IDENTIFICATION AND CHARACTERIZATION OF

Stevia rebaudiana EXTRACTION FOR TOTAL

PHENOLIC COMPOUND USING SOXHLET

EXTRACTION AND MICROWAVE-ASSISTED

EXTRACTION TECHNIQUES

ERNA WATI IBNU HAJAR

Thesis submitted in fulfilment of the requirements

For the award of the degree of

Master of Engineering in Chemical

Faculty of Chemical and Natural Resources Engineering

UNIVERSITI MALAYSIA PAHANG

December 2013

iv

ABSTRACT

This study focused on identifying extraction condition (e.g. sample concentration,extraction time) of total phenolic compound (TPC) from leaves, stems and flowers ofStevia rebaudiana. Two method processes were used: 1) using Soxhlet Extraction (SE)and 2) Microwave-Assisted Extraction (MAE). Possible mechanisms of productivityenhancement by both methods were investigated. The extract were analyzed with anappropriate Gas Chromatography-Mass Spectrometry (GC-MS) and FourierTransform Infrared Spectroscopy (FTIR). The reproducibility of extraction and ofchromatographic analysis was proved.

The identification of extraction in both extraction technique has been intensivelystudied. In comparison of these two techniques to extraction of TPC in leaves, stemsand flowers of Stevia rebaudiana, MAE had a higher extraction efficiency withreduces extraction time. The yield of TPC in leaves of Stevia rebaudiana was 100.385mg GAE/g dw and in stems was 18.702 mg GAE/g dw at concentration 0.1 g/ml.Meanwhile, the yield TPC in flowers was 64.461 mg GAE/g dw at the concentration0.06 g/ml after 12 min running using MAE.

The qualitative GC-MS analysis of the extract from leaves, stems and flowers ofStevia rebaudiana showed that total ion chromatogram (TIC) of Soxhlet and MAEshows some valuable constituents of extracts such as Octadecanoic acid, n-Hexadecanoic acid, 1-Octadecene, Taraxasterol, Tridecanoic acid, D-allose,Morpinan, Benzene rings, Stigmasterol, β-Sitosterol, γ-Sitosterol, α-Amyrin,Longifolenaldehyde, Allo-inisitol, Neo-inositol, Azulene, and Phenol. The resultssuggest that Stevia rebaudiana can also be consider as a potential source of essentialoil for the use of cosmeceutical, medicinal and pharmacological application.

FTIR analysis of raw and extracted from leaves, stems, and flowers of Steviarebaudiana showed a great abundance of alcohols, phenols, aromatic rings, carboxylicacids, ketones, aldehydes, alkanes, ethers, ester, acetylenic as well as olefinic.

Heavy metals accumulation in the Stevia rebaudiana from leaves, stems, and flowersis reported. The analysis of sample was carried out by using an Inductively CoupledPlasma Mass-Spectrophotometer (ICP-MS) to analyze the Arsenic (As), Cadmium(Cd), Chromium (Cr), Copper (Cu), Iron (Fe), Magnesium (Mg), plumbum (Pb),Selenium (Se), Zinc (Zn), aluminium (Al), silver (Ag), Cobalt (Co), calcium (Ca),manganese (Mn) and nickel (Ni). The accumulation of heavy metals in leaves, stems,and flowers of Stevia rebaudiana contain fifteen heavy metals below tolerance limit inplant and can be used as food products.

Keywords: Soxhlet Extraction, Microwave-assisted extraction, Stevia rebaudiana,GC-MS, FTIR, ICP-MS, Heavy metals.

vi

TABLE OF CONTENTS

Page

SUPERVISOR’S DECLARATION i

STUDENT’S DECLARATION ii

ACKNOWLEDGEMENTS iii

ABSTRACT iv

ABSTRAK v

TABLE OF CONTENTS vi

LIST OF TABLES x

LIST OF FIGURES xii

LIST OF SYMBOLS xiv

CHAPTER 1 INRTODUCTION

1.1 Background 1

1.2 Problem Statement 5

1.3 Objectives of the Research 6

1.4 Research Scope 6

1.5 Rationale and Significant of Research 6

CHAPTER 2 LITERATURE REVIEW

2.1 Introduction 8

2.2 Medicinal Plants 8

2.3 Stevia rebaudiana 10

2.4 Antioxidant 12

2.5 Phenolic Compound 13

2.6 Heavy Metals 15

2.7 Extraction Techniques 18

2.8 Soxhlet Extraction 20

vii

2.9 Microwave-assisted Extraction 22

2.10 Microwave Theory 23

2.11 Factors Effecting Microwave-assisted Extraction 25

2.11.1 Solvent Nature 26

2.11.2 Extraction Time 27

2.11.3 Microwave Power 28

2.11.4 Matrix Characterization 29

2.11.5 Temperature 30

2.12 Optimization 31

CHAPTER 3 MATERIALS AND METHODS

3.1 Introduction 33

3.2 Materials 33

3.3 Preparation of Sample 34

3.4 Soxhlet Extraction 34

3.5 Microwave-assisted Extraction 35

3.6 Gallic acid standard Calculation 36

3.6.1 Reagents 36

3.6.2 Procedure 36

3.7 Total Phenolic Compound (TPC) Analysis by Folin-Ciocaltue Method 37

3.8 FTIR analysis 38

3.8.1 Solid Sampling 38

3.8 2 Liquid Sampling 38

3.9 GC-MS Analysis 39

3.10 ICP-MS Analysis 40

3.10.1 Preparation Sample 40

3.10.2 Analysis of Heavy Metals 41

3.11 Data Collection and Analysis by Statistical 43

CHAPTER 4 RESULTS AND DISCUSSION

4.1 Introduction 44

4.2 Optimization of TPC using Soxhlet extraction and Microwave-assisted 44

viii

extraction

4.3 Characterization of Stevia rebaudiana using SE and MAE analyzed by 60

GC-MS and FTIR

4.3.1 GC-MS Analysis 60

4.3.2 FTIR Analysis 71

4.4 Assessment of heavy metals using microwave digestion system analyzed 78

by ICP-MS

CHAPTER 5 CONCLUSION AND RECOMMENDATIONS

5.1 Introduction 85

5.2 Conclusions 85

5.3 Recommendations for the Future Research 87

REFERENCES 88

APPENDICES

A.1 Experimental Flowchart 105

A.2 TPC in Stevia rebaudiana by Folin - Ciocaltue Method 106

A.3 Output of TPC in Leaves of Stevia rebaudiana using SE by Statistics 121

A.4 Output of TPC in Stems of Stevia rebaudiana using SE by Statistics 127

A.5 Output of TPC in Flowers of Stevia rebaudiana using SE by Statistics 134

A.6 Output of TPC in Leaves of Stevia rebaudiana using MAE by Statistics 137

A.7 Output of TPC in Stems of Stevia rebaudiana using MAE by Statistics 143

A.8 Output of TPC in Flowers of Stevia rebaudiana using MAE by Statistics 150

A.9 GC-MS Profile Releated Results of Stevia rebaudiana using SE 153

A.10 GC-MS Profile Releated Results of Stevia rebaudiana using MAE 159

B.1 FTIR Spectra of Raw Material Stevia rebaudiana 167

B.2 FTIR Spectra of Stevia rebaudiana Extract using SE 168

B.3 FTIR Spectra of Stevia rebaudiana Extract using MAE 169

B.4 Principle IR Absorbance for Certain Fungtional 170

B.5 Accumulation Heavy Metals in Stevia rebaudiana Extract by ICPMS 172

Agilent 7500a

ix

B.6 Accumulation Heavy Metals in Stevia rebaudiana Extract by ICP-MS 176

Elan 9000

C Published 185

x

LIST OF TABLES

Table No. Title Page

2.1 Chemical and physical properties of phenol 14

2.2 Total concentration of heavy metals normal range in plants, normal range 17

In plants, normal range in soils and toxic soils for plants

2.3 Dissipation factor and dielectric constants for same solvents commonly 25

used in microwave-assisted extraction

2.4 Boiling temperatures and temperatures under microwave at 175 psig of 31

different solvents

3.1 Details of UV-Vis Spectrophotometer equipment 37

3.2 Details of FTIR Spectrometer equipment 38

3.3 Details of GC-MS equipment and column 39

3.4 Details of ICPMS Elan 9000 equipment 42

3.5 Operating conditions for ICP-MS Agilent 7500a 42

4.1 TPC yield in leaves of Stevia rebaudiana using SE 48

4.2 TPC yield in stems of Stevia rebaudiana using SE 48

4.3 TPC yield in flowers of Stevia rebaudiana using SE 48

4.4 TPC yield in leaves of Stevia rebaudiana using MAE 49

4.5 TPC yield in stems of Stevia rebaudiana using MAE 49

4.6 TPC yield in flowers of Stevia rebaudiana using MAE 49

4.7 The volume rate of heat generation at 450 Watt for 2-12 min 58

4.8 The identified components present in leaves extract using SE 66

4.9 The identified components present in stems extract using SE 63

4.10 The identified components present in flowers extract using SE 63

4.11 The identified components present in leaves extract using MAE 70

4.12 The identified components present in stems extract using MAE 70

4.13 The identified components present in flowers extract using MAE 71

4.14 Stuctural features of raw material from Stevia rebaudiana (leaves, stems 73

xi

and flowers) by FTIR

4.15 Stuctural features of leaves, stems and flowers extracts from Stevia 76

rebaudiana using SE by FTIR

4.16 Stuctural features of leaves, stems and flowers extracts from Stevia 78

rebaudiana using MAE by FTIR

4.17 The accumulation of heavy metals in Stevia rebaudiana (leaves, stems 79

Flowers) by using ICP-MS Elan 9000 and Agilent 7500a

xii

LIST OF FIGURES

Figure No. Title Page

2.1 Stevia rebaudiana Bertoni 11

2.2 Phenol chemical structure 14

3.1 Sample of Stevia rebaudiana purchase from Malacca Malaysia 33

3.2 Soxhlet Extraction (SE) 34

3.3 Microwave-assisted extraction (MAE) 35

3.4 The Spectrum 100 FTIR Spectrometer 38

3.5 GC-MS an Agilent 5973N Mass Selective Detector 39

3.6 (a) The vessels (b) Microwave digestion System 40

3.7 Inductively Coupled Plasma Mass – Spectrophotometer Elan 9000 41

3.8 Inductively Coupled Plasma Mass – Spectrophotomete Agilent 7500a 42

4.1 Gallic acid standard curve 45

4.2 TPC yield in leaves and stems of Stevia rebaudiana at concentration 50

0.01 g/ml using SE

4.3 TPC yield in leaves and stems of Stevia rebaudiana at concentration 50

0.02 g/ml using SE

4.4 TPC yield in leaves and stems of Stevia rebaudiana at concentration 51

0.04 g/ml using SE

4.5 TPC yield in leaves, stems and flowers of Stevia rebaudiana at concen- 51

tration 0.06 g/ml using SE

4.6 TPC yield in leaves and stems of Stevia rebaudiana at concentration 52

0.08 g/ml using SE

4.7 TPC yield in leaves and stems of Stevia rebaudiana at concentration 52

0.1 g/ml using SE

4.8 TPC yield in leaves and stems of Stevia rebaudiana at concentration 53

0.01 g/ml using MAE

4.9 TPC yield in leaves and stems of Stevia rebaudiana at concentration 53

xiii

0.02 g/ml using MAE

4.10 TPC yield in leaves and stems of Stevia rebaudiana at concentration 54

0.04 g/ml using MAE

4.11 TPC yield in leaves, stems and flowers of Stevia rebaudiana at concen- 54

0.06 g/ml using MAE

4.12 TPC yield in leaves and stems of Stevia rebaudiana at concentration 55

0.08 g/ml using MAE

4.13 TPC yield in leaves and stems of Stevia rebaudiana at concentration 55

0.1 g/ml using MAE

4.14 The volume of heat generation vs. rate of temperature 58

4.15 The volumetric rate of heat generation at 450 Watt for 2 - 12 min 59

4.16 GC-MS TIC chromatography of Stevia rebaudiana extracts using SE 61

(a) leaves, (b) stems and (c) flowers

4.17 GC-MS TIC chromatography of Stevia rebaudiana extracts using MAE 67

(a) leaves, (b) stems and (c) flowers

4.18 FTIR spectra of raw material from Stevia rebaudiana (a) leaves, (b) stems 72

and (c) flowers

4.19 FTIR spectra of Stevia rebaudian extract using SE (a) leaves, (b) stems 75

and (c) flowers

4.20 FTIR spectra of Stevia rebaudian extract using MAE (a) flowers, (b) lea- 77

ves and (c) stems

xiv

LIST OF SYMBOLS

Ag = Silver

Al = Aluminium

As = Arsenic

Ca = Calcium

Cd = Cadmium

Co = Cobalt

Cr = Chromium

Cu = Copper

Fe = Iron

FTIR = Fourier Transforms Infra-Red

GAE = Gallic acid equivalent

GC-MS = Gas Chromatography – Mass Spectrometry

ICP-MS = Inductively Couple Plasma – Mass Spectrophotometer

MAE = Microwave-assisted extraction

Mg = Magnesium

Mn = Manganese

Ni = Nickel

Pb = Plumbum

Se = Selenium

SE = Soxhlet extraction

SEM = Scanning Electron Microscopy

TIC = Total ion chromatogram

TPC = Total phenolic compound

UV = Ultra violet

Zn = Zinc

1

CHAPTER 1

INTRODUCTION

1.1 Background

Stevia is a genus of about 240 species of herbs and shrubs in the sunflower

family (Asteraceae), native to subtropical and tropical regions of Western North

America to South America. The species Stevia rebaudiana, commonly known as

sweet leaf, sugar leaf, or simply stevia, is widely grown for its sweet leaves. As a

sweetener and sugar substitute, stevia's taste has a slower onset and longer duration

than that of sugar, although some of its extracts may have a bitter aftertaste at high

concentrations.

With its steviol glycoside extracts having up to 300 times the sweetness of the

sugar. Stevia has garnered attention with the rise in demand for low-carbohydrate,

low-sugar food alternatives. Stevia has a good potential for the human health

especially for negligible effect on blood glucose, it is attractive as a natural sweetener

for people on carbohydrate-controlled diets.

Stevia rebaudiana is an outstanding herb bearing leaves of very refreshing

sweet taste and remarkable health qualities. The sweetness of Stevia leaves is largely

due to its complex molecules called stevioside and the second compound called

rebaudioside. Stevia leaves have a taste that is unique and very sweet with a slight

bitter aftertaste. The sweetness of Stevia leaves is much different from the sweetness

of other natural sweeteners, sugar, or artificial sweeteners, but it is delicious. Stevia

has many favorable and exciting health benefits and it is completely non-toxic. The

herb is nutrient rich, containing substantial amounts of protein, calcium, phosphorous,

sodium, magnesium, zinc, rutin, vitamin A, vitamin C, and other nutrients, yet has no

caloric value.

2

The availability of Stevia rebaudiana varies from country to country. In a few

countries, it has been available as a sweetener for decades or centuries; for example,

Stevia rebaudiana is widely used as a sweetener in Japan where it has been available

for decades. In some countries, Stevia is restricted or banned. In other countries,

health concerns and political controversies have limited its availability; for example,

the United States banned stevia in the early 1990s unless labeled as a dietary

supplement, but in 2008 approved rebaudioside extract as a food additive. Over the

years, the number of countries in which Stevia is available as a sweetener has been

increasing (McCaleb, 1997). Approximately 80% of the world populations depending

exclusively on plants for their health and healing. Whereas in the developed world,

reliance on surgery and pharmaceutical medicine is more usual however in the recent

years, more and more people are complementing their treatment with natural

supplements (Dursum et al., 2004).

Recently there has been an upsurge of interest in the therapeutic potentials of

plants, as antioxidants in reducing free radical induced tissue injury. Although several

synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated

hydroxytoluene (BHT), are commercially available, but are quite unsafe and their

toxicity is a problem of concern. Hence, strong restrictions have been placed on their

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

antioxidants. Natural plant based antioxidants especially phenolics and flavonoids

have been exploited commercially either as antioxidant additives or as nutritional

supplements (Schuler, 1990). Also many other plant species have been investigated in

the search for novel antioxidants (Chu et al., 2000). However there is still a demand

to find more information concerning the antioxidant potential of plant species as they

are safe and also bioactive. Therefore, in recent years, considerable attention has been

directed towards the identification of plants with antioxidant ability.

Many herbs and some common medicinal plants are good sources of

antioxidant compounds. Many of the biologically active substances found in plants,

including phenolic compounds are known to possess potential antioxidant properties.

The antioxidant activity of medicinal plants depends on the concentration of

individual antioxidant entering into the composition (Larson, 1988).

3

The protective effects of plant products are due to the presence of several

components which have distinct mechanisms of action, some are enzymes and

proteins, other are low molecular weight compounds such as vitamins, carotenoids

and phenolic compounds (Tadhani et al., 2007; Zhang and Wang, 2002). The

beneficial health-related effects of some phenols or their potential antioxidant

properties, especially when these compounds are present in large quantities in foods,

are important to be consumers. Therefore, it is of great interest to evaluate the total

phenolic compounds of Stevia rebaudiana. For this purpose, leaves, stems and

flowers of Stevia rebaudiana were selected in the present study for the analysis of the

total phenolic compounds.

More than 100 compounds have been identified in Stevia rebaudiana, the best

known of which are the steviol glycosides, particularly stevioside and rebaudioside A,

being the most abundant (Kennelly, 2002). Many review articles on the composition

of Stevia rebaudiana have been published. In general, it has reported that Stevia

rebaudiana is nutrient rich, containing substantial of protein, vitamins, antioxidants

and other important nutrients has made this very interesting field for chemical

investigations of Stevia rebaudiana extract or essential oil. Stevia rebaudiana are

being intensively investigated because the search for new compounds is still ongoing.

According to the literature, many of these compounds are known for their

antioxidant proprieties (pinene, carvacrol, caryophyllene) also the essential oil

constituents of the plants varied to several factors: geographical, culture conditions,

collect time, altitude, climate, different type and nutritional status; all these factors

can influence their composition (Kapoor et al., 2009, Ozkan et al., 2010). Stevia

rebaudiana has become rather widespread over a wide range of climatic location

around the world and can apparently be successfully grown under different

cultivation condition. Since the chemical composition of the Stevia rebaudiana

extract is dependent on the applied condition of plant cultivation, thus, they have

become the subject of many research projects.

It has been observed that agricultural soils have been contaminated due to use

chemical fertilizers, pesticides, irrigation water and disposal of chemicals nearby.

4

Thus, the environmental conditions of the region and the agricultural practices decide

the level of heavy metals that would accumulate in the plant of Stevia as herbal raw

materials are also often used as food, functional food, nutritional or dietary

supplements. It is clear that quality control of herbal materials and medicinal plants is

very important, not only for the safety of the herbal medicines themselves, but also

for food safety. It should be borne in mind that although controlling the contaminants

in plant of the herb has certain similarities, there may also be many differences.

Therefore, more research is needed in order to establish the scientific evidence

criteria for herbal medicines.

Stevia has become rather widespread over a wide range of climatic location

around the world and can apparently be successfully grown under different

cultivation condition, although often by seedling establishment in a greenhouse

before planting in the field. Stevia plant is easily contaminated during growth,

development and processing, and for this, an extensive research is needed to explore

the characteristics of the heavy metal produced by a plant of Stevia. The heavy metals

produced from the herb and its tolerance of heavy metals in Stevia plant is not well

documented, and scientific evidence is limited to establishing Stevia plant as a

medicinal plant. Although there is a great concern about heavy metal contamination

of herbal raw materials, information from the World Health Organization (WHO)

regarding permissible limit is available only for Pb and Cd. Since heavy metal

accumulation of the Stevia rebaudiana extract is dependent on an obtained heavy

metals from the soil and water. Heavy metals from plant sources may be also vary

from place to place because soil heavy metals content varies geographically, thus,

they have become the subject of many research projects.

Classical techniques for the solvent extraction of nutraceuticals from plant

matrices are based on the choice of solvent coupled with the use of agitation and/or

heat. Traditional extraction methods include maceration, Soxhlet extraction, and

percolation; however, they are often time-consuming, require relatively large

quantities of solvents, and the active compounds sometimes degrade (De Castro and

Garcia-Ayuso, 1998).

5

In order to increase the productivity and extraction performance, several

intensification techniques like ultrasonic waves, supercritical fluids or microwaves

were associated with extraction of plant compounds will be implemented to improve

the yield and quality of extracted products (Wang and Weller, 2006). From these,

ultrasound and microwave assisted-extractions emerged as two promising techniques

from an economical point of view, being inexpensive, simple and efficient. These

procedures increase at least one of the major parameters governing extraction: the

kinetic, through the partial mass transfer rates, the interfacial area or the driving force

(Cravotto et al., 2008). Microwave-assisted extraction widely used to selectively

extract target compounds from various raw materials. The principle of microwave-

assisted extraction is based on two processes, which is ionic conduction and dipole

rotation. The heating mechanism of microwave-assisted extraction can significantly

reduce the extraction time as compared to soxhlet extraction.

Despite the great variety of extraction applications already in use, it is difficult

to interpret and transfer theory to application at the industrial scale. Even in recent

application of extraction, optimal extraction or separation condition is obtained

empirically. One primary goal of food process engineering is finding out how an

extraction process works best achieving the optimum in respect of yield, quality, or

cost and consequently finding adjustable processing factor value that produces the

optimum.

Optimization of extraction processes is needed either for extraction processes

that are already in use or for future process designs for certain applications in food

engineering. Consequently, it is important for the food industry to optimizations

(yield, quality, operation time, and cost) using appropriate method techniques.

1.2 Problem Statement

1. Long time extraction and procedure by using conventional methods.

2. There are few reports on the chemical composition of extract Stevia

rebaudiana leaves, but there is no report on examining the chemical

composition of the extracts from stems and flowers of Stevia rebaudiana.

6

3. The heavy metals produced from the herb and its tolerance of heavy

metals in Stevia rebaudiana plant is not well documented and scientific

evidence is limited to establishing Stevia rebaudiana plant as a medicinal

plant.

1.3 Objectives of the Research

1. To investigate the various effects of different concentration and time to

optimize the yield of total phenol compound from the leaves, stems and

flowers of Stevia rebaudiana using soxhlet extraction and microwave-

assisted extraction techniques.

2. To characterize the chemical composition of extracts from leaves, stems

and flowers of local Stevia rebaudiana.

3. To investigate the heavy metal accumulation from leaves, stems and

flowers of Stevia rebaudiana extracts.

1.4 Research Scope

1. Investigate the various effects of different concentration and time on

phenol yields from leaves, stems and flowers of Stevia rebaudiana using

UV-Spectrophotometer from soxhlet extraction and microwave-assisted

extraction techniques.

2. Optimization of total phenolic compounds from leaves, stems, and flowers

of Stevia rebaudiana.

3. Characterization the chemical composition of extracts from leaves, stems,

and flowers of Stevia rebaudiana by FTIR, GC-MS and SEM.

4. Investigate the heavy metal accumulation of leaves, stems and flowers of

Stevia rebaudiana extract using ICP-MS.

1.5 Rationale and Significant of Research

1. Governments Malaysia focuses on growth Stevia rebaudiana plants and

the investigation about Stevia rebaudiana plants as raw materials for

phenol has been done continuously.

7

2. In an attempt to explore such untapped sources of good nutrition, the

present investigation has been undertaken to identify the chemical

composition of the extracts in the Malaysia species Stevia rebaudiana

Bertoni.

3. Stevia rebaudiana stems and flowers discarded as unused materials,

whereas the stems and flowers could be processed as an alternative raw

material for natural supplements.

4. Separations of total phenolic compounds from leaves, stems and flowers

of Stevia rebaudiana using soxhlet extraction and microwave assistance-

extraction has never been done, whereas these methods could be applied

for phenol and give economical value.

8

CHAPTER 2

LITERATURE REVIEW

2.1 Introduction

This chapter would contents the related literature of the previous studies on

the subject of interest which are optimization and characterization of Stevia

rebaudiana extracts for total phenolic compound using sohxlet extraction and

microwave-assisted extraction techniques.

2.2 Medicinal Plants

Medicinal plants as herb medicine, are plants that have at least one of their

part are leaves, stems, barks, roots , tubers and flowers used for therapeutic purpose

and active principles in medicinal plants plays a strategic role in the phytochemical

investigation of crude plant extract and very important to their potential

pharmacological effects (Pascual et al., 2002). Medicinal plants are of great

importance to the health of individuals and communities, and this plant lies in some

chemical substances that produce a definite physiological action on the human body.

The most important of these bioactive constituents of plants are alkaloids, flavonoids,

tannins, and phenolic compounds (Edeoga et al., 2005).

Medicinal plants have been used in almost all cultures as a source of

medicine. The use of traditional medicine and medicinal plants in most developing

countries as a normative basis for the maintenance of good health has been widely

observed (Hoareau and DaSilva, 1999). Moreover, in the nowadays motivation of

people towards herbs is increasing due to the concern about the side effects of

synthetic chemical drugs. People want to concern their own health rather than

submitting themselves to the impersonal health care system.

9

Tadhani and Subhash (2006a) subjected leaves of Stevia rebaudiana to

qualitative phytochemical screening for the identification of various classes of active

chemical constituents. The powdered leaf subjected to preliminary phytochemical

screening using chemical methods showed the most abundant compounds in the leaf

extract to be tannins and alkaloids, followed by cardiac glycosides, saponins, sterols

and triterpenes, reducing compounds and anthraquinones. Tests for cyanogenetic

glycosides, however, showed negative results.

The World Health Organization (WHO), estimated that eighty percent (80%)

of the population of developing countries rely on traditional medicine, most plant

drug, for their primary health care need medicinal plants being natural, non-narcotic,

having no side effect of arrange of safe, cost effective, preventive and curative

therapies could be useful in achieving the goal of health for all, in the coat effective

manner (Tewari, 2000). The World Health Organization (WHO) has now recognized

that stevioside is not genotoxic and assigned a temporary acceptable daily intake for

steviol glycosides of 0–2 mg/kg body weight (Beneford et al., 2006). Given the

impact that steviol glycosides have had on society, and the plant’s remarkable

metabolic capability the study of their biosynthesis is more than warranted.

Stevia rebaudiana, native of Paraguay, has been used as an herbal sweetener

for over 1500 years. Extracts of Stevia rebaudiana are part in weight-loss programs

because of its ability to reduce the cravings for sweet and fatty foods, to treat the

diseases' diabetes, hypoglycaemia, candidasis, high blood pressure, skin abrasions

and inhibiting growth and reproduction of bacteria-like plaque. Stevia's greatest

appears to be a natural alternative to artificial sweeteners (such as aspartame or

sodium saccharin). The sweetness in Stevia rebaudiana is mainly attributed to two

glycoside compounds: stevioside (3-10% of dry leaf weight) and rebaudioside A (1-

3%) which can be up to 250 times sweeter than sucrose (Duke, 2006).

The Paraguayan herb Stevia rebaudiana (Bertoni), also known by natives as

Kaa-He-é o “hierba dulce," has captured the growing interest as a potential source of

natural non caloric sweeteners for use as a possible substitute for synthetic

sweeteners. Its use has been approved in Brazil, Argentina, and Paraguay, as well as

10

in China, Korea, and Japan (Kolb et al., 2000). Nowadays, Stevia rebaudiana plant

and stevioside are being used as sweeteners in South America, Asia, Japan, China,

and in some countries in Europe. In Canada and the USA, Stevia products have been

used as dietary supplements since 1990 and 1995, respectively (Geuns, 2003).

As the Malaysian herbal medicine market is experiencing an extraordinary

growth, the research approaches taken have recently included activities to develop

herbal medicine into quality efficacious and safe products for human consumption.

More people are turning to herbal products as alternatives to the conventional therapy

medicine or as nutritional and dietary supplements. To date, in past five decades of

medicinal plant research in Malaysia, efforts to discover new bioactive agents from

flora for the use as chemicals have taken the lead in the development. In Malaysia,

the medicinal plants use as chemical templates to produce new candidates for drugs is

developing herbal medicine into quality, efficacious and safe products for human

consumption. Furthermore, the tropical rain forest plants are biologically and

chemically diverse resources as they syntheses various chemicals, as defense agents

against diseases and predator (Ibrahim, 2004).

2.3 Stevia rebaudiana

In 1899 Swiss botanist Moisés Santiago Bertoni, while conducting research in

eastern Paraguay, first described the plant and the sweet taste in detail (Bertoni,

1899). Initially called Eupatorium rebaudianum, its name changed to Stevia

rebaudiana (Bertoni) Bertoni in 1905. The sweet principle was first isolated in 1909

and only in 1931 was the extract purified to produce stevioside, the chemical structure

of which was established in 1952 as a diterpene glycoside. Stevioside is described as

a glycoside comprising three glucose molecules attached to an aglycone, the steviol

moiety. During the 1970s, other compounds were isolated, including rebaudioside A,

with a sweetening potency even higher than stevioside (Barriocanal et al., 2008).

Stevia rebaudiana (Bertoni) as shown in Figure 2.1 family Asteraceae is an

herbaceous perennial plant indigenous to Paraguay and Brazil where its leaves are

used by the local Guarani Indians as a natural sweetener for hundreds of years. About

150 stevia species are known, among them Stevia rebaudiana is the only one with

11

significant sweet-tasting properties (Soejarto et al., 1983). This plant is of worldwide

importance today because its leaves are used as non-nutritive high-potency sweetener

primarily in Japan, Korea, China and South America. The consumption of Stevia

extracts in Japan and Korea is about 200 and 115 tons/year, respectively (Kinghorn et

al., 2000).

Figure 2.1: Stevia rebaudiana Bertoni

Classification of Stevia rebaudiana plant is:

Kingdom : Plantae

Subkingdom : Tracheobionta

Superdivisio : Spermatophyta

Divisio : Magnoliophyta

Class : Magnoliopsida

Sub-class : Asteridae

Ordo : Asterales

Familia : Asteraceae

Genus : Stevia Cav

All sweeteners within the leaves of Stevia rebaudiana contain steviol (ent-13-

hydroxykaur-16-en-19-oic acid) as the sole aglycone contributor, which has two

hydroxyl groups, one attached to the C-19 of the C-4 carboxyl and the other attached

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to the C-13, both of which in theory can be glycosylated. Elaboration of the aglycone

steviol in Stevia rebaudiana by various glycosyltransferases creates up to at least

eight different steviol glycosides and may vary from 4% to 20% of the leaf dry

weight, depending on variety and growth conditions, although most field crops

contain around 10% by leaf dry weight. Besides sweet glycosides, sterebins A to H,

flavonoids, triterpenes, volatile oil constituents, pigments, gums, and inorganic matter

have been isolated from Stevia rebaudiana (Brandle et al., 1998).

2.4 Antioxidant

Antioxidants are compounds that have gained importance in recent years due

to their ability to neutralize free radicals (Devasagayam et al., 2004). Antioxidants

have been reported to prevent oxidative damage caused by free radicals. They can

interfere with the oxidation process by reacting with the free radicals, chelating

catalytic metals and also acting as oxygen scavengers (Buyukokuroglu et al., 2001).

The antioxidant compounds present in edible plants have recently been promoted as

food additives since they display little or no toxic side effects (Seong et al., 2004).

Many of the biologically active substances found in plants, including phenolic

compounds (flavonoids, phenolics) are known to possess potent antioxidant

properties. The antioxidant activity of medicinal plants depends on the concentration

of the individual antioxidant entering into the composition (Shukla et al., 2011).

Stevia leaf extract exhibits a high degree of antioxidant activity and has been

reported to inhibit hydroperoxide formation in sardine oil with potency greater than

that of either DL-α-tocopherol or green tea extract. The antioxidant activity of Stevia

leaf extract has been attributed to the scavenging of free radical electrons and

superoxides (Thomas and Glade, 2010). A recent study assessing the in vitro potential

of ethanolic leaf extract of Stevia rebaudiana indicates that it has a significant

potential for use as a natural antioxidant (Shukla et al., 2009). However there is still a

demand to find more information concerning the antioxidant potential of plant species

as they are safe and also bioactive. Therefore, in recent years, considerable attention

has been directed towards the identification of plants with antioxidant potential

(Shukla et al., 2011).

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Cioni et al (2006) examined the composition of the essential oil on the area

parts of five different Stevia rebaudiana genotypes from Brazil and Paraguay

cultivated in the coastal area of Tuscany (Italy). Forty different components were

identified and the main constituents in all the samples were spathulenol,

caryophyllene oxide, β-caryophyllene and β-pinene.

Yoda et al (2003) used supercritical fluid extraction with carbon dioxide to

investigate the active compounds in Stevia. They identified six classes of compounds,

i.e., sesquiterpenes, alcohols, labdanic diterpenes, aliphatic hydrocarbons, sterols and

triterpenes. In the investigated extract, the main compounds were diterpenes, of which

the most abundant was austroinulin. The other labdanic diterpene represented at a

higher level was jhanol. Significant portions of the contents of non-polar components

belonged to the hydrocarbons n-tetracosane and n-pentacosane.

2.5 Phenolic Compound

Phenolic compounds are secondary plant metabolites, which are important

determinants in the sensory and nutritional quality of fruits, vegetables and other

plants. (Tomas-Barberan et al., 2000). In vascular plants, more than 4000 phenolic

and polyphenolic compounds were the most antioxidants which have been identified,

namely, phenolic acids, tannins, coumarins, anthraquinones, flavonoids, phenolic

diterpens and anthocyanin (Middleton and Kandaswami, 1993; Trease and Evans,

1989), which have the ability to scavenge free radicals, donate hydrogen atoms or

electrons, or chelate metal cations (Pietta et al., 1998). A high correlation was

reported between the antioxidant capacity and total phenol and flavonoides contents

of plants (Silva et al., 2007; Tawaha et al., 2007). Besides antioxidant capacity,

phenolic compounds exhibit a wide range of biological activities, including anti-

carcinogenic, anti-inflammatory, anti-viral, anti-allergic, estrogenic, immune-

stimulating agents, antiallergenic, anti-artherogenic, anti-inflammatory, anti-

microbial, anti-thrombotic, anti-stress, anti-hyperglycemia, cardioprotective and

vasodilatory effects (Balasundram et al., 2006). Therefore, supplementing a food

product with antioxidant plant phenols may provide a health benefit as well.