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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 antiinflammatory 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, nondiscolouring, 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: [email protected]

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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

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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.

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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.

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Stevia rebaudiana: Beyond sweetness

Table 1. Percentage of major glycosides and other biochemical constituents of Stevia. Component Stevioside

Sweetening* (times) 150-300

Value (gm/100gm dry leaf weight) 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; 100-250 Steviolbioside; Rubusoside

˃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 [1-13C] glucose and NMR spectroscopy showed that main precursor steviol is synthesized via the plastid localized methylerythritol 4-phosphate (MEP) pathway (Figure 4) [28].

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Madhumita Kumari & Sheela Chandra OPP OPP KS

CPS

Geranylgeranyl diphosphate

(-)-copalyl diphosphate

KO

OH

O-glc UGT85C2

KAH COOH

COOH

Steviolmonoside

Kaurene

COOH

Steviol

(-)-Kaurnoic acid

UGT

Kaurenoic acid 7-oxidase

O-glc-glc

COOH

COOH COOH

Steviolbioside

GA12 UGT74G1

glc O-glc-glc

O-glc-glc

Gibberllins UGT76G1

COO-glc

Stevioside

COO-glc

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

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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].

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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

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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

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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

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Stevia rebaudiana: Beyond sweetness

Table 2. List of countries where Stevia is approved by regulatory agencies [58-61]. Regulatory agency approved Food additive Country

year

Food additive and dietary supplement Forms of Stevia

Country

year

Forms of Stevia

Australia, 2008 New Zealand Brazil 1986

Steviol glycosides extracts

Japan

1970

All steviol glycosides, leaves

Stevioside extracts

European Union

2011

Steviol glycosides

Hong Kong 2010

Steviol glycosides

United States 2008

Israel

2012

Steviol glycosides

Indonesia

2012

Mexico

2009

Mixed steviol glycosides

Canada

2012

Reb A, Stevia leaves Steviol glycosides, dried leaves Steviol glycosides, dried leaves

Norway

2012

Steviol glycosides

Russian 2008 Federation

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].

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Equal, $45.9m Private Label, $125.9m

Sweet 'N low, $74.5m

Truvia, $90.6m

Splenda, $287.7 m

Other, $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

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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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