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Handbook of Medicinal Plants and their Bioactive Compounds, 2014: 11-26

ISBN: 978-81-308-0548-1 Editor: Nidhi Gupta

2. Stevia rebaudiana: Beyond sweetness

Madhumita Kumari and Sheela Chandra
Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi-835215
Jharkhand, India

Abstract. Stevia rebaudiana (Bert.) is a name that flourishes in

the medicinal world from hundreds of years. Stevia plant has
applications in diverse fields. Leaves of Stevia contains diterpene
glycosides (Stevioside and rebaudiosides), which are well known
for intense sweetness. Stevioside is approx 300 times sweeter than
sucrose but have zero calories. Besides sweetness, steviol
glycosides can be used as potential therapeutics against numerous
diseases and are proving their potential far better compared to
established drugs in the market. Steviosides have antihypertensive,
antitumour and vasodilator activity. Isosteviol possess
neuroprotective activity. Rebaudioside A and Dulcoside A have
been found to be similar in acitivity with Hydrocortisone as anti-
inflammatory drug. Rebaudioside C acts on 10 to 100 times lower
doses as compared to Indomethacin, an anti-inflammatory drug.
Similarly isosteviol is comparable with Nimodipine, a neuroprotective
drug. In comparision to artificial sweetners available in market,
steviosides are 100% natural, zero calories, heat stable, non-
discolouring, and have no other side effects. It can be added to tea
or coffee and cooked or baked. In India, prevalence of diabetes is
rising rapidly, and more than half of the patients have poor
glycemic control with vascular complications. So there is a need
to develop novel therapeutic agent with multipotential activities.

Correspondence/Reprint request: Dr. Sheela Chandra, Department of Biotechnology, Birla Institute of

Technology, Mesra, Ranchi-835215, Jharkhand, India. E-mail: schandra@bitmesra.ac.in
12 Madhumita Kumari & Sheela Chandra

India has suitable climate for Stevia cultivation. Inspite of this, Stevia cultivation has
not been taken up on a large scale and China is dominating the market. Lack of
awareness among the farming fraternity on medicinal values and the commercial
prospects of the crop are lacking. Aim of this review is to create awareness by
exploring the hidden therapeutic potential of steviol glycosides beyond its sweetness
value.

Introduction
Stevia rebaudiana (Bert.) is natural sweetener plant and flourishes in the
medicinal world from years. It is a perennial herb belongs to Asteraceae
family. About 200 species of Stevia are known but Stevia rebaudiana (Bert.)
emerged distinct on the ground of its sweet nature [1].
Stevia is native of Paraguay. The word Stevia originates against the
name of Spanish botanist P.J. Stevus, who first studied the different species
of Stevia genus. In 1888 M.S. Bertoni first discovered the plant and its sweet
taste. The plant was scientifically named as Stevia rebaudiana in 1905 after
a Paraguayan chemist Dr.Rebaudi. It is also known as sweet herb of
Paraguay, honey leaf, sweet leaf, sweet herb and candy leaf [2].In 1931, two
chemists isolated the compounds responsible for its sweet taste [3]. These
compounds, Steviosides and Rebaudioside A are diterpene glycosides and
are 250-300 times sweeter than sucrose. These are heat-stable, pH-stable,
and not fermentable[4].
Stevia rebaudiana is a short day plant (Figure 1).It grows easily on
tropical and subtropical areas between the temperature range of 21 to 40
degree with semi humid environment and well-draining soil in pH range of
6.5 to 7.5[5]. First crop of Stevia was domesticated at Japan and used as an
alternative sweetener. Later on, extensive studies on Stevia revealed its
useful effects in human body and this favour its commercialization in several
countries including Latin America, Canada, China, Japan, Indonesia, USA
[6, 7]. In India, Stevia is being cultivated successfully in the states of
Rajasthan, Maharashtra and Kerala.
These natural high intensity sweeteners are non-fermentable,
non-discoloring nature, maintaining heat-stability at 100C and feature a
long shelf life. The product can be added to tea and coffee, cooked or baked
goods, processed foods and beverages. It is used as a table top sweetener, in
soft drinks, baked goods, pickles, fruit juices, tobacco products,
confectionery goods, jams and jellies, candies, yogurts, pastries, chewing
gum and sherbets [8,9].
Stevia rebaudiana: Beyond sweetness 13

Figure 1. Stevia rebaudiana (Bertoni)

In India, prevalence of diabetes is raising rapidly, due to urbanisation,

population growth and increase of obesity and physical inactivity. The
International Diabetes Federation (FDI) estimated that around 50.8 million
Indians are currently suffering from diabetes and by 2030 expected rise up to
87.0 million. Studies in India indicate that more than half of the patients
have poor glycemic control and have vascular complications [10, 11]. So the
immediate objective in maintaining diabetes mellitus is to attain near normal
glycemia. Therefore, there is an urgent need to develop novel therapeutics
such as Stevia and derived products which are recommended for diabetics
and have been extensively tested on animals and used by humans with no
side effects [12].
Lot of work has been done on importance of plant as a sweetener and its
management aspects but is less explored for its potential therapeutics. Recent
studies showed that besides sweetness it has therapeutic effect against
number of maladies. It regulates the blood glucose level by stimulating
insulin secretion [13, 14]. Stevioside can also be used as an
antihyperglycaemic[15], antihypertensive [16], anti-tumour[17], vasodilator
[18] drug. This chapter includes a comprehensive review on current
understanding of Stevia as potential therapeutics beyond its sweetness and
current market scenario of Stevia products.
14 Madhumita Kumari & Sheela Chandra

Chemical constituents of Stevia rebaudiana (Bert.)

The sweet diterpene glycosides of Stevia have been the subject of a
number of reviews [9, 19]. The leaves of Stevia rebaudiana contain at least
eight diterpene glycosides viz. stevioside and rebaudioside. After recognition
of sweet taste of Stevia, several substances have been isolated from the plant
including stevioside and steviol. In 1931, Isolation of stevioside was done by
Bridel and Lavieille. In 1952, the chemical structure of stevioside (Figure 2)
was established and described as an aglycon, steviol with glycoside of three
glucose molecule [20]. During the 1970s, other compounds were isolated,
including rebaudioside A (Figure 3), also known as rebtose, with a sweet
potency even higher than stevioside [21].
Besides steviol glycosides, other diterpenoids also present in leaves of S.
rebaudiana such as manoyl oxide and labdanescareol. Manoyl oxide shows
anti-inflammatory and anti- parasitic action whereas labdanesclareol, has
anti-tumorous and cytotoxic properties [22].
Wild Stevia leaves have been evaluated for typical proportion of major
glycoside and other biochemical constituents on dry weight basis. Among
steviol glycosides, stevioside have highest proportion and than rebaudioside
A and other glycosides. In addition to steviol glycosides, other minerals,
protein, fat, carbohydrate and ash content has also been estimated (Table 1)
[23].

Figure 2. Stevioside. Figure 3. Rebaudioside A.

Stevia rebaudiana: Beyond sweetness 15

Table 1. Percentage of major glycosides and other biochemical constituents of

Stevia.

Component Sweetening* Value (gm/100gm dry

(times) leaf weight)
Stevioside 150-300 414%

Rebaudioside A 250-350 24%

Rebaudioside C 50-150 12%
Dulcoside A 50-150 0.4-0.7%
Rebaudioside D,E,F; 100-250 0.4%
Steviolbioside;
Rubusoside
Carbohydrates 35.2
Proteins 12.020.42
Lipids 2.74.34

Ash 13.12
*Sweetness is tasted at a series of dilutions to determine the concentration that is as sweet as a
given percent sucrose reference. Taste panellists usually are trained to quantitate sweetness on a
15 cm line scale, using 2-15% sucrose solutions as references. For example, if a 1% solution of
sweetener X is as sweet as a 10% sucrose solution, then sweetener X is said to be 10 times as
potent as sucrose [24].

Stevia leaves also contain numerous all-natural nutrients that are

medically and commercially important, including chromium, magnesium,
manganese, potassium, selenium, zinc, and vitamin B3(Niacin).
Phytochemical screening has showed that tannins are present in higher
concentrations followed by alkaloids, glycosides, saponins, sterols, and
triterpenes, anthraquinones, and other reducing compounds [25].

Biosynthetic pathway of steviol glycosides

Steviol glycosides biosynthesis is currently an endless area of research
because not much is known about the pathway and it shares some common
steps with GA (Gibberellic acid) biosynthesis [26]. Steviol glycoside
biosynthesis occurs in leaves and transported to different parts [27]. In vivo
labeling with [1-13C] glucose and NMR spectroscopy showed that main
precursor steviol is synthesized via the plastid localized methylerythritol
4-phosphate (MEP) pathway (Figure 4) [28].
16 Madhumita Kumari & Sheela Chandra

OPP
OPP
CPS KS

Geranylgeranyl diphosphate (-)-copalyl diphosphate Kaurene

KO
OH
O-glc

UGT85C2 KAH

COOH COOH
COOH
Steviolmonoside Steviol
(-)-Kaurnoic acid
UGT Kaurenoic acid
O-glc-glc 7-oxidase

COOH COOH
Steviolbioside COOH
GA12
UGT74G1 glc
O-glc-glc O-glc-glc
Gibberllins
UGT76G1

COO-glc COO-glc
Stevioside Rebaudioside A

Figure 4. Biosynthetic pathway of Steviol glycosides (Redrawn from Brandle and

Telmer, 2007).[26]
[Abbreviations: copalyl diphosphate synthase (CPS), kaurene synthase (KS), kaurene
oxidase (KO), kaurenoic acid 13-hydroxylase (KAH)]

2-C-methyl-D-erythritol-4-phosphate (MEP) pathway

Initial steps of steviol biosynthesis are common with MEP pathway and
synthesize isopentenyl diphosphate (IPP) and dimethylallyl diphosphate
(DMAPP) [29] following with the Geranylgeranyl diphosphate (GGDP)
synthesis.
Like many other diterpenes, steviol glycosides are derived from GGDP.
From GGDP, next upto synthesis of kaurenoic acid in steviol biosynthesis
has identical steps with GA biosynthesis pathway (Figure 4, [30]). For
Stevia rebaudiana: Beyond sweetness 17

steviol, GGDP is first converted by protonation initiated cyclization to (-)-

copalyldiphosphate (CDP) by CDP synthase (CPS). Next, ionization
dependent cyclization of CDP by kaurene synthase (KS) produces kaurene.
These enzymes from GA biosynthetic pathway has been identified and
characterized from number of plants including Stevia [26]. Gene expression
of both CPS and KS genes revealed that both occur in leaf parenchyma [31]
and from this we can conclude that early steps in pathway are limited to
green tissue.
Kaurene is then oxidized in a three step reaction to kaurenoic acid by
kaurene oxidase (KO), a novel P450 monooxygenase, similar in GA
biosynthesis [32]. Stevia KO was found to be highly expressed in leaves,
succulent stems, flowers and seedling shoots [33].
Steviol biosynthesis diverges from gibberellins biosynthesis with
hydroxylation of kaurenoic acid by kaurenoic acid 13- hydroxylase (KAH)
to form steviol[34] whereas in GA biosynthesis hydroxylation occurs at C-7
position [35], so this is the first committed step of steviol glycoside
biosynthesis.

Glycosylation
Aglycone steviol is glycosylated by various glucosyltransferases in
cytoplasm. Steviol has two hydroxyl groups, one at C-19 of C-4 carboxyl
and other at C-13. Glycosylation starts at C-13 by UGT85C2 which
produces steviolmonoside. Steviolmonoside is then glycosylated to produce
steviolbioside. UGT of this step is not yet identified. Finally Stevioside is
produced by UGT74G1 by glucosylation at C-19 position (Figure 4, [36]).
Rebaudioside A is synthesized by glucosylation at C-13 of Stevioside by
UGT76G1 [26].

Bioactivities of Steviol glycosides beyond sweeteness

Sweet nature of steviol glycosides and high protein content of Stevia are
responsible for its acceptance in food industry. Now, numerous medicinal
importances of its biochemical constituents have been identified.
Stevia leaf extracts have probably been used in traditional medicine and
as sweetener by native people before being described; however stevioside
has achieved worldwide attention due to its potent sweetness recently [37].
Steviol glycosides are presently used in several countries in the form of
different products and it has been tested clinically to demonstrate that its use
is safe for humans [38].
18 Madhumita Kumari & Sheela Chandra

Antihypertensive
Stevioside is emerged as very effective antihypertensive compound. It
reduces arterial blood pressure when administered orally or intravenously
[39, 40]. Efficiency of intravenously administered Stevioside is completely
established [41] whereas efficiency of oral administration is not completely
established due to low gastrointestinal absorption [42, 16].The intravenous
administration of this compound could be useful in hypertensive
emergencies,as it induces hypotension by causing dilation of peripheral
vessels [43]. Additional advantage is consumption of as much as 1000
mg/day of rebaudioside A produced no clinically important changes in blood
pressure in healthy adults with normal and low-normal blood pressure [44].
Studies in humans showed that continued consumption of Stevioside
(750mg/day) for one year in mild and moderate hypertension reduces both
systolic and diastolic pressure and no significant side effects observed on
lipid or fasting glucose [45]. Studies with increased dose of Stevioside
(1500mg/day) showed same results with no change in body mass index,
blood biochemistry values and left ventricular mass index [40].
Bornia et al (2008) also studied the activity of the nitric oxide (NO)
synthesis pathway, as it is an important factor in vascular relaxation [18],
they investigated the effects of stevioside in aortic ring preparation of rats
pre-contracted by either norepinephrine or KCl and the effects of treatment
with inhibitors of NO synthesis and concluded that the stevioside-induced
vasodilatation is not dependent on the activities of NOS and guanylate
cyclase when the vascular endothelium is damaged, but that it depends on
the activities of these enzymes when the endothelium of the aortic ring
preparations is intact.
The precise mechanism of Stevioside action is still under study.

Antioxidant
The radical scavenging capacity of methanolic extract of stevia
rebaudiana was evaluated by the DPPH test [46]. Methanolic extract have
higher percent inhibition of DPPH radical with ethanolic extract [47].

DNA damage preventive

At 0.1 mg/mL, the ethyl acetate extract (EAE) of the crude 85% methanolic
extract (CAE) of Stevia rebaudiana leaves exhibited preventive activity against
DNA strand scission by OH generated inFentons reaction on pBluescript II SK
() DNA. Its efficacy was observedbetter than that of quercetin [46].
Stevia rebaudiana: Beyond sweetness 19

Antimicrobial
Chemical extracts of Stevia rebaudiana at the concentration of 1000g/ml
showed antibacterial activity against Serratia marcescens, Klebsiella
pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, Bacillussubtilis,
Alcaligenesdenitrificans and Salmonella typhimurium[48]. Most effective
zone of inhibition (12mm) was for Bacilluscereus which serves for the
application of stevioside in foods to increase their shelf life. In addition,
Stevia leaves extracts have antimicrobial potential against some pathogenic
food spoiling fungus (Alternaria solani, Helminthosporium solani,
Aspergillus niger, Penicillium chrysogenum) and also some other pathogenic
bacteria (Escherichia coli, Enterococcus faecalis, Proteus mirabilis,
Staphylococcus aureus) [49]. Minimum concentration (250g/ml) of
petroleum ether extract was sufficient enough to completely inhibit the
growth of E. Coli.

Anti-inflammatory
Sufficient evidences are there for anti-inflammatory effect of Stevioside.
For example four different steviol glycosides, stevioside, rebaudiosides A
and C, and dulcoside A, showed strong inhibitory activity against
12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice
[50]. Isosteviol inhibits DNA polymerases and human DNA topoisomerase
II, cellular targets for pharmacotherapy of cancer as well as inflammatory
diseases. Moreover, isosteviol also retards growth of three different types of
human cancer cells and inhibits inflammation induced by TPA [51].

Neuroprotective
Stevioside and isosteviol have neuroprotective activity. Stevioside
shows antiamnesic effect on scopolamine (drug for motion sickness) treated
rats. On pre-treatment Stevioside suppresses the scopolamine induced
learning and memory deficit. It also constrict scopolamine induced high
acetylcholine activity and oxidative stress level in brain. So stevioside have a
memory preservative effect in cognitive deficits of rats [52].
Isosteviol have protective effects against ischemia-reperfusion (IR)
after cerebral ischemia. Occlusion of cerebral artery damages the brain
even after reperfusion. Isosteviol is tested in different concentration
(5mg/kg to 20mg/kg) and compared with Nimodipine (drug for prevention
of cerebral ischemia) to determine its potential in preventing IR injury in
20 Madhumita Kumari & Sheela Chandra

brain. It is found that isosteviol is as effective as Nimodipine. Isosteviol

acts by reducing imfarct volume, ameliorated cell death and infilteration
of neutrocytes and finally improved neurolocomotor activity observed
[53].

Antidiarrhoeal activity
Diarrhoea is most commonly caused by pathogenic bacteria or viruses
by either direct invasive damage to intestine or deranged intestinal function
[54].Different types of diarrhoea can occur according to their source, it may
be secretory, osmotic, motility related or exudative diarrhoea [55].Currently
in antidiarrheal drug discovery main focus is on rehydration therapyand
antibiotic treatment, but the antibiotic treatment is not effective in case of
antibiotic resistance.
Application of Stevioside as a therapeutics of diarrhoea originates from
its bactericidal effect [56] as it has antimicrobial activity against broad range
of food borne pathogenic bacteria including enterohemorrhagic E.coli,
known to cause diarrhoea. It also inhibits rotavirus which causes
gastroenteritis in children. [57]
Stevioside has an inhibitory effect on intestinal smooth muscle
contraction, stimulation of which results in hypermotility-associated
diarrhoea. Stevioside inhibits CaCl2 induced contraction of isolated guinea
pig ileum by 40% [13]. The mechanism was related to its inhibitory effect on
Ca2+ influx into muscle cells. Thus Stevioside may be useful in treatment of
diarrhoea resulting from intestinal hypermotility.

Global market of Stevia

Stevia is one of the fastest growing industries in world because of its
good taste of food and drink without any calories or health risks. Its zero
calories do not cause any health problem or tooth decay. Now a days Stevia
is approved as food additive or dietary supplement in several countries
(Table 2). It is also popular among food manufacturing companies and
distributed over the world. Cargill and Coca Cola companies distribute their
products under the brand name Truvia and PepsiCo Inc. distributes by the
name Pure Via.
In view of relatively early stage of Stevia market, global sale of Stevia
varies. Data from Leatherhead Food Research valued the world Stevia
market (including both crude extracts and high purity products such as Reb A)
at US$100m in 2010, up by nearly 27% from $79m in 2009. During this
Stevia rebaudiana: Beyond sweetness 21

Table 2. List of countries where Stevia is approved by regulatory agencies [58-61].

Regulatory agency approved

Food additive Food additive and dietary supplement

Country year Forms of Stevia Country year Forms of Stevia

Australia, 2008 Steviol glycosides Japan 1970 All steviol

New extracts glycosides, leaves
Zealand
Brazil 1986 Stevioside extracts European 2011 Steviol glycosides
Union

Hong Kong 2010 Steviol glycosides United States 2008 Reb A, Stevia
leaves
Israel 2012 Steviol glycosides Indonesia 2012 Steviol glycosides,
dried leaves
Mexico 2009 Mixed steviol Canada 2012 Steviol glycosides,
glycosides dried leaves

Norway 2012 Steviol glycosides

Russian 2008 Stevioside

Federation

Singapore 2005 Steviol glycosides

In some other countries Stevia is available in different forms but not verified by regulatory
agencies, which includes Argentina, Chile, China, India, Colombia, Korea, Malaysia, Paraguay,
Peru, Philippines, Saudi Arabia, Taiwan, Thailand, Turkey, United Arab Emirates, Uruguay,
and Vietnam [61].

time, volume sales rose from less than 2,300 tonnes to 2,400 tonnes, with
crude extracts accounting for up to 80% of this figure. Separate data from
Zenith International (another UK-based consultancy) suggests that global
market value reached $285m in 2010, with volume sales worth in the
region of 3,500 tonnes. A report from Packaged Facts of the US estimates
the world Stevia market in 2011 is between $800m and $2bn, up from
just $20m in 2008. For this growth, credit goes to Stevia as it gained
regulatory approval in the large sized US market, where sales of intense
sweeteners such as sucralose and aspartame remain above the global
average [62].
22 Madhumita Kumari & Sheela Chandra

Equal,
$45.9m Sweet 'N
low,
Private $74.5m
Label,
$125.9m

Truvia,
$90.6m

Splenda, Other,
$287.7 m $111.9 m

Pure Via,
$5.9m

Figure 5. Sales of sugar substitutes in US in 2012.

Figure 5 (ChicagoBusiness.com) shows sales and market share for sugar

substitutes. Splenda is the clear leader but Truvia with sales of $90.6 million
is second and is growing the fastest. Market analysts predict that Truvia
could eventually lead the industry with sales of $300 million plus [63].
According to Zenith International, the global market for Stevia is
estimated to reach 11,000 tonnes by the middle of the current decade,
equivalent to $825m on sale. PureCircle CFO William Mitchell in a
predicted that world demand for Stevia leaves would exceed 8m tonnes by
2020, while global sales of Reb A may reach as high as $10bn over the next
few years. It has the potential to penetrate up to 25% of the world sugar
market [64].
Stevia's growing popularity as a natural sweetener has drawn global
beverage makers such as Coca-Cola Co. to introduce it in leading brands
such as Coca-Cola and Sprite, as companies aim to offer reduced-calorie soft
drinks that don't taste like diet drinks.
The industry is making serious efforts to further develop the world
Stevia market for example, 2010 saw the establishment of the Global
Stevia rebaudiana: Beyond sweetness 23

Stevia Institute, which aims to promote and provide scientific information

about Stevia and its benefits to health professionals, consumers and food and
drink producers. From a geographical perspective, new markets for
Stevia-based sweeteners are expected to open up in India and parts of the
Middle East in the near future.
There are various reasons for popularity of Stevia such as, trouble in
market of commercial sugar substitutes by health concerns. For example,
Saccharin was banned in Canada in 1977 after studies raised possible links
to cancer. Aspartame is now known to contain three metabolites (aspartate,
phenylalanine, and methanol) that act as powerful neurotoxins when
ingested in high concentrations. The market share leader, Splenda has been
known to cause dizziness, cramping, and rashes. And these side effects of
Splenda are not particularly surprising as its chief compound, sucralose, was
originally designed as an insecticide [65].
In contrast to this, Stevia rises in separate class. Clinical studies in rats
have shown that Stevia extract reduces blood pressure, diuresis, and
natriuresis, without any adverse effects.

Conclusion
Sweeteners were developed because of their possible benefits in special
diets, health and economy; they can be used in the formulation of foods and
beverages without affecting the quality of the product. Keeping in view the
global economic aspects for Stevia cultivation and by comparing it with
Indian scenario, it appears that it may be suitable as a source of
comparatively cheaper sweetener with plenty of bioactivities. India has still
long way to go to create awareness about medicinal importance of Stevia
plant. It needs more exploration. Most of the studies related to bioactive
compounds of Stevia have been performed on experimental animals or
cultured tissues. Fewer studies have been carried out in humans.
Comprehensive clinical studies in humans are needed to develop
pharmaceutics related to bioactive compound of Stevia.

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