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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 time’s 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

Madhumita Kumari & Sheela Chandra 12

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 100°C 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.

Madhumita Kumari & Sheela Chandra 14

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*

(times)

Value (gm/100gm dry

leaf weight)

Stevioside 150-300 4–14%

Rebaudioside A 250-350 2–4%

Rebaudioside C 50-150 1–2%

Dulcoside A 50-150 0.4-0.7%

Rebaudioside D,E,F;

Steviolbioside;

Rubusoside

100-250 ˃0.4%

Carbohydrates 35.2

Proteins 12.0–20.42

Lipids 2.7–4.34

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 [1-13

C] glucose and NMR spectroscopy showed that main

precursor steviol is synthesized via the plastid localized methylerythritol

4-phosphate (MEP) pathway (Figure 4) [28].

Madhumita Kumari & Sheela Chandra 16

OPP

CPS

OPP

Geranylgeranyl diphosphate (-)-copalyl diphosphate

KS

Kaurene

COOH

KO

COOH

OH

COOH

O-glc

COOH

O-glc-glc

O-glc-glc

COO-glc

(-)-Kaurnoic acidSteviolSteviolmonoside

Steviolbioside

Stevioside

O-glc-glc

glc

KAHUGT85C2

UGT

UGT74G1

UGT76G1

COOHCOOH

GA12

Gibberllins

Kaurenoic acid7-oxidase

Rebaudioside A

COO-glc

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].

Madhumita Kumari & Sheela Chandra 18

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 inFenton’s 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 1000µg/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 (250µg/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

Madhumita Kumari & Sheela Chandra 20

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 day’s 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,

New

Zealand

2008 Steviol glycosides

extracts

Japan 1970 All steviol

glycosides, leaves

Brazil 1986 Stevioside extracts European

Union

2011 Steviol glycosides

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

glycosides

Canada 2012 Steviol glycosides,

dried leaves

Norway 2012 Steviol glycosides

Russian

Federation

2008 Stevioside

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].

Madhumita Kumari & Sheela Chandra 22

Equal,

$45.9m Sweet 'N

low,

$74.5m

Truvia,

$90.6m

Other,

$111.9 m

Pure Via,

$5.9m

Splenda,

$287.7 m

Private

Label,

$125.9m

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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