepalese medicinal

plant used to treat several diseases, showed significant inhibitory activity against rat in-

testinal α-glucosidase and porcine pancreatic α-amylase. This extract was fractionated

for the isolation of novel active compounds which were further screened for activity

against the same enzymes. (-)-3-O-galloylepicatechin and (-)-3-O-galloylcatechin were

isolated as potent antidiabetic compounds which showed dose-dependent enzyme inhi-

Chapter 1 19

bition (Bhandari et al., 2008). Chebulanin, chebulagic acid and chebulinic acid isolated

from Terminalia chebula have been shown to possess potent inhibitory activity against

α-glucosidase (Gao et al., 2007). Furthermore, chebulagic acid from Terminalia chebula

was later shown to have good antidiabetic activity (Gao et al., 2008b).

Tussilago farfara L. is a common plant in China used in folk medicine. The aqueous

methanolic extract of flower buds of this plant and the isolated compounds 3,4-

dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid and rutin

showed good maltase inhibitory activity of rat intestinal α-glucosidase, and thus may

help in reduction of postprandial hyperglycemia (Gao et al., 2008a).

Flour made from unripe banana (Musa paradisiaca L.) can be used to make pasta or

spaghetti with increased undigestible carbohydrates and increase the antioxidant con-

tent. Green, unripe banana is rich in proanthocyanidins, polyphenolic compounds and

vitamins and thus possesses significant antioxidant activity. Moreover, the flour is con-

sidered to be of low glycemic index food, high in resistant starch and non-starch poly-

saccharides and possesses slow carbohydrate absorption which could be a good strategy

to prevent diabetes (Ovando-Martinez et al., 2009). An aqueous extract of unripe plan-

tain (Musa paradisiaca) was shown to possess hypoglycemic activity, as it reduced glu-

cose levels in normal and alloxan-induced diabetic rats (Oloyede, 2008).

MC 2-1-5, a water soluble peptide purified from Momordica charantia L.Var. Abbrevi-

ata Ser., has significant hypoglycemic potential. When studied in alloxan-induced dia-

betic mice, it significantly reduced the blood glucose level (Yuan et al., 2008). The hot

water extract of chamomile, Matricaria chamomilla L., and the isolated compounds,

esculetin and quercetin, help in prevention of hyperglycemia and the reduction of dia-

betic complications in diabetes patients. It was suggested that daily consumption of

chamomile tea can prevent hyperglycemia and diabetic complications (Kato et al.,

2008).

Six groups of flavonoids – flavones, flavonol, flavanone, isoflavone, flavan-3-ol, and

anthocyanidins (Figure 2) – were screened for inhibitory activity on α-amylase and α-

glucosidase enzymes and the chemical structures responsible (structural activity rela-

tionship) for these activities were evaluated. The basic structure of flavonoids consists

Chapter 1 20

of benzopyran (A & C rings) and a phenyl group (B ring). The six groups of flavonoid

are classified on the basis of variation in the C ring and linkage between the benzopyran

and phenyl groups. Inhibitory activities of 4-hydroxylated, 4,5-dihydroxylated and

3,4,5-trihydroxylated flavonoids in the same flavonoid group were compared and it was

found that activity was increased with increase in number of hydroxyl group on the B

ring. The inhibitory activity was found to be increased by the unsaturated C ring, 3-OH,

4-CO, linkage of B ring at position 3 and hydroxyl substitution on the B ring. For ex-

ample, 2,3-double bond (isoflavone, flavones, and flavonol > flavanone and flavan-3-

ol), 5-OH of flavonol or isoflavone (quercetin > fisetin; genistein > daidzein), linkage of

the B ring at the 3 position (genistein > apigenin) and hydroxyl substitution on the B

ring increased the inhibitory activity (genistein > luteolin). It was found that A, B and C

rings structures were related to inhibitory activity (Tadera et al., 2006).

1.4.2. Glycosides

Chrysophanol-8-O-β-D-glucopyranoside and chrysophanol anthraquinones from Rhu-

barb rhizome showed good antidiabetic properties (Lee and Sohn, 2008). Rhaponticin

and rhein isolated from Rhei Rhizoma improved glucose tolerance by inhibiting α-

glucoamylase activity, increasing insulin sensitivity and delaying carbohydrate diges-

tion in STZ-induced diabetic mice. In vitro studies also showed improvement in insulin

sensitivity (Choi et al., 2006). Dolichandroside A, a new phenylpropanoid glycoside

isolated from Dolichandrone falcate Seem, is a novel α-glucosidase inhibitor, while sa-

ponarin II, isolated from the same plant, is a very effective α-glucosidase inhibitor hav-

ing the same potency as acarbose (Aparna et al., 2008). Dendrobium chrysotoxum

Lindl. is a traditional Chinese herb. Polysaccharides isolated from this plant have been

found to significantly reduce blood glucose levels in alloxan-induced diabetic mice as

well as having good antioxidant activity (Zhao et al., 2007). Lupinoside isolated from

Pureria tuberosa helps in prevention of palmitate-induced impairment of insulin (Dey

et al., 2007).

Chapter 1 21

Figure 2 - Structures of six groups of flavonoids

1.4.3. Alkaloids

Adhatoda vasica Nees, a common Indian Ayurvedic plant, was screened for activity

against α-glucosidase and α-amylase. The aqueous methanolic extract of its leaves

showed high sucrase inhibitory activity and enzyme assay-guided fractionation led to

discovery of vasicine and vasicinol. Both the compounds showed high sucrase inhibito-

ry activity by reversible inhibition of sucrose hydrolyzing activity of rat intestinal α-

glucosidase. The enzymatic inhibition of α-glucosidase was studied for the first time in

this plant, although it is well known for other pharmacological activities (Gao et al.,

2008c). Two new active compounds, uniflorines A and B, have been isolated from the

leaves of Eugenia uniflora L. and showed reduction in plasma glucose levels in sucrose

tolerance tests on mice and inhibited α-glucosidase enzyme (Matsumura et al., 2000).

Chapter 1 22

1.4.4. Essential oils

A mixture of oleanolic acid and ursolic acid in a ratio of 2:1 isolated from Phyllanthus

amarus was screened for α-amylase inhibition and found to exhibit significant inhibito-

ry activity (Ali et al., 2006). Roselle tea extract is made from the dried flowers of Hibis-

cus sabdariffa Linn. and is a popular beverage in Thailand. Hibiscus acid and its 6-

methyl esters isolated from a Roselle tea extract showed significant inhibition of por-

cine pancreatic amylase (Hansawasdi et al., 2000). The essential oils from the wood of

Juniper oxycedrus showed good activity against α-amylase, while the wood and berries

of the same plant possessed significant antioxidant activity (Loizzo et al., 2007). A

diterpenoid, andrographolide, isolated from the ethanolic extract of Andrographis pa-

niculata (Burm.f.) Nees (Acanthaceae) showed significant α-glucosidase inhibition

(Subramanian et al., 2008a). Swietenine, a tetranortriterpenoid, isolated from Swietenia

macrophylla seeds showed significant in vivo hypoglycemic and hypolipidemic activity

in Type 2 diabetic rats (Dewanjee et al., 2009).

Two new compounds, 7’-(3’, 4’-dihydroxyphenyl)-N-((4-methoxyphenyl) ethyl)

propenamide and 7’-(4’-hydroxy, 3’-methoxyphenyl)-N-((4-butylphenyl) ethyl) propen-

amide, isolated from Cuscuta reflexa Roxb. showed strong inhibition for α-glucosidase

(Anis et al., 2002). Pipataline, pellitorine, sesamine, brachystamide B and guineensine

were isolated from Piper longum by bio-activity (α-glucosidase enzyme inhibition)

guided fractionation and found to possess potent inhibitory activity (Pullela et al.,

2006).

From the above scientific evaluation of phyochemicals, it is clearly seen that the majori-

ty of foods traditionally used to reduce hyperglycemia and related disorders (e.g. obesi-

ty) are rich in polyphenolic compounds and flavonoids.

In this project, seven Australian Aboriginal plant extracts and nineteen Indian Ayurve-

dic plant extracts were studied. The main aim was to evaluate Australian Aboriginal

plants for potential use in the management of type 2 diabetes. The Australian Aboriginal

plants have been used for general illness but have never been used for diabetes, thus this

is the first study to investigate their use in the management of type 2 diabetes. The In-

Chapter 1 23

dian Ayurvedic plants were studied, by contrast, because of their long held holistic me-

dicinal benefit.

Many of the plants screened here have been used as food or food supplements, suggest-

ing that they are safe to take orally. Seeds and gums of Acacia species are edible and, as

this plant grows in harsh environments, it is commonly known as “dead finish”, Santa-

lum lanceolatum (SL) has sweet fruits which are eaten fresh and the decoction of the

inner bark of S. spicatum (SS) is taken orally to relieve coughs (Anonymous, 1988).

Fruits of Eugenia jambolana (EJ), called blackberries in English, are eaten fresh, are

rich in polyphenols, are widely distributed in India and are known to reduce glucose

(Sagrawat et al., 2006). Seeds of Mucuna pruriens (MP), also known as velvet beans,

are cooked or can be eaten raw (Taylor, 2005) and in Central America the roasted and

ground seeds are used as a substitute for coffee (Sathiyanarayanan and Arulmozhi,

2007). Tuberous roots of Curculigo orchioides (CO) are eaten to maintain vitality,

strength and have aphrodisiac effects (Chauhan and Dixit, 2007). Tribal people of West

Bengal eat Boerrhaavia diffusa (BD) as a vegetable, while in the Assam state of India,

this plant is also cooked and eaten (Awasthi and Verma, 2006). Bulbs of Allium sativum

(AS) has been used as a food and spice in many countries (Pittler and Ernst, 2007). Ripe

fruits of Solanum nigrum (SN) are used in Africa for bed wetting in children and the

whole plant is used in India as food (Jain et al., 2011).

Table 1 and Table 2 shows the ethnobotanical uses of the plants used in this study.

Chapter 1 24

1.5. Brief overview of plants used in this study

Plant name Family Part used Traditional Uses References

Acacia kempeana F. Muell. Mimosaceae Leaves Chest infection, severe cold and gen-

eral sickness

(Palombo and Semple, 2001a,

O’Connell et al., 1983)

Acacia tetragonophylla F. Muell. Mimosaceae Stem Cough, treatment of circumcision

wounds and dysentery

(Reid and Betts, 1979)

Acacia ligulata Cunn. ex Benth. Mimosaceae Leaves Cough, cold and chest infection (Webb, 1959, O’Connell et al.,

1983, Latz, 1995)

Beyeria leschenaultii (DC.) Baillon Euphorbiaceae Leaves

and stem

General sickness and fever (Webb, 1959)

Euphorbia drummondii Boiss. Euphorbiaceae Whole

plant

Skin sores, genital sores, fever and

dysentery

(Palombo and Semple, 2001a)

Santalum lanceolatum R. Br. Santalaceae Leaves Cold, malaise, sore throat, venereal

diseases and painful urination

(Palombo and Semple, 2001a)

Santalum spicatum (R. Br.) A. DC. Santalaceae Leaves Cough (Webb, 1959)

Table 1 - Ethnobotanical uses of Australian Aboriginal plants

Chapter 1 25

Plant name Family Part used Traditional Uses References

Andrographis paniculata

Nees.

Acanthaceae Herb Warts, Febrifuge, tonic, anthelmintic, useful in debility,

dysentery and dyspepsia

(Ram et al., 2004; Ahmad

et al., 1998)

Anacyclus pyrethrum

DC.

Compositae Roots Aphrodisiac, rejuvenation, rubefacient, tooth ache, ap-

noea. sciatica, paralysis, hemiplegia and amenorrhoea

(Sharma et al., 2009,

Gautam et al., 2011)

Allium sativum Liliaceae Bulbs Cough and fever (Ahmad et al., 1998)

Bacopa monnieri Scrophularia-

ceae

Herb Leaves of plant are used to treat epilepsy, insanity and

other nervous disorders

(Dabur et al., 2008)

Boerhaavia diffusa Linn.

Nyctaginaceae Herb Diuretic, anti-inflammatory, antifibrinolytic, anticonvul-

sant, antibacterial, antihepatotoxic and antidiabetic

(Awasthi & Verma, 2006;

Pari & Amarnath

Satheesh, 2004)

Boswellia serrata Roxb. Bruseraceae Oleo-

gum-resin

Diarrhoea, dysentry, urinary disorders, gonorrhoea and

bronchitis

(Kumar et al., 2006)

Chlorophytum borivilianum Liliaceae Tubers Roots are used to treat diarrhoea and dysentery and also

used as demulcent and galactogogue

(Dabur et al., 2008)

Commiphora mukul Hook.

Bruseraceae Resin Antiseptic, urinary disorders, skin diseases, gonorrhoea,

nasal catarrh, bronchitis, Astringent, antiseptic, expecto-

rant, carminative, enriches the blood and used for scalp

lesions

(Kumar et al., 2006,

Ahmad et al., 1998)

Convolvulus pluricaulis

Choisy

Convolvulaceae Herb Nervine tonic, memory enhancer, anthelmin-

tic,dysentery, brain and hair tonic, cures skin ailments

(Sethiya and Mishra,

2010)

Chapter 1 26

Curculigo orchioides

Gaertn.

Amaryllidaceae Rhizomes Demulscent, diuretic, aphrodisiac, asthma and jaundice (Chauhan and Dixit,

2007, Madhavan et al.,

2007)

Eugenia jambolana Lam.

Myrtaceae Seeds Bronchitis, asthma, sore throat, diabetes, dysentery, an-

tibacterial and antioxidant

(Sagrawat et al., 2006,

Grover et al., 2000,

Teixeira et al., 1997)

Mucuna pruriens Linn.

Fabaceae Seeds Antiparkinson, hypoglycemic, hypo-cholestrolemic, an-

tioxidant, antitumour and antimicrobial

(Katzenschlager et al.,

2004a, Sathiyanarayanan

and Arulmozhi, 2007)

Nardostachys jatamansi

DC.

Valerianaceae Roots Cholera, flatulence and leprosy (Kumar et al., 2006)

Plumbago zeylenica Linn. Plumbaginaceae Roots Skin disease, Skin diseases, diarrhoea, piles and leprosy (Kumar et al., 2006,

Ahmad et al., 1998)

Pterocarpus marsupium

Roxb.

Fabaceae Wood Antidiabetic, anticataract, cardiotonic and hepatoprotec-

tive

(Devgun et al., 2010,

Abesundara et al., 2004)

Puereria tuberosa DC. Fabaceae Tuberous

roots

Aphrodisiac, antirheumatic, diuretic, demulcent, galac-

tagogue and purgative

(Sumalatha Gindi, 2010)

Solanum nigrum Linn. Solanaceae Fruits Wound healing, antioxidant, antibacterial, anticancer

and CNS depressant

(Mohamed Saleem et al.,

2009)

Tinospora cordifolia Willd. Menispermeace Stem Fever and jaundice (Ahmad et al., 1998)

Chapter 1 27

Valeriana wallichii Valerianaceae Rhizomes Anti-spasmodic, stimulant, carminative, antiseptic and

sedative,

(Jawaid et al., 2011)

Table 2 - Ethnobotanical uses of Indian Ayurvedic plants

Chapter 1 28

Natural inhibitors from dietary sources have shown lower inhibitory effects against α-

amylase activity and stronger inhibitory activity against α-glucosidase, which are bene-

ficial properties that can reduce postprandial hyperglycemia with minimal side effects

(McCue et al., 2005).

Lifestyle modifications and proper diet management are also important factors in the

treatment and prevention of diabetes mellitus and its related complications. Diabetes

patients should include wholegrain products, vegetables, fruits, low fat milk, food high

in fibre, meat products and other appropriate sources of proteins, soft margarines and

vegetable oils rich in monounsaturated fatty acids and foods with low glycemic index in

their diets (Mann, 2002).

Nuts and peanuts are beneficial in maintaining glucose and insulin homeostasis. Ome-

ga-3-fatty acids and regular consumption of fish helps in diabetes by reducing the

chances of developing cardiovascular diseases and cinnamon may also have some affect

in reducing blood glucose (Rudkowska, 2009). Exercise and physical activity are the

other important factors to help manage diabetes by increasing energy expenditure, as

physical inactivity and a sedentary lifestyle are associated with metabolic disorders such

as diabetes, obesity and other cardiovascular disorders (Astrup, 2007).

Therefore, the research on plants can lead to the identification of metabolites that pro-

mote health, reduce the risk of chronic diseases and lead to the developmental tools to

assay the health-promoting effects of these metabolites which will positively contribute

in preventing the social and economic burdens of chronic disease globally (Martin et al.,

2011).

Hence, in the present investigation, attempts have been made to study the traditional

plants as antidiabetic agents.

The overall objective of the studies in this thesis were to determine the potential use of

Australian Aboriginal plant and Indian Ayurvedic plant extracts in the management of

type 2 diabetes and related disorders.

Chapter 1 29

1.6. The specific aims

To investigate the antibacterial and antifungal activity in Australian Aboriginal

and Indian Ayurvedic plant extracts against two Gram-negative and two Gram-

positive bacteria and yeast.

To determine the inhibitory concentration (IC50) antioxidant activity of Australi-

an Aboriginal and Indian Ayurvedic plant extracts against free radicals 1, 1-

diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-3-ethylbenzothiazoline-6-

sulfonate (ABTS) and ferric reducing power assay (FRAP). The total phenolic

and flavonoid contents were also determined for the same plant extracts.

To determine the activity of Australian Aboriginal and Indian Ayurvedic plant

extracts against -amylase and -glucosidase, both of which are involved in the

metabolism of carbohydrates, and angiotensin converting enzyme-I, which is

linked to hypertension.

To investigate the mechanism of action and possible role of selected plant ex-

tracts in the management of type 2 diabetes by measuring glucose uptake and

role in adipogenesis using the 3T3-L1 cell lines. Cytotoxicity was also measured

in 3T3-L1, MDCK, HeLa and A549 cells.

Chapter 2 30

Chapter 2. Antimicrobial activity of plants

Chapter 2 31

2.1. Abstract

The antimicrobial potential of 26 plant extracts was evaluated against four bacteria (Ba-

cillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa)

and a yeast (Candida albicans) using the disk diffusion assay. Ten extracts, Androgra-

phis paniculata (AP), Convolvulus plauricaulis (CP), Mucuna pruriens (MP), Plumba-

go zeylenica (PZ), Pterocarpus marsupium (PM), Curculigo orchioides (CO), Eugenia

jambolana (EJ), Valeriana wallichii (VW), Allium sativum (AS) and Nardostachys

jatamansi (NJ), showed broad spectrum activity against all the four bacterial strains

used. Most of the plant extracts showed varied levels of antimicrobial activity against

one or more test bacteria. Anticandidal activity was detected in only seven plant ex-

tracts. The lowest concentration of the extract which inhibited any visual microbial

growth was considered to be the minimum inhibitory concentration (MIC). Minimum

bactericidal concentration (MBC) was also determined. Acacia kempeana (AK) and

Santalum lanceolatum (SL) showed mild activity against B. cereus. None of the Aus-

tralian Aboriginal plants showed any activity against other bacteria.

Chapter 2 32

2.2. Introduction

Medicinal plants produce bioactive molecules which react with other organisms in the

environment to help inhibition of bacterial or fungal growth. The substances that can

inhibit pathogens and are less toxic to host cells are considered candidates for develop-

ing new antimicrobial drugs (Sengul et al., 2009).

Infectious diseases are the major cause of death worldwide with more than 50% of the

deaths occurring in tropical countries according to the World Health Organization

(WHO) (Rice et al., 2000). Treatment of such diseases is complicated due to resistance

in microorganisms to the commonly used antibiotics and the low income of the popula-

tion in the developing countries. Therefore, about 80% of the world’s population re-

mains dependent on plant-based drugs. Scientific experiments involving the antimicro-

bial properties of plants were first documented in the late 19th century with many plants

now having been shown to produce antimicrobial and bactericidal compounds which

have been utilized as effective treatments for infectious diseases (Shah et al., 2004).

There is a need, however, for many more avenues to fight diabetes, propelling increased

research into the discovery of new therapeutic agents based on herbal medicines (Kuete

et al., 2011).

Patients with diabetes are more susceptible to infections, particularly soft tissue infec-

tions like diabetic foot ulcers (mainly caused by group A Streptococcus, Staphylococcus

aureus, aerobic Gram-positive cocci, Gram-negative rods and anerobes). Urinary tract

infections are mainly due to Candida albicans. Invasive (malignant) otitis media, which

is an uncommon but life-threatening infection, is almost exclusively due to Pseudomo-

nas aeruginosa in diabetic patients. Since neutrophil function, chemotaxis and phagocy-

tosis are depressed in people with diabetes, bactericidal activity and cell-mediated im-

munity is also compromised. Thus, it is a good strategy to manage diabetes in a holistic

fashion with plants which show good enzyme inhibitory, antioxidant and anti-microbial

activities (Joshi et al., 1999). Therefore, the aim of this study was to screen traditional

Australian Aboriginal plant and Indian Ayurvedic plant extracts to determine the poten-

tial against various bacteria and yeast.

Chapter 2 33

2.2.1. Chapter Aims

The experiments described in this chapter were designed to evaluate the antimicrobial

activity of plant extracts against

Two Gram-positive (Bacillus subtilis and Staphylococcus aureus)

Two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa)

Yeast (Candida albicans).

Chapter 2 34

2.3. Materials and methods

All the plants were extracted in ethanol. Plant materials used in this study were as follows:

Plant name Codes

used

Common name Family Part used Voucher

number Acacia kempeana F. Muell. AK Witchetty Bush Mimosaceae

Bark and leaves AD99732178

Acacia tetragonophylla F. Muell. AT Dead finish Mimosaceae

Stem and leaves AD99732184

Acacia ligulata Cunn. ex Benth. AL Umbrella bush Mimosaceae

Bark and leaves AD99732187

Beyeria leschenaultii (DC.) Baillon BL Turpentine bush Euphorbiaceae Leaves and stem AD99732200

Euphorbia drummondii Boiss. ED Caustic weed Euphorbiaceae Whole plant AD99732190

Santalum lanceolatum R. Br. SL Northern sandal-

wood

Santalaceae Bark, stem and

leaves

AD99732181

Santalum spicatum (R. Br.) A. DC. SS Australian sandal-

wood

Santalaceae Bark AD99732191

Table 3 - Australian Aboriginal plants used in this study

Chapter 2 35

Plant name Codes

used

Common name Family Part used Voucher

number Andrographis paniculata Nees. AP Kalmegh Acanthaceae Herb PROM/AP-25

Anacyclus pyrethrum DC. Ana. Akarkara Compositae Roots PROM/ANPY-56

Allium sativum AS Garlic Liliaceae Bulbs PROM/AS-10

Bacopa monnieri BM Brahmi Scrophulariaceae Herb PROM/BRM-12

Boerhaavia diffusa Linn. BD Punarnava Nyctaginaceae Herb PROM/BD-43

Boswellia serrata Roxb. BS Shallaki Bruseraceae Oleo-gum-resin PROM/BSW-02

Chlorophytum borivilianum CB Safed musli Liliaceae Tubers PROM/CHBR-09

Commiphora mukul Hook CM Guggul Bruseraceae Resin PROM/CMKL-10

Convolvulus pluricaulis Choisy CP Shankhpushpi Convolvulaceae Herb PROM/CP-06

Curculigo orchioides Gaertn. CO Kali musli Amaryllidaceae Rhizomes PROM/CUOR-15

Eugenia jambolana Lam. EJ Jamun Myrtaceae Seeds PROM-EUJA-10

Mucuna pruriens Linn. MP konch Fabaceae Seeds PROM/MUPR-05

Nardostachys jatamansi DC. NJ Jatamansi Valerianaceae Roots PROM/NAJT-03

Plumbago zeylenica Linn. PZ Chitrak Plumbaginaceae Roots PROM/PZ-13

Pterocarpus marsupium Roxb. PM Vijayasaar Fabaceae Wood PROM/PTMA-01

Puereria tuberosa DC. PT Patalkumra Fabaceae Tuberous roots PROM/PT-19

Solanum nigrum Linn. SN Makoi Solanaceae Fruits PROM/SONI-08

Tinospora cordifolia Willd. TC Guduchi Menispermeace Stem PROM/TC-18

Valeriana wallichii VW Tagar Valerianaceae Rhizomes PROM/VAWA-21

Table 4 - Indian Ayurvedic plants used in this study

Chapter 2 36

2.3.1. Preparation of extracts

Five grams of Indian plant powders were soaked overnight in 50 ml absolute ethanol

and filtered with Whatmann filter paper No. 1. The filtrates were concentrated in vacuo

in a rotary evaporator at 55 °C and reconstituted in ethanol at 250 μg/μl. These stock

solutions were used to make working solutions of various concentrations.

Australian Aboriginal plants were obtained in solution of ethanol and used as such.

2.3.2. Media used

Materials and media were sterilized in a Siltex 250D autoclave (Siltex, Australia) at 121

°C for 20 minutes prior to use. Aliquots of agar were prepared in 20 ml McCartney bot-

tles and aliquots of broth were prepared in 5 ml plastic vials and stored at room temper-

ature. Circular sterile petri dishes (80 mm diameter, Techno-Plas, Australia) were used

for the agar diffusion assays. Microtitre (96 well) round bottom plates were used in the

broth dilution assays.

2.3.3. Anti-bacterial assay

Two Gram-positive bacteria, Bacillus subtilis (ATCC 6051) and Staphylococcus aureus

(ATCC 12600), and two Gram-negative bacteria (Escherichia coli (ATCC 11775),

Pseudomonas aeruginosa (ATCC 10145) were used. The cultures were part of a collec-

tion located at Swinburne University of Technology, were maintained on nutrient agar

slants and recovered by culturing in nutrient broth for 18 hours at 37 ºC. The disk diffu-

sion method was used to determine the antibacterial activity of plants (Kalemba and

Kunicka, 2003). The bacterial cultures were plated over nutrient agar plates with sterile

swab sticks. The plates were dried and sterile discs were dipped in the plant extracts

(250 mg/ml) then placed onto the centre of nutrient agar plates. The plates were incu-

bated for 18 hours at 37 ºC. Control plates were also prepared in which ethanol was ap-

plied to discs instead of plant extracts. The diameters of zones of inhibition were meas-

ured after incubation and the results were compared with the control.

Chapter 2 37

Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations

(MBC) were also determined for the plant extracts that showed antibacterial activity.

The initial concentration was 250 mg/ml, two-fold serial dilutions were prepared in 96

well microplates and each well was inoculated with overnight bacterial cultures. The

microplates were incubated at 37 ºC for 18 hours. The visual turbidity was compared

with the control well which did not have the plant extract. The MIC was determined as

the lowest concentration which completely inhibited bacterial growth. The MBC was

also determined by sub-culturing the samples from the wells of microplate with no visi-

ble growth onto nutrient agar plates. The lowest concentration of the extract which did

not show any visible growth was recorded as the MBC (Chandrasekaran and

Venkatesalu, 2004).

2.3.4. Anti-fungal assay

The fungal strain Candida albicans was used to evaluate the antifungal activity of plant

extracts. Cadida albicans (FRR5580) was cultured in Potato Dextrose Broth (PDB,

Difco) with an incubation at 30 ºC for 48 hours. The culture was part of a collection lo-

cated at Swinburne University of Technology and was maintained on Potato Dextrose

Agar (PDA, Difco) slants.

The anti-fungal assay used was similar to that described for the antibacterial assay. A

sterile cotton-tipped swab was dipped into a PDB fungal suspension of Candida albi-

cans and streaked in two directions across PDA plates. The plates were dried for 2-3

min, and subsequently, pre-sterilised discs (6mm) were placed onto the agar surface and

5 µl of the plant extracts were individually added to the discs. The seeded plates were

incubated at 30 ºC for 48 hours. Control plates were also prepared in which ethanol was

applied to discs instead of plant extracts. The diameters of zones of inhibition were

measured after incubation and the results were compared with the control.

Chapter 2 38

2.4. Results and Discussion

The anti-microbial activity of 26 ethanolic plant extracts was investigated against four

common bacteria and yeast.

Specifically, the following microorganisms were used to detect antimicrobial activity;

E. coli is the best-known member of the normal microbiota of the human intes-

tine and a versatile gastrointestinal pathogen,

P. aeruginosa is an opportunistic pathogen which causes urinary tract infections,

respiratory system infections, dermatitis and soft tissue infections,

B. subtilis is representative of endospore-forming bacteria, some of which are

food-borne pathogens.

S. aureus is a pyrogenic bacterium known to play a significant role in invasive

skin diseases including superficial and deep follicular lesions, and rapidly de-

velops resistance to many antimicrobial agents,

C. albicans was chosen for this study since it is one of the most pervasive path-

ogenic fungi, especially infecting immuno-compromised hosts, in which it can

invade various tissues and causes serious systemic infections, including oppor-

tunistic infections in diabetic patients and those infected with HIV (Paiva et al.,

2003).

Antibacterial activity was assessed by the disc diffusion method which determines the

average diameter of inhibition zones of microbial growth around the disc. The Australi-

an Aboriginal plant extracts have been previously screened for antibacterial activity and

it was found that the majority of the extracts were active against B. cereus but none was

active against the other micro-organisms tested here (Palombo and Semple, 2001).

Chapter 2 39

Plant

name

E. coli B. cereus P. aeru-

ginosa

S. aureus C. albi-

cans

AK - 9 - - -

AT - 6 - - -

AL - - - - -

BL - 6 - - -

ED - - - - -

SL - 7 - - 16

SS - - - - -

Table 5 - Zones of inhibition by Australian Aboriginal plant extracts

(diameter in mm).

None of the seven Australian Aboriginal plant extracts showed any activity against

Gram negative bacteria (Table 5). The disc used here was of 5mm diameter and control

ethanol did not give any zone of inhibition. Of the seven, only four plant extracts

showed some activity against the Gram positive bacterium B. cereus but not against S.

aureus. Santalum lanceolatum (SL) was the only plant extract which showed biofungal

activity against C. albicans with a 16 mm zone of inhibition. Table 6 shows the zone of

inhibition of Indian Ayurvedic plant extracts. Ethanol (100%) was used as a control and

showed no zone of inhibition. This suggested that the solvent used for plant extraction

did not contribute to the inhibitory activity of plants against the growth of bacteria.

All the Indian Ayurvedic plant extracts investigated showed activity against both the

Gram-negative bacteria and both the Gram-positive bacteria, except for Pueraria tuber-

osa (PT) which did not show any activity against any tested bacteria (Table 6)

Chapter 2 40

Plant name E. coli B. subtilis P. aeruginosa S. aureus C. albicans

AP 11 12 15 12 8

Ana. 9 - - 10 -

AS 10 11 7 - 10

BM - 8 17 - 10

BD 8 8 12 - 10

BS 7 7 7 - -

CB 12 8 - 9 -

CM 7 8 9 8 -

CP 17 7 10 16 10

CO 14 8 13 12 -

EJ 10 9 9 7 -

MP 15 10 11 13 11

NJ 10 9 9.5 18 -

PZ 14 12 19 11 -

Chapter 2 41

PM 15 8 14 18 -

PT - - - - 10

SN 8 9 - 7 -

TC 9.5 8.5 12 13 -

VW 8 - 8 13 -

Table 6 - Zone of inhibition by Indian Ayurvedic plant extracts (diameter in mm)

Chapter 2 42

Bacteria Plant Name MIC (mg/ml) MBC (mg/ml)

E. coli AS 1.95 3.90 TC 1.95 15.6 PM 1.95 125 CP 7.81 15.6 EJ 7.81 62.5 PZ 7.81 15.6 VW 7.81 31.5 MP 15.61 125 CO 15.61 62.5 BD 15.61 125 AP 31.5 62.5 BS 31.25 62.5 CB 31.25 62.5 NJ 62.5 125

Ana. 62.5 125 SN 62.5 125 CM 250 250

PT - -

BM - -

P.aeruginosa PZ 1.95 7.81 AP 1.95 15.6 CP 1.95 31.25 VW 3.12 62.5 EJ 7.81 62.5 CO 7.81 62.5 MP 7.81 62.5 BM 7.81 7.81 AS 7.81 62.5 BD 7.81 125 SN 15.62 15.62 NJ 15.62 62.5 PM 15.6 125 TC 62.5 62.5 CM 125 250 BS - - CB - - PT - -

Ana. - -

B.subtilis CP 1.95 31.25 AP 3.90 15.6

CM 3.90 62.5 PZ 6.25 31.5

TC 7.81 62.5

Chapter 2 43

Table 7 - Minimum inhibitory concentrations (MIC) and minimum bactericidal concen-trations (MBC) of plant extracts

SN 7.81 125 PM 7.81 >250 EJ 15.6 >250 CO 15.6 >250 MP 15.6 >250 BS 15.62 125 BM 31.25 62.5 VW 31.25 62.5 CB 62.5 62.5 NJ 62.5 62.5 AS 62.5 125

Ana. - - PT - - BD ND ND

S.aureus AS 3.90 7.81 NJ 3.90 3.90 AP 3.125 25

PM 3.90 62.5 CP 7.81 15.6 TC 7.81 31.9 PZ 7.81 31.9

EJ 7.81 62.5 VW 15.62 62.5 CO 15.62 62.5 MP 15.62 125 CB 31.25 62.5 SN 62.5 125

Ana. 125 125 CM 125 250 BS - - PT - - BD - - BM - -

Chapter 2 44

Most of the extracts showed bactericidal activity at concentrations greater than the re-

spective MIC (Table 7). However, an MBC against Bacillus subtilis was unable to be

determined, indicating that the extracts were unable to inhibit this species, possibly due

to the production of endospores.

Among all the plants screened, AS, TC, PM, AP, CP, MP, PZ, CO, EJ, VW and NJ

showed broad spectrum activity against all the four bacterial strains used.

Among these plant extracts, AS, TC and CP showed the strongest inhibitory effect on

the growth of E. coli with MIC of 1.95, 1.95 and 7.81 mg/ml and MBC of 3.90, 15.6,

15.6 mg/ml, respectively.

The MIC of AS was 3.90 mg/ml with bactericidal concentration 7.81 mg/ml against S.

aureus. Otunola et al. (2010) found that garlic has high levels of saponins, alkaloid, tan-

nin, carotenoids, flavonoids while steroids and cardenolides are present in trace

amounts. The consumption of garlic has the potential to reduce arterial plaque and the

bulb possesses antioxidant properties against skin cancer. Garlic and other spices are

good sources of nutrients, mineral elements and phytochemicals which could be ex-

ploited for the development of drugs and/or nutritional supplements (Otunola et al.,

2010).

It has been reported that the pharmacological actions of garlic (antimicrobial, anti-

cancer, antioxidant, ability to reduce cardiovascular diseases and antidiabetic activity)

are mainly due to allicin (diallyl-thiosulfinate), which is one of the major organo-sulfur

compounds in garlic and a chemically very reactive and unstable compound (Rahman,

2007).

In vivo studies showed that infections induced by injecting S. aureus, E. coli, C. albi-

cans or Klebsiella pneumoniae into immunosuppressed animals were prevented by ex-

tracts of TC (Panchabhai et al., 2008).

Chapter 2 45

NJ showed an MBC of 3.90 mg/ml against S. aureus but the value was 62.5 mg/ml

against P. aeruginosa and B. subtilis, whereas, the same plant showed very high MBC

(125 mg/ml) against E. coli (Table 6). This is a relaxing plant with established effec-

tiveness for mental health, mainly used for anxiety and insomnia (Samy et al., 2008).

The antiseptic properties are due to essential oils present in the same plant (Jadhav et

al., 2009).

PZ and AP showed an MBC of 25 mg/ml against E. coli, B. subtilis and S. aureus but

against P. aeruginosa both of the plants exhibited an MBC of 12.5 mg/ml. Other studies

have also reported the antimicrobial activity of both these plants. The extracts of both

species exhibited maximum inhibition against P. aeruginosa and S. aureus at lower

concentrations (Ram et al., 2004).

Previous studies have reported that aqueous and alcoholic extracts from roots of PZ

showed antibacterial activities against S. aureus, P. aeruginosa, B. subtilis, Proteus vul-

garis, and against the yeast C. albicans. Plumbagin exhibited relatively specific antimi-

crobial activity. The isolated naphthoquinone, plumbagin, could be the main constituent

from roots responsible for its activity. The growth of S. aureus and C. albicans was

completely inhibited. However, it was ineffective against the Gram-negative bacteria E.

coli and S. typhimurium, demonstrating the specificity of plumbagin activity (Paiva et

al., 2003).

CO showed antibacterial activity with an MIC of 7.81 mg/ml and an MBC of 62.5

mg/ml against P. aeruginosa in our study. Previous studies were also carried out with

different extracts and showed antibacterial activities against P. aeruginosa, S. aureus

and K. pneumoniae. Out of different extracts tested, the methanolic extract was found to

be the most active against P. aeruginosa followed by S. aureus and K. pneumonia. The

antimicrobial (and antitumor) activities were found to be due to presence of saponins,

which are the glycosides present in the plant (Singh and Gupta, 2008).

CP showed broad spectrum activity against all the tested microrganisms in this study.

This plant is traditionally used as a nerve tonic, for learning and memory, and for treat-

ment of neurodegenerative disorders (Sethiya and Mishra, 2010). However, a study by

Nautiyal et al. (2007) showed that airborne bacteria in the environment can be reduced

Chapter 2 46

by medicinal smoke which is produced from natural substances. The smoke is a kind of

Havana in Indian culture, agnihotra–yagnas to purify the environment as described in

Rigveda—by sublimating the havana sámagri (mixture of mango wood and odoriferous

and medicinal herbs) in the fire accompanied by the chanting of Vedic mantras. The re-

port showed that medicinal smoke emanated from havan sámagri has very interesting

inhibition effects on the aerial bacterial population. CP was one of the ingredients of

Havana Samagri (Nautiyal et al., 2007) which explains the braod spectrum antibacterial

properties of the same plant.

PT extract did not show any activity against any bacterial strains but showed a 10 mm

zone of inhibition against C. albicans. The findings supported the previous work by

Ratnam and Raju (2009) that ethanolic extracts of PT exhibited the least antimicrobial

activity when tested against a range of Gram positive and Gram negative bacteria

whereas the ethyl acetate extract showed promising results (Ratnam and Raju, 2009).

Extracts of EJ also showed broad spectrum activity against all the bacterial strains tested

with an MIC of 7.81 and MBC of 62.5 mg/ml against E. coli, P. aeruginosa and S. au-

reus whereas the MBC was greater than 250 mg/ml against B. subtilis. Previous re-

search conducted on crude methanolic seed extracts of EJ against beta-lactamase-

producing drug-resistant bacteria and further fractionation and phytochemical analysis

indicated that the most promising fraction revealed activity due to the presence of sapo-

nins as the active phytoconstituent (Jasmine et al., 2010).

Previous reports investigated on aqueous and methanolic extracts of BD have shown

good activity against S. aureus, K. pneumoniae, B. species, E. coli and P. aeruginosa

which supports the use of this plant since ancient times as a useful in the treatment of

infectious diseases like diarrhoea, intestinal tract, throat and ear infections, fever and

skin diseases (Girish and Satish, 2008). They have also shown anticandidial activity in

the current study but only mild activity against Gram negative bacteria. Other plant ex-

tracts which showed activity against C. albicans were AS, BM, MP, CP and AP.

Chapter 2 47

In brief, out of twenty six plant extracts screened here, seventeen plant extracts showed

broad spectrum activity against different microbial strains. In classifying the antibacte-

rial activity against Gram-positive or Gram-negative bacteria, it would generally be ex-

pected that a greater number would show activity against Gram-positive rather than

Gram-negative bacteria species. However, in this study, a large number of the extracts

were active against both Gram-positive and Gram-negative bacteria. The activity

against both the types of bacteria may be indicative of the presence of broad spectrum

antibiotic compounds or general metabolic toxins (Srinivasan et al., 2001).

In general, plant antibiotic compounds appear to be inhibitor towards Gram-positive

microorganisms than Gram-negative. Even Penicillin and some of the other prominent

antibiotic agents of fungal origin are moderately selective in their inhibitory action,

most of them being inhibitory to Gram-positive organisms. The major component of the

outer surface of Gram-negative bacteria is the lipopolysaccharide layer along with pro-

teins and phospholipids. Thus, access of most compounds to the peptidoglycan layer of

the cell wall is hindered by the outer lipopolysaccharide layer. This explains the general

resistance of Gram-negative strains to the lytic action of plant extracts exhibiting activi-

ty (Kumar et al., 2006).

Most of the plant extracts showed relatively high inhibitory and bactericidal concentra-

tions. However, the antibacterial activity of plant extracts is strain specific and depends

on bioactive compounds present in the extract and this property can be expressed in dif-

ferent ways. Mishra et al. (2009) reported that two types of S. aureus had different re-

sponses against the extract of Andrographis paniculata. The antibacterial property of

medicinal plants also depends on different parts of the plant used which contain greater

or fewer antibacterial bioactive compounds. In additon, the reaction of these compounds

individually or together (synergism) also influences antibacterial property of extracts

(Mishra et al., 2009).

Previous literature on the plant extracts used in this study also showed variable results

which may be explained by differences in the composition of bioactive compounds due

to different geographic conditions and different method of extractions. For example, the

antimicrobial activity of garlic powder against most bacteria was greater than for the

plant or crude drug (Ross et al., 2001). The solvent use can also affect the activity. Stud-

Chapter 2 48

ies have demonstrated that species such as Helichrysum italicum or Phytolacca dodec-

andra showed moderate activity against E. coli when the diethyl extract obtained after

extraction of the aqueous suspension of the drug powder was tested. However, no active

fractions were identified when a sequential extraction with petroleum ether, dichloro-

methane, dichloromethane–methanol (9:1) and methanol was used (Nostro et al., 2000).

The pH of compounds in dilutions can also modify the results, for example when phe-

nolic or carboxylic compounds are present in the extract. Not only do ionisable com-

pounds change the activity, but it has been reported that the different effects of neutral

essential oil depend on the pH. Thus, for example, anise oil had higher antifungal activi-

ty at pH 4.8 than at 6.8, while the oil of Cedrus deudora was most active at pH 9. The

composition of the growth medium could also influence the activity of the tested ex-

tracts or compounds. The antimicrobial activity of garlic oil was found to be greater in

media lacking tryptone or cysteine, which led to the hypothesis that the effects may in-

volve sulfhydryl reactivity (Rios and Recio, 2005).

This study is a preliminary evaluation of antimicrobial activity of the plant extracts. It

indicates that several extracts have the potential to generate novel antimicrobial metabo-

lites. In particular, the crude extracts demonstrating anticandidal activity could result in

the discovery of novel anticandidal agents. The plants demonstrating broad spectra of

activity may help in the discovery of new chemical classes of antibiotics that could

serve as selective agents for the maintenance of animal or human health and provide

biochemical tools for the study of infectious diseases.

2.5. Conclusions

The above results show that the crude extracts of Plumbago zeylenica, Andrographis

paniculata, Allium sativum, Tinospora cordifolia and Convolvulus pluricaulis exhibited

broad spectrum antimicrobial activity and properties that support their folkloric use in

the treatment of infectious diseases. This probably explains the use of these plants by

the indigenous people against a number of infections for many generations and suggests

that some of the plant extracts possess compounds with antibacterial properties that can

be used as antimicrobial agents for the treatment of infectious diseases caused by patho-

Chapter 2 49

gens. The most active extracts can be subjected to isolation of the therapeutic antimi-

crobials and undergo further pharmacological evaluation.

Though the inhibitory and bactericidal concentrations obtained in this study were high,

modifying the solvent and the extraction system may both influence the final results

such that their efficiency in the field may be higher, or lower, than their efficiency when

isolated in the laboratory. Previous literature on the plant extracts used show variable

results which could be explained, for example, by differences in bioactive compounds

due to the collection of plants from different geographic conditions, different method of

extractions and use of solvent can modify the results. It can be concluded, therefore,

that further study of medicinal plants as antimicrobial agents is warranted and may re-

sults in insights into their real value as well as validation of their traditional medicinal

use.

Chapter 3 50

Chapter 3. Antioxidant activity of plants

Chapter 3 51

3.1. Abstract

The ethanolic extracts of twenty-six traditionally used Australian Aboriginal and Indian

Ayurvedic plants were screened for their antioxidant free radical scavenging properties

using ascorbic acid and butylated hydroxy toluene (BHT) as standard antioxidants. Free

radical scavenging activity was evaluated using diphenyl picryl hydrazyl (DPPH) radi-

cal, 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS), and ferric reducing anti-

oxidant power (FRAP). The overall free radical scavenging activity was strongest in

Acacia ligulata, followed by Acacia kempeana, Beyeria leshnaultii and Santalum lan-

ceolatum. Among the Indian plant extracts, Bacopa moneirrei, Andrographis paniculata

and Pterocarpus marsupium showed good free radical scavenging activity. The phenol-

ic and flavonoid concentrations were also determined. The 50% inhibition (IC50) values

were also calculated for the same plant extracts. This is the first study on the antioxidant

potential of Australian Aboriginal plant extracts. The Australian Aboriginal plant ex-

tracts were found to be more potent in comparison to Indian Ayurvedic plant extracts.

Chapter 3 52

3.2. Introduction

Oxidation is the important pathway of generating free radicals and can play a vital role

in diverse biological phenomenon. Oxidation, in general, plays a dual role in our body;

it is essential for life but can also aggravate and damage the cells (Sen et al., 2010). Free

radicals are continuously formed inside the human body when cells use oxygen to gen-

erate energy. These free radicals are reactive oxygen species (ROS) such as superoxide

anion (O2•), hydroxyl radical (OH•), hydrogen peroxide (H2O2), peroxyl (ROO•) and

nitric oxide (NO•) and result from cellular redox processes (Pandey et al., 2010). There

is evidence that ROS and reactive nitrogen species (RNS) can be damaging to cells

leading to cellular dysfunction and disease. The adverse effects of free radicals were

discovered in the last few decades. In low or moderate concentrations, free radicals are

involved in normal physiological functions but excess production leads to oxidative

stress due to the high reactivity of these radicals forming a chain reaction (Sen et al.,

2010). These are generated as part of the body’s normal metabolic process and the chain

reactions are usually produced in the mitochondrial respiratory chain, from atmospheric

pollutants, and from transitional metal catalysts, drugs and xenobiotics. In addition,

chemical mobilization of fat stores under various conditions like lactation, exercise, fe-

ver, infection and fasting, can result in increased free radicals in our body. Stress hor-

mones secreted by the adrenal glands during excessive emotional states also give rise to

free radicals (Atawodi, 2005).

Free radicals also arise due to radiation, chemicals, toxins, deep fried foods and physical

stress. They cause depletion of the immune system, changes in gene expression and in-

duce abnormal proteins (Pourmorad et al., 2009). ROS can cause great damage to cell

membranes and DNA. The resulting oxidation causes membrane lipid peroxidation

which in turn stimulates glycation of protein, inactivation of enzymes and alteration in

the structure and function of collagen basement membranes. This, in turn, plays a role in

the long-term complication of diabetes, decreased membrane fluidity and other DNA

mutations that leads to cancer, cardiovascular diseases, autoimmune and neurodegener-

ative disorders and ageing (Subhasree et al., 2009).

Chapter 3 53

When the rate of ROS production overwhelms antioxidant defence mechanisms, oxida-

tive damage can lead to the onset of many chronic and degenerative diseases in humans.

The process of oxidation can also affect foods, where it is one of the major causes of

chemical spoilage, causing rancidity, deterioration of nutritional quality, colour, flavour,

texture and safety of foods (Antolovich et al., 2002).

The formation of ROS is prevented by an antioxidant system containing low molecular

mass antioxidants and enzymes which generate reduced (less active) forms of antioxi-

dants. Antioxidants such as ascorbic acid, glutathione, tocopherols and ROS‐interacting

enzymes such as superoxide dismutase (SOD), peroxidases and catalases help to termi-

nate the harmful chain reaction caused by free radicals. Many phenolic compounds, fla-

vonoids, tannins and lignin precursors found in plants are potential antioxidants

(Blokhina et al., 2003).

Antioxidants scavenge free radicals and prevent the damage caused by them. These

compounds are endogenous as well as exogenous in nature. Beta-carotenes and some

micronutrient elements such as zinc and selenium are found in food sources (Subhasree

et al., 2009). Results from biological and phytochemical studies have indicated the ex-

istence of a wide range of complex and effective defence systems of antioxidants such

as secondary metabolites in plants which can overcome this oxidative stress (Valyova et

al., 2009). Therefore, antioxidants are those elements that deactivate and scavenge the

free radicals. Antioxidants inhibit the effect of oxidants by donating the hydrogen atom

or chelating metal and prevent oxidation of biomolecules (Koksal et al., 2011). Antioxi-

dants can also be used in food products to lengthen the shelf life of the food and reduc-

ing nutritional loss and formation of harmful substances (Moein and Moein, 2010).

Synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxy-

toluene (BHT) have been used as food additives to prevent oxidation of lipids. Howev-

er, these compounds are undesirable due to their toxic and harmful effects and are sus-

pected of being responsible for liver damage and carcinogenesis (Nguyen and Eun,

2011, Koksal et al., 2011).

Many studies have confirmed that plants and foods rich in polyphenolic contents are

effective scavengers of free radicals, thus helping to prevent diseases through their anti-

Chapter 3 54

oxidant activity (Nabavi et al., 2009). Antioxidants which are present in plants, herbs

and dietary sources help in preventing vascular diseases in diabetic patients

(Buyukbalci, 2008). Tannins and flavonoids are secondary metabolites in plants and are

considered to be the best natural source of antioxidants which prevent destruction of β-

cells and diabetes-induced ROS formation (Aslan et al., 2010).

Medicinal plant parts (roots, leaves, branches/stems, barks, flowers, and fruits) are

commonly rich in phenolic compounds, such as flavonoids, phenolic acids, stilbenes,

tannins, coumarins, lignans and have various biological effects and antioxidant activity

(Surveswaran et al., 2007). Their traditional use often indicates that their toxic affects

are minimal known or can be ameliorated. There is therefore much interest in the dis-

covery of natural antioxidants from plants that can be safe and effective (Ebrahimzadeh

et al., 2009).

3.2.1. Chapter Aims

This study aims to evaluate the antioxidant effects of ethanolic extracts of 26 plants by

evaluating free radical scavenging activity with in vitro assays using

1,1-diphenyl-2-picrylhydrazyl (DPPH)

2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS)

determining ferric reducing antioxidant power (FRAP)

The total phenolic and flavonoid contents of the extracts were also determined.

3.3. Materials and methods

The plant materials used in this study have been detailed in Table 3 and 4 in Chapter 2

on page 34-35.

All chemicals were purchased from Sigma-aldrich Australia. The chemicals used were

DPPH, ABTS, Folin-Ciocalteu reagent, gallic acid, sodium bicarbonate (Na2CO3), sodi-

um nitrite (NaNO2), aluminum chloride, sodium hydroxide (NaOH), butyl hydroxyl tol-

Chapter 3 55

uene (BHT), potassium persulfate, ascorbic acid, quercetin, potassium ferricyanide (III)

powder, trichloroacetic acid and ferric chloride.

3.3.1. Determination of total phenolic

The total phenolic content was quantified using a modified version of the assay de-

scribed by Singleton et al. (1977) using Folin-Ciocalteu reagent. 20 µl of plant sample

or gallic acid (standard phenolic compound) was diluted with 1580 µl of distilled water

and then mixed with 100 µl of 2N Folin-Ciocalteu reagent. The mixture was shaken and

kept for 6 minutes, after which 300 µl of 5% aqueous Na2CO3 solution was added and

mixed properly. The mixture was incubated for 2 hours at 20 ºC. The absorbance was

measured for all the samples at 765 nm. A standard curve was prepared using 0 - 1000

mg/l of gallic acid. The total phenolic values were expressed in terms of gallic acid

equivalents (µg/mg of dry mass). The (distilled water) was treated in the same way as

the samples and the dilution factor was taken into account for the samples where dilu-

tion was performed.

3.3.2. Determination of total flavonoids

The total flavonoids were determined using a modification of the assay described by

Dixit et al. (2009). Plant samples (250 µl) were diluted with 1250 µl of distilled water

and 75 µl of 5 % NaNO2 was added to the sample together with 150 µl of 10 % alumi-

num chloride. After mixing and incubation for 5 minutes, 500 µl of 1 M NaOH was

added to the reaction mixture and a total volume of 2500 µl was made up with distilled

water. Following vigorous mixing, the absorbance was measured at 510 nm. A standard

calibration curve was prepared using 0 - 1000 mg/l of quercetin. Results were expressed

as µg of quercetin equivalents per milligram of dry mass of the plant extract.

3.3.3. Antioxidant activity determined by 1,1-diphenyl-2-

picrylhydrazyl (DPPH) radical inhibition

The DPPH scavenging activity was determined by an assay modified from Kwon et al.

(2007b). To 150 µl of 0.1 mM DPPH in methanol, a volume of 50 µl of plant extract

Chapter 3 56

was mixed and kept in the dark at room temperature for 60 minutes. After incubation,

the absorbance was recorded at 490 nm. The results were compared with the negative

control which contained 50 µl of ethanol instead of plant extracts. The positive controls

were BHT and ascorbic acid in the concentration range 3.12 to 250 µg/ml. The antioxi-

dant activity was expressed as a percentage (%);

( –

) .

Chapter 3 57

3.3.4. Antioxidant activity determined by 2, 2′-azinobis-3-

ethylbenzothiazoline-6-sulfonate (ABTS) radical inhibition

The free radical scavenging activity of plant extracts was also studied using ABTS radi-

cal cation. The decolorization assay is based on reduction of ABTS +• radical by plant

extracts having antioxidant capacity (Dudonn et al., 2009). ABTS radical was dis-

solved in deionized water to make 7 mM solution and 2.45 mM potassium persulfate

solution was added to the same. To produce ABTS free radical cation, the mixture was

allowed to stand in the dark at room temperature for 12 to 16 hours. The free radical so-

lution of ABTS was diluted with ethanol to an absorbance of 0.7 at 734 nm for the as-

say. To 990 µl of ABTS free radical solution, a volume of 10 µl of plant extract (range 1

to 1000 µg/ml) was mixed and kept in the dark at room temperature for 60 minutes. Af-

ter incubation, the absorbance was recorded at 734 nm. The results were compared with

the negative control which contained 10 µl of ethanol in place of plant extracts. The

positive controls were BHT and ascorbic acid in concentration range 1.56 to 250 µg/ml.

The antioxidant activity was expressed as percentage (%)

( –

) .

3.3.5. Ferric reducing antioxidant power assay

The ferric reducing power assay was carried out as described by Fawole (Fawole et al.,

2010) using 30 µl plant extracts and standards (BHT and ascorbic acid) of different

concentrations (range 25 to 1000 µg/ml) added to 96 well plates along with 40 µl of

0.2M potassium phosphate buffer (pH 7.2) and 40 µl of potassium ferricyanide (1%

w/v). The reaction mixtures were incubated at 50 ºC for 20 minutes. After incubation,

40 µl of trichloroacetic acid (10% w/v), 150 µl distilled water and 30 µl of ferric chlo-

ride (0.1 % w/v) were added and the reaction mixture again incubated for 30 minutes at

room temperature in the dark. Absorbance was recorded at 630 nm using a microplate

reader and the positive controls were BHT and ascorbic acid whereas the negative con-

Chapter 3 58

trol was buffer. The absorbance of each sample was plotted against concentration and

compared with the standards.

3.3.6. Statistical analysis

All samples were analyzed in triplicates. Data are presented as mean ± standard error

mean (SEM). Differences were evaluated by one-way analysis of variance (ANOVA)

test completed by a Bonferroni’s multicomparison test. Differences were considered

significant at p < 0.001. The concentration giving 50% inhibition (IC50) was calculated

by non-linear regression with the use of GraphPad Prism Version 5.0 for Windows

(GraphPad Software, San Diego, CA, USA) (www.graphpad.com). The dose–response

curve was obtained by plotting the percentage inhibition versus concentration (Loizzo et

al., 2008).

3.4. Results

The antioxidant capacities, flavonoid and phenolic contents of 26 ethanolic extracts of

Australian Aboriginal and Indian Ayurvedic medicinal plant species were systematical-

ly assessed. The results of three in vitro assays (ABTS, DPPH and FRAP) for antioxi-

dant properties of the 26 plant samples are given in the figures and tables below.

3.4.1. Determination of total flavonoids and phenolic content

The total phenolic contents (TPC) of these samples were estimated using the standard

Folin–Ciocalteu colorimetric method which is based on the chemical reduction of a rea-

gent. It was found that TPC of the 26 plant samples varied from 0.42 to 1.47 µg of gallic

acid equivalents (GAE) / mg for Australian aboriginal plant extracts whilst total flavo-

noid content ranged from 0.51 to 1.78 µg of quercetin equivalents (QE) / mg (Table 8).

TPC was 1.04 to 30.27 µg of gallic acid equivalents (GAE) / mg and total flavonoid

content ranged from 0.20 - 32.94 µg/mg quercetin equivalents for the Indian Ayurvedic

plant extracts (Table 9).

Chapter 3 59

Table 8 - Total phenolic and flavonoid contents of Australian Aboriginal plant extracts

Plant name TPC

(µg GAE/mg ex-

tract)

TFC

(µg QE/mg extract)

AK 1.47 ± 0.07 1.78 ± 0.08

AT 0.95 ± 0.06 1.51 ± 0.20

AL 0.56 ± 0.17 0.94 ± 0.07

BL 0.42 ± 0.03 1.33 ± 0.12

ED 0.93 ± 0.01 0.51 ± 0.06

SL 1.28 ± 0.03 1.35 ± 0.10

SS 0.87 ± 0.11 1.36 ± 0.06

Chapter 3 60

Ta- ble 9 - Total

phenolic and flavonoid contenst of Indian Ayurvedic plant extracts

Plant name TPC

(µg GAE/mg ex-

tract)

TFC

(µg QE/mg extract)

AP 7.57 ± 0.12 8.11 ± 0.06

Ana. 3.09 ± 0.10 2.89 ± 0.23

AS 28.31 ± 0.22 23.06 ± 0.48

BM 5.54 ± 0.24 3.58 ± 0.35

BD 11.80 ± 0.07 8.52 ± 0.25

BS 1.90 ± 0.15 0.52 ± 0.46

CB 1.04 ± 0.14 0.24 ± 0.30

CM 20.58 ± 0.68 22.93 ± 15.79

CP 15.17 ± 0.42 22.17 ± 0.25

CO 7.76 ± 0.13 12.32 ± 0.32

EJ 6.98 ± 0.24 5.32 ± 0.53

MP 5.83 ± 0.57 13.25 ± 2.13

NJ 9.50 ± 0.22 9.14 ± 0.11

PZ 30.27 ± 0.88 32.93 ± 1.87

PM 1.61 ± 0.12 0.20 ± 0.33

PT 7.34 ± 0.19 8.84 ± 0.40

SN 2.88 ± 0.03 3.01 ± 0.26

TC 10.24 ± 0.12 7.96 ± 0.09

VW 10.12 ± 0.13 12.89 ± 0.58

Chapter 3 61

3.4.2. 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical inhibition

Antioxidant studies were carried out with the seven Australian Aboriginal and nineteen

Indian Ayurvedic plant extracts. The free radicals scavenging ability of extracts were

evaluated by studying the reaction of DPPH radicals with extracts using in vitro colori-

metric assays.

The IC50 values were calculated, IC50 value is defined as the concentration which shows

50% of maximum inhibitory activity. The lesser the IC50 value for a compound the more

effective (Kumanan et al., 2010). IC50 values of standard compounds, ascorbic acid and

BHT, were determined as 12.37 µg/ml and 41.3 µg/ml, respectively.

DPPH is a radical, purple in colour, which is reduced to the yellow coloured diphe-

nylpicrylhydrazine by plant extracts. This antioxidant assay is based on the reduction of

alcoholic DPPH solution in the presence of hydrogen-donating antioxidant due to the

formation of the non-radical form, DPPH-H (Sunil and Ignacimuthu, 2011).

Out of seven Australian aboriginal plants tested (Figure 3), extract AL showed an IC50

value of 6.98 µg/ml, whereas extracts AK, SL and ED had IC50 in the range of 14 - 15

µg/ml. Extracts BL and AT showed quite high IC50 as compared to other Australian

plant extracts tested in this study.

Out of the 19 Indian plant extracts tested, only BM (14.95 µg/ml), AP (14.95 µg/ml)

and TC (29.49 µg/ml) showed an IC50 value less than the control, BHT. For ease of

comparison, the Indian plants were divided into three categories as based on IC50 values;

(a) IC50 less than 200 µg/ml, (b) IC50 between 200 - 400 µg/ml and (c) IC50 greater than

400 µg/ml (Figure 4). The majority of the tested Indian plants had IC50 values greater

than 200 µg/ml.

Chapter 3 62

Figure 3 - DPPH radical scavenging IC50 of Australian Aboriginal plant extracts.

Each value represents the mean ± SEM of triplicates experiments. No significant

difference between tested conditions P > 0.05, one-way ANOVA

Chapter 3 63

a

b

c

Chapter 3 64

Figure 4 - DPPH radical scavenging IC50 of Indian Ayurvedic plant extracts.

a) IC50 value less than 200 µg/ml, b) IC50 between 200 - 400 µg/ml and c) IC50 more

than 400 µg/ml. Each value represents the mean ± SEM of triplicates experiments. No

significant difference between tested conditions P > 0.05, one-way ANOVA.

c

Chapter 3 65

3.4.3. 2, 2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS) radical

inhibition

The free radical scavenging capacity of plant extracts was also studied using the ABTS

radical cation decolorization assay, which is based on the reduction of ABTS+• radical

by antioxidants of the plant extracts tested. ABTS is a stable free radical, bluish green in

colour. Two standard antioxidants (ascorbic acid and BHT) were used to compare the

antioxidant potential. Of the seven Australian Aboriginal plants tested, AK showed sig-

nificantly lower IC50 as compared to BHT (Figure 5). AK, which had shown IC50 of

14.07 µg/ml in the DPPH assay, showed IC50 value of 8.86 µg/ml in this assay. AL, SL

and AT showed ten, five and three fold rise, respectively in their IC50 values of DPPH

assay, whereas the remaining plants showed similar IC50 as obtained in the DPPH assay.

IC50 values (Figure 5) for positive controls, ascorbic acid and BHT, were found to be

30.20 and 88.24 µg/ml, respectively. Out of seven Australian Aboriginal plant extracts

screened for ABTS radical scavenging activity, AK showed significantly lower IC50 as

compared to BHT. With the exception of SL and AT, the remainder of the plant extracts

tested showed IC50 value equal or less than the positive controls.

None of the Indian plant extract tested showed significant activity in comparison to se-

lected standards. The positive outcome from the ABTS assay was that findings were

consistent with IC50 obtained by DPPH assay. Indian plants were divided into three cat-

egories a) IC50 less than 200 mg/ml b) IC50 between 200 to 400 mg/ml and c) IC50

greater than 400 mg/ml.

Chapter 3 66

Figure 5 - ABTS radical scavenging IC50 of ethanolic extracts of Australian Aboriginal

plants.

Each value represents the mean ± SEM of triplicates experiments.

Chapter 3 67

a

b

Chapter 3 68

Figure 6 - ABTS radical scavenging IC50 of ethanolic extracts of Indian Ayurvedic

plants.

a) IC50 values less than 200 µg/ml, b) IC50 between 200 - 400 µg/ml and c) IC50 more

than 400 µg/ml. Each value represents the mean ± SEM of triplicates experiments. No

significant difference between tested conditions P > 0.05, one-way ANOVA.

3.4.4. Ferric-reducing antioxidant power assay (FRAP)

Antioxidant activities of these plant extracts were assessed through their ability to re-

duce the Fe3+/ferricyanide complex to the ferrous (Fe2+) form. The ferrous ion was mon-

itored by measuring the formation of Perl’s Prussian blue at 630 nm (Moyo et al.,

2010). The reducing power of all the plant extracts, ascorbic acid and BHT increased

with increasing concentration.

The reducing power of BHT was significantly more pronounced relative to all the Aus-

tralian Aboriginal plant extracts and ascorbic acid (Figure 7)

c

Chapter 3 69

However, the antioxidant potencies of ascorbic acid and plant extracts were compara-

ble. AK and ED plant extracts had similar activity to BHT, thus suggesting that they

have similar capacity to act as electron donors, indicating their potential to react with

free radicals which they can convert to more stable products.

The phenolic content of AK and ED was 10-fold higher than that of BL extract, and a

similar trend was observed in the ferric-reducing antioxidant power assay. For example,

an absorbance reading of 0.6 corresponded to a dose level of approximately 100 mg/ml

for AK, ED and 1000 mg/ml for BL. Thus, to achieve a similar extent of reducing pow-

er as AK and ED extracts, a 10-fold dose level is required for BL extract.

Many previous studies have reported a direct relationship between reducing power ef-

fects of plant extracts and the content of phenolic compounds and it has been postulated

that high total phenol content represents the primary source of antioxidant activity

(Moyo et al., 2010). Due to the high total phenolic content of PM and AS extracts, it is

postulated that they represent the primary source of this antioxidant activity. The rest of

extracts tested did show dose-dependent activity but not as high as BHT and ascorbic

acid which could be attributed to moderate phenolic content. Overall, it was seen that

most of the extracts possessed promising antioxidant activity (Figure 8).

Chapter 3 70

Figure 7 - Ferric ion-reducing power effects of Australian Aboriginal plant extracts.

Chapter 3 71

Chapter 3 72

Chapter 3 73

Figure 8 - Ferric ion-reducing power effects of Indian Ayurvedic plant extracts.

3.5. Discussion

Plants which contain high levels of phenolics are good source of antioxidants and there-

fore it is important to quantify the total phenolics and total flavonoids in plant extracts

as they might have some beneficial effects on health (Gorinstein et al., 2004). Gallic

acid and hydroxycinnamic acids are phenolic acids commonly found in plants. Phenolic

acids are known to be chain-terminating antioxidant agents contributing the scavenging

activity due to their hydrogen-donating capacity. Their antioxidant activity depends on

the number and position of hydroxyl groups in the molecule and is also affected by gly-

cosylation of aglycones and other groups (-NH and -SH). Previous studies have also

found that there is a direct relationship between antioxidant activity and total phenolic

content (Cai et al., 2004).

There is considerable evidence suggesting that plant flavonoids may be health promot-

ing and disease-preventing dietary compounds. Of current interest is the presence of po-

tent antioxidant flavonoids in red wine, the cause of the so-called French paradox. De-

spite an intake of a high-fat diet, the low incidence of coronary heart disease among the

Chapter 3 74

French people has been attributed to the consumption of red wine with meals. Flavo-

noids have been shown in both in vitro and in vivo experimental systems over several

decades to possess anti-allergic, anti-inflammatory, antiviral and anticancer properties

(Leopoldini et al., 2011).

The current study has revealed that a wide range of total antioxidant activities and asso-

ciated phenolic content exist among the 26 plant extracts assayed. A highly significant,

positive correlation was found between antioxidant capacity and phenolic content, indi-

cating that phenolic compounds are a major contributor to antioxidant activity in the

medicinal plants. The medicinal plants with the strongest antioxidant capacity were AK,

SL, BM, AP, TC, VW, PM and AS.

The Australian Aboriginal plant extracts showed very good potential for antioxidant ac-

tivity and, to our knowledge; no data are currently available on antioxidant studies with

these plants. However, the family Acaciaceae and other species of Acacia have been

shown various pharmacological properties and also contain very high levels of carote-

noids, tocopherols and sterols (Nasri et al., 2012).

Previous studies also evaluated the protective effect of Bacopa monnieri on the tissue

antioxidant defense system and lipid peroxidative status in streptozotocin-induced dia-

betic rats. In their study, an aqueous ethanolic extract of BM was administered orally at

doses 50, 125 and 250 mg/kg (body weight) once a day for 15 days to diabetic rats. Sig-

nificant increase in activity of antioxidant enzymes such as superoxide dismutase

(SOD), catalase and glutathione peroxidase (GPx) were observed along with increased

levels of glutathione (GSH). The study showed significant reversal of disturbed antioxi-

dant status and peroxidative damage which indicated that extract of BM modulates anti-

oxidant activity and enhances the defense against ROS generated damage in diabetic

rats (Kapoor et al., 2009) which supports the present study in revealing its potential to

play a crucial role in defense against free radical damage.

Arabinogalactan polysaccharides (G1-4A) and (1, 4)-alpha-d-glucans extracted from the

stems of Tinospora cordifolia have been shown to induce tolerance against endotoxic

shock by modulating activity of cytokines and activation of macrophages. Thus, poly-

saccharides of the same have been shown to provide effective protection against lipid

Chapter 3 75

peroxidation and metastasis. The other compounds having immunomodulatory proper-

ties are cordioside, cordiofolioside A and cordiol (Krishna et al., 2009) which might al-

so be responsible to show antioxidant activity in the present study.

TC has been shown to be effective in killing cancer cells (cultured HeLa cells) in vitro

and inhibiting skin carcinogenesis in mice and experimental metastasis. It may offer an

alternative treatment strategy for cancer in combination with gamma radiation. TC has

also been reported to possess heptoprotective activity and reduces the damage induced

by radiations to liver cells (Upadhyay et al., 2010) .

Valeriana officinalis has been used as a sedative for the treatment of insomnia and rest-

lessness throughout the world since the second century and many clinical trials have

reported that valerian extract decreases sleep latency, improves sleep quality and hence

is useful in treating insomnia (Stevinson and Ernst, 2000). The other uses which have

been reported for this plant are anxiolytic, antimicrobial, antispasmodic, analgesic, anti-

inflammatory, antidepressant and neuroprotective (Chopra et al., 2010).

However, very limited data are available on Valeriana’s antioxidant potential, even

though insomnia may also occur due to oxidative stress and anxiety; therefore, it can be

hypothesised that this plant exerts its pharmacological effects in treatment of insomnia

via modulation of oxidative stress because of its good antioxidant potential. Further-

more, alcoholic extracts of valerian roots have been considered promising pharmacolog-

ical agents against lipid peroxidation (LPO) in previous studies (Sudati et al., 2009). In

vitro models have shown PM, AP and AS to have potent antioxidant activity, presuma-

bly linked to the phenolics and flavonoids in these plants (Maruthupandian and Mohan,

2011, Banerjee et al., 2003, Akbar, 2011) which also supports the present study that

demonstrated good antioxidant potential.

Many studies have revealed that intake of natural antioxidants is correlated with low

incidence of cancer, heart disease, diabetes, and other diseases associated with ageing.

Phenolics may inhibit carcinogenesis by affecting the molecular events in the initiation,

promotion, and progression stages of free radical chain reaction (Cai et al., 2004).

Chapter 3 76

It has been observed flavonols and flavones containing a catechol group in ring B are

found to be highly active, with flavonols more potent than the corresponding flavones

because of the presence of the 3-hydroxyl group. An additional hydroxyl group in ring

B (pyrogallol group) seems to further enhance the antioxidant capacity, as exemplified

by myricetin (Leopoldini et al., 2011).

3.6. Conclusions

In summary, this study has revealed that many of the 26 plant extracts investigated

demonstrated promising antioxidant activity. This activity correlated with the presence

of phenolic compounds which are known to contribute to antioxidant activity. Systemat-

ic evaluation of a large number of plants is useful for understanding their functionality

and chemical constituents, and also supports the view that they can be potential sources

of potent natural antioxidants. Among Australian Aboriginal plants AK, BL, ED and SS

whereas, only AP and BM among Indian Ayurvedic plants were found to possess very

good free radical scavenging potential as they have shown IC50 less than 50 µg/ml

against ABTS and DPPH free radicals. The plants can be further exploited and utilized

for drug discovery.

Chapter 4 77

Chapter 4. Inhibition of enzymes relevant to the control of

hyperglycemia and other diabetes-related conditions

Chapter 4 78

4.1. Abstract

The work described in this chapter addressed the hypothesis that some Indian Ayurve-

dic plants commonly used in the management of diabetes can reduce glucose by inhibit-

ing key carbohydrate hydrolyzing enzymes. Extracts of these and Australian Aboriginal

plants were evaluated for α-amylase, α-glucosidase and angiotensin converting enzyme

inhibitory effects. Among the Australian Aboriginal plants, Santalum spicatum extract

was found to inhibit α-amylase with IC50 5.43 µg/ml. Extracts of Chlorophytum

borivilianum, Plumbago zeylenica, Solanum nigrum and Pterocarpus marsupium were

found to inhibit α-amylase activity with IC50 values of 4.17, 4.36, 4.87 and 5.16 µg/ml

respectively whereas, the positive control (acarbose) showed IC50 of 7.81 µg/ml. The

Australian Aboriginal plants significantly inhibited (p < 0.001 ) α-glucosidase enzyme

as compared to acarbose with IC50 of 0.48 µg/ml for Beyeria leshnaulti and the other

plants also exhibited significant IC50 (p < 0.001 ) in the range of 1 to 1.83 µg/ml.

Among the Indian Ayurvedic plants, Chlorophytum borivilianum and Mucuna pruriens

showed significant inhibition (p < 0.001) with IC50 of 0.58 and 0.80 µg/ml, respectively.

However, all tested plant extracts showed very high IC50 as compared to Captopril

against angiotensin converting enzyme. These findings provide evidence for the hypo-

glycemic action of these plants and indicate that they may represent alternative therapies

in the management of diabetes. The Australian Aboriginal plant extracts were found to

be more potent in comparison to Indian Ayurvedic plant extracts. This is the first study

of the enzyme inhibition potential of Australian Aboriginal plant extracts in the context

of management of type 2 diabetes and the related condition of hypertension.

Chapter 4 79

4.2. Introduction

Medicinal plants are not only used for the management of mild diseases such as the

common cold, headaches or gastrointestinal ailments but also in the long-term treatment

of chronic or incurable diseases, such as diabetes, hypertension or cancer. The anti-

hyperglycemic activity of plants is due to their ability to restore pancreatic tissue func-

tion causing an increase in insulin output or inhibition of intestinal glucose absorption

(Patel et al., 2012). Diabetes mellitus is a multi-factorial disease. It is an endocrine and

metabolic disorder characterized by chronic hyperglycemia. There is a need to develop

strategies for the management of diabetes to decrease its incidence and deaths from re-

lated complications. One therapeutic approach for management of type 2 diabetes is to

reduce glucose absorption by inhibiting the enzymes α-amylase and α-glucosidase

which are involved in degradation of starch. Various berries have been investigated for

their potential use by inhibiting starch-degrading enzymes, consumption of soft fruits,

Brazilian native fruits, strawberries and raspberries have been proved effective in man-

aging type 2 diabetes. Natural sources of inhibitors of α-amylase and α-glucosidase

would be beneficial to avoid potential gastrointestinal side-effects caused by commer-

cial α-glucosidase inhibitors (Johnson et al., 2011).

Carbohydrates constitute the major part of the human diet and play a main role in the

energy supply. The complex components of dietary carbohydrates must be broken down

to monosaccharides since only the monomers can be absorbed from the intestinal lumen

and transported into the blood. Inhibition of α-amylase and α-glucosidase can consider-

ably prolong the overall carbohydrate digestion time and decrease the postprandial in-

crease of blood glucose level after a mixed carbohydrate diet. This can therefore be an

important strategy in the management of postprandial blood glucose levels linked to

type 2 diabetes (Nickavar and Yousefian, 2011). Thus, the retardation of the α-

glucosidase and α-amylase enzymes by inhibitors might be one of the most effective

approaches to control type 2 diabetes. At present, the commercial enzyme inhibitors

acarbose and voglibose are used either alone or in combination with insulin to control

post-prandial hyperglycaemia.

Chapter 4 80

The modern medicines currently available for management of diabetes exert serious

side effects such as hepatotoxicity, abdominal pain, flatulence, diarrhoea and hypogly-

caemia. Traditional herbal medicines have been used throughout the world for a range

of diabetes and related complications. The search for improved and safe natural antidia-

betic agents is underway, and the World Health Organization has also recommended the

development of herbal medicines in this context (Ghosh et al., 2011).

It is important to note that there are no reports about the antidiabetic activity of Austral-

ian Aboriginal plants. Similarly, no research has been carried out to identify the targets

that can confirm and support the antidiabetic potential of these plants. Hence, this inves-

tigation will evaluate the inhibitory effect of various extracts on commercially available

porcine pancreatic α-amylase and glucosidase. Furthermore, the in vitro antidiabetic po-

tential of the active plant extracts was determined in 3T3L1 cells line as described in the

next chapter (Chapter V).

4.2.1. Chapter Aims

The study aimed to evaluate the effect of ethanolic extracts of 26 plants against specific

enzymes using in vitro assays.

Preliminary -amylase enzyme inhibition assay by disc diffusion method

-amylase enzyme inhibition using spectrophotometric assay

-glucosidase enzyme inhibition using spectrophotometric assay

Angiotensin converting enzyme inhibition using High performance liquid

chromatography (HPLC)

4.3. Materials and methods:

The plant materials used in this study have been described in Table 3 and Table 4 in

chapter 2 (page 34-35).

All chemicals were purchased from Sigma-Aldrich (Australia) as follows: porcine pan-

creatic α-amylase (PPA) (EC 3.2.1.1) type VI B, 3, 5-dinitro salicylic acid reagent

(DNS), acarbose, yeast α-glucosidase (EC – 2328898), p-nitrophenyl α-D-

Chapter 4 81

glucopyranoside (p-NPG), rabbit lung (ACE-I) angiotensin converting enzyme (EC –

3.4.15.1), hippuryl-L-histydyl-L-leucine (HHL), hippuric acid and captopril.

4.3.1. Alpha-amylase enzyme inhibition

The α-amylase inhibitory assay was modified from Correia et al. (2004a). Twenty µl of

porcine pancreatic α-amylase solution (EC 3.2.1.1; equivalent to 3000 U in 50 mM

phosphate buffer, pH 6.9) were mixed with 15 µl of plant extract and incubated at 37 ºC

for 45 minutes. After incubation, the plant - enzyme mixture was applied to a sterile pa-

per disc and placed onto the centre of petri plates containing medium consisting of 1%

(w/v) agar and 1% (w/v) starch in distilled water. Plates were allowed to stand for 3

days at 25 ºC then stained with iodine and allowed to stand for 15 min. The diameter of

the clear zone was measured and used to calculate the amylase inhibitory activity. As a

control, the enzyme was mixed with the solvent in which the plants were extracted (eth-

anol) and applied onto the sterile disc. Results were expressed as:

( –

)

4.3.2. Alpha-amylase enzyme inhibition by quantitative starch hydrol-

ysis

The -amylase inhibitory activity was determined (Kwon et al., 2008a) using porcine

pancreatic α-amylase (EC 3.2.1.1) type VI B. To 125 µl of plant extracts at different

concentrations (range 1.56 to 500 µg/ml), α-amylase solution (0.5 mg/ml in 0.02M so-

dium phosphate buffer) was mixed and the reaction mixture was pre-incubated for 10

minutes at room temperature. After pre-incubation, 25 µl of 1% starch solution was

added every 5 seconds for a total of 125 µl. The reaction mixture was again incubated

for 10 minutes at room temperature. The reaction was terminated by adding 250 µl of 3,

5-dinitro salicylic acid reagent. The tubes were placed in a boiling water bath for 5

minutes and then cooled at room temperature. The reaction mixture was diluted by add-

ing 5000 µl of distilled water. The generation of maltose was quantified by measuring

the absorbance at 540 nm of 3-amino-5-nitrosalicylic acid (from reduction of 3, 5-

Chapter 4 82

dinitrosalicylic acid (Loizzo et al., 2008) using a UV-visible spectrophotometer. The

control was buffer treated in the same way as plant samples. The standard used was

acarbose (concentration range 1.56 to 500 µg/ml). Results were expressed as:

( –

)

4.3.3. Alpha-glucosidase enzyme inhibition

The α-glucosidase inhibition assay was modified from Apostolidis et al. (2007) using

yeast α-glucosidase (EC – 2328898). A volume of 25 µl of plant extract (range 0.35 to

100 µg/ml) was mixed with 50 µl of α-glucosidase enzyme (0.1 U/ml in 0.1 M potassi-

um phosphate buffer solution, pH 6.9) in 96 well plates and incubated at 37 ºC for 30

minutes. After pre-incubation, 25 µl of 5 mM pNPG in 0.1M phosphate buffer were

added to each well and the reaction mixture was incubated again at 37 ºC for 30

minutes. Thirty µl of 0.1 M sodium carbonate solution were added to the above reaction

mixture and incubated again for 20 minutes at 37 ºC. Before and after incubation, the

absorbance was measured at 405 nm and compared to the control that contained 25 µl of

buffer solution instead of plant extract. The standard used was acarbose (concentration

range 0.35 to 100 µg/ml). The α-glucosidase activity was determined by measuring re-

lease of p-nitrophenol from p-nitrophenyl α-D-glucopyranoside (Hogan et al., 2010).

Chapter 4 83

The α-glucosidase inhibitory activity was expressed as:

( –

)

4.3.4. Angiotensin converting enzyme inhibition

The angiotensin converting enzyme (ACE) inhibition activity was determined (PEGG et

al., 2007) using rabbit lung angiotensin converting enzyme (EC – 3.4.15.1). A volume

of 100 µl of plant extract (range 50 to 1000 µg/ml) was mixed with 100 µl of ACE (0.25

U/ml in 0.05 M sodium borate buffer solution, pH 8.2) and 100 µl of 5 mM hippuryl-L-

histydyl-L-leucine (HHL) and incubated at 37 ºC for 30 minutes without mixing. After

pre-incubation, the reaction was removed from the incubator, mixed and again incubat-

ed at 37 ºC for 30 minutes. The reaction was terminated by adding 300 µl of 1 M hydro-

chloric acid. The liberated hippuric acid was detected and quantified by UHPLC (Shi-

madzu). The negative control used was buffer solution instead of plant extract. The

standard used was the commercially available ACE inhibitor, captopril. Ten µl of sam-

ple was injected by auto sampler. The calibration curve of hippuric acid was constructed

using 0.5, 0.4, 0.3, 0.2 and 0.1mmol/l solutions made from a 2mmol/l stock solution. An

isocratic method was used to detect the hippuric acid peak. The mobile phase used was

6% acetonitrile in 0.025 M KH2PO4 (pH 3) with a flow rate 0.6 ml/min. The column

used was a ResteK Ultra II ® C18 1.9 micron, 100 X 2.1 mm (Catalog No. 9604212).

The ACE inhibitory activity was expressed as:

( –

)

Chapter 4 84

4.3.5. Statistical analysis

All samples were analyzed in triplicates. Data are presented as mean ± standard error

mean (SEM). Differences were evaluated by one-way analysis of variance (ANOVA)

test completed by a Bonferroni’s multicomparison test. Differences were considered

significant at p < 0.001. The concentration giving 50% inhibition (IC50) was calculated

by non-linear regression with the use of GraphPad Prism Version 5.0 for Windows

(GraphPad Software, San Diego, CA, USA) (www.graphpad.com).

Chapter 4 85

4.4. Results

4.4.1. Alpha-amylase enzyme inhibition by disc diffusion

a

b

c

Figure 9 - Disc diffusion assay of α-amylase for starch hydrolysis by plant extracts.

Where 9 a) is the negative control i.e. ethanol mixed with α -amylase enzyme where the

starch was hydrolysed as is evident after staining with iodine solution. Fig. 9 b) shows

complete inhibition of α -amylase i.e. the starch was not hydrolysed by the plant extract.

Fig. 9 c) shows the partial inhibition of α -amylase enzyme by the plant extract.

Chapter 4 86

The percentage inhibition of -amylase by plant extracts is shown in Table 10 and Ta-

ble 11 for Australian Aboriginal and Indian Ayurvedic plant extracts, respectively. All

of the Australian Aboriginal plant extracts showed complete inhibition of α-amylase

such that no hydrolysis of starch was evident.

Plant name Concentration (mg/ml) Percent inhibition

AK 321 100

AT 43 100

AL 75 100

BL 242 100

ED 188 100

SL 326 100

SS 120 100

Table 10 - Inhibition of α-amylase by Australian Aboriginal plant extracts.

The Australian plant extracts were received in random concentrations and used as such

for this disk diffusion assay since this was the qualitative assay.

Chapter 4 87

Plant name Percent inhibition

EJ 100

CO 100

VW 57.8

CP 55.6

PM 51.42

MP 51.42

CM 51.42

BD 48.57

NJ 45.71

AP 45.71

SN 42.85

TC 42.85

CB 35.45

PZ 28.57

BS 28.57

AS 27.28

PT 22.85

Ana. 20

BM 14.28

Table 11 - Inhibition of α-amylase by Indian Ayurvedic plant extracts.

Chapter 4 88

Among the Ayurvedic Indian plant extracts, which were all tested at 250 mg/ml, only

EJ and CO showed complete inhibition and considered to be highly potent.

Extracts with 50 – 75 % inhibitory effect

In this study, the ethanolic extracts of VW, CP, CM, MP and PM extracts showed par-

tial inhibition. The plants which showed percentage inhibition in range of 50-75 % are

considered to be moderately potent.

Extracts with 25 – 50 % inhibitory effect

The extracts of BD, NJ, AP, SN, TC, CB, PZ, BS and AS showed amylase inhibitory

activity of 25 - 50 % and are thus considered to have lower potency.

Extracts with less than 25 % inhibitory effect

PT, Ana. and BM showed percentage inhibition of 22.85%, 20% and 14.28%, respec-

tively, among the 26 plant extracts screened. These plant extracts are considered to have

minimal inhibitory effect on the amylase enzyme (Ahmad Gholamhoseinian, 2008).

While the majority of the extracts demonstrated potent amylase inhibiting activity, some

of the Australian plant extracts were particularly active (AL and AT) and showed activi-

ty at concentrations lower than those of the other extracts.

4.4.2. Alpha-amylase activity determined by spectrophotometric assay

Much research has focused on glucosidase inhibitors to control hyperglycemia, but

many forms of starch are also digested as rapidly as glucose absorption (Ghavami et al.,

2001). Slowing the digestion and breakdown of starch may have beneficial effects on

insulin resistance and glycemic index control in people with diabetes. Of the seven Aus-

tralian Aboriginal plants screened for inhibitory activity against - amylase (Figure 10),

no significant differences were observed compared to acarbose. However, the plant ex-

tract SS showed a particularly low IC50 value of 5.43 µg/ml while AL, BL and SL

showed IC50 values of 11.52, 13.27 and 13.41 µg/ml respectively, which compare fa-

vourably with acarbose.

Chapter 4 89

Figure 10 - Inhibitory activity of Australian Aboriginal plants against α- amylase.

Each value represents the mean ± SEM of triplicate experiments. No significant

difference between tested conditions p > 0.05, one-way ANOVA.

Most of the herbs used in traditional Indian medicine for the treatment of diabetes melli-

tus are known to contain rosmarinic acid and quercetin. Therefore, the health benefits of

these herbs against diabetes mellitus may be due to the amylase-inhibiting activity of

the phenolic compounds (McCue and Shetty, 2004). Hence, 19 Indian Ayurvedic plant

extracts were screened for -amylase inhibitory activity (Figure 11). Five of the select-

ed plants showed an IC50 value less than 10 µg/ml, of which extracts CB, PZ, SN, PM

and BS showed IC50 value of 4.17, 4.36, 4.87 5.16 and 7.25 µg/ml respectively (Figure

11 a).

Chapter 4 90

Chapter 4 91

Figure 11 - Inhibitory activity of Indian Ayurvedic plants against α-amylase.

a) IC50 less than 10 µg/ml b) IC50 of 10-40 µg/ml c) IC50 greater than 40 µg/ml.

Each value represents the mean ± SEM of triplicate experiments. No significant

differences between test ed conditions p > 0.05, one-way ANOVA.

4.4.3. Glucosidase inhibition assay

Glucosidases of the intestine are known to play key roles in carbohydrate digestion and

inhibitors against these enzymes will be effective in retarding glucose absorption to

suppress post prandial hyperglycemia (PPHG). Thus working on the same principle, the

seven Australian Aboriginal and 19 Indian Ayurvedic plants were investigated for their

inhibitory potential of glucosidase enzyme. From Figure 12, it can be observed that all

of the selected Australian plants showed significantly lower IC50 as compared to acar-

bose. BL extract showed an IC50 of 0.48 µg/ml whereas the rest of the plants showed

values of between 1 to 2 µg/ml.

Out of the 19 Indian Ayurvedic plants tested, extracts CB and MP showed IC50 of 0.58

and 0.80 µg/ml, respectively, whereas PM, PZ, BD and TC showed IC50 values of 1.06,

1.16, 1.72 and 1.86 µg/ml respectively (Figure 13). Figure 13 b shows that plant extract

Chapter 4 92

SN, EJ, BS, AP, PT, NJ and BM showed similar IC50 to acarbose and highest IC50

against glucosidase enzyme was shown by CO at 10 µg/ml.

Figure 12 - Inhibitory activity of Australian Aboriginal plants against α-glucosidase.

Each value represents the mean ± SEM of triplicate experiments. **** p < 0.001

indicate a significant difference between acarbose and plant extracts, one-way ANOVA

post-hoc Bonferroni's multiple comparison test

Chapter 4 93

Chapter 4 94

Figure 13 - Inhibitory activity of Indian Ayurvedic plants against α- glucosidase.

a) IC50 less than 2 µg/ml b) IC50 between 2 – 6 µg/ml c) IC50 greater than 6 µg/ml. Each

value represents the mean ± SEM of triplicate experiments. **** p < 0.001 indicate a

significant difference between acarbose and plant extracts, one-way ANOVA post-hoc

Bonferroni's multiple comparison test.

4.4.4. ACE inhibition assay

Angiotensin converting enzyme (ACE) is a key component in the renin angiotensin al-

dosterone system (RAAS) which regulates blood pressure. As the over expression of

RAAS is associated with vascular hypertension, ACE inhibition has become a major

target for the control of hypertension. New alternatives have been explored extensively

as replacements of current therapeutic drugs to minimise the side effects of dizziness,

coughing and angioneuretic edema related to these drugs. Most of the research have

been targeted at bioactive compounds from natural resources such as peptides, anthocy-

anins, flavonols and triterpenes (Balasuriya and Rupasinghe, 2011).

The objective of this experiment was to assess whether the selected plant extracts have

the potential to be used as ACE inhibitors. Of the seven Australian Aboriginal plants

Chapter 4 95

screened for ACE inhibitory potential, none of extracts showed significant activity as

compared to the commercially available ACE inhibitor, captopril (Figure 14). Captopril

showed an IC50 value of 12.31 µg/ml and plant extracts AK, AL and ED showed five

times the IC50 of this positive control. The remaining plant extracts showed nearly ten-

fold higher IC50 values compared to captopril. Flavonoids are the largest group of poly-

phenolic compounds found in higher plants. Thus, working on the same hypothesis, 19

Indian Ayurvedic plants (Figure 15) were investigated for potential in management of

hypertension by inhibiting ACE. Of the 19 plant extracts screened, only seven showed

IC50 value less than 200 µg/ml but none of them were as effective as captopril.

Figure 14 - Inhibitory activity of Australian Aboriginal plants against ACE.

Each value represents the mean ± SEM of triplicate experiments. No significant

difference between tested conditions p > 0.05, one-way ANOVA.

Chapter 4 96

Chapter 4 97

Figure 15 - Inhibitory activity of Indian Ayurvedic plants against ACE.

Plants were divided into three categories based on their IC50 values: (a) IC50 value less

than 200 µg/ml; (b) IC50 value between 200 – 400 µg/ml; (c) IC50 value greater than 400

µg; (c) IC50 value greater than 400 µg/ml. Each value represents the mean ± SEM of

triplicate experiments. No significant difference between tested conditions p > 0.05,

one-way ANOVA.

4.5. Discussion

In the present study, medicinal plant extracts were investigated for their possible appli-

cation in the control of type 2 diabetes and hypertension. Specifically, the extracts were

tested for their ability to inhibit the enzymes amylase, glucosidase and angiotensin con-

verting enzyme - I. This investigation is a novel approach to identify the agents in-

volved in lowering the glycaemic index and controlling of post-prandial hyperglycemia.

In this study, the detailed biochemical basis of the potential antidiabetic activity was

presented which involved assessing the inhibition of enzymes involved in carbohydrate

Chapter 4 98

metabolism, which are the key players in the development of type 2 diabetes, and an

enzyme related to the associated condition of hypertension.

The data obtained showed that the different plant extracts exhibited variable inhibitory

effects on α-amylase activity in vitro (Figures 10 and 11). In this study, SS showed an

IC50 of 5.43 μg/ml. This plant bears sweet fruits which are eaten fresh and the decoction

of the inner bark is taken orally to relieve cough (Anonymous, 1988). The other Aus-

tralian Aboriginal plant extracts tested here (AL, BL and SL) were found to inhibit α-

amylase with an IC50 less than 15 μg/ml. Seeds and gums of Acacia species are edible

and, as this plant grows in harsh environments, it is commonly known as “dead finish”.

Many of the Australian Aboriginal plants screened here have been used as either food or

food supplements, suggesting that they are safe to be consumed. (Anonymous, 1988).

The natural occurring compounds in the plants such as flavonoids, alkaloids, triterpe-

noids, saponins, steroidal glycosides, amino acids have been reported to possess hypo-

glycaemic action (Narender et al., 2011). BL contains several di- and triterpenoids

(Baddeley et al., 1964) which might have contributed towards inhibition of α-

glucosidase (IC50 of 0.48 μg/ml).

Among the Indian Ayurvedic plant extracts, CB was a good inhibitor against porcine

pancreatic amylase as well as glucosidase with IC50 of 4.17 µg/ml and 0.58 µg/ml (Fig-

ure 11 a and 13 a), respectively. Previous studies detected the antidiabetic activity of

aqueous extracts of Chlorophytum borivilianum roots in streptozotocin (STZ)-induced

hyperglycemia in rats (Mujeeb, 2009). Among all the species of Chlorophytum present

in India, CB produces the highest yield of roots along with the highest saponin content.

Several steroidal sapogenins and glycosides have been isolated with various pharmaco-

logical actions (Kaushik, 2005). These possess great aphrodisiac and adaptogenic ac-

tions and are hence considered as alternatives to Viagra (Thakur et al., 2009).

MP inhibited glucosidase with IC50 0.80 µg/ml whereas it showed IC50 of 25.10 µg/ml

against -amylase. The powder of MP seeds is a rich source of dietary fibre and con-

tains both organic and inorganic minerals. The aqueous extract is reported to signifi-

cantly reduce blood glucose in STZ-induced diabetic rats after 21 days of daily oral ad-

ministration (Bhaskar et al., 2008). The current study also indicated its potential in in-

Chapter 4 99

hibition of carbohydrate hydrolyzing enzymes. It also contains L-dopa and hence can be

used in management of Parkinson’s diseases. The clinical efficacy of the plant was as-

sessed in a double-blind randomised clinical trial and was compared with standard L-

dopa/carbidopa (LD/CD). The rapid onset of action demonstrated in the study suggested

that this natural source of L-dopa might possess advantages over conventional L-dopa

preparations in the long-term management of Parkinson’s disease (Katzenschlager et al.,

2004b).

PM is one of the important widely used plants in traditional Indian Ayurvedic medicine

for the treatment of diabetes mellitus (Maruthupandian and Mohan, 2011). The heart

wood of Pterocarpus has been used for many years to treat diabetes. Traditionally, peo-

ple would soak water overnight in mugs made up of heart wood and drink the same wa-

ter (known as Beeja wood water) next morning to manage diabetes. The presence of

pterostilbene, marsupin and other phenolics known to be present in PM might be con-

tributing to the hypoglycaemic effect of the plant (Devgun et al., 2010) which might be

the reason of inhibition of amylase and glucosidase in our study with IC50 5.16 and 1.06

µg/ml respectively.

PZ possessed amylase inhibition at IC50 4.36 µg/ml and glucosidase inhibition with an

IC50 of 1.16 µg/ml in our study. It is reported to have hypoglycaemic action by increas-

ing hepatic hexokinase activity and decreasing hepatic glucose-6-phosphatase as previ-

ously studied in oral administration of an ethanolic extract (100, 200 mg/kg) in STZ-

induced diabetic rats. (Zarmouh et al., 2010).

The findings of the current study indicated that all of the Australian Aboriginal plant

extracts showed potent amylase and glucosidase inhibitory activity and one of the

mechanisms by which these extracts could exhibit a hypoglycemic effect would be by

inhibition of these carbohydrates-hydrolysing enzymes leading to retardation of starch

hydrolysis, eventually lowering post-prandial hyperglycemia (PPHG). Since, this is the

first study on anti-hyperglycemic activity of these plants, the nature of the phytochemi-

cals responsible for enzyme inhibition and the active principles need to be isolated,

characterized and further tested through in vitro and in vivo studies. The Indian Ayurve-

dic plant extracts also showed potent amylase and glucosidase inhibition. Though many

Chapter 4 100

of the plants traditionally have been used to treat diabetes, limited information was

available on their mechanism of action.

ACE plays an important role in the renin–angiotensin system, which regulates arterial

blood pressure as well as salt and water balance. In the cardiovascular system, ACE

converts angiotensin I to angiotensin II, a potent vasoconstrictor, and degrades brady-

kinin, a vasodilator. Angiotensin II also stimulates the synthesis and release of aldoste-

rone, which increases blood pressure by promoting sodium retention in the distal tu-

bules. Therefore, inhibition of ACE by drugs such as captopril and natural ACE inhibi-

tory peptides has been shown to result in an antihypertensive effect. Lifestyle modifica-

tions and diet therapy are two of the most important tools for effective lowering of

blood pressure. Moreover, even small decreases in blood pressure result in significantly

lower risks for cardiovascular diseases. A higher dietary protein intake, a diet rich in

fruits, vegetables and low-fat dairy products, is associated with an effective reduction in

blood pressure (Vermeirssen et al., 2004). Hypertension is one of the long-term compli-

cations of diabetes, therefore inhibition of this enzyme is considered a useful therapeutic

approach in the treatment of high blood pressure in both diabetic and non-diabetic pa-

tients (Kwon et al., 2007c) .

In the current study, none of the plant extracts showed ACE inhibitory activity compa-

rable to captopril. Previous studies have shown that flavonoids possess higher IC50 val-

ues for ACE when compared with antihypertensive drugs, as most flavonoids are found

to be competitive inhibitors of ACE (Balasuriya and Rupasinghe, 2011). Therefore, fur-

ther studies using animal models are required to confirm their ACE inhibitory proper-

ties.

It has been observed in previous studies that enzymatic inhibition is the basis for the

management of type 2 diabetes by controlling glucose absorption and decreasing the

risk of hypertension. This is due to the presence of high fibre and phenolic content

(Kwon et al., 2008a). Plants have a wide array of phytochemicals that are known to

produce a large variety of pharmacological activities. In type 2 diabetes, excessive he-

patic glycogenolysis and gluconeogenesis are associated with underutilization of glu-

cose by tissues causing hyperglycemia. The glucosidase inhibitors inhibit α-1,6-

glucosidase of glycogen-debranching enzymes in the liver and reduce the glycogenolyt-

Chapter 4 101

ic rate which increases the glycogen stores in the liver. Hence, inhibition of these en-

zymes decreases the blood glucose levels in diabetic patient (as a short-term effect) and

shows a small reduction in haemoglobin A1c level (as a long-term effect) (Bhat et al.,

2011b).

Plant extracts are known to be potent enzyme inhibitors due to their rich phenolic con-

tent that bind to the reactive sites of enzymes, thus altering the catalytic activity. It has

been suggested that the mechanism of inhibition of α-amylase may occur through the

direct blockage of the active centre at several subsites of the enzyme (Ghosh et al.,

2011). A main drawback with acarbose is the development of side effects including ab-

dominal distention and flatulence which might be due excessive inhibition of pancreatic

-amylase resulting in the abnormal bacterial fermentation of undigested carbohydrates

in the colon by the anaerobic microbial community. This would increase the concentra-

tion of gases and butyrate produced in the colon (Kwon et al., 2007c). These side effects

are well-tolerated in comparison to the other oral hypoglycaemic agents that can cause

severe hypoglycaemia and weight gain.

A review of the scientific literature shows that a significant contribution has been made

regarding α-glucosidase inhibitors like acarbose, miglitol, voglibose and dexynojirimy-

cin (discovered from roots of mulberry trees) (Kuriyama et al., 2008). These were the

first drugs developed to more effectively control postprandial glucose compared to sul-

fonylureas and biguanides which were previously the only available oral antidiabetics.

Inhibition of this enzyme can significantly decrease the postprandial increase of blood

glucose after a mixed carbohydrate diet and therefore can be an important strategy in the

management of hyperglycaemia (Mahomoodally et al., 2012).

Chapter 4 102

4.6. Conclusions

Plants have been suggested as a rich, as yet unexplored, source of potentially useful an-

tidiabetic drugs. However, only a few have been subjected to detailed scientific investi-

gation due to a lack of mechanism-based in vitro assays. There are several reports on

antidiabetic plants in traditional use. However, many of these plants are used for the

treatment of diabetes mellitus with no mechanistic basis known. The present investiga-

tion showed that the antidiabetic property of some of the plants can be related to their

enzyme inhibitory effects. Therefore, they can be considered as potential candidates for

further studies aimed at isolating the enzyme inhibitors. These efforts may provide new

alternative treatments for diabetes and validate the use of traditional medicinal plants

having anti-diabetic activity.

Chapter 5 103

Chapter 5. Assessing the Anti-Diabetic and Anti-Cancer Potential of Plant Extracts using Cell-Based

Assays

Chapter 5 104

5.1. Abstract

Plant-derived compounds have been used clinically to treat type 2 diabetes for many

years a decision often made easier because they can also exert beneficial effects on var-

ious other disorders. In the present study, the possible mechanism of anti-diabetic ac-

tivity of selected Australian Aboriginal and Indian Ayurvedic plant extracts (based on

antioxidant and enzyme inhibition assays) was investigated by determining if they could

significantly improve glucose uptake in murine 3T3-L1 adipocytes. The plants were al-

so investigated for their role in adipogenesis. Of the seven Australian Aboriginal plant

extracts tested, only Acacia Kempeana and Santalum spicatum stimulated glucose up-

take in adipocytes. Among the five Indian Ayurvedic plant extracts, only Curculigo or-

chioides enhanced glucose uptake. With respect to adipogenesis, the Australian plants

Acacia tetragonophylla, Beyeria leshnaultii, Euphorbia drumondii and the Indian plants

Pterocarpus marsupium, Andrographis paniculata and Curculigo orchioides reduced

lipid accumulation in differentiated adipocytes. Cytotoxicity studies were also carried

out against two cancerous cell lines, HeLa and A549, to investigate the potential anti-

cancer activities of the extracts. Extracts of Acacia kempeana and Acacia tetragono-

phylla showed potent and specific activity against HeLa cells suggesting they may be

useful candidates for the development of chemotherapeutic agents for the treatment of

cervical cancer.

Chapter 5 105

5.2. Introduction

Type 2 diabetes has become a major health problem in both developed and developing

countries. Readily-available high calorie foods and sedentary lifestyles are major factors

for obesity which contribute to insulin resistance and type 2 diabetes. Insulin resistance

is defined as defective insulin signalling and decreased insulin efficiency to induce glu-

cose transport from the blood into key target cells (Boden, 1997). Obesity, mainly vis-

ceral fat, contributes to insulin resistance (Lazar, 2005). Most antidiabetic drugs pro-

mote long-term weight gain. Thus, these drugs treat one of the key symptoms, hyper-

glycemia, but exacerbate weight gain and obesity which further contributes to the pro-

gression of type 2 diabetes. Therefore, while these drugs are beneficial over the short-

term, they are not optimal for long-term health of type 2 diabetic patients (Chan et al.,

2012). The most desirable situation would be the development of new types of antidia-

betic drugs that are either hypoglycaemic or anti-hyperglycemic without the side effect

of promoting weight gain (Klein et al., 2007). The activity of numerous plants has been

evaluated and confirmed in animal models which suggest that herbal remedies could

represent culturally relevant complementary and alternative treatments, as well as serve

in the search for new anti-diabetic agents (Baldea et al., 2010). Reducing obesity can

slow down the rate of occurrence of type 2 diabetes (Tom Yates, 2011). The side effects

associated with currently available therapies discourage correct and complete compli-

ance to medication protocols by patients, with the concomitant progression of diabetes

and its associated complications. Therefore, it is highly desirable to find new anti-

diabetic agents that stimulate glucose uptake by adipose or muscle cells but, unlike thia-

zolidinediones or insulin, do not induce obesity or other side effects (Alonso-Castro and

Salazar-Olivo, 2008). Obesity and weight gain increase the risk of type 2 diabetes and

hypertension which increases the number and size of adipocytes (Kahn and Flier, 2000).

The increase in adipocyte lipid content can influence adipocyte function by reducing

adiponectin secretion which promotes adipocyte differentiation, insulin sensitivity and

lipid accumulation in vivo (Yeo et al., 2011a). Low levels of circulating adiponectin

have been linked to insulin resistance and an increased risk of diabetes. Secondary plant

metabolites such as saponin glycosides, triterpenes and phenolic compounds have been

Chapter 5 106

reported to influence adipocyte differentiation in cultured 3T3-L1 cells, a murine fibro-

blast cell line that is often used as a model for adipocyte metabolism (Yeo et al., 2011b).

Green and Kehinde (1975) established several cloned lines of mouse 3T3 fibroblasts

which are capable of differentiating into adipocyte-like cells in vitro. The most fre-

quently employed adipocytes cell lines are 3T3-F442A and 3T3-L1. They were clonally

isolated from Swiss 3T3 cells derived from disaggregated 17 to 19-day mouse embryos

(Green and Kehinde, 1975). Cell lines have been used as model systems to understand

various mechanisms of plants in animal and human health as they provide a continuous

source of large numbers of cells necessary for proliferation and differentiation. The

3T3-L1 cell line was selected for this study because it diplays relevant features includ-

ing lipid storage and glucose homeostasis. These murine adipocytes have been used ex-

tensively to study the regulation of glucose transporters, cell proliferation and insulin

signalling. During differentiation, 3T3-L1 pre-adipocytes become adipocytes with a 20-

fold increase in the number of insulin receptors and acquire the ability to utilize glucose

in response to insulin (Frost and Lane, 1985).

Many studies have exploited the Sprague-Dawley rat model (SD model) for in vitro

evaluation of hypoglycemic activity. This is normally time-consuming, restricted to lim-

ited animal sources and involves sacrificing of animals. Therefore, the differentiated

3T3-L1 adipocyte model (3T3-L1 model) was developed as an alternative to the SD

model and is used by researchers to evaluate hypoglycaemic and anti-adipogenic effects

and establish the mechanisms of action. Wu et al. (2011) screened yeast extracts for hy-

poglycemic activity with the 3T3-L1 model, compared results with the SD model and

found that the two models were highly correlated (Wu et al., 2011).

In the search for novel treatments, attention should be given to the many traditional

herbal medicines for diabetes which have been employed by various ethnic groups

throughout the world. One region which contains a rich flora and fauna is Australia.

However, Australian Aboriginal plants have never been screened for their use in diabe-

tes. Therefore, in this work, the well-characterized 3T3-L1 model was used to investi-

gate the role of Aboriginal Australian and selected Indian Ayurvedic plant ethanolic ex-

tracts for their anti-diabetic mechanisms and ability to inhibit lipid accumulation.

Chapter 5 107

As all seven Australian Aboriginal plants and particular Indian plants, namely AP, BM

and PM, showed good in vitro radical scavenging activity, the anti-cancer activity of

these extracts against cervical carcinoma HeLa cells and lung adenocarcinoma A549

cells was also investigated.

5.2.1. Chapter Aims

The overall aim of this study was to further evaluate the anti-diabetic mechanisms of

ethanolic extracts of 12 traditional medicinal plants. This was achieved by:

1. Evaluating glucose uptake in 3T3-L1 mouse pre-adipocytes

2. Assessing inhibition of lipid accumulation in 3T3-L1 mouse pre-adipocytes

In addition, cytotoxicity against MDCK cells, 3T3-L1 cells and human cancer cell lines

(cervical carcinoma HeLa cells and lung adenocarcinoma A549 cells) was evaluated by

establishing the cytotoxic concentrations of the extracts using MTT assays.

5.3. Materials and Methods

Dulbecco’s modified Eagle medium (DMEM), Dulbecco’s modified Eagle medi-

um/Ham’s nutrient mixture F12 (DMEM/F12), fetal bovine serum (FBS), insulin, 2-[N-

(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), tryp-

sin/EDTA and penicillin-streptomycin were purchased from Invitrogen Australia. Bo-

vine serum albumin (BSA), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, 3-(4,

5 dimethylthiazol- 2-yl)-2, 5 diphenyltetrazolium bromide (MTT), d-biotin, rosiglita-

zone and oil red O were obtained from Sigma-Aldrich, Australia.

5.3.1. Cell lines

Madin-darby canine kidney epithelial cells (MDCK) were procured from the American

Type Cell Culture (ATCC). A549, HeLa and 3T3-L1 cells were provided by Monash

University, Victoria, Australia. The cells were routinely passaged as described below.

Chapter 5 108

5.3.2. Passaging of cell lines

Cells were routinely cultivated as monolayers in disposable 25 cm2 flasks (Corning) in

DMEM supplemented with 10% (v/v) FBS, 1% (v/v), penicillin-streptomycin (10,000

U/ml pencillin and 10,000 μg/ml streptomycin in 0.85% saline) and passaged when 70-

80% confluent. The media was aspirated from the confluent cells using a sterile pipette

and cells were washed with approximately 5 ml sterile 1X PBS solution, which was

subsequently aspirated. Trypsin/EDTA solution (2.5 ml) was added to the flask to cov-

er the cell monolayer and the flask was incubated at 37 °C for 3 minutes to allow the

cells to detach. Fresh media (3 ml) was used to resuspend the detached cells and neutral-

ize the action of trypsin. The cell suspension was centrifuged at 200 rpm for 5 min at 20 oC. The supernatant was discarded and cell pellet was resuspended in 5 ml of fresh me-

dia. Cell counts were carried by the trypan blue dye exclusion method. Cells were seed-

ed at a density of 150000/flask and incubated at 37 °C in 5% CO2 atmosphere.

5.3.3. Cryopreservation of cells

To prepare cells for storage, a confluent monolayer was detached from the tissue culture

flask with trypsin/EDTA solution as described above. The suspended cells were trans-

ferred to a sterile 15 ml falcon tube, centrifuged at 200 rpm for 10 min, and the superna-

tant aspirated with a pipette. The cell pellet was resuspended into DMEM with 80%

FBS and 10% DMSO. A cell count was performed and 150000 cells per vial were ali-

quoted into 1.8 ml cryogenic vial using a sterile pipette. The vials were packed in cotton

wool and gradually frozen at -80 °C for long term storage in liquid nitrogen.

5.3.4. MTT cytotoxicity studies

Cell viability was determined by the MTT (3-(4, 5 dimethylthiazol- 2-yl)-2, 5 diphenyl-

tetrazolium bromide) method. Cells were cultured in 96-well plates (5000 cells/well)

containing 100 µl medium, prior to the treatment with plant extracts or vincristine (anti-

cancer drug) at 37 °C for 24 hrs. The cells were exposed to 100 µl of each test solution

(containing various concentrations of plant extracts or vincristine) and incubated for a

further 72 hrs at 37 °C. Plant extract (1 – 500 µg/ml) and vincristine (0.001 – 200

Chapter 5 109

µg/ml) in media containing 2% FBS were freshly prepared prior to each experiment.

MTT (5 mg/ml) was dissolved in PBS and the solution was freshly prepared every time,

filtered through a 0.2 m filter and used immediately. The test solutions were then re-

moved and the cells were washed in PBS and 50 μl of media was added into each well.

Then, 5 μl of MTT solution (5 mg/ml PBS) was placed into each well and incubated at

37 °C. After 4 hrs, 25 μl of cells were removed, 50 μl DMSO was added and the mix-

ture incubated at 37 °C for 10 min. The absorbance at 540 nm was measured using a

microplate reader (Bio-Rad Laboratories). Each experiment was performed three times

with each sample tested in triplicate. The data represent the mean cell viability of the

test solutions, calculated as a percentage of the total cell viability found with the control

well (which contained no test solutions).

5.3.5. Adipocyte differentiation of 3T3-L1 cells

3T3-L1 cells (ATCC; CL-173) represent a subclone of the 3T3 cells which is able to

undergo adipocyte differentiation. Cells were cultured and differentiated as described

previously (Huang et al., 2006), Kühn et al., 2011) with minor amendments. 3T3-L1

cells at passage 9 or 10 were seeded in 96-well plates (5000 cells/well) for Oil red O

staining and glucose uptake measurements using DMEM/F12 medium with 10% FBS.

DMEM/F12 is a serum free media which is supplemented with a defined combination

of nutrients, growth factors, and hormones to culture variety of cells. Two days after

reaching confluence, the medium was changed to differentiation medium- DMEM/F12

+ 2% FBS containing 10 μg/ml insulin, 0.5 mM (IBMX) 3-isobutyl-1-methylxanthine

and 1.0 μM dexamethasone. 3T3-L1 cells when treated with a combination of dexame-

thasone, isobutylmethylxanthine (IBMX) and insulin adopt a rounded phenotype and

within 5 days begin to accumulate lipids intracellularly in the form of lipid droplets

(Wise and Green, 1979). Cells remained in the differentiation medium for four days

with media replenished every 48 hrs. Thereafter, differentiation medium was replaced

by DMEM/F12 + 2% FBS in which cells remained for the respective experiments.

Chapter 5 110

5.3.6. Glucose uptake measurements

At day 9 of differentiation, adipocytes were incubated for 24 hrs with the respective test

solutions. Ethanol was used as a negative control whereas 10 μM rosiglitazone was used

as a positive control. Next day, the cells were rinsed with PBS and incubated for 60 min

at 37 °C with exclusion of light in DMEM containing 80 μM of the fluorescent glucose

analogue, 2-NBDG, again in the presence of the extracts for basal glucose uptake meas-

urement. As a second positive control, cells were treated with 100nM insulin during the

2-NBDG incubation to measure the insulin-stimulated glucose uptake. The reaction of

2-NBDG uptake was terminated by washing the cells with pre-cooled PBS. The remain-

ing fluorescence activity in the cells was measured by using fluorescence microplate

reader (POLARStar Omega, BMG Labtech, Germany) at an excitation wavelength of

485 nm and an emission wavelength of 535 nm. Fluorescence activity in the absence of

2-NBDG was subtracted from all values.

5.3.7. Lipid accumulation inhibition assay and Oil Red O staining of

intracellular triglycerides

Lipid accumulation inhibition assay was carried out as per standard protocols with mi-

nor amendments (Fang et al., 2008). 3T3-L1 cells were differentiated into adipocyte as

described above. To quantify the effect of plant extracts on lipid accumulation in 3T3-

L1 cells, the cells were treated with plant extracts in DMEM supplemented with 2%

FBS from day 2 till day 10 of differentiation (Roh and Jung, 2012). On day 10 of differ-

entiation, media was removed and the cells treated with and without plant extracts were

washed with PBS and fixed with 10% formalin for 30 minutes. Cells were rinsed with

deionized H2Oand then incubated with Oil Red O solution (0.25% w/v in 60% isopro-

panol) for 1 hr at room temperature. Finally, the dye retained in the 3T3-L1 cells was

eluted with isopropanol and quantified by measuring the optical absorbance at 540 nm

(BioRad Plate Reader). Cells were also imaged under a light microscope.

Chapter 5 111

5.3.8. Statistical analysis

All samples were analysed in triplicates. Data are presented as mean ± standard error

mean (SEM). The dose–response curve was obtained by plotting the percentage inhibi-

tion versus concentration (Loizzo et al., 2008). For the final evaluation of the glucose

uptake assay, fluorescence activities measured for the negative control (solvent ethanol)

were set to 100% and values for test extracts and positive controls were calculated ac-

cordingly. In the case of lipid inhibition assays, cells treated with inducers set to 100%

and values for tested extracts were calculated accordingly. Differences were evaluated

by one-way analysis of variance (ANOVA) test completed by a Bonferroni’s multicom-

parison test. Differences were considered significant at p < 0.001. The concentration

giving 50% inhibition (IC50) was calculated by non-linear regression with the use of

GraphPad Prism Version 5.0 for Windows (GraphPad Software, San Diego, CA, USA)

(www.graphpad.com).

Chapter 5 112

5.4. Results

5.4.1. Cytotoxicity studies

This study examined the cytotoxicity and anti-tumour activity of seven Australian Abo-

riginal plant extracts. The ethanolic extracts were tested for cytotoxic effects on A549,

HeLa and MDCK cells. The cytotoxicity and selectivity of the Australian Aboriginal

plant extracts against the selected cancerous cell lines are summarized in. According to

the standard National Cancer Institute (NCI) criteria, crude extracts possessing an IC50

of <30 μg/ml are considered active against the tested cancer cells (Itharat et al., 2004).

Of the seven extracts tested, only two extracts, AK and AT, showed activity according

to NCI criteria with IC50 of 13.73 μg/ml and 27.00 μg/ml respectively (Table 12) against

HeLa cells. Vincristine, a chemotherapeutic drug used for some cancer types, had cyto-

toxic effects on MDCK, A549 and HeLa with values of 145.83 µg/ml, 0.6 and 0.39

ng/ml, respectively. The five Indian Ayurvedic plant extracts were tested against select-

ed leukemic cell lines.

None of the Indian Ayurevedic plant extracts showed promising effects (Table 13)

against the cells used in this assay. The Australian Aboriginal plant extracts showed

IC50 values in the range of 158.81 - 386.95 µg/ml and Indian plant extracts showed IC50

values in in range of 171.45 - 394.03 μg/ml against 3T3-L1 cells. Thus, two concentra-

tions, 10 and 100 µg/ml, were selected for evaluating the effects of the extracts on adi-

pogenesis and glucose uptake.

Chapter 5 113

Table 12 - IC50 values (μg/ml) of Australian Aboriginal plant extracts on two cancer cell lines and the non-cancerous MDCK and 3T3-L1 cell lines. Data are expressed as mean ± SEM of independent experiment (n = 3). * denotes IC50 less than 30 μg/ml which is considered as active extract against

cancer cells.

Plant

name

Cell Lines

3T3-L1 MDCK A549 HeLa

AK 202.36 ± 21.44 550.03 ± 36.20 75.17 ± 6.25 13.73 ± 1.51*

AT 240.70 ± 65.22 398.51 ± 31.26 298.6 ± 44.45 110.61 ± 35.82

AL 306.46 ± 65.56 567.38 ± 52.59 210.85 ± 30.82 27.00 ± 14.28*

BL 386.95 ± 56.32 331.33 ± 30.90 84.08 ± 24.37 229.11 ± 40.57

ED 256.34 ± 59.53 460.25 ± 20.96 91.26 ± 19.84 142.50 ± 29.27

SL 173.14 ± 24.86 406 ± 24.31 162.95 ± 27.34 186.66 ± 62.21

SS 158.81 ± 25.53 283.66 ± 12.64 112.28 ± 13.28 110.11 ± 11.91

Chapter 5 114

Plant name Cell line

3T3-L1 MDCK A549 HeLa

CO 214.73 ± 42.89 403.28 ± 12.64 457.6 ± 65.93 287.61 ± 42.86

MP 256.34 ± 59.53 441.33 ± 59.12 301.73 ± 48.71 397.41 ± 87.83

PM 171.45 ± 17.06 491.95 ± 35.80 416.15 ± 108.93 380.73 ± 72.88

AP 352.18 ± 41.27 449.55 ± 23.13 351.31 ± 59.06 487.86 ± 47.64

BM 394.03 ± 25.95 540.3 ± 37.66 320.36 ± 22.08 366.05 ± 37.63

Table 13 - IC50 values (μg/ml) of Indian Ayurvedic plant extracts on two cancer cell lines, MDCK and 3T3-L1 cell line. Data are expressed as mean ± SEM of independent experiment (n = 3).

Chapter 5 115

5.4.2. Glucose uptake assay

The seven Australian Aboriginal and five Indian Ayurvedic plant extracts were all test-

ed at 10 and 100 µg/ml to assess their impact on basal and insulin-stimulated glucose

uptake into differentiated 3T3-L1 adipocytes. After incubation for 28 hrs, the Australian

aboriginal (Figure 16) and Indian Ayurvedic plant (Figure 18) failed to enhance basal

and insulin-stimulated glucose uptake at concentration of 10 µg/ml. However, when the

same extracts were tested at 100 µg/ml, it was observed that AK, AT and SS moderately

enhanced basal glucose uptake by 19, 19 and 16%, respectively, as compared to the eth-

anol control (Figure 17). In contrast, rosiglitazone was enhanced basal glucose uptake

by nearly 43 % as compared to the control. Of the Indian Ayurvedic plant extracts tested

at 100 µg/ml, only CO was able to enhance basal glucose uptake by nearly 19% as

compared to control (Figure 19).

AK and SS at 100 µg/ml were able to enhance insulin-stimulated glucose uptake by 45

and 47%, respectively, which approached the 65% enhancement observed for rosiglita-

zone (Figure 17). AT enhanced glucose uptake in the presence of insulin by 34% which

was approximately half that of rosiglitazone (Figure 17). CO was able to enhance glu-

cose uptake by 48% in the presence of insulin (Figure 19).

Chapter 5 116

Figure 16 - Effects of Australian Aboriginal plant extracts at 10 µg/ml on basal and

insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Cells were treated with the individual extract for 24 hrs followed by incubation for 60

min in serum- and glucose-free media containing 80 μM 2-NBDG. Ethanol was used as

a negative control, while rosiglitazone and insulin were used as positive controls. Cells

received insulin only during 2-NBDG uptake. After incubation, fluorescence activity

remaining in the cells was measured by a fluorescence microplate reader. Fluorescence

activity in the absence of 2-NBDG was subtracted from all values. Data shown are

means ± SD of at least three independent experiments performed in triplicates.

Significance against ethanol control (= 100%): *** p < 0.001. Significance against

ethanol + 100 nM insulin control: +++ p < 0.001.

Chapter 5 117

Figure 17 - Effects of Australian Aboriginal plant extracts at 100 µg/ml on basal and

insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

Cells were treated with the individual extract for 24 hrs followed by incubation for 60

min in serum- and glucose-free media containing 80 μM 2-NBDG. Ethanol was used as

a negative control, while rosiglitazone and insulin were used as positive controls. Cells

received insulin only during 2-NBDG uptake. After incubation, fluorescence activity

remaining in the cells was measured by a fluorescence microplate reader. Fluorescence

activity in the absence of 2-NBDG was subtracted from all values. Data shown are

means ± SD of at least three independent experiments performed in triplicates. Signifi-

cance against ethanol control (= 100%): ** p < 0.01, *** p < 0.001. Significance against

ethanol + 100 nM insulin control: + p < 0.05, ++ p < 0.01, +++ p < 0.001.

Chapter 5 118

Figure 18 - Effects of Indian Ayurvedic plant extracts at 10 µg/ml on basal and insulin-

stimulated glucose uptake in 3T3-L1 adipocytes.

Cells were treated with the individual extract for 24 h followed by incubation for 60 min

in serum- and glucose-free media containing 80 μM 2-NBDG. Ethanol was used as a

negative control, while rosiglitazone and insulin were used as positive controls. Cells

received insulin only during 2-NBDG uptake. After incubation, fluorescence activity

remaining in the cells was measured by a fluorescence microplate reader. Fluorescence

activity in the absence of 2-NBDG was subtracted from all values. Data shown are

means ± SD of at least three independent experiments performed in triplicates. Signifi-

cance against ethanol control (= 100%): *** p < 0.001. Significance against ethanol +

100 nM insulin control: +++ p < 0.001.

Chapter 5 119

Figure 19 - Effects of Indian Ayurvedic plant extracts at 100 µg/ml on basal and

insulin-stimulated glucose uptake in 3T3-L1 adipocytes:

Cells were treated with the individual extract for 24 h followed by incubation for 60 min

in serum- and glucose-free media containing 80 μM 2-NBDG. Ethanol was used as a

negative control, while rosiglitazone and insulin were used as positive controls. Cells

received insulin only during 2-NBDG uptake. After incubation, fluorescence activity

remaining in the cells was measured by a fluorescence microplate reader. Fluorescence

activity in the absence of 2-NBDG was subtracted from all values. Data shown are

means ± SD of at least three independent experiments performed in triplicates. Signifi-

cance against ethanol control (= 100%): * p < 0.05, ** p < 0.01, *** p < 0.001. Signifi-

cance against ethanol + 100 nM insulin control: ++ p < 0.01, +++ p < 0.001.

Chapter 5 120

5.4.3. Inhibition of lipid accumulation in 3T3-L1 cells

Adipocyte differentiation of 3T3-L1 cells is a highly-controlled process that can be in-

duced under a hormonal cocktail of insulin, dexamethasone and IBMX (Liu et al.,

2001). Intracellular lipid accumulation is commonly monitored as a general marker to

indicate the extent of adipogenesis in 3T3-L1 cells (Yeo et al., 2011a). 3T3-L1 pre-

adipocytes were differentiated in the presence of seven selected Australian Aboriginal

plant extracts and five Indian plants extracts for 8 days.

Figure 20 - Micrographs showing effects of plant extracts on lipid accumualtion

Effect of AT (b) and CO (c) extracts on fat droplet formation in 3T3-L1 cells as

compared to control (a).

Pre-adipocytes were differentiated with 100 μg/ml of AT and CO extract treatment for 8

days, then stained with Oil Red O dye and examined using a light microscope.

Chapter 5 121

Figure 20 shows reduction in lipid accumulation in adipocytes treated with selected ex-

tracts. Three Australian plant extracts, AT, BL and ED, were found to significantly re-

duce lipid accumulation in 3T3-L1 adipocytes, suggesting anti-obesity activity. AT was

able to significantly reduce lipid accumulation by 51 and 82 % at 10 and 100 µg/ml re-

spectively (Figure 21). Lipid accumulation was reduced by 34 and 35 % at concentra-

tion of 10 µg/ml BL and ED, respectively, and was further reduced by 74 and 65 % re-

spectively, by the same extracts at 100 µg/ml (Figure 21). Indian Ayurvedic plants test-

ed failed to reduce lipid accumulation at 10 µg/ml but CO, PM and AP were able to

moderately reduce lipid accumulation by 31, 30 and 28 % respectively at 100 µg/ml

(Figure 22).

Chapter 5 122

Figure 21 - Effect of Australian Aboriginal plant extracts on Oil Red O staining in

cultured 3T3-L1 adipocytes.

(A) Effect of 10 µg/ml extract and (B) Effect of 100 µg/ml extract on fat droplet for-

mation in 3T3-L1 cells. Values are expressed as mean ± standard deviation of at least

three independent experiments. Values are means ± SE (n = 3), significance against

control (without plant extract) (= 100%): *** p < 0.001 and * p < 0.05.

Chapter 5 123

Figure 22 - Effect of Indian Ayurvedic plant extracts on Oil Red O staining in cultured

3T3-L1 adipocytes.

(A) Effect of 10 µg/ml extract and (B) Effect of 100 µg/ml extract on fat droplet for-

mation in 3T3-L1 cells. Values are expressed as mean ± standard deviation of at least

three independent experiments. Values are means ± SE (n = 3), significance against

control (without plant extract) (= 100%): * p < 0.05.

Chapter 5 124

5.5. Discussion

Plants have played an important role as a source of effective anti-cancer agents, and it is

important to note that over 60% of the currently used anti-cancer agents are originally

derived from natural sources, including plants, marine organisms and micro-organisms.

The modern scientifically based search for anti-cancer agents from plant sources started

as early as the 1950s with the discovery of the alkaloids vinblastine and vincristine from

Vinca rosea and the isolation of cytotoxic podophyllotoxins from Podophyllum (Ukiya

et al., 2002). The phytochemicals present in plants possess strong antioxidant activities

that may prevent and help cure cancer by protecting healthy cells from damage caused

by the highly reactive oxygen species known as ‘free radicals’ (Schafer and Buettner,

2001). Thus, consuming a diet rich in antioxidant plant foods will provide a milieu of

phytochemicals that possess health protective effects, provide therapeutic actions to all

cells with low cytotoxicity and are beneficial in producing nutrient repletion to immune-

compromised people (Reddy et al., 2003). Strong and consistent epidemiology evidence

also indicates that a diet rich in antioxidants significantly reduces the risk of many can-

cers (Dai and Mumper, 2010).

In the present study, plants previously shown to display good antioxidant activity

(Chapter 3) were assessed for their cytotoxicity against cancerous (HeLa, derived from

a human uterus carcinoma, and A549, derived from human small lung carcinoma) and

non-canceous (MDCK, normal epithelium) cell lines. The cells were exposed to the ex-

tracts and the viability of cells was measured and expressed in terms of the relative ab-

sorbance of extract-treated cells, in comparison with control cells.

The results of cytotoxicity testing of Australian Aboriginal and Indian Ayurvedic plant

extracts (Tables 1 and 2) were assessed according to the US NCI plant screening pro-

gram, where a crude extract is generally considered to have in vitro cytotoxic activity if

the IC50 value is less than 30 µg/ml. Among all the plant extracts screened here, two ex-

tracts, AK and AT, showed particularly potent activity with IC50 values of 13.73 μg/ml

and 27.00 μg/ml, respectively, against HeLa cells. Other extracts showed moderate ac-

tivity. None of the Indian Ayurvedic plant extracts are likely candidates for anticancer

drug development as all showed IC50 values of more than 200 µg/ml against HeLa and

Chapter 5 125

A549 cells. None of the Australian Aboriginal plant extracts had IC50 values of less than

30 µg/ml against A549 cells, although AK, BL, ED, SS and SL had IC50 values of less

than 200 µg/ml so they have moderate anticancer activity. However, only two cell lines

were tested in this study and further testing against other cancer cells may reveal addi-

tional anticancer activity.

Based on the IC50 of the plant extracts, they can be divided into three groups:

1. Those with IC50 values of less than 30 µg/ml can be considered as potential can-

didates for further development as cancer therapeutic agents; such as AK and

AT against HeLa cells.

2. Those with IC50 values between 30 and 200 µg/ml have moderate potential to be

developed into cancer therapeutic agents.

3. Those with IC50 more than 200 µg/ml are unlikely candidates for development

into cancer therapeutic agents.

An important observation was that the activity against HeLa cells exhibited by extracts

AK and AT was specific as no cytotoxity was observed against the non-cancer cell line,

MDCK. Therefore, these extracts may be promising candidates for the development of

chemotherapeutic agents targeting cervical cancer with minimal side effects against

normal cells.

The well-characterized murine pre-adipose 3T3-L1 cell line was used to investigate the

mechanisms of action by which plant extracts exert their anti-diabetic effects. Since

obesity is a side effect of some anti-diabetic drugs, the effects of plants on adipogenesis

was also evaluated.

The impact of plant extracts on basal and insulin-stimulated glucose uptake into 3T3-

L1 adipocytes was examined, using the nonradioactive method of measuring 2-NBDG

uptake. Of the seven Australian Aboriginal plant extracts tested, six were able to en-

hance insulin-stimulated glucose uptake at a concentration of 100 µg/ml. By contrast,

only AK, AT and SS were able to enhance basal glucose uptake by 19.71, 19.19 and

16.82 % respectively. It is well known that thiazolidinediones have beneficial effects on

hyperglycemia in type 2 diabetes, but the molecular mechanism is still to be elucidated.

Chapter 5 126

These drugs stimulate glucose uptake either by enhancing synthesis of the insulin inde-

pendent (basal) glucose transporter GLUT-1 or by increasing expression or transloca-

tion of the insulin-dependent/sensitive glucose transporter GLUT-4 (Kim et al., 2007,

Kühn et al., 2011).

Upon the completion of adipogenesis, spindle-shaped pre-adipocytes were transformed

into round-shaped cells that accumulated lipids and acquired the metabolic mechanisms

to facilitate glucose uptake in response to insulin, synthesize fatty acids, accumulate tri-

glyceride and secrete a wide variety of hormones and cytokines (Yeo et al., 2011a).

Therefore, intracellular lipid accumulation is commonly monitored as a general marker

to indicate the extent of adipogenesis in 3T3-L1 cells (Avram et al., 2007). The results

of this study showed that the three Australian plant extracts AT, BL and ED were able

to significantly reduce lipid accumulation (by 82, 74 and 65 % respectively when tested

at 100 µg/ml) in 3T3-L1 adipocytes when compared to control, suggesting anti-obesity

activity which is a desirable property for an anti-diabetic drug.

Morphological observations of cells stained with Oil Red O, a lipid stain, showed a de-

crease in cellular lipid content in cells treated with plant extracts. Among the Indian

Ayurvedic plant extracts, CO, PM and AP (31, 30 and 28 % respectively at 100 µg/ml),

were able to moderately reduce lipid accumulation. Thus, these plants have the potential

for the management of obesity as they inhibited adipogenesis. A number of studies have

demonstrated that natural compounds like EGCG, genistein, esculetin, berberine,

resveratrol, guggulsterone, capsaicin, baicalein and procyanidins inhibited adipogenesis

(Rayalam et al., 2008). 3T3-L1 cells are widely used models of adipocyte function. In

vivo, excessive triglyceride accumulation by the adipocyte has been linked to an in-

creased risk of a variety of metabolic disorders (Popovich et al., 2010). The presence of

phenolic compounds, tannins, alkaloids, procyanidins and cyanogenic glycosides have

been attributed to the hypoglycaemic action of various plants (Alonso-Castro and

Salazar-Olivo, 2008). Tannins, catechins and epicatechins are the most active antioxi-

dant constituents and are found to enhance the glucose uptake and inhibit adipogenesis

in differentiated adipocytes (Deutschländer et al., 2011, Muthusamy et al., 2008).

Chapter 5 127

5.6. Conclusions

The results of the current study showed that plant extract AT probably exerts its anti-

diabetic properties by stimulating glucose uptake in adipocytes with significant inhibi-

tion of adipogenesis. Plant extracts AK, SS and CO were also observed to enhance basal

and insulin-stimulated glucose uptake. BL, ED, MP and PM inhibited lipid accumula-

tion but should be further studied using anti-lipase activity assays and Western blot

analysis to confirm their anti-adipogenic effect. The ability of AT to enhance glucose

uptake in insulin-resistant adipocytes, in addition to its anti-adipogenic effects, suggests

that this extract could be useful in the treatment of type 2 diabetes. Future studies

should address the molecular mechanisms by which these plants and their active com-

pounds regulate glucose uptake by adipose and muscle tissues.

The ability of existing therapies to target various aspects of the insulin resistance syn-

drome induces other metabolic abnormalities, chiefly those involved in lipid metabo-

lism. Therefore, glucose-lowering drugs with minimal adipogenic activity are desirable

and this study has demonstrated that phytochemicals from traditional medicinal plants

have such properties. However, the pharmaceutical significance of the present results

will depend on further exploration including lead molecule identification and in vivo

studies.

Chapter 6 128

Chapter 6. General Discussion

Chapter 6 129

6.1. Discussion

This chapter provides a general overview of the work presented in the thesis and its im-

plications. It also provides possible future directions that will help in the identification

of novel drug targets for the management of type 2 diabetes.

It has been estimated that 275 Australians develop diabetes every day. The 2005 Aus-

tralian AusDiab Follow-up Study (Australian Diabetes, Obesity and Lifestyle Study)

showed that 1.7 million Australians have diabetes but up to half of the cases of type 2

diabetes remain undiagnosed and it is estimated that by 2033 nearly 3.5 million Austral-

ians will have type 2 diabetes (Cameron et al., 2003). Therefore there is a great need to

develop new drugs for diabetes. Part of this drug discovery research effort will be to

identify plant species that can potentially be applied in management of type II diabetes

and related complications of weight gain, hypertension and immune-suppression.

The strategy used in this thesis was to investigate the traditional plants used by Austral-

ian Aboriginal people for general health and medicinal plants utilised by Indians for the

management of diabetes which are described in ancient Indian texts of Ayurveda. The

initial part of this study involved screening the plants for bioactivity by testing their

ability to inhibit common microbes i.e. two Gram-positive and two Gram-negative bac-

teria and a fungus, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudo-

monas aeruginosa and Candida albicans, respectively, using the disk diffusion assay.

MIC and MBC were also determined. Results from disk diffusion assays with these mi-

crobes showed that many extracts exhibited broad spectrum activity. However, the MIC

and MBC values indicated that the plant extracts were not very potent. Cos (Cos et al.,

2006) have suggested that strict criteria should be used to assess the potential applica-

tion of natural products. In the context of anti-infective agents, MIC levels less than 100

μg/ml are indicative of useful bioactivity for natural product extracts. None of the ex-

tracts showed MIC less than 100 μg/ml against any bacteria tested in our study, which

was unexpected, the poor result may be simply due to natural variations in chemical

components due to factors such as difference in geographic conditions of the plants. De-

spite the poor MIC and MBC results, most of the Indian Ayurvedic plants showed broad

spectrum antimicrobial activity. However, SL and MP showed good activity against

Chapter 6 130

Candida albicans, suggested that these extracts can be potential candidates as antifungal

agents, while AS, BD, BM, CP and PT demonstrated moderate antifungal activity. The

mode of extraction, solvent used for extraction, the nature of bioactive compounds and

the geographical sources of plants affect the presence of bioactive compounds and thus

the activity (Rios and Recio, 2005). These factors, individually or collectively, may ex-

plain the low antimicrobial activity observed for the extracts.

The antioxidant activity of the plants was evaluated against free radicals. Free radicals

can damage biomolecules in our body such as lipids, nucleic acids and proteins. They

also cause cellular membrane peroxidation and attract various inflammatory mediators

(Leopoldini et al., 2011) as shown in Figure 23. Phenolic compounds and flavonoids

are known to have antitumor properties, antiproliferative effects and induce apoptosis in

different cancer cell lines. They are free radical scavengers, and flavonoids in particular

inhibit invasion and metastasis (Čipák et al., 2003). Polyphenols are able to interact

with the cytochrome P450 (CYP1) family enzymes which promote the initiation of car-

cinogenesis. These interactions act via two mechanisms: to inhibit the procarcinogen

activation and to be a substrate for the release of inhibitors of tumour cell growth

(Sawadogo et al., 2012). Several studies have described that the anticancer activity of

phytochemicals is due to their antioxidant compounds such as vitamins, minerals, poly-

phenols, flavonoid, terpenoids, lignins, xanthones and polysaccharides (Pandey and

Madhuri, 2009). Therefore, antioxidants are considered to be good source of anticancer

agents. In this study, the free radical scavenging activity was strongest in AL, AK, BL

and SL. Among Indian Ayurvedic plant extracts, AP, BM and PM demonstrated potent

free radical scavenging activity against the diphenyl picryl hydrazyl (DPPH) radical,

2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS) and ferric ions. It was there-

fore decided to target these plants for screening against cancer cells to investigate the

anticancer activity.

Of the total twenty six plant extracts initially included in this study, only twelve plant

extracts (all seven Australian Aboriginal and five Indian Ayurvedic) were selected

based on their results with antioxidant and enzyme inhibition assays for further studies

with cell culture experiments. The cancer cell lines used were cervical carcinoma HeLa

cells and lung adenocarcinoma A549 cells. According to the US NCI plant screening

Chapter 6 131

program, a crude extract is generally considered to have in vitro cytotoxic activity if the

IC50 value is less than 30 µg/ml. Among the plant extracts screened, only two extracts,

AK and AT, showed cytotoxic activity (IC50 of 13.73 μg/ml and 27.00 μg/ml, respec-

tively) against HeLa cells as assessed by the MTT assay. None of the other plant ex-

tracts showed IC50 values less than 30 μg/ml against A549. However, it is possible that

these extracts possess activity against other cancer cell lines. Thus, further testing is

warranted.

Figure 23 - Physiological pathway representing the generation of free radicals and their

contribution to various disorders which can be moderated or prevented by antioxidants

found in plant species.

Chapter 6 132

Phenolic components found in vegetables, fruits, spices and medicinal herbs might pre-

vent cancer through antioxidant action and/or the modulation of several protein func-

tions. They may inhibit carcinogenesis by affecting the molecular events in the initia-

tion, promotion and progression stages of free radical chain reactions (Cai et al., 2004).

Phenolics have also been reported to demonstrate agonism and/or antagonism of carcin-

ogenesis-related receptors such as the arylhydrocarbon receptor, epidermal growth fac-

tor and estrogen receptor β. They modulate the secretion of protein kinases in tumour

cell proliferation and induce the expression of anticarcinogenic enzymes or inhibit in-

duction of cancer-promoting enzymes (Sakakibara et al., 2002).

The use of natural products as anticancer agents has a long history that began with folk

medicine and has been incorporated into modern medicine. Several drugs currently used

in chemotherapy were isolated from plant species such as alkaloids from Vinca, vinblas-

tine and vincristine isolated from Catharanthus roseus, etoposide and teniposide, the

semisynthetic derivatives of epipodophyllotoxin isolated from species of the genus

Podophyllum, the naturally derived taxanes isolated from species of the genus Taxus

and the semisynthetic derivatives of camptothecin, irinotecan and topotecan isolated

from Camptotheca acuminate as detailed in the review by Costa (Costa-Lotufo et al.,

2005).

The plant extracts were also assessed for their ability to inhibit enzymes involved in

carbohydrate metabolism, which is an important feature of diabetes control or preven-

tion. Enzyme inhibition activity was evaluated against starch hydrolysing enzymes α-

amylase and α-glucosidase. Hydrolysis of dietary carbohydrates such as starch is the

major source of glucose in the blood. This hydrolysis is carried out by a group of hydro-

lytic enzymes that includes pancreatic α-amylase and intestinal α-glucosidases. There-

fore, the inhibition of these enzymes is believed to be a good and important strategy for

management of type 2 diabetes (Apostolidis and Lee, 2010). α-glucosidase is abundant-

ly found in the brush border of the small intestine and can delay carbohydrate digestion

and absorption, and thereby diminish post-prandial hyperglycaemia (PPHG) (Tiwari et

al., 2008). Recently, there had been widespread interest in these enzymes because of

their potential as therapeutic targets. In particular, inhibition of α-glucosidase had been

found to help control postprandial blood glucose levels in diabetic patients (Ryu et al.,

Chapter 6 133

2009). Clinical trials showed that the α-glucosidase inhibitors improved long-term gly-

caemic control as measured by decreased haemoglobin A1c (HbA1c) in patients with

type 2 diabetes and delay the development of type 2 diabetes in patients with impaired

glucose tolerance (Du et al., 2006).

The commercial inhibitors acarbose and miglitol are drugs that inhibit α-glucosidase

present in the epithelium of the small intestine and have been demonstrated to decrease

post-prandial hyperglycaemia and improve impaired glucose metabolism without pro-

moting insulin secretion in NIDDM patients (Subramanian et al., 2008b). Several food

components and plants have also been reported to inhibit these enzymes. Therefore,

natural α-glucosidase and α-amylase inhibitors from plant sources offer an attractive

strategy for the control of postprandial hyperglycaemia (Bhat et al., 2011a). Similarly,

angiotensin converting enzyme (ACE) in the rennin–angiotensin system is known to

increase blood pressure by the hydrolysis of the rennin-induced decapeptide, angioten-

sin I to octapeptide angiotensin II, a potent vasoconstrictor. This reaction stimulates al-

dosterone secretion in adrenal/cardiovascular tissue which promotes sodium and water

retention and causes an increase in rennin generation in the kidneys (Kurtz and

Pravenec, 2004). Therefore, the inhibition of ACE may prevent hypertension, other car-

diovascular and renal diseases and oxidative stress-associated diseases. The synthetic

ACE inhibitors are captopril, benazepril, enalapril, and lisinopril. Tannins, flavonoids,

xanthones, terpenoids, peptides and caffeoylquinic acid derivatives are the natural com-

pounds that have been reported to have anti-hypertensive action through ACE-inhibition

(Jung et al., 2006a).

Extracts of CB, PZ, SN and PM were found to inhibit α-amylase activity with IC50 val-

ues 4.17, 4.36, 4.87 and 5.16 µg/ml; whereas acarbose showed an IC50 of 7.81 µg/ml.

SS was found to inhibit α-amylase with an IC50 of 5.43 µg/ml. Among the Australian

plants, AL, AK, AT, BL, ED, SS and SL showed an IC50 of 1.01, 1.34, 1.38, 0.48, 1.06,

0.90 and 1.83 µg/ml respectively and were significantly more potent inhibitors of α-

glucosidase compared to acarbose (p < 0.001). Among the Indian Ayurvedic plants, CB

and MP showed significant inhibition (p < 0.001) with IC50 values of 0.58 and 0.80

µg/ml respectively against α-glucosidase. These findings provide evidence for the hy-

poglycaemic action of these plants and indicate that they may represent alternative ther-

Chapter 6 134

apies for the management of diabetes. Interestingly, the Australian Aboriginal plant ex-

tracts were found to be more potent compared to Indian Ayurvedic plant extracts. Alt-

hough the Australian plants investigated have no recorded traditional use in the treat-

ment of diabetes, this study shows that ethno medicinal knowledge alone cannot predict

the therapeutic potential of medicinal plants, especially when the ethnic group did not

naturally experience the disease under investigation.

All tested plant extracts showed very high IC50 as compared to captopril against ACE,

suggesting that these could be used as preventative nutraceuticals against hypertension

rather than therapeutic drugs for the treatment of the condition. Alternatively, they could

perhaps help in managing the side effects by modern anti-hypertensive drugs.

The plant extracts were selected on the basis of their enzyme inhibition (Chapter 4) ac-

tivity and were further evaluated by cell culture assays to assess their mode of action.

The ability of the selected plant extracts to stimulate glucose uptake in 3T3-L1 adipo-

cytes was examined and compared with that of rosiglitazone. In addition, the effects of

extracts on differentiation of pre-adipocytes into adipocytes, a process induced by addi-

tion of an insulin/3-isobutyl-1-methylxanthine/dexamethasone (IS-IBMX-DEX) cock-

tail, were also investigated. These studies were designed to characterize the effects of

extracts in 3T3-L1 cells at the cellular level and to identify extracts that may be used for

prevention and treatment of obesity and NIDDM without the undesirable side effects of

insulin therapy (Liu et al., 2001) .

Antidiabetic drugs such as insulin or thiazolidinediones (TZD) up-regulate both glucose

transport and lipid biosynthesis in adipocytes. Weight gain is a frequent side effect of

insulin therapy in type 2 diabetic patients (Laville and Andreelli, 2000). Therefore,

drugs with glucose-lowering activity, but lacking adipogenic activity, are highly desira-

ble. The extracts of AK, SS and CO found to stimulate glucose uptake in mature adipo-

cytes with moderate inhibition on adipogenesis. AT, BL and ED were found to signifi-

cantly reduce lipid accumulation whereas CO, PM and AP were able to moderately re-

duce lipid accumulation at 100 µg/ml. Thus, an understanding of the mechanism of ac-

tion will be valuable for the study, prevention and treatment of obesity, insulin re-

sistance and type 2 diabetes (Liu et al., 2001) if these extracts were to be further investi-

gated. The use of plants with good antidiabetic potential, which help reduce lipid accu-

Chapter 6 135

mulation and have anti-oxidant activity, would be an excellent strategy to control hy-

perglycaemia and related complications of diabetes. Strengthening the immune system

and controlling weight gain and obesity would assist in the overall management of dia-

betes and related conditions, as depicted earlier in Figure 23.

To our knowledge, this is the first study of the potential use of Australian Aboriginal in

the management of diabetes and related complications. The Australian medicinal plants

investigated in this study (SS, AL, ED, BL, AK, AT and SL) have been used for general

illness, cold, cough and pain (Palombo and Semple, 2001b). The traditional hunter-

gatherer lifestyle and diet of Aboriginal people, which was high in carbohydrates, fibre,

proteins and nutrients but low in fat and sugars, meant that cardiovascular diseases and

diabetes were not common in these people. After the settlement of Europeans, the diet

became Westernized with high sugar and fat content and the lack of essential nutrients,

vitamins, minerals, proteins and fibre. This has increased the disease risk and these dis-

orders are now prevalent in indigenous populations with the incidence of type 2 diabetes

rapidly increasing (Diamond, 2003, Gulati et al., 2012).

This thesis has evaluated the biochemical and cellular properties of a number of plant

extracts with potential application in the management, treatment and prevention of dia-

betes and related conditions. Tables 14 and 15 summarize the results obtained with dif-

ferent bioassays. In addition, Figure 24 summarizes the activities of these extracts.

Chapter 6 136

Table 14 - Results summary of Australian Aboriginal plant extracts

For antibacterial assays: + denotes activity against 1 species, ++ against 1-3 species and +++ >3 species; for antioxidant and enzyme inhibition assay: + denotes IC50 >200 µg/ml, ++ de-notes IC50 value 50-200 µg/ml and +++ IC50 value <50 µg/ml; for glucose uptake assay: + denotes glucose uptake is stimulated by 10-30 %, ++ denotes 30 to 50 % and +++ denotes >50 %; for adipogenesis assay: + denotes lipid accumulation is reduced by 0-30 %, ++ between 30 and 60 % and +++ >60 %; for antileukmic assay: + denotes IC50 >200 µg/ml, ++ IC50 be-tween 30 and 200 µg/ml and +++ IC50 <30 µg/ml

Plant name

Assays

Antimicrobial Antioxidant Enzyme Inhibition Glucose

uptake

Adipogenesis Antileukemic

DPPH ABTS α-

amylase

α-

glucosidase

AK + +++ +++ ++ +++ ++ + +++

AT + ++ + +++ +++ ++ +++ +++

AL - +++ ++ +++ +++ ++ + +

BL + +++ +++ +++ +++ + +++ ++

ED - +++ +++ ++ +++ - +++ ++

SL ++ +++ ++ +++ +++ - ++ ++

SS - +++ +++ +++ +++ ++ ++ ++

Chapter 6 137

Plant name

Assays

Antimicrobial Antioxidant Enzyme Inhibition Glucose

uptake

Adipogenesis Antileukemic

DPPH ABTS α-

amylase

α-

glucosidase

AP +++ +++ +++ +++ +++ - ++ +

Ana. ++ + + ++ +++ n.d n.d n.d

AS +++ + + ++ +++ n.d n.d n.d

BM ++ +++ +++ ++ +++ - + +

BD +++ + + +++ +++ n.d n.d n.d

BS ++ + + +++ +++ n.d n.d n.d

Chapter 6 138

CB ++ + + +++ +++ n.d n.d n.d

CM +++ + + ++ +++ n.d n.d n.d

CP +++ + + +++ +++ n.d n.d n.d

CO +++ + + +++ +++ ++ ++ +

EJ +++ + + +++ +++ n.d n.d n.d

MP +++ + + +++ +++ - + +

NJ +++ + + +++ +++ n.d n.d n.d

PZ +++ + + +++ +++ n.d n.d n.d

PM +++ ++ ++ +++ +++ - ++ +

PT + + + +++ +++ n.d n.d n.d

Chapter 6 139

SN ++ + + +++ +++ n.d n.d n.d

TC +++ +++ ++ +++ +++ n.d n.d n.d

VW ++ ++ ++ +++ +++ n.d n.d n.d

Table 15 - Results summary of Indian Ayurvedic plant extracts

The symbols used are as for Table 14; n.d = not done

Chapter 6 140

Figure 24 - The proportion of active Australian Aboriginal and Indian Ayurvedic plants

investigated by various assays.

6.2. Future directions

The conclusions and recommendations outlined in this chapter relate to the original

aims and outlines of the study. A summary of the study findings was included in the in-

dividual chapter.

The present study details an investigation of the bioactive properties of traditional me-

dicinal plant extracts. This included activity against common microbes and free radical

scavenging properties, however, the main focuses was the potential role in the manage-

ment of hyperglycaemia. The results of this study demonstrated that many of the plants

had broad-spectrum antimicrobial activity, good anti-oxidant activity and promising an-

ti-diabetic potential. Further studies should include in vivo experiments to examine the

effectiveness of the extracts in animal models.

On the basis of results of this study, extracts from AK (Acacia kempeana), AL (Acacia

ligulata), AT (Acacia tetragonophylla), SS (Santalum spicatum), BL (Beyeria

Australian Aboriginal plants

DPPH

ABTS

Glucosidase

Amylase

ACE

Anticancer

Gluocse uptake

Adipogensis

Indian Ayurvedic plants

DPPH

ABTS

Glucosidase

Amylase

ACE

Anticancer

Gluocse uptake

Adipogensis

Chapter 6 141

leschnaultii), ED (Euphorbia drumondii) and CO (Curculigo orchioides) were deemed

to be the most active. Therefore, these plants should be further explored and detailed

phytochemical analysis, isolation of active compounds and their characterisation

through bio-activity testing should be carried out with cell culture assays and animal

models. In particular, given the promising anti-diabetic potential of these plants, ex-

tracts or purified compounds should be examined in diabetes mouse models (Cheng et

al., 2012, Kolb, 1987). Encouraging results would then allow the bioactive compounds

to be developed as drug candidates and used in human clinical trials.

The other approach with moderately active plant extracts such as SL (Santalum lanceo-

latum), AP (Andrographis paniculata), BM (Bacopa moneirrei), PM (Pterocarpus mar-

supium) and MP (Mucuna pruriens) could be to develop them into nutraceuticals.

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