OPTIMIZATION OF REBAUDIOSIDE A YIELD

FROM STEVIA REBAUDIANA CULTIVATED ON

BRIS SOIL AND ITS BIOLOGICAL ACTIVITIES

ASRUL AFANDI BIN AB HALIM

MASTER OF SCIENCE

2015

Optimization of Rebaudioside A Yield From Stevia

Rebaudiana Cultivated On BRIS Soil And Its Biological

Activities

by

Asrul Afandi bin Ab. Halim

A thesis submitted in fulfillment of requirements for the degree of

Master of Science

Faculty of Agro Based Industry

UNIVERSITI MALAYSIA KELANTAN

2015

i

THESIS DECLARATION

I hereby certify that the work embodied in this thesis is the result of the original

research and has not been submitted for a higher degree of any other University or

Institution.

OPEN ACCESS I agree that my thesis is to be made immediately

available as hardcopy or on-line open access (full text)

EMBARGOES I agree that my thesis is to be made available as hardcopy

or on-line (full text) for a period approved by the Post

Graduate Committee.

Dated from until .

CONFIDENTIAL (Contains confidential information under the Official

Secret Act 1972)*

RESTRICTED (Contains restricted information as specified by the

organization where research was done)*

I acknowledge that Universiti Malaysia Kelantan reserves the right as follows.

1. The thesis is the property of Universiti Malaysia Kelantan.

2. The library of Universiti Malaysia Kelantan has the right to make copies for the

purpose of research only.

3. The library has the right to make copies of the thesis for academic exchange.

SIGNATURE SIGNATURE OF SUPERVISOR

IC/ PASSPORT NO. NAME OF SUPERVISOR

Date: Date:

iv

ACKNOWLEDGEMENT

Alhamdulillah, praise to Allah S.W.T, because of His blessing and mercy, thus

I managed to complete this thesis. I would like to thank all those who made this thesis

possible and enjoyable experience for me.

Firstly, I would like to express my deepest gratitude and appreciation to my

supervisor, Dr. Shamsul bin Muhamad and my previous supervisor, Dr. Ranajit Kumar

Shaha for their guidance, constructive, encouragement and support throughout the

study of Maximization of yield of rebaudioside A from Stevia rebaudiana cultivated on

BRIS soil.

Special thanks to Mr. Mohamed, Mr. Suhaimi and Mr. Fathrio for their

guidance in the laboratory and help throughout of this study. My appreciation to my

friends especially all post graduate students for their support. My deepest gratitude to

my colleagues, Ms. Sarah, Ms. Ain and Ms. Mardiah for their favour and support

throughout this study. My appreciation to Mr. Hanif , Mr. Wahyudi Yusra and Ms.

Salmi for their kindness and support.

Finally, I would like to thank Universiti Malaysia Kelantan especially Faculty

of Agro Based Industry for the research facilities and Majlis Amanah Rakyat (MARA)

for the scholarship provided to me. My special thanks goes to the director, MARDI,

Bachok, Kelantan, for the collaborating research and supply S. rebaudiana leaves

(Variety MSR007 and MSR0012) at different time of intervals to carry out this

experiments. Last but not least, it is my pleasure to thank all others who have

contributed either directly or indirectly give their support and guidance.

Sincerely,

Asrul Afandi Ab. Halim

iii

TABLE OF CONTENTS

PAGE

THESIS DECLARATION i

ACKNOWLEDGEMENT ii

TABLE OF CONTENTS iii

LIST OF TABLES vii

LIST OF FIGURE viii

LIST OF ABBREVIATIONS ix

LIST OF SYMBOLS x

ABSTRAK xi

ABSTRACT xii

CHAPTER 1: INTRODUCTION

1.1 Background of Study 1

1.2 Significance of Study 2

1.3 Research Objectives 4

1.4 Scope and Limitation of Study 5

CHAPTER 2: LITERATURE REVIEW

2.1 Introduction 6

2.2 Species description and morphology 8

2.3 Phytochemical constituents 10

2.3.1 Steviol glycosides 10

2.3.2 Other Phytochemical constituents 15

2.4 Cultivation of S. rebaudiana 16

2.4.1 Crop cultivation on BRIS soil 16

2.4.2 Effect of photoperiod on biomass yield of S. rebaudiana 19

2.5 Extraction 21

2.5.1 Solvent extraction 21

2.5.2 Extraction of steviol glycosides 22

2.6 Analytical Method of Compound Analysis 24

iv

2.6.1 Thin Layer Chromatography (TLC) 24

2.6.2 High Performance Liquid Chromatography (HPLC) 25

2.7 Biological Activity 26

2.7.1 Antioxidants 27

2.7.2 Toxicity of S. rebaudiana Extract 29

2.8 World market 30

CHAPTER 3: MATERIALS AND METHODS

3.1 Materials and Chemical 32

3.2 Plant sample 32

3.3 Preparation of Samples 33

3.4 Proximate analysis of S. rebaudiana leaves 35

3.4.1 Estimation of moisture content 35

3.4.2 Estimation of ash content 36

3.4.3 Total sugars 36

3.4.4 Reducing sugar and Non-reducing sugars 37

3.4.5 Estimation of total soluble carbohydrate 38

3.4.6 Determination of total chlorophylls and carotenoids 39

3.5 Extraction of rebaudioside A from S. rebaidiana leaves 40

3.6 Purification and isolation of rebaudioside A 40

3.6.1 Partial purification of rebaudioside A 40

3.6.2 Isolation of rebaudioside A 41

3.7 Analysis of the purified compound 42

3.7.1 Identification and Quantification of rebaudioside A by HPLC 42

3.7.2 Identification of the compound by TLC Plate 43

3.8 Biological activity 46

3.8.1 Antioxidant activity 46

3.8.1.1 DPPH test expressed as the ability to scavenge DPPH 46

radical (IP %)

3.8.1.2 DPPH test expressed as vitamin C equivalent 47

3.8.1.3 ABTS test 48

3.8.2 Brine shrimp lethality test (BSLT) 48

v

3.9 Data Analysis 50

CHAPTER 4 : RESULTS

4.1 Introduction 51

4.2 Proximate analysis of dried S. rebaudiana leaves at different maturity 53

stage

4.2.1 Moisture, ash, reducing sugar, non-reducing sugar and total 53

carbohydrate content of S. rebaudiana leaves extract

4.2.2 Total pigments contents in S. rebaudiana leaves. 53

4.3 Optimized extraction of rebaudioside A 54

4.3.1 Effect of solvent polarity on the extraction of rebaudioside A 55

4.3.2 Effect of temperature on the extraction of rebaudioside A 60

4.3.3 Effect of material ratio on the extraction of rebaudioside A 61

4.3.4 Effect of extraction time on the rebaudioside A 63

4.3.5 Effect of number of extractions on rebaudioside A 64

4.4 Isolation and purification of rebaudioside A 64

4.5 Identification and quantification of rebaudioside A 67

4.5.1 Identification of rebaudioside A from different stage of 67

purification by Thin Layer Chromatography (TLC)

4.5.2 Identification and Quantification of rebaudioside A by High 69

Performance Liquid Chromatography (HPLC)

4.6 Bioactivity screening of purified rebaudioside A 74

4.6.1 Antioxidant activity analysis 74

4.6.2 Brine Shrimp Lethality Test (BSLT) 75

CHAPTER 5: DISCUSSION

5.1 Introduction 78

5.2 Proximate analysis 80

5.3 Optimization of rebaudioside A 84

5.4 Isolation and purification of rebaudioside A 87

5.5 Biological activities of S. rebaudiana extract 90

5.5.1 Antioxidant potential of S. rebaudiana 90

5.5.2 Cytotoxicity of S. rebaudiana extract 92

vi

CHAPTER 6: CONCLUSION

6.1 Conclusion 94

6.2 Recommendation for future work 95

REFERENCES 97

APPENDIX A 108

APPENDIX B 112

APPENDIX C 117

vii

LIST OF TABLES

NO. PAGE

2.1 Structural derivatives of stevioside and its related compounds and 14

sweetness intensity compared to sugar (sucrose)

2.2 Quantitative composition of rebaudioside A and stevioside in various 15

samples of S. rebaudiana leaves

3.1 Solvent system and visualizing agent for identification of compound 45

using TLC plate.

4.1 Chemical composition of dried three growing stages of mature S. 56

rebaudiana leaves

4.2 Chlorophyll (A and B), carotenoids, total pigment and rebaudioside A 57 contents of fresh and sun dried S. rebaudiana leaves (MSR007) at different growing stages.

4.3 Effect of polarity of solvent on extract yield of rebaudioside A (mg/g 59

dried leaves) from dried S. rebaudiana leaves ms007 and ms012.

4.4 Effect of sample/solvent ratio on rebaudioside A content at T=50◦C. 61

4.5 Effect of methanol extraction and purification on the recovery and purity of 66 rebaudioside A produced.

4.6 Antioxidant activities of the S. rebaudiana leaves purified extract. 74

4.7 Brine shrimp lethal toxicity test of crude and purified extract of S. 75

rebaudiana leaves.

viii

LIST OF FIGURES

NO. PAGE

2.1 Stevia rebaudiana (Bertoni) plant 9

2.2 Macroscopical Character: Morphological description of 10

S.rebaudiana

2.3 Chemical backbone of stevioside and rebaudioside A 13

2.4 Map of peninsular Malaysia showing BRIS soil along the 18

shoreline of Kelantan, Terengganu, Pahang and Johore.

3.1 Schematic diagram of isolation of rebaudioside A from the two 34

years old S. rebaudiana leaves

3.2 TLC plates with label 45

4.1 S. rebaudiana plant at MARDI, Bachok grown on BRIS soil 52

(a) Variety MSR007 (b) Variety MSR012

4.2 Chromatogram of (a) stevioside and (b) rebaudioside A from 58

standard solution

4.3 Effect of extraction temperature on rebaudioside A content by 60

absolute

4.4 HPLC Chromatogram of stevioside (Rt=3.3min) and rebaudioside 62

A (Rt=4.1min) of sample extract at different material ratio

4.5 Effect of extraction time in hour. (T=500C, 1:10 mass of dry leaves 63

powder: solvent ratio and three times extract)

4.6 Effect of number of extraction (constant T=500C and 1:10 mass: 64

solvent ratio)

4.7 TLC profiling of S. rebaudiana extract from different stage of 68 purification via mobile phase

4.8 HPLC chromatogram of crude extract of S. rebaudiana 70

4.9 HPLC chromatogram of partial purified extract 71

4.10 HPLC chromatogram of purified extract after re-Crystallization 72

4.11 HPLC chromatogram of commercial product 73

4.12 Determination of LC50 values 77

5.1 Schematic diagram of production of sweetener from S. 83

rebaudiana

ix

LIST OF ABBREVIATIONS

HPLC High Performance Liquid Chromotography

TSS Total Soluble Solids

DPPH 2,2-diphenyl-1-picrylhydrazyl

HCL Hydrochloric acid

TLC Thin Layer Chromatography

N Normal

NaOH Sodium Hydroxide

RPM Revolutions per minute

Na2SO3 Sodium Sulphate

FeCl3 Ferum Chloride

x

LIST OF SYMBOLS

cm Centimeter

% Percentage

g Gram

m Meter

mm Millimeter

mg Milligram

°C Degree celsius

ml Milimeter

µm Micrometer

nm Nanometer

µl Microliter

µg Microgram

(v/v) Volume over volume

(w/v) Weight over volume

xi

Pengoptimuman Hasil Rebaudioside A Daripada Stevia Rebaudiana Yang

Ditanam Di Tanah BRIS Dan Aktiviti Biologinya

ABSTRAK

Stevia rebaudiana adalah herba manis yang telah ditanam secara meluas di

serata dunia untuk diterpene glikosida. S. rebaudiana telah digunakan sebagai

pengganti untuk sukrosa, untuk rawatan penyakit kencing manis, obesiti, tekanan darah

tinggi dan mencegah kerosakan gigi. Antara diterpene glikosida itu, rebaudioside A

menunjukkan kepahitan yang paling rendah dan rasa yang menghampiri sukrosa.

Dalam kajian ini dua jenis variati S. rebaudiana iaitu MSR012 dan MSR007 diperolehi

daripada MARDI, Bachok, Kelantan yang ditanam di atas tanah bris telah dikaji untuk

memaksimumkan hasil rebaudioside A. Dalam usaha untuk mencari masa penuaian

yang terbaik S. rebaudiana, analisis proksimat telah dijalankan ke atas daun S.

rebaudiana pada peringkat kematangan yang berbeza, iaitu, sebelum berbunga (30-

35days), masa berbunga (50-60 hari) & lebih matang (70 - 90days) dari batang apikal

baru tumbuh dari pokok asal berumur 2 tahun. Kaedah pemisahan oleh pengekstrakan

pelarut diikuti oleh penghabluran semula rebaudioside A dalam bentuk amorfus telah

dilakukan untuk mendapatkan pemanis yang terdiri terutamanya sebatian bioaktif,

rebaudioside A. Kelembapan, abu, reducing sugar, non reducing sugar dan jumlah

karbohidrat didapati maksimum dalam variati MSR007 sebelum berbunga peringkat

dengan 8.45%, 7.17%, 4.45%, 8.45% dan 13.65% masing-masing. Peratusan

rebaudioside A didapati maksimum sebelum peringkat berbunga dengan 3.45% diikuti

oleh peringkat lebih matang dengan 2.20% dan semasa berbunga dengan 1.82%. Hasil

maksimum dicapai apabila serbuk daun yang telah diekstrak dengan metanol mutlak

pada suhu 500C dengan nisbah 1:10 (w / v) yang juga diulangi dua kali untuk tempoh

pengekstrakan satu jam. Keputusan penulenan menunjukkan bahawa kandungan

rebaudioside A maksimum yang diperolehi ialah 2.88% (g/100g daun). Tambahan

pula, ekstrak S. rebaudiana menunjukkan aktiviti antioksidan yang tinggi dalam

ekstrak mentah dan penurunan ketara kapasiti antioksidan (p<0.05) selepas peringkat

penulenan. Nilai-nilai IC50 untuk ekstrak mentah, ekstrak separa tulen, penghabluran

semula dan produk komersial adalah 2.9μg/ml, 19.459μg/ml, 135.219μg/ml dan

112.449μg/ml. Ujian ketoksikan menggunakan brine shrimp menunjukkan bahawa

semua peringkat penulenan S. rebaudiana yang diuji mempunyai LC50> 100 μg/mL

yang mungkin membuktikan S. rebaudiana adalah tidak beracun. Kesimpulannya S.

rebaudiana variati MSR007 daripada pemotongan apikal ditanam pada tanah bris

mempunyai potensi untuk dikomersialkan sebagai pengganti gula di Malaysia.

Penyelidikan lanjut mengenai proses untuk mengurangkan penggunaan bahan kimia

dan aliran sisa serta menimbangkan penggunaan bahan kimia selamat adalah sangat

diperlukan.

xii

Optimization of Rebaudioside A Yield From Stevia Rebaudiana Cultivated On

BRIS Soil And Its Biological Activities

ABSTRACT

Stevia rebaudiana is a sweet perennial herb which has been widely cultivated

through the world for the sweet diterpene glycosides. S. rebaudiana has been applied

as substitutes for sucrose, for treatment of diabetes mellitus, obesity, hypertension and

caries prevention. Among the diterpene glycoside, rebaudioside A showed the lowest

bitterness and the taste is close to sucrose. In this study two varieties MSR012 and

MSR007 obtained from MARDI, Bachok, Kelantan were studied to maximize the yield

of rebaudioside A from S. rebaudiana grown on BRIS soil. In order to find the best

harvesting time of S. rebaudiana, proximate analysis were done on S. rebaudiana

leaves from different maturity stage, i.e., before flowering (30-35days), flowering time

(50-60 days) & over mature (70- 90days) of new apical stem grown on 2 year old plant.

Separation method by solvent extraction followed by crystallization and re-

crystallization of rebaudioside A in amorphous form were done in order to obtain

sweetener which consist mainly bioactive compound, rebaudioside A. Moisture, ash,

reducing sugar, non-reducing sugar and total carbohydrate were found maximum in

variety MSR007 before flowering stage with 8.45%, 7.17%, 4.45%, 8.45%, and

13.65% respectively. Percentages of rebaudioside A were found maximum just before

flowering stage with 3.45% followed by over matured stage with 2.20% and during

flowering time with 1.82%. The maximum yield was achieved when the leaves powder

were extracted with absolute methanol at 500C with ratio 1:10 (w/v) which were also

repeated two times for one hour extraction duration. Results from the purification

indicate that the maximum rebaudioside A content obtained was 2.88% (g/100g

leaves). Furthermore, the S. rebaudiana extract shows high antioxidant activity in

crude extract and significant decrease of antioxidant capacity (p<0.05) after

purification stage. The IC50 values for crude extract, partial purified extract, 1st

re-

crystallization and commercial product were 2.9µg/ml, 19.459µg/ml, 135.219µg/ml

and 112.449µg/ml respectively. The brine shrimp toxicity assay test indicate that all of

the S. rebaudiana purified stage tested had LC50>100 µg/mL which might be suggested

that S. rebaudiana were practically non‐toxic. In conclusion S. rebaudiana variety MSR007 from apical cutting plants cultivated on BRIS soil has the potential to commercialize as sugar substitute in Malaysia. Further research on improving the

process to reduce the consumption of chemicals and waste stream as well as

considering the used of safe chemical were very demanding.

xiii

CHAPTER 1

INTRODUCTION

1.1 Background of Study

The demand for a sweetener substitute to sugar has increases every year

because of the surprisingly increasing diabetic patients (Marc, 2008). In the last

decades, the increasing of diabetic has encouraged people to take less sugar

content in their diets. Nowadays artificial sugars such as saccharine, aspartame,

sucralose, neotame, and acesulfame are used as substitute to sugar (Marc,

2008). These compounds are high intensity sweeteners, but they are reported to

have some toxic effect, so the people is now being focused on finding natural

sweeteners without health risk.

In the recent years Stevia rebaudiana, a non caloric sweetener has

found to have high potential to became substitute to sucrose (Madan et al.,

2010). S. rebaudiana (Family: Asteraceae) is commonly known as sweet

leaves, sweet herb or honey herb (Joshi et al., 2006). Similarly in Malaysia, it

has been called by several names such as daun manis, daun gula and tea manis.

According to Varanuj et al., (2009) S. rebaudiana have anti-

hyperglycemic, anti-hypertensive, diuretic, anti-tumor, anti-diarrheal, anti-

inflammatory which is also does not affect blood sugar metabolism. S.

rebaudiana also has the potential to reduces plasma glucose levels in normal

2

adults (Richard, 2006). The sweet compounds pass through the digestive

process of the body without broken the chemical structure, thus making S.

rebaudiana safe for consumption and will reduce the sugar content in human

blood (Tejo et al., 2013).

Toxicological studies have shown that S. rebaudiana extract does not

shows any sign of carcinogenic, mutagenic and teratogenic effects and no

allergic reaction for human consuption (Flamm et al., 2003; Geuns et al., 2003;

Brusick, 2008; and Carakostas et al., 2008). S. rebaudiana is safe and have

been applied in treatment of hypertention, diabetes, obesity, and caries

prevention (Jaroslav et al., 2007).

S. rebaudiana has been used as a low-calorie sweetener for years in

South America, Asia, Japan, and China, and in some countries of the European

Union. Currently, Japan and Korea are the largest markets for S. rebaudiana.

However, China is the largest S. rebaudiana grower in the world, and about

80% of their product is exported (Mohammed et al, 2014).

1.2 Significance of study

As a replacement sugar, S. rebaudiana have trapped more consideration

to the researchers. Though total chemical composition of S. rebaudiana is still

unavailable, a range of S. rebaudiana species has been studied by biochemists

and biotechnologists for its chemical constituents and out of 110 species, only

3

18 were found having sweet compound (Soejarto et al., 1982; Ahmed et al.,

2011).

Furthermore, Stevioside had slight bitterness, some astringency and

showed low acceptability but rebaudioside A showed the lowest bitterness and

astringency and highest acceptability (Abelyan et al, 2010). Hence, more

research interest to isolate these compounds. However, other Stevia glycosides

have very similar chemical structure as they have same diterpene skeleton and

slightly differ only in the types, quantities, and structural of glycoside moieties

(Yang et al, 2011). As a result, it is still difficult to obtain high purity

rebaudioside A at an industrial scale (Yang et al, 2011).

Despite remarkable advancement in extraction and purification of

natural products, it is still a challenging task in isolation of water soluble

compounds from plants without impurities. Many conventional

processes(chemical/physical) used long procedures in isolation and ion

exchange purification steps, thus leaving noxious residues and impurities in the

final product, which are responsible for the quality and taste of the sweet

glycosides (Kutowy et al., 1999; Zhang et al., 2000; Midmore& Rank, 2006;

JECFA, 2007; Abelyan et al., 2010). Repeated trials showed the past steps

cannot fully separate rebaudioside A from other Stevia glycosides, which have

very similar structures (Jackson et al., 2006).

Extensive researches had already been done regarding the isolation of

stevioside and rebaudioside A, but there are always more potential findings

that can be discovered. As of current time studies, there are no studies reported

4

on the potential isolation of sweetener from S. rebaudiana leaves grown in

Malaysia and also lack of studies on physiochemical properties of S.

rebaudiana grow on BRIS (Beach Ridges Interspersed with Swales) soil.

Moreover, Abdullateef et al., (2011) reported that Malaysia still lacks suitable

varieties and mass production technologies for commercialization purpose. The

target variety should naturally contain high rebaudioside A content.

Thus, this study was conducted to maximize the yield of rebaudioside

A from S. rebaudiana varieties grown on BRIS soil in Malaysia and shed more

light on the extraction and purification process of rebaudioside A from S.

rebaudiana. There is a need of improving the process for preparing an organic

certifiable S. rebaudiana extract with high sweetness which less costly, high

antioxidant and safe for consumption.

1.3 Research Objectives

Objectives of the study are:

i. To investigate the maximum nutritional properties and rebaudioside A

contents of S. rebaudiana grown on BRIS soil at different maturity

stage focusing on variety MSR007 and MSR012.

ii. To maximize the extracted yield of sweetened compound (rebaudioside

A) recovered during extraction and purification stage.

iii. To determine antioxidant capacity and toxicity of purified S.

rebaudiana extract.

5

1.4 Scope and limitation of study

The research focused on the S. rebaudiana plant from variety MSR007

and MSR012 which cultivated on BRIS soil at Telong, Bachok, Kelantan.

6

CHAPTER 2

LITERATURE REVIEW

2.1 Introduction

S. rebaudiana is a natural sweetener plant having medicinal and

commercial importance. It is belongs to the (Asteraceae) Compositae family.

Stevia is native to Paraguay and Brazil and it is often referred to as „„the sweet

herb of Paraguay‟‟. It is also known as “honey yerba”and “honey leaf” and by

some other variations of these names (Ahmed et al., 2011). The mature plant

grows up to 65-centimetres (26 inches) to as tall as 180 cm (72 inches) when

cultivated or growing naturally in fertile soil. It is a short day plant and

flowering from January to March in the southern hemisphere. It prefers a sandy

soil, requiring a warm sunny position. The suitable natural climate is

semihumid subtropical with temperature extremes from 21 to 43°C and

average 24°C (Huxley, 1992).

S. rebaudiana were originally from mountainous regions of Brazil and

Paraguay (Soejarto, 2002). For centuries, S. rebaudiana leaves were used by

the natives of Paraguay and Brazil to sweeten tea (Soejarto, 2002) and to mask

the bitter taste of various plant-based medicines and beverages (Elkins, 1997).

S. rebaudiana was rediscovered by Europeans in Paraguay in 1888 by Dr. M.

S. Bertoni. In 1905, Dr. bertoni named the plant in honor of Paraguayan

7

chemist Dr. Rebaudi (Midmore & Rank, 2002). Bertoni also describe about the

incredible sweetening power of the plant, which is superior to sugar and

saccharin because it is nontoxic and has significant therapeutic benefits. It

sweetens with high potency and can be used in its natural state (Elkins, 1997).

In 1931, French chemists extracted stevioside, the main sweet

component in the form of white crystalline compound. Japanese developed a

new method in 1970, which could better refine the stevioside contained in the

S. rebaudiana leaf (Elkins, 1997). As reported by Midmore & Rank, (2002),

Japanese started planting S. rebaudiana in 1971. Six years later they

commercialized and marketed a sweetener extracted from the S. rebaudiana

leaves.

S. rebaudiana has been carried to many countries and grown

commercially in Japan, Taiwan, Korea, Thailand and Indonesia (Mosettig et

al., 1963). It was introduced to Malaysia as a potential alternative natural

sweetener to sugar in the early year of 2000 (Raji et al., 2012).

The main stevioside producing countries are China and Paraguay. Japan

is the main user of Stevioside which supplied by China, the main S. rebaudiana

supplier (Elkins, 1999). As reported by Pankaj et al., (2010), over 80% of

World‟s S. rebaudiana leaf grown and supplied by China. In most other

countries S. rebaudiana were used directly, rather than commercially.

According to Midmore & Rank, (2002), domestic users utilize dried leaves,

liquid extracts, crystals or powder to their drinks and cooking as an “herbal”

supplement.

8

2.2 Species description and morphology

Stevia is a small perennial shrub with green leaves that belongs to the

aster (asteraceae) or chrysanthemum family of plants (Antonie et al., 2000).

According to Soejarto et al., (1983); Elkins, (1999), there are more than 200

species in the genus Stevia but S. rebaudiana was the only species which

has significant steviol glycoside (sweet compound) concentrations. Figure 2.1

and figure 2.2 shows the picture of S. reabudiana.

Normally S. reabudiana can achieve to a height of 60-70 cm. The

flowers are sessile, opposite to lanceolate leaves and leaves are serrated.

The flowers are white and arranged in irregular cyme small with the size (7-15

mm),. S. rebaudiana start flowering after the formation of minimum of four

leaves. The flowers are arranged in a cyme of corymbs with five white

tubular flowers. Corymbs are arranged in loose panicles (Goettemoeller et al.,

1999). The seed is an achene with a feathery pappus (Sekihashi et al., 2002).

According to Southward et al., (2004), S. rebaudiana can produce more than

500 inflorescences in a single plant. According to Kulasekaran et al., (2006),

the growth pattern of S. rebaudiana were divided into four stages;

germination, grand growth period, flowering and seed maturity.

There are about 90 varieties of S. rebaudiana scattered throughout

the world and were developed for different climatic requirements

(Kulasekaran et al.,2006). According to Zaidan et al., (1980), steviol

glycosides concentration is highest in leaves, followed by inflorescence and

9

stem. Normally S. rebaudiana leaves contain about 5-10 % stevioside and 3-

5% rebaudioside A of leaf dry weight (Brandle et al., 1992).

Figure 2.1: Stevia rebaudiana plant

Figure 2.2: Macroscopical Character: Morphological Description of S.rebaudiana

(Somporn, 2008).

10

2.3 Phytochemical constituents

S. rebaudiana is rich in terpenes and flavonoids (Geuns, 2003). The

phytochemicals present in S. rebaudiana are steviol, stevioside, austroinullin,

b-carotene, riboflavin, dulcoside, nilacin, rebaudi oxides, and thiamine

(Jayaraman et al., 2008). S. rebaudiana extracts were also contained sterebins

A to H, triterpenes, volatile oil, pigments, gums and inorganic constituents

(Geuns et al., 2003).

2.3.1 Steviol glycosides

Glycosides are compounds containing a carbohydrate molecule (sugar)

bound to a non-carbohydrate moiety. They are named specifically by the type

of sugar that they contain, as fructosides (fructose), glucosides (glucose) and

pentosides (pentose) (Bernal et al., 2011).

S. rebaudiana leaves naturally contain steviol glycosides which are

diterpenes as describe by Geuns, (2003). He successfully isolated and

identified 9 steviol glycosides from S. rebaudiana leaves namely stevioside,

steviolbioside, rebaudioside A, B, C, D, E, F and dulcoside. These

compounds, which are also known as S. rebaudiana sweeteners, are the

glycosides of the diterpenesteviol, ent-13-hydroxykaur-16-en-19-oic acid

(Chaturvedula et al., 2011). According to Shibata et al., (1995), the diterpenoid

11

glycosides have the same chemical backbone structure (steviol) but differ in

the residues of carbohydrate at positions C13 and C19.

As reported by Makapugay et al., (1984), Stevioside was the major

Steviol glycoside (4–13% w/w) found in the S. rebaudiana leaves. It is

followed by rebaudioside A (2–4% w/w), rebaudioside C (1–2% w/w) and

dulcoside A(0.4–0.7% w/w). Steviolbioside, rebaudioside B, D, E and

rebaudioside F were also identified in the leaf extracts, but as minor

constituents (Geuns et al., 2003). The molecular structure of stevioside

derivatives and its sweetness fold compared to sucrose is reported by Crammer

and Ikan, (1986); Geuns et al., (2003) which are enlisted in table 2.1.

Along with sweetness, S. rebaudiana leaves extract has some bitter

after taste. It is probably because of the presence of some tannins, essential oils

and flavonoids (Madan et al., 2010). Stevioside and rebaudioside A were also

responsible for the bitter after taste. However rebaudioside A is less bitter than

other steviol glycoside (Madan et al., 2010).

Steviol glycosides are highly stable in aqueous solutions with a broad

range of pH and temperature (Abou-Arab et al., 2010). Stevioside also

thermally stable at the temperature of below 80oC. Stevioside was decomposed

under strong acidic conditions (pH 1.0) after incubation at a temperature of

80oC for 2 hours (Abou-Arab et al., 2010). Stevioside possess excellent heat

stability up to100oC for 1 h ranging from pH 3 to pH 9, but rapid

decomposition occurs at pH level greater than 9 ( Kroyer, 2010).