IDENTIFICATION AND CHARACTERIZATION OF Stevia rebaudiana...
Transcript of IDENTIFICATION AND CHARACTERIZATION OF Stevia rebaudiana...
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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
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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.
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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
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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
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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
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B.6 Accumulation Heavy Metals in Stevia rebaudiana Extract by ICP-MS 176
Elan 9000
C Published 185
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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
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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
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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
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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
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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
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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.
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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).
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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.
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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).
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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.
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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.
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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.
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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.
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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
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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
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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.