CHAPTER 2

Review of
Literature
 Review of Literature

Stevia rebaudiana has been used throughout the world since ancient times for

various purposes such as a sweetener and a medicine. The literature has been retrieved up

to 2012 from various databases to reflect upon functional and sensory properties of high-

potency sweetener. Stevia is likely to become a major source of high-potency sweetener

for the growing natural food and therapeutic market in the future. Complete review on

stevioside is beyond the purview of this chapter; however brief description about various

aspects is presented below.

2.1. Stevia rebaudiana plant

Stevia is a genus of about 200 species of herbs and shrubs in the sunflower family

(Asteraceae). The Stevia genus comprises at least 110 species (Rajbhandari and Roberts

1983) but there may be as many as 300. Different species of Stevia contain several

potential sweetening compounds, with S. rebaudiana Bertoni being the sweetest of all

(Soejarto et al., 1982; Kinghorn et al., 1984).

2.1.1. Botanical description and Cultivation

It is a small perennial herb, with an extensive root system and brittle stems

producing small, elliptic leaves (Shock, 1982). It grows up to 1 m tall (Mishra et al., 2010)

as shown in Fig. 2.1. Stevia is a semi-humid subtropical plant that can be grown easily like

any other vegetable crop even in the kitchen garden. Stevia will grow well on a wide range

of soils given a consistent supply of moisture and adequate drainage. Its habitat extends

from the southwestern United States to the Brazilian highlands (Soejarto et al., 1982).

10
 Fig 2.1: Field photograph of Stevia rebaudiana Bertoni

It is cultivated as a perennial shrub in subtropical regions including parts of the

United States. The plant is indigenous to the northern regions of South America and grows

wild in the Highlands of Amambay and near the source of the river Monday (a border area

between Brazil and Paraguay). It is being cultivated in continental China, Taiwan,

Thailand, Korea, Brazil and Malaysia. Besides the above-mentioned countries, Stevia is

also grown in Israel, the Ukraine, the UK, the Philippines, Canada, Hawaii, California and

all over South America (Sivaram and Mukundam, 2003). Stevia must be cultivated as an

annual plant in mid-to high-latitude regions, where longer days favour leaf yield and

stevioside contents. Oddone (1997) considers clear seeds to be infertile. Poor seed

germination is one of the factors limiting large-scale cultivation. Poor percentages of

viable seeds in Stevia were reported (Duke 1993; Camerio et al., 1997). The soil should be

in the pH range 6.5-7.5; well-drained, red and sandy loam soil. Saline soils should be

avoided to cultivate this plant.

Propagation by seeds does not allow the production of homogeneous populations,

resulting in great variability in important features like sweetening levels and composition

(Nakamura and Tamura, 1985). Stevia is therefore usually propagated by stem cuttings
which root easily, but require high labour inputs. The vegetative propagation is further

limited by the lower number of individuals that can be obtained simultaneously from a

single plant. Due to the above-mentioned difficulties, tissue culture would be the best

alternative for rapid mass propagation of Stevia plants (Sivaram and Mukundam, 2003).

Publications have appeared describing the possibility of establishing the cultivation of S.

rebaudiana in additional countries such as Canada (Amzad-Hossain et al., 2010), the

Czech Republic (Nepovim et al. 1998), India (Goyal et al., 2010), and Russia (Dzyuba and

Vseross 1998).

There are about 90 varieties of S. rebaudiana developed all around the world

depending upon the different climatic requirements (Ibrahim et al., 2008; Singh and Rao,

2005). The land sites are ploughed and/or cultivated twice to prepare a fairly smooth firm-

planting surface. However, different climatic conditions would influence Stevia

cultivation, so it is advisable to carry out trials in each planting zone to establish adequate

plant population density for that particular area (Ramesh et al., 2006). The Stevia plants

appear to have low nutrient requirements; generally, the plant requires frequent shallow

irrigation. Normally, irrigation is applied at least one time per week, if the stem tips are

drooping (Kaushik et al., 2010).

Stevia has been successfully cultivated in recent years in many areas of Indian

states such as Rajasthan, Maharashtra, Kerala, Orissa and Punjab. The increasing demands

for natural sweeteners have driven the farmers in India toward large-scale Stevia

cultivation. Diterpene glycosides are the group of natural sweeteners that have been

extracted from Stevia. The quantity of dry leaves that can be harvested varies from 15 to

35 g per plant (Mishra et al., 2010). One planted hectare can produce between 1000 and

1200 kg of dried leaves that contain 6070 kg stevioside, which is considerable a low yield

compared to sugarcane or sugar beet. However, 70 kg stevioside, which is 300 times

1
2
sweeter than saccharose, is equivalent to a yield of 21,000 kg sugar per hectare (Serio,

2010).

2.1.2. In vitro propagation

Plant biotechnology offers a great opportunity to exploit plant cell culture

techniques to produce a whole range of secondary metabolites. However, the fact remains

that, at present success in production on a commercial scale has been achieved for only a

few compounds. Majority of the cultivars rely upon plants raised in tissue culture (micro

propagation) laboratories which includes optimization of a sub-culturing and rooting

medium for developing the explants. Generally, the explants are collected from several

days old in vitro generated S. rebaudiana. Different rooting mediums comprising of

optimum combinations of growth hormones (i.e. MS+IBA+NAA and MS+IBA) would be

checked for growth of roots. The best subculturing and rooting medium for the in vitro

culture of S. rebaudiana is followed in routine to raise thousands of plants in commercial

laboratories. The hardened plants are transferred into fields before the explants develop

into small plants (Debnath, 2008; Das et al., 2010).

2.2. Phytochemical constituents

The complete chemical composition of Stevia species is not yet available.

However, a variety of Stevia species has been tested for their chemical compositions. The

useful part of this shrub is the leaves. Out of 110 species tested for sweetness, only 18 were

found to possess this characteristic (Goyal et al., 2010).

2.2.1. Diterpene glycosides

Glycosides are compounds containing a carbohydrate molecule (sugar) bound to a

non-carbohydrate moiety. These compounds are mainly found in plants, and they can be

converted, by hydrolytic cleavage, into a sugar and a non-sugar component (aglycone).

13
They are named specifically by the type of sugar that they contain, as glucosides (glucose),

pentosides (pentose), fructosides (fructose), etc. (Bernal et al., 2011).

The natural sweeteners of Stevia leaves, called steviol glycosides, are diterpenes,

isolated and identified as stevioside, steviolbioside, rebaudioside A, B, C, D, E, F and

dulcoside (Geuns, 2003; Abou-Arab et al., 2010). Stevioside was reported to be the most

abundant stevia glycoside (510% of total dry weight) found in the plant leaves. It is

followed by rebaudioside A (24% w/w), rebaudioside C (12% w/w) and dulcoside A

(0.40.7% w/w) (Makapugay et al., 1984), as noted in Table 2.1 with other details of the

glycosides. Steviolbioside, rebaudioside B, D, E and F were also identified in the leaf

extracts, but as minor constituents (Geuns, 2003). On average, the sweetness of the steviol

glycosides is 250300 times greater than that of saccharose, with low water solubility and

high melting points (Crammer and Ikan, 1986).

The amount of the sweet glycosides present in the leaves of Stevia depends on

growing conditions (Pl et al., 2007b), as well as on the adoption of modern agronomical

techniques (Nepovim et al., 1998; Geuns, 2003). The content of rebaudioside B is

negligible in comparison to that of stevioside (Pl et al., 2007a). Conversely, purified

extracts obtained from Stevia leaves and offered on the market contain mainly stevioside

(>80%) or rebaudioside A (>90%) (Gardana et al., 2010). Some evidence exists that

rebaudioside B and steviolbioside are not native constituents of S. rebaudiana, but are

formed by partial hydrolysis during extraction (Prakash et al., 2008), being thus artifacts of

the extraction procedure (Kennelly, 2002).

 Table 2.1: Comparison of sweet glycosides present in S. rebaudiana

Diterpene Content (%) Relative Molecular Reference

glycoside sweetening Mass
power (g/mol)

Stevioside 5.0-10.0 250-300 804.87 Bridel and Lavieille,

1931
Rebaudioside A 2.0-4.0 350-450 967.01
Rebaudioside B <<1.0 300-350 804.87
Rebaudioside C 1.0-2.0 50-120 951.01 Sakamoto et.al.,
(Dulcoside B) 1977a
Rebaudioside D <<1.0 200-300 1129.15 Sakamoto et.al.,
1977b

Rebaudioside E <<1.0 250-300 967.01

Rebaudioside F <<1.0 ND 936.99
Steviolbioside <<1.0 100-125 642.73 Kohda et.al., 1976
Dulcoside A 0.4-0.7 50-120 788.87 Kobayashi et.al.,
1977
ND not defined

All diterpene glycosides isolated from S. rebaudiana leaves have the same steviol

backbone (Fig. 2.2) and differ mainly in the content of carbohydrate residues, mono-, di-,

and trisaccharides containing glucose and/or rhamnose at positions C13 and C19 (Kochikyan

et al., 2006). The sweetness of rebaudiosides increases with increasing amount of sugar

units bonded to the steviol aglycone. However, their content in the plant material decreases

at the same time (Kovylyaeva et al., 2007). The edulcorant properties of those glycosides,

however, differ from one another.

 H OH HO OH
O
O H H H
HO H H H O
H
H OH OH
OH O H
HO OH OH
HOH O
H OH
O H O
H H
H H
H OH H
OH
O HO OH
O
H
H H
H
HO OH O HO OH H HO HO
O O
O O O

H H H
H H H
H
H
HO H HO HO
OH

Stevioside Rebaudioside A

HO OH
O
H H H
H O
OH
H H OH OH H
H H
OH
OH OH O O
H O
H H
OH
O H
O H O H HO H
H H
HH H
OH H
OH H HO
H OH
O

HO
O

HO
Dulcoside Rebaudioside B
A Dulcosidde A
HO OH
O
H H
H
O
H
OH
OH H
OH H HO OH O
OH H H
OH
H H
H O O O H H H
H H O H
O H
H CH3 OH
H O HO OH
OH O
O H
O H H H
OH H
O H HO
OH
HO OH
O O
H H
H
O H
H
HO
HO OH
O O HO OH O
O
H
HH H H
OH H
OH H OH H
OH
Dulcoside B
Rebaudioside D
 HO OH
O
H H H
O
H
HO OH
H
OH
H O
OH
O H
H H H

OH O
HO O O

H H
H
H H
OH
HO OH O O
H H
H
H OH H
OH

Rebaudioside E

Fig. 2.2: Chemical structure of major glycosides found in S. rebaudiana Bertoni

2.2.2. Structure and properties of Stevioside

Stevioside has the chemical formula of a diterpene glycoside (C38H60O18) and as an

active component in Stevia leaves is responsible for the edulcorant properties. Structurally,

stevioside (13-[2-O-D-glucopyranosyl--glucopyranosyl) oxy] kaur-16-en- 19-oic-acid -

D- glucopyranosyl ester) is a glycoside with a glucosyl and a sophorosyl residue attached

to the aglycone steviol, which has a cyclopentanonhydrophenanthrene skeleton. In

addition to stevioside, several related sweet compounds such as steviobioside, rebaudioside

A, B, C, D, E and ducoside A were isolated from S. rebaudiana leaf. All other isolated

diterpenoid glycosides possess an ent-kaurene diterpene steviol skeleton (ent-13-hydroxy

kaur- 16-en-19-oic acid) but differ in the residues of carbohydrate at position C13 and C19

(Kenelly, 2002; Kolb et al., 2001). Stevioside and its specification were shown in Fig 2.3.
 What is Stevioside?
Specification
White crystalline powder, plant-derived high-
potency sweetener

Potency 200-300X sugar

Stevioside content >97% dry basis
Other glycosides <3%
Empirical formula C38H60O18
Molecular weight 804.88
Density 0.390 to 0.420
g/ml 5

R1:Glu; R2: two Glu unit

Fig 2.3: Structure of Stevioside and its specification

Stevioside, the most abundant steviol glycoside in the leaf of the plant, has become

well known for its intense sweetness (250300 times sweeter than solutions containing

0.4% saccharose), and is used as a non-caloric sweetener in several countries

(Chatsudthipong and Muanprasat, 2009; Gardana et al., 2010). Its use has been approved

in Brazil, Argentina and Paraguay as well as in China, Korea and Japan. These molecules

are highly stable in aqueous solutions within a broad range of pH and temperature

(Virendra and Kalpagam, 2008; Abou-Arab et al., 2010). Stevioside show remarkable

stability in aqueous solution over a wide range of pH values and temperatures. Under

thermal treatment in a pH range of 110 over 2 h at 60C, hardly any degradation of

stevioside was observed with slight losses up to 5% (pH 2 and 10) were determined on

heating to a temperature of 80C. Under strong acidic conditions (pH 1.0) forced

decomposition of stevioside was observed which resulted in total decomposition after

incubation at a temperature of 80C for 2 h (Abou-Arab et al., 2010). Similar results were
observed by Buckenhuskers and Omran (1997) who showed that the stevioside possess

excellent heat stability up to 100C for 1 h at pH range 39, but rapid decomposition

occurs at pH level greater than 9 under these conditions. It has been reported recently that

stevioside was thermostable even at 200C, making them suitable for use in cooked foods

(Lemus-Mondaca et al., 2012).

2.2.3. Other constituents

S. rebaudiana Bertoni contains stigmasterol, b-sitosterol and campesterol

(DAgostino et al., 1984). In addition to these compounds, Stevia extracts were also

reported to contain flavonoids, sterebins A to H, triterpenes, volatile oil components,

pigments, gums and inorganic constituents (Geuns, 2003). These other secondary plant

constituents include labdanes, sterols, triterpenoids, chlorophylls, organic acids, mono-

disaccharides, and inorganic salts (Gardana et al., 2010). Tadhani and Subhash (2006a)

subjected leaves of S. rebaudiana to qualitative phytochemical screening for the

identification of various classes of active chemical constituents. The presence of

biologically important secondary plant products in Stevia leaf contributes to its medicinal

value, since they exhibit physiological activity (Sofowara, 1993). The most important of

these bioactive compounds of plants are alkaloids, flavonoids, tannins and phenolic

compounds (Edeoga et al., 2005). Savita et al. (2004a) found a high percentage of anti-

nutritional factors in extracts of Stevia leaf dissolved in water: oxalic acid and tannins,

2295.0 and 0.010 mg/100 g respectively. Oxalic acid may hinder the bio-availability of

calcium, iron and other nutrients as in the case of green leafy vegetables. Tannins have

been reported to have several pharmacological activities such as spasmolytic activity in

smooth muscle cells (Tona et al., 1999). It has also been reported that they have free

radical scavenging properties (Bharani et al., 1995) and antioxidant activity. Saponins,

which are amphipathic glycosides, have also been studied in seeds, plants and cereals.
19
Saponins can stimulate muscle growth and raise testosterone levels and they can also show

anti-bacterial, immunological and anti-diabetic properties (Bernal et al., 2011).

According to Sharma et al. (2006), the fresh Stevia leaves contain a large amount

of water between 80 and 85%. The other constituents present in Stevia were ascorbic acid,

-carotene, chromium, cobalt, magnesium, iron, potassium, phosphorous, riboflavin,

thiamin, tin, zinc and so forth. The other chemicals found in Stevia include apigenin,

austroinulin, avicularin, -sitosterol, caffeic acid, compesterol, caryophyllene,

centaureidin, chorogenic acid, chlorophyll, cynaroside, daucosterol, di-terpene glycoside,

dulcosides A and B, foeniculin, formic acid, gibberellic acid, gibberellin, indole-3-

acetonitrile, isoquercitrin, isosteviol, kaempferol, kaurene, lupeol, luteolin,

polysatachoside, quercetin, quercitrin, scooletin, stigmasterol, umbelliferone and

xanthophyllus (Fujita et al., 1977; Yoda et al., 2003).

Other chemicals with no sweet taste are also found in Stevia species and some may

even be bitter in taste. Stevisalioside A (from the roots of Stevia salicifolia) (Mata et al.,

1992), longipinane derivatives in the roots of Stevia connata (Sanchez-Arreola et al.,

1995), epoxylabdane diterpenes and a clerodane derivative in the leaves of Stevia

subpubescens (Roman et al. 2000), flavonoids from the leaves of S. rebaudiana (Soejarto

et al., 1982), Stevia nepetifolia (Rajbhandari and Roberts 1983), Stevia microchaeta, Stevia

monardifolia, Stevia origanoides (Rajbhandari and Roberts 1985) and Stevia procumbens

(aerial parts) (Sosa et al., 1985) and sesquiterpene lactones from the aerial parts of S.

procumbens and the leaves of S. origanoides (Calderon et al., 1987) are in this group.

2.3. Extraction of stevioside

In the preceding paragraph, conventional approach (comprising physical and

chemical processes) followed for the extraction of stevioside is discussed.

2.3.1. Conventional approach

Conventionally the leaves of S. rebaudiana Bertoni are ground, defatted, treated

with an organic extractant, filtered, the resultant filtrate reduced to syrup and the syrup

thereafter treated by one or more steps to form crystals of stevioside (Persinos, 1973). The

extraction patents have been categorized into those based on solvent (Haga et al., 1976),

ion exchange (Unesh et al., 1977), adsorption chromatography (Itagaki and Ito, 1979) and

solvent plus a decolorizing agent (Ogawa et al., 1980). An improved method for the

recovery of stevioside from S. rebaudiana plants is provided which does not require the

use of dangerous chemicals or separation equipment such as ion exchange

chromatography. In this process, the raw material, preferably in comminuted form, is first

extracted with water. The resulting aqueous extract is treated with a di- or tricarboxylic

acid chelating agent to remove metallic and other impurities. Subsequently, a calcium

containing agent is added to precipitate out other impurities. The aqueous extract is

essentially neutralized with an acid and is then subjected to extraction with a water-

immiscible solvent. Purified stevioside crystals are recovered by cooling the water layer

obtained from said solvent extraction step. Other conventional extraction methods for

stevioside from S. rebaudiana leaves involve aqueous or alcohol extraction followed by

precipitation, coagulation and crystallization (Phillips, 1989). The most favoured extraction

processes involve four steps: aqueous or solvent extraction, ion exchange, precipitation or

coagulation with filtration, then followed by crystallization and drying. A process

involving pre-treatment of the extract with lime and the use of ion exchange columns have

been reported (Giovanetto, 1990).

A Japanese patent provides information on supercritical fluid extraction with

carbon dioxide and co-solvents such as methanol, ethanol and acetone for the production of

Stevia glycosides (Tan et al., 1983). A similar US patent includes extraction of plant parts
of S. rebaudiana with a solvent to provide an extract and subjecting the extract to an

extraction with a supercritical gas to obtain an extraction residue which is freed from

undesired and taste-impairing constituents (Kienle, 1992). The quality of the glycosidic

fraction with respect to its capacity as sweetener was better for the SCFE (Supercritical

Fluid Extraction) with CO2 and a co-solvent as compared to the extract obtained by the

conventional process (Pasquel et al., 2000). Selective adsorptions on zeolites X and A was

studied with S. rebaudiana extract. Ionic exchange was made with calcium and barium

ions in zeolites NaX and NaA and the effect of contact of the aqueous extract of dry S.

rebaudiana leaves with CaX, BaX, CaA and BaA zeolites was evaluated. Stevia extract in

contact with the zeolite CaX showed the highest clarification (Moraes and Machado,

2001). S. rebaudiana contains glycosides which are insoluble in carbon dioxide and

soluble in mixtures of carbon dioxide and a polar solvent. The glycosides from Stevia

leaves were extracted using supercritical fluid extraction by a two-step process: (i) CO2

extraction at 200 bar and 30C, and (ii) CO2 plus water extraction. The chemical

compositions of the extracts were analyzed by GC-FID, GC-MS, TLC and HPLC. The

overall extraction curves for the system Stevia+CO2 had a typical shape and were

successfully described by the Sovova's model (Yoda et al., 2003).

Pressurized fluid extraction using water or methanol was employed for the

extraction of stevioside from S. rebaudiana. Methanol showed better extraction ability for

isolation of stevioside from Stevia leaves than water within the range of 110160C.

However, water represents the green alternative to methanol (Pol et al., 2007a,b). Modern

extraction techniques such as pressurized liquid extraction, pressurized hot water

extraction (PHWE), supercritical fluid extraction and microwave assisted extraction

(MAE) have been used for extracting bioactive compounds (Kovylyaeva et al., 2007). The

glycoside composition of S. rebaudiana leaves was optimized using supercritical fluid

extraction (SFE). A Box-Behnken statistical design was used to evaluate the effect of

various values of pressure (150350 bar), temperature (4080C) and concentration of

1
ethanol-water mixture (70:30) as co-solvent (020%) by CO2 flow rate of 15 g min for 60

min. The most effective variables were found to be co-solvent concentration (P<0.005) and

temperature (P 0.005) for stevioside extraction (Erkucuk et al., 2009). On using the

ultrasound assisted extraction, the yield of extracts increased by a factor of 1.5 versus

classical extraction procedures. The results indicated the optimal extraction conditions to

be an extraction temperature of 68C, a sonic power of 60 W, and an extraction time of 32

min (Liu et al., 2010). The work done on the extraction of stevioside in the last decade is

summarized in Table 2.2.

Table 2.2. Comparison of different methods used for Stevioside extraction from S.
rebaudiana
Method Yield Treatment Reference
Time (min)
a c
Supercritical Fluid Extraction 2.51 Nd Choi et. al., 2002
a
SFE (CO2- methanol, 80:20) 3.56
SFE (CO2-methanol-water, 2.36
80:16:4)
Organic solvent
a d
Chromatographic Separation 0.06 24 Pol et. al., 2007a
(Hexane, dicholroethane, ethyl
acetate, methanol)
Pressurized Fluid Extraction 50 Pol et. al., 2007b
(PFE)
b
Methanol 5.2
b
Water 4.7
a
PHWE (Pressurized hot water 13.90 50 Teo et. al., 2009
extraction) 20
a
MAE (Microwave assisted 21.37
extraction)
b
Microwave 8.64 1 Jaitak et.al., 2009
b
Ultrasound 4.20 30
a
Supercritical fluid extraction 36.66 100 Erkucuk et al.,
(SFE) 48.6 2009
Ethanol
b
Ultrasonic assisted extraction 43.62 32 Liu et. al., 2010
a b c d
mg/g, %, not defined, h.
 A rapid and efficient method has been developed for the extraction of stevioside

and rebaudioside A in optimum yields using a microwave assisted extraction (MAE)

procedure. Conventional cold extraction was performed at 25C for 12 h while ultrasound

extraction was carried out at a temperature of 355C for 30 min. MAE was carried out at

different power levels ranging from 20 to 160W with extraction time ranging between 30 s

and 5 min with a temperature range of 1090C. MAE had an optimum yield at a power

level of 80W for 1 min at 50C (Jaitak et al., 2009). The proposed methods, namely

pressurized hot water extraction (PHWE) and microwave assisted extraction (MAE),

showed that stevioside and rebaudioside A could be extracted at an elevated temperature

using water without the addition of an organic modifier/solvent. It was observed that the

extraction efficiency of PHWE and MAE was comparable or higher compared to heating

under reflux. The main parameters which influence its extraction efficiency are

temperature, extraction time, flow rates and addition of modifiers/additives. In case of

stevioside, the extraction was carried out at 100C for 15 min and the flow rate was set at

1.5 mL/min (Teo et al., 2009). All of the above processes have complex steps and contain

no information about the effect of extraction method on the contents of stevioside and the

yield obtained.

2.3.2. Limitations/drawbacks of conventional approach

Although several different systems have been operated, as discussed in the

foregoing paragraphs, certain limitations of conventional methods for stevioside extraction

have come to the forefront.

(i) The raw material cannot be used as such; prior treatment is required.

(ii) Adsorption columns are usually regenerated with dilute alkali solution and

this may affect organoleptic properties and final quality of compound.

(iii) The chemicals used in certain cases cannot be recycled, thereby rendering
 extraction and disposal of pollutant cost-prohibitive.

(iv) The methods through chemical reaction(s) may alter composition of the

stevioside.

(v) The methods introduce batch-to-batch variation.

(vi) The methods of extraction and finishing affect the yield, quality and

characteristics of stevioside produced.

(vii) Emergence of bitter aftertaste with the use of chemically extracted

stevioside.

(viii) The heated water was not able to penetrate into the sample core to release

more target compounds and thus resulted in lower extraction efficiency.

(ix) The capital cost investment requirement for equipment like solvent recovery

used in fluid extraction.

(x) The high energy requirement in elevating temperature during hot water

extraction.

Because of the various drawbacks, the capabilities of non-enzymatic extraction

technologies are limited.

2.3.3. Enzymatic extraction

Currently, procedures for isolation of stevioside from S. rebaudiana leaves on a

pilot-scale mostly involve liquid extraction with such solvents as chloroformmethane,

glycerol and propylene glycol, followed by refinement with polar organic solvents

(Chatsudthipong and Muanprasat, 2009). The mentioned limitations/drawbacks (listed in

Section 2.3.2) warranted the necessity to look for innovative technique(s) using

biotechnological approaches for stevioside production.

Enzymes are ideal catalysts to assist in the extraction, modification or synthesis of

complex biofunctional substances of natural origin. With the successful applications of

25
enzymes in the extraction of carotenoids from marigold flower (Barzana et al., 2002),

vanillin from vanilla green pods (Ruiz-Teran et al., 2001), polysaccharide from sterculia

(Wu et al., 2007), oil from grape seed (Passos et al., 2009) and polyphenols (Yang et al.,

2010), it appeared feasible and cost effective to develop comparable processes for

stevioside production. We recently optimized the use of enzymes for increasing release of

flavonoids from citrus peel waste (Puri et al., 2011b), thus paving a way for improved

stevioside extraction. In addition, enzymatic processes also minimize pollution during

processing. Further, enzyme systems have resulted in yield improvement when used in the

vegetable and fruit waste processing (Kaur et al., 2010).

Enzyme-assisted extraction aids cell wall degrading enzymes such as cellulase and

pectinase to break down the plant cell wall, thus rendering intracellular materials more

accessible for extraction. To minimize the usage of organic solvents, enzymatic extraction

offers a feasible green option. This has worked in many cases where molecules closely

related to stevioside have been successfully extracted. For an example, lycopene is a

natural carotenoid pigment and a high value nutraceutical having wide use. Enzyme-aided

extraction of lycopene from whole tomatoes under optimised conditions resulted in an

increase in the lycopene yield (Choudhari and Ananthanarayan, 2007). The valuable

quantities of lycopene pigment in tomatoes, which is lost as waste in processing, could be

recovered in high yields by extraction using cellulases and pectinases. Additionally,

grapefruit peel waste is usually dried, pelletized and sold as a low value cattle feed.

Cellulose, pectin, and hemicellulose in grapefruit peel waste can be hydrolysed by

pectinase, rhamnosidase and cellulase enzymes to monomeric sugars, which can then be

used by microorganisms to produce ethanol and other fermentation products (Wilkins et

al., 2007; Puri et al., 2011b,c,2012). A more in depth knowledge of the polysaccharide

structure of the vegetal substrate and the use of more specific enzymes would contribute to

26
the unravelling and better penetration of the enzymes to the active sites. The use of

hydrolytic enzymes such as cellulases, hemicellulases and pectinases and their

combinations during maceration of the plant material before the extraction facilitate

breaking of plant cell wall thereby enhancing the extraction of metabolites from plants. We

have recently observed that enzyme treatment disrupts the pectincellulose complex in

citrus peel powder and enhances flavonoid production (Puri et al., 2011c).

Enzymes commonly used in the food industry catalyse a variety of hydrolytic

reactions and a high percentage act on cell wall polymers improving extraction yield of

juices, oils and sugars. In food processing, protease enzymes are industrially employed in

the extraction, clarification and concentration of blackcurrant juices (Landbo et al., 2006),

extraction of pectinpolysaccharide from mangosteen (Gan and Latiff, 2011), flavours and

pigments from plant materials (Sowbhagya and Chitra, 2010). Enzyme incorporation in oil

extraction processes produces a high content of antioxidant compounds in olive oil

(Aliakbarian et al., 2008), defatted meal of borage oil (Soto et al., 2008). The quality of

oils obtained by enzyme treatment is better than the hexane-extracted oils. An increase of

phenolic compounds extracted from citrus peel and grape pomace by means of enzyme

incorporation has recently been observed (Li et al., 2006). Enzyme assisted extraction of

protein from soybean has recently been regarded as a green alternative to hexane

extraction (Campbell and Glatz, 2010). Additionally, the digestion using trienzyme

including protease, -amylase and conjugase (glutamyl hydrolase) has recently been

optimized for the extraction of foliate from cereal grain products using response surface

technology. The method resulted in shortened digestion timings, cost saving and

minimized the foliate loss possible with prolonged digestions (Cho et al., 2010).

Such studies have not been investigated on stevioside. So our work was focussed

on the feasibility of enzyme-assisted extraction of stevioside in giving better yields as

compared to the solvent (conventional) method of extraction.

2.4. Estimation and purification of stevioside

2.4.1. Method of Estimation

The glucose units hydrolysed from the stevioside upon acid hydrolysis which take

part in the anthrone reaction and read at 625nm. The concentration of glucose was

measured against glucose standard and was multiplied by a factor of 1.64 (on the basis of

molecular weight). This estimation method using glucose standard plot was given by

Metivier and Viana (1979). A wide range of analytical techniques have been employed to

assess the distribution and level of sweet diterpenoid glycosides in S. rebaudiana. These

include thin layer chromatography (Tanaka, 1982), droplet counter-current

chromatography (Kinghorn et al., 1982), over-pressured layer chromatography (Fullas et

al., 1989) and capillary electrophoresis (Liu and Li, 1995).

A simple enzymatic method is described for the determination of stevioside in S.

rebaudiana. The method is based on the hydrolysis of stevioside with crude hesperidinase.

The reaction is followed by monitoring the production of glucose with a glucose oxidase

peroxidase-2, 2-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) system (Mizukami et al.,

1982).

2.4.2. Purification of stevioside

The different techniques used for purification of stevioside can be classified in

various categories, those based on solvent extraction (Morita et al., 1978; Bondarev et al.,

2001), chromatographic adsorption (Ahmed and Dobberstein, 1982; Makapugay et al.,

1984; Striedner et al., 1991; Kolb et al., 2001), ion exchange (Fuh and Chiang, 1990;

Giovanetto, 1990; Payzant et al., 1999), selective precipitation (Kumar, 1986), membrane

processes (Fuh and Chiang, 1990; Giovanetto, 1990; Shi et al., 2000) and supercritical
28
fluids (Kienle, 1992).

The most common analytical method has been high performance liquid

chromatography. However, separation has also been achieved by using silica gel

(Nikolova-Damyanova et al., 1994), hydroxyapatite (Kasai et al., 1987), hydrophilic

(Hashimoto et al., 1978), and size exclusion (Ahmed and Dobberstein, 1982)

chromatography procedures. Stevioside and rebaudioside A have also been analyzed by

HPLC after conversion to their bromophenacyl esters (Ahmed et al., 1980). Amino

columns have also been used to measure stevioside and related glycosides in foods and

beverages (Chang and Cook, 1983). An improved analytical method was developed which

may be applied to (a) quality control of stevioside and rebaudioside A contents in dried

leaves of S. rebaudiana before processing; (b) searching for plants of higher yield in

diterpene glycosides content; or (c) when a large number of samples are sent to the

laboratory for analysis. The procedure developed involves two steps: solvent extraction

followed by an isocratic HPLC analysis (Kolb et al., 2001).

A simple method for stevioside analysis based on hydrophobic chromatography

(Sep-Pak C18 cartridges) was described where a linear gradient of acetonitrile in water

(Vanek et al., 2001). Optimal conditions were developed for the HPLC-ESI-MS method

and supercritical fluid extraction of stevioside from S. rebaudiana leaves. The extraction

yield by CO2methanolwater (80:16:4) was found to be 150% improved versus

conventional organic extraction (Choi et al., 2002). A simple and highly sensitive reversed

phase HPLC method (RP-HPLC) has been developed for the determination of steviol using

dihydroisosteviol (DHISV) as internal standard (IS) (Minne et al., 2004).

Rank and Midmore (2006) classified the refining methods of stevioside into solvent

partition extraction mainly methanol or water extraction and solvent partition extraction,

incorporating mainly in situ precipitation with calcium hydroxidecarbon dioxide to

29
remove impurities, similar to the purification process in the sugar industry. They also

reported different methods of purification, such as adsorption, chromatography, ion-

exchange, plasmid gel or adsorption by activated carbon. Stevioside profiles in ex vitro and

in vitro leaves and callus cultures of S. rebaudiana were investigated by HPLC analysis

and further confirmed by MS using mass spectral fragmentation data obtained from LC-

MS-ESI studies. The studies confirmed the biosynthesis of stevioside in callus and

suspension cultures (Rajasekaran et al., 2008).

A high-performance thin-layer chromatographic (HPTLC) method was developed

for simultaneous quantification of three steviol glycosides, i.e. steviolbioside, stevioside

and rebaudioside A in S. rebaudiana leaves. For achieving good separation, mobile phase

of ethyl acetate: ethanol: water (80:20:12, v/v/v) on pre-coated silica gel 60 F254 HPTLC

plate has recently been used. The densitometric quantification of steviol glycosides carried

at =510 nm in reflectionabsorption mode showed better results after spraying with acetic

anhydride: sulphuric acid: ethanol reagent. The calibration curves were linear in the range

of 160960 ng/spot for steviolbioside, 16 g/spot for stevioside and 0.53 g/spot for

rebaudioside A (Jaitak et al., 2009). Recently, a semi-quantitative determination of steviol

glycosides was also performed by desorption electrospray ionization mass spectrometry

(Jackson et al., 2009).

Three steviol glycosides, stevioside, rebaudioside A and rebaudioside C were

successfully isolated and purified from the extract of leaves of S. rebaudiana by high-

speed counter-current chromatography (HSCCC) (Huang et al., 2010). Based on the

principle of the partition coefficient values (k) for target compounds and the separation

factor between targets compounds, the two-phase solvent system, which contains n-

hexane-n-butanol-water at an optimized volume ratio of 1.5:3.5:5 (v/v/v), was selected for

the HSCCC separation. The lower phase was employed as the mobile phase in the head to
tail elution mode. In a single operation 200 mg of the crude extract yielded pure stevioside

(54 mg), rebaudioside A (36 mg) and rebaudioside C (13 mg).

The determination of stevioside, rebaudioside A and steviol was carefully pursued

through different methods as indicated in the scientific literature, including enzymatic

hydrolysis and chemical detection, GC, overpressure TLC, densitometry, HPLC and

capillary electrophoresis (Gardana et al., 2010). HPLC technology and a near infrared

(NIR) spectroscopy model were established to directly measure the stevioside glycosides

(rebaudioside A and stevioside) content in the leaves of S. rebaudiana Bertoni. This model

can be applied directly to measure the content of rebaudioside A and stevioside in the

leaves of S. rebaudiana Bertoni, and resolved the problem of high cost and complex

operation in using the current chemical laboratory methods (Yu et al., 2011).

Table 2.3: Different methods of stevioside estimation

Method Details References

TLC Chloroform-methanol-glacial Sherma and Norfolk., 1992
acetic acid
(90:60:12)
Hydrophobic
chromatography Sep-Pak C18 cartridges Vanek et al., 2001
HPLC
HPLC-ESI-MS CO2+Methanol+Water (80:16:4) Choi et al.,2002

RP-HPLC DHISV as internal standard Minne et al., 2004

HPLC NH2-MS (4.6mm150mm5m) Kolb et al., 2001;

column, with a mobile phase Erkucuk et al., 2009;
comprised of water and Teo et al., 2009
acetonitrile (mention ratios).
C18 (15063.9 mm, 5 ml),the
mobile phase comprised water
and methanol (what ratios??).
Jaitak et al., 2009
HPTLC Ethyl acetate: ethanol: water
(80:20:12, v/v/v)
2.5. Safety aspects of stevioside

One of the most obvious indications of the safety of Stevia is that there have never

been any reports of ill effects in over 1500 years of continuous use by Paraguayans

(Kroger et al., 2006). The first official investigation of possible toxicity from Stevia was

performed in South America (Pomaret and Lavieille, 1931). Stevioside, when passed

through the human alimentary canal, wasnt altered by digestive processes. However,

stevioside and rebaudioside A were both degraded to steviol by rat intestinal microflora

(Wingard et al., 1980). Steviol is one of the metabolites that could do serious harm to

humans. The stevioside was incubated for 24 days in a specially prepared solution

containing the contents of the rat cecum. Under these conditions, conversion was almost

100%. However, as Kinghorn and Soejarto (1985) have pointed out, there are two things

wrong with extrapolating these results to humans. First, humans do not have a cecum, as

does the rat; therefore, a critical step in the conversion process has no equivalent physical

location in which to occur. Second, there is no good reason to believe that the microflora of

the human intestinal tract contain the same microorganisms as does the rat cecum.

However, various concerns have been expressed over the safety of Stevia.

Stevioside has been used to treat a range of ailments, including diabetes, high blood

pressure, digestive disorders and skin defects by indigenous populations in various

countries. These are incredible claims made in the literature which are not based on peer

review process, however needs validation by conducting robust analysis. Its use as a

sweetener in herbal preparations can provide a new vista to the pharmaceutical industry,

i.e., a product considered to be sweet, acceptable and palatable by diabetic consumers.

A comprehensive review on the toxicity of stevioside and related compounds

emphasizing its therapeutic benefits was reported elsewhere (Chatsudthipong and

Muanprasat, 2009). The pharmacokinetics, safety evaluation and associated clinical trials
of stevioside are well documented in a recent article (Brahmachari et al., 2011).

Most studies demonstrated that oral stevioside, at an acceptable daily intake (5 mg/kg

BW), is safe to use. In the US, the FDA only granted the first no objection letters for Reb-

As (Rebaudioside A) use in foods in December 2008 to the companies Cargill and

Merisant. Until then, dried Stevia leaves and extracts could only be used in dietary

supplements (since 1995). In Europe, the market is not yet wide open. Switzerland

currently allows Stevia-based sweeteners to be used in foods and beverages, but with

cumbersome pre-market approval of finished products. France gave the go-ahead for

temporary approval in 2010. The European Food Safety Authority (EFSA) is expected to

give its safety opinion, which could pave the way for pan-EU approval, by the end of 2010.

In the US, multinational beverage manufacturers have rolled out new product lines

sweetened with Stevia sweeteners. The worldwide regulatory status of stevia and its

glycosides were shown in Fig. 2.4.

Fig 2.4: Regulatory status of Stevia and its glycosides

2.6. Applications of stevioside
Some important applications where stevioside has been used are described below
and also shown in Fig 2.5.

Stevia rebaudiana

Fig 2.5: Applications of Stevia and its glycosides

2.6.1. Microbicidal property

Stevia is thought to inhibit the growth of certain bacteria and other infectious

organisms (Patil et al., 1996; Sivaram and Mukundam, 2003). Some people even claim that

using Stevia helps to prevent the onset of colds and flu. The ability of Stevia to inhibit

growth of certain bacteria helps to explain its traditional use in treating wounds, sores and

gum disease. It may also explain why the herb is advocated for anyone who is susceptible
to yeast infections or reoccurring streptococcal infections, two conditions that seem to be

aggravated by white sugar consumption (Debnath, 2008).

In some studies the antimicrobial activity of various extracts of S. rebaudiana (with

water, acetone, chloroform, methanol, ethyl acetate or hexane as solvents) have been

investigated and its effect on some selected microorganisms such as Salmonella typhi,

Aeromonas hydrophila, Vibrio cholerae, Bacillus subtilis, Staphylococcus aureus and

others have been examined (Tadhani and Subhash, 2006b; Debnath, 2008; Ghosh et al.,

2008; Jayaraman et al., 2008; Seema, 2010;). The biological activity for Stevia compounds

has been studied by Tomita et al. (1997); they studied the bactericidal activity of a

fermented hot-water extract from S. rebaudiana Bertoni towards enterohaemorrhagic

Escherichia coli and other food-borne pathogenic bacteria. Other microorganisms like

Salmonella typhimurium, B. subtilis, and S. aureus has also been found to be inhibited by

the fermented leaf extract (Debnath, 2008; Ghosh et al., 2008).

We have recently observed that purified stevioside from S. rebaudiana showed

reduction in number of pathogenic bacteria. These food borne pathogenic bacteria such as

Bacillus cereus, Klebsiella pneumonia and Pseudomonas aeruginosa are the root cause of

many food-borne diseases such as enteric fever, diarrhea etc. (Puri and Sharma, 2011d). It

is anticipated that usage of stevioside in various food formulations i.e. herbal tea, bakery,

confectionary items, tooth paste, mouth refreshers, candies, chewing gums etc. may protect

from mentioned bacterium. Different microbicidal properties of stevioside were given in

Table 2.4.
 Table 2.4: Microbicidal properties of Stevioside

Application Details Reference

Prevention of dental Caries Streptococcus mutans Das et al., 1992;

(inhibition of dental plaque)
Antiviral
Enterohemorrhagic E. coli
Zanela et al., 2002;
de Slavutzky, 2010.
Neuralgia and lumbago Dozono, 1993
Without affecting normal Tomita et al., 1997;
intestinal flora Ghosh et al., 2008.

Antimicrobial activity Four bacteria were inhibited Debnath, 2008

by the S. rebaudiana extracts
prepared in various solvents
although only a few fungi
showed inhibition to the leaf
extract.
Human rotavirus Hot water extracts of Stevia Takahashi et al., 2001
leaf inhibit the replication of
human rotavirus in vitro by
blocking viral attachment to
cells

2.6.2. Food Formulation

About 115 metric tons of stevioside were consumed in Korea in 1995, with the

majority of this being used in the sweetening of the alcoholic beverage- soju and produced

from leaves of S. rebaudiana grown in the Peoples Republic of China (Kinghorn et al.,

2001). S. rebaudiana yields a sweet aqueous extract containing various glycosides. Coca-

Cola uses Stevia in Japan for its Diet Coke and has filed 24 patents applications in 2007

concerning extracting the tastiest parts of the Stevia plant. It is seeking exclusive rights to

develop and market rebina for use in drinks (Prakash and Upreti, 2009). At present, the

Coca-Cola Company and Cargill, Inc. have initiated the development and

commercialization of the S. rebaudiana derived sweetener rebaudioside A. They have

performed stability tests with Rebiana (Stevia-derived sweetener), and traded it as Truvia.
Further, no significant photodegradation in acidic beverages containing rebaudioside A or

stevioside, when exposed to light, has been reported.

Cargill, one of the world's largest agribusiness and trading companies, has been

marketing stevioside for its use in food such as yoghurt, cereals, ice cream and sweets

(Etter and McKay, 2007). It has spent the past three years developing Stevia plantations in

China, Paraguay and Argentina. However, this company acknowledges that they face

regulatory troubles since Stevia has been banned in the US and EU after a 1985 medical

study linked the plant to liver problems. They aim to market it first in countries where

Stevia is not banned, such as Japan and South America, and Cargill seeks to help

regulatory approval in the US by sponsoring more scientific studies. The potential to

process Stevia without any chemical treatment during production - should enhance the

acceptance of stevioside as a green sweetener.

Crammer and Ikan (1986) exhibited stability of stevioside at 95C which was found

to be suitable additive to cooked or baked foods. Clos et al. (2008) challenged the Chang

and Cook, 1983 article which had suggested that rebaudioside A is unstable to sunlight

exposure, while the structurally homologous stevioside is stable. They found rebaudioside

and stevioside to be light stable, and degradation of the sweeteners to be acid-promoted

(Clos et al., 2008). Recently, the stability of stevioside during different processing and

storage conditions has been evaluated in tea and coffee beverages. Stevioside at elevated

temperature for 1 h showed good stability up to 120C. In aqueous solution stevioside is

remarkably stable in the pH range of 210. This knowledge seems to be essential for its

effective application in hot coffee and tea beverages (Kroyer, 2010). The leaves, as well as

the pure stevioside extracts, can be used in their natural state or cooked and are

thermostable at temperature up to 200C (Serio, 2010). The list of various formulations in

which stevioside was used so far was given in Table 2.5.

 Table 2.5: Application of Stevioside in Food formulation

Uses in Food Reference

Yoghurt, cereals, ice cream and sweets Etter and McKay, 2007
Tea and coffee beverages Kroyer, 2010

Soft drinks (Coca-Cola) or fruit drinks Goyal et al., 2010;

Jayaraman et al., 2008
Prakash and Upreti, 2009
Some of the teas containing stevia include Acai Deoudes, 2010
Mango Iced Tea, Echinacea Complete Care,
Powerfully Plum, White Tea Antioxidant Plum C,
Tangerine Orange Sweet Zinger Ice Tea,
Antioxidant Green Tea and Raspberry Iced Tea.
Ready-to-eat cereals, pickles, yoghurt, candies, Wallin, 2007;
soju, soy sauce, seafoods Koyama et al.,2003;
Amzad-Hossain et al., 2010;
Tadhani and Subhash, 2006a;
Koyama et al.,2003;
PepsiCo has introduced zero-calorie versions of Bogle, 2011
Sobe Lifewater in three flavors: Yumberry
Pomegranate, Fuji Apple Pear and
Blackberry/Blueberry by using Stevia glycosides
Tropicana's Trop 50 is a mid-calorie formulation Anthony, 2012
which is an orange juice product with less than
half the calories of regular orange juice prepared
by using stevia is blending with sugar

Stevioside can be used as an alternative to table sugar (sucrose). Stevia-based

sweeteners fit well into this space, offering a combination of favourable attributes: high

intensity and zero calories. There are two distinct markets: specifically used for diabetic

patients as a low calorie/dietetic segment and other as a nutritive sweetener which can be

used as an alternative to marketed sugar (i.e. corn syrup, fructose, glucose, sucrose etc.).

The established uses for Stevia products cover all those of artificial low-calorie (non-

sucrose) sweeteners, aspartame etc. as well as for most other purposes for which sugar can

be used. The primary use is as a sweetener to enhance the palatability of foods and drinks.

38
Unlike aspartame, Stevia sweetener is heat-stable up to 200C, acid-stable and non

fermentable, making it suitable for use in a wide range of products including baked/cooked

foods. Stevioside has been used in various food formulations such as jam, ckikki, basen

ladu, biscuits, milk shake, grape juice, fruit custard, jam, tea and milk shake (Savita et al.,

2004b). These are incredible claims made in the literature which are not based on the peer

review process, however needs validation by conducting robust analysis.

2.6.3. Medicinal property

Another interesting area is derived from studies on the effects of Stevia sweeteners

on carbohydrate metabolism. Based on the research on the hypoglycemic activity of Stevia

extracts, the leaves, flowers, and stems are now used in popular medicine in Paraguay as a

remedy for diabetes (Soejarto and Kinghorn, 1985). Stevioside stimulates insulin secretion

from mouse islets and INS-1 cells. Stevioside seems to possess anti-hyperglycemic effects

that may be important in the treatment of type 2 diabetes. Stevioside and steviol appear to

have an inherent advantage over the classic sulfonylurea, since the action of the diterpenes
+
is not mediated via K ATP sensitive channels. Furthermore, the lack of insulin stimulatory

effects at subnormal glucose levels may reduce or eliminate the risk of hypoglycemia

(Jeppesen et al., 2000).

Stevioside has a significant hypotensive effect in different strains of hypertensive

rats, is relatively non-toxic, and can possibly be developed into a complimentary therapy

for hypertension (Hsu et al., 2002). It is also an approved herbal medicine for diabetics in

China. Although Stevia has just achieved the status of a dietary supplement by the FDA in

North America, it could also be a nutraceutical product with great potential. Stevioside

inhibited inflammatory ear edema and also tumor-promoting activities induced by TPA in

mouse skin. This is of great importance from the view point of the chemo-prevention of

cancer (Yasukawa et al., 2002). The inhibitory effects of stevioside was greater than those
39
of glycyrrhizin, which is a known antitumor-promoter isolated from licorice root (Takasaki

et al., 2009).

However, supplementation of the diet with Stevia leaves or stevioside caused a

decrease in the total concentration and a change in the profile of short chain fatty acids

(SCFA), suggesting not only a change in number but also a change in the types of

microbes that may be present in the ceca. The finding implicates stevioside, as a

component of Stevia leaves that may depress SCFA production in the chicken ceca as a

result of changes in microbial population (Atteh et al., 2008).

Stevioside has no effect on weight and triglycerides, but lowers glucose and insulin

levels. Stevioside treatment has been associated with improved insulin signaling and

antioxidant defense in both adipose tissue and the vascular wall, leading to inhibition of

atherosclerotic plaque development and inducing plaque stabilization (Geeraert et al.,

2010). Other list of therapeutic application of stevioside was listed in Table 2.6 as shown

below. In addition, we have recently observed that stevioside has antiamnesia properties

(Sharma et al., 2010). Pretreatment with stevioside significantly reversed scopolamine-

induced learning and memory deficits along with attenuation of scopolamine-induced rise

in brain acetylcholine esterase (AChE) activity and brain oxidative stress levels. We

concluded that stevioside exerts a memory preservative effect in cognitive deficits of rats

possibly through its multiple actions (Sharma et al., 2010).

A recent study has been conducted on the effect of stevioside on plaque formation.

Authors observed that accumulation of dental plaque after rinsing with stevioside was

57.82% less than under rinsing with sucrose. The results proposed that stevioside can

effect on diminishing plaque formation (Blauth de Slavutzky, 2010). However, these

observation needs further assessment as leaving out sugar from any diet would probably

have the same effect. Hence, Stevia and its glycosides can largely be used in the areas

40
where sucrose has previously been used, e.g., foods, beverage, and as a medicine.

Table 2.6: Therapeutic application of Stevioside

Application Details Reference
Glucoregulation Stevioside enhances both insulin Jeppesen et.al.,2003;
secretion and insulin sensitivity. Lailerd et al., 2004;
Stevioside stimulates insulin secretion Chen et al., 2006;
by isolated mouse pancreatic islets but Yang et al., 2009.
does not affect fasting insulinemia.

Blood pressure Stevioside induces vasorelaxation. Lee et al., 2001;

regulation Stevioside exhibited significant decrease Hsieh et al., 2003
in systolic and diastolic blood pressures.

Bowel disease Stevioside exerted anti-inflammatory Shiozaki et al., 2006;

effects on colonic epithelial cells. Chatsudthipong and
Steviosides inhibitory effect on Muanprasat, 2009
intestinal smooth muscle contraction and
stimulation was linked to hypermotility-
associated diarrhoea.

Chemopreventio Stevioside, the stevia leaf aglycones, Akihisa et al., 2004;

n of cancer and steviol and isosteviol, and their Boonkaewwan et al.,
inhibition of metabolites inhibited tumor promotion 2006;
tumor by blocking Epstein-barr virus early Takasaki et al., 2009.
promotion and antigen (EBV-EA) induction.
initiation. Stevioside inhibited inflammatory ear
edema and tumor-promoting activities
induced by TPA in mouse skin.

Atherosclerotic Stevioside treatment associated with Geeraert et al., 2010

plaque improved insulin signaling and antioxidant
inhibition and defense in both adipose tissue and the
inducing plaque vascular wall.
stabilization.

Antioxidant Stevia leaf extract exhibited a high degree Ghanta et al., 2007;
of anti-oxidant activity and reported to Shukla et al., 2009;
inhibit hydroperoxide formation in sardine Kim et al., 2011.
oil with potency greater than that of either
DL--tocopherol or green tea extract.
 Stevia-derived sweeteners have increased in popularity since December 2008, when

the United States Food and Drug Administration (USFDA) approved the Stevia-derived

sweetener Reb A as generally recognized as safe (GRAS) for its use in foods and

beverages. GLG Life Tech has signed a memorandum of understanding (MOU) with

Indian-based Global AgriSystem Private Limited, to pursue the commercialization of

Stevia-based sweetener in India. The process would include a push to develop the market

for Stevia-derived sweeteners in India and agricultural development of growing regions

that would be suitable for Stevia plants. The leading supplier, Pure Circle, currently has a

total of about 15,000 ha contracted in Kenya, Paraguay, Columbia, Indonesia, Vietnam,

Thailand and China, for growing Stevia plantations. The companies are also considering

constructing joint extraction facilities in India as the demand builds up. Currently, the

United States is the biggest market for the zero-calorie sweetener. However, India with its

population of 1.14 billion and a growing rates of obesity, offers an untapped market

which creates a significant opportunity for Stevia suppliers (Caroline, 2010).