Czech J. Food Sci., 34, 2016 (4): 281–299 Review doi: 10.17221/335/2015-CJFS

Stevioside and Rebaudioside A – Predominant Ent-Kaurene Diterpene Glycosides of Therapeutic Potential – a Review

Ena GUPTA1, Shalini PURWAR1, Shanthy SUNDARAM1, Prashasti TRIPATHI 2 and Gyanendra RAI 1,2

1Centre for Biotechnology and 2Centre of Food Technology, University of Allahabad, Uttar Pradesh, India

Abstract

Gupta E., Purwar S., Sundaram S., Tripathi P., Rai G. (2016): Stevioside and rebaudioside A – predomi- nant ent-kaurene diterpene glycosides of therapeutic potential – a review. Czech J. Food Sci., 34: 281–299.

Stevia rebaudiana (Bertoni) is a Paraguayan perennial herb of the family Asteraceae. The leaves contain a great amount of secondary metabolites with a wide range of important biological activities commonly known as steviol glycosides which differ in their molecular configuration, power of sweetness and their taste profile. Out of various steviol glyco- sides, the main compounds of interest are diterpenoid glycosides of ent-kaurene type extracted from the leaves of this plant as non-toxic, thermally stable, low-calorie natural sweeteners stevioside and rebaudioside A. These glycosides are a high-quality sugar substitute or dietary supplement with diverse applications in the medicinal world along with the food and beverage industry. This review article is aimed at the chemistry of stevioside and rebaudioside A, possible biosynthetic pathways, their metabolism and acceptable daily intake along with a broad spectrum of pharmacological and therapeutic applications.

Keywords: Stevia rebaudiana; secondary metabolites, therapeutic applications; sweetener

Stevia rebaudiana (Bertoni) (Asteraceae) is a per- the North and South American genus S. rebaudiana ennial herb native to Paraguay and Brazil. This plant has been found to produce these sweet tasting steviol is used all over the world as it possesses medicinal glycosides at high concentration levels. Stevioside and commercial importance. It contains a significant (triglucosylated steviol) being the most predominant amount of important nutrients and minerals neces- ent-kaurene type diterpene glycoside, approximately sary for regulating and maintaining various metabolic 3–8% of dried leaves, was first isolated in the first dec- processes in the body (Lemus-Mondaca et al. 2012). ade of the twentieth century in impure form (Bertoni The leaves of S. rebaudiana are extensively studied 1905, 1918) but its final structure was not elucidated as a source of high potency sweet tasting. Natural for nearly sixty years (Mosetting et al. 1963). Later, constituents of the plant are ent-kaurene diterpene (Chaturvedula & Prakash 2011) isolated a new glycosides – stevioside, rebaudiosides A, B, C, D, and diterpenoid glycoside, 13-[(2-O-β-d-glucopyranosyl- E, dulcoside A, and steviolbioside. The foremost active β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid- sweet tasting, non-toxic tetracyclic diterpene steviol (4-O-(2-O-α-d-glucopyranosyl)-α-d-glucopyranosyl- glycosides of particular interest are stevioside and β-d-glucopyranosyl) ester (1) from the commercial leaf rebaudiosides A. The structure of these glycosides extract of Stevia rebaudiana together with the important consists of a diterpene ent-kaurene skeleton, linked sweet ent-kaurene diterpene glycosides stevioside, to a number of glucose units. Out of 230 species in rebaudioside A-G, rubusoside, and dulcoside A.

Supported by the Department of Science and Technology (DST), New Delhi, India, under a Women Scientist Project Scheme (WOS-A), vide letter No. SR/WOS-A/LS-668/2012. The authors have declared no conflict of interest. 281 Review Czech J. Food Sci., 34, 2016 (4): 281–299

doi: 10.17221/335/2015-CJFS

These non-caloric natural products taste intensely glycosides which are also present in the leaf extract sweet and are estimated to be 150 to 300 times sweeter contributing to low-calorie sweetness and bioactive than sucrose on a weight basis (Richman et al. 1999; properties. Potzel & Brouns 2012). Rebaudioside A exhibits Besides sweetness, various medicinal activities more sweetness and palatable taste profile than other of stevioside and rebaudioside A have been identi- associated steviol glycosides. The Joint FAO/WHO fied which include antihyperglycaemic (Jeppesen Expert Committee on Food Additives (JECFA) in et al. 2002; Gregersen et al. 2004), antineoplastic 2007 laid down specifications that steviol glycoside (Mizushina et al. 2005), anticariogenic (Das et al. sweeteners should consist of at least 95% of the 1992), antihypertensive (Chan et al. 2000), anti- known steviol glycosides (JECFA 2007). inflammatory (Yasukawa 2002), and antioxidant In Europe, United States, China, Japan, Russia, activity (Žlabur et al. 2015). Currently, numerous Korea, and Brazil stevioside and rebaudioside A have reviews have been published concerning the valu- been approved for use as a sweetener (Kinghorn able and abundant potential of steviol glycosides as a & Soejarto 1985). A difference in the structure sweetener in beverages and variety of food products of stevioside and rebaudioside A is having one less including soy sauce, sea foods, pickled vegetables, glucose moiety. Recent development in the field of and confectionary or bakery products. The main research of these glycosides and their derivatives focus area of this article is to review systematic for extracting, isolating, purifying, and modifying literature and to summarise the comparative study them is an ever growing field. The published lit- on the structural features, biosynthetic pathways, erature reported different analytical techniques for safety evaluation, metabolic, pharmacological, and determination of steviol glycosides in S. rebaudiana therapeutic applications of the major steviol glycoside which include an electrospray ionisation technique sweeteners stevioside and rebaudioside A existing used in mass spectrometry (ESI-MS) (Jackson et in S. rebaudiana leaves. al. 2009), analysis of infrared light interacting with a molecule via infrared spectroscopy (Dacome et al. 2005; Hearn & Subedi 2009), liquid chromatogra- Chemistry of steviol glycosides – stevioside phy/mass spectrometry (Wang et al. 2004), capillary and rebaudioside A electrophoresis (Mauri et al. 1996; Dacome et al. 2005), high-performance thin layer chromatography The effort to explain the chemical structures of (HPTLC) (Chester 2012; Londhe & Nanaware S. rebaudiana sweeteners started at the beginning of 2013) and one of the most reliable and simplest the twentieth century, which was from 1901 to 2000; methods recommended by the FAO/WHO Joint but the progress was not fast. In different species of Expert Committee on Food Additives (JECFA) at Stevia plants almost thirty ent-kaurene diterpene gly- the 73rd Meeting (JECFA 2010) is high-performance cosides have been isolated that are commonly known liquid chromatography (HPLC) (Woelwer-Rieck as steviol glycosides – the aglycone part of such gly- et al. 2010; Bergs et al. 2012; Tada et al. 2013). On cosides is steviol (ent-13-hydroxy kaur-16-en-19-oic the other hand, these analytical methods essentially acid) which is involved in constructing a C19-ester focused on the quantification of either stevioside linkage between the C19-carboxylic function and a or rebaudioside A and to a lesser extent on minor glucose unit, along with the formation of ether linkages

Table 1. The chemical identity of stevioside and rebaudioside A

Steviol Common Chemical Chemical Formula C.A.S. glycosides name name formula weight number 13-[(2-O-β-d-glucopyranosyl-β-d-gluco-

Stevioside stevioside pyranosyl)oxy]kaur-16-en-18-oic acid C38H60O18 804.88 57817-89-7 β-d-glucopyranosyl ester

13-[(2-O-β-d-glucopyranosyl-3-O-β-d- glucopyranosyl-β-d-glucopyranosyl)- Rebaudioside A rebaudioside A C H O 967.03 58543-16-1 oxy]kaur-16-en-18-oic acid β-d-gluco- 44 70 23 pyranosyl ester

282 Czech J. Food Sci., 34, 2016 (4): 281–299 Review doi: 10.17221/335/2015-CJFS

Figure 1. The structures of sweet ent-kaurene diterpene glycosides: aglycon steviol, stevioside, and rebaudioside A using its C13-hydroxy group with combinations of in the leaves, a slight amount is in the stem and an glucose, xylose, and rhamnose moieties. Stevioside and untraceable amount in the roots (Pól et al. 2007). rebaudioside A are two sweet tasting diterpene steviol The primary seven steps involved in biosynthesis glycosides found naturally in the leaves of the Stevia of steviol glycosides are similar to the MEP (2-C- plant similar in structure to rebaudioside A having methyl-d-erythritol-4-phosphate) pathway when iso- one more glucose moiety as compared to stevioside. pentenyl diphosphate (IPP), dimethylallyl diphosphate The chemical identities and key chemical identifiers (DMAPP), and geranylgeranyl diphosphate (GGDP) for the two main sweetener components of the genus are synthesised (Wanke et al. 2001). Subsequently, Stevia are presented in Table 1. the next four steps are similar to the gibberellic acid The glucosyl and sophorosyl residue is present (GA) biosynthesis pathway which involves synthesis in the stevioside attached to the aglycon steviol, fi- of kaurenoic acid from geranylgeranyl diphosphate nally showing a cyclopentanoperhydrophenanthrene (GGDP). The last five steps include the steviol glyco- skeleton. Individually the C4 and C13 of steviol are side (SG) biosynthesis pathway (Figure 2) (Hanson linked to the β-glucosyl and β-sophorosyl group. & White 1968). The rebaudioside A has the same structure as that of In the presence of ent-copalyl diphosphate (CDP) stevioside, the only difference is that glucosyl-(1-3)- synthase (CPS) the GGDP is initially converted sophorosyl residue is present in place of sophorosyl through protonation initiated cyclisation to (–)-co- residue. The chemical structures of the Stevia sweet- palyl diphosphate for steviol. Subsequently, kaurene eners stevioside and rebaudioside A and their similar synthase (KS) produces kaurene by ionisation depend- core and metabolite, steviol are shown in Figure 1. ent cyclisation of CDP. Further, via the three-step reaction kaurene is oxidised to kaurenoic acid by a novel kaurene oxidase (KO), like in GA biosynthesis Biosynthetic pathway (Helliwell et al. 1999). Kaurene oxidase (KO) was found to be extremely high in flowers, leaves, suc- Sweetness in Stevia leaves is due to the presence culent stems, and seedling shoots of S. rebaudiana of secondary metabolites steviol glycosides (SGs). (Humphrey et al. 2006). Finally, hydroxylation of Stevioside and rebaudioside A are the vital metabo- kaurenoic acid occurs to produce steviol by means lites among various steviol glycosides. The biosyn- of the kaurenoic acid 13-hydroxylase (KAH) enzyme. thetic pathway of steviol glycosides involves 16 steps At this step, steviol glycoside biosynthesis diverges which are catalysed by numerous enzymes such as from the gibberellic acid (GA) biosynthesis pathway four UDP-glycosyltransferases (UGTs) identified as (Kim et al. 1996; Brandle & Telmer 2007). UGT85C2, UGT74G1, and UGT76G1 and kaure- In the cytoplasm the aglycone steviol is glycosylated noic acid 13-hydroxylase (KAH) (Yadav & Guleria by different glucosyltransferases. There are two 2012). It is operated in the leaves and transported to hydroxyl groups present in steviol – one at C-19 of various parts of the Stevia rebaudiana plant which C-4 carboxyl and the other at C-13. The glycosylation synthesises the important sweetening compounds starts at C-13 by UGT85C2 producing steviol which very much related to the gibberellic acid biosynthetic brings about steviolmonoside. The formation of steviol- pathway (Humphrey et al. 2006). According to dif- bioside takes place by glycosylation of steviolmonoside. ferent studies steviol glycosides are mostly present UGT catalysing this step has not been yet identified. As 283 Review Czech J. Food Sci., 34, 2016 (4): 281–299

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Figure 2. Biosynthetic pathway of steviol glycosides (stevioside and rebaudioside A) (source Brandle & Telmer 2007) CPS – copalyl diphosphate synthase; KS – kaurene synthase; KO – kaurene oxidase; KAH – kaurenoic acid 13-hydroxylase a final point, glycosylation of steviolbioside occurs at is degraded by the bacterial flora of the caecum or C-19 position by UGT74G1 to form stevioside (Shibata colon producing free steviol which is further con- 1991). Rebaudioside A is synthesised by glucosylation at verted into its glucuronide derivative in the liver and C-13 of stevioside catalysed by the enzyme UGT76G1 excreted from the body through urine (Geuns et al. (Brandle & Telmer 2007). 2007). An in vitro method of digesting steviosides by various digestive enzymes was studied by Hutapea et al. (1997) and it was found that none of the enzymes Biotransformation digested the stevioside but the microflora present in the intestinal tract hydrolysed the stevioside to Researches on the biotransformation of major ent-kau- further compounds steviol and steviol-16,17alpha- rene diterpene glycosides showed that stevioside and epoxide. Eventually, steviol-16,17alpha-epoxide was rebaudioside A are safely metabolised by the body and then completely transformed back into steviol and do not differ in elimination though similar pathways excreted as steviol glucuronide from the body in urine followed in both humans and animals (Roberts & (Chatsudthipong & Muanprasat 2009). Renwick 2008). Stevioside owing to its high mo- Rebaudioside A is metabolised by microbes in the lecular weight is not readily absorbed from the upper colon of the digestive tract to stevioside, which is fur- small intestine of the human body and no digestive ther transformed to produce the end product glucose enzymes from the gastro-intestinal tract can break- molecule and steviol. According to Renwick and down stevioside to steviol. Alternatively, stevioside Tarika (2008) the bacterium or Bacteroides sp. in the 284 Czech J. Food Sci., 34, 2016 (4): 281–299 Review doi: 10.17221/335/2015-CJFS colon use the released glucose molecule which is not On 20th September 2012, at the 10th Meeting of Food absorbed into the blood stream and the metabolised Authority held at FDA Bhavan, New Delhi, the use components essentially leave the body. Koyama et of steviol glycoside was also approved as an artificial al. (2003) studied the human digestive tract and ob- sweetener in a variety of foods. served through human faeces that steviol is the end Worldwide, for any marketed food ingredient cor- product of Stevia metabolism and it is not altered at rect and reliable specifications are important for high or low concentrations. The major steviol glyco- commercial, regulatory, and safety reasons. For the sides (stevioside and rebaudioside A) are absorbed comparison of intake and safety limits, the entire steviol and glucuronidated in the liver and a small part of glycosides are converted to their steviol equivalents glucuronidate that stays behind the colon is excreted (JECFA 2007). On the basis of relative molecular through faecal matter and the newly bonded glucuron- weights, 0.33 is multiplied by rebaudioside A quan- ide is released in the blood and filtered via the kidneys tities and 0.40 is multiplied by stevioside quantities into urine. Wingard et al. (1980) also reported the so that both can be converted to steviol equivalents. conversion rate of stevioside and rebaudioside A in The current Joint FAO/WHO Expert Committee on animals (rats) and humans; it was recognised that in Food Additives (JECFA) conducted a systematic scien- individual species the conversion from stevioside to tific review on steviol glycosides at the 58th, 63rd, and steviol is more rapid than that of rebaudioside A to 68th Meetings and established both temporary speci- stevioside. Furthermore, quantitative and qualitative fications and ADI for steviol glycosides of 0–2 mg/kg similarities have been found in the gut (microflora) of BW/day on a steviol equivalent basis and it corre- both the rat organism and the human body. sponds to 0–6 mg of rebaudioside A/kg BW/day using this molecular weight conversion. The predictable permanent ADI for rebaudioside A is 0–12 mg/kg Acceptable daily intake (ADI) BW/day based on an expected permanent JECFA and ADI for steviol equivalents of 0–4 mg/kg BW/day. After Numerous renowned food safety and regulatory getting sufficient information, the European Commis- agencies have made their apprehension accurately sion’s Scientific Committee on Food (SCF) reviewed with Stevia based ingredients (SCF 1985; JECFA 1999; and recommended that it would decrease the safety FDA 2007). S. rebaudiana has been reported as the factor to 100 and make the ADI permanent (SCF 1985, first natural non-caloric sweetener with medicinal 1999a, b). JECFA also concluded that natural sweeteners properties which is safe for people of all ages (Gupta made from Stevia rebaudiana are not dangerous and et al. 2013). Extracted forms of Stevia usually have are safe for use in foods and beverages. a high percentage of the principal sweetening gly- In 2010 The European Food Safety Authority (EFSA) cosides stevioside and rebaudioside A which can be established an ADI of steviol glycosides from Stevia marketed as sweeteners after meeting the regulatory and assessed their safe use. The steviol equivalents purity criteria laid down in Commission Regulation are expressed as ADI of 4 mg/kg BW (Carakostas (EU) No 231/2012 of 9th March 2012. et al. 2008). On 11th November 2011, the European The US FDA has approved the most important Commission permitted the wide-scale usage of steviol steviol glycosides as a safe dietary supplement and glycosides as a food additive in Europe (Stoyanova considered rating in the USA (GRAS Notification 287 et al. 2011). for steviol glycosides with stevioside and rebaudioside A as main components) as appears to have an adequate daily intake (ADI) of approximately 7.9 mg/kg BW Therapeutic applications (body weight) in humans and 25 mg/kg BW in rats (Xili et al. 1992; Geuns et al. 2003). Although the Stevioside and rebaudioside A are the most common authors did not test the concentrations of stevioside active principles of the plant S. rebaudiana. These higher than 793 mg/kg BW, this ADI is supposed compounds have achieved universal attention due to to be considered as a minimum value. In 1994 the their potent sweetness and exhibit diverse activities United States passed the Dietary Supplement Health which play an important part in the medicinal world and Education Act (DSHEA), which permitted the as traditional medicine and have been recommended use of steviol glycosides (SGs) as an ingredient in as a treatment against different chronic and non- dietary supplements (Williams & Burdock 2009). chronic diseases like diabetes, cancer, cardiovascular 285 Review Czech J. Food Sci., 34, 2016 (4): 281–299

doi: 10.17221/335/2015-CJFS disease, high blood pressure, renal disease, fertility, 1 mg of stevioside reduced the postprandial blood and teratogenicity. glucose levels by 18% relative to the control (1 mg of maize starch) and promoted the insulin to glucose ratio in serum by 40%. Glucoregulation The role of rebaudioside A in the stimulation of pan- creas for the secretion of insulin in mouse islets in a dose, The isolation of active principles from medicinal glucose, and Ca2+-dependent manner was studied by plants is a traditional practice for the treatment of Abudula et al. (2008); it was noticed that the presence diabetes mellitus (Kujur et al. 2010). A number of of 16.7 mM glucose and a high concentration of 10–9 M clinical studies have suggested that stevioside and of rebaudioside A significantly increased the ATP/ADP rebaudioside A are now recognised as glucogonostatic, ratio without changing the intracellular cAMP level insulinotropic, and antihyperglycaemic. It may offer and by reducing the ATP-sensitive potassium channel therapeutic benefits for subjects with type 2 diabe- [K(ATP)] conductance in a glucose-dependent manner. tes mellitus and have a direct effect on the β-cells Additionally, rebaudioside A stimulates the secretion of the islets of Langerhans of pancreas to produce of insulin from MIN6 cells in a dose and glucose de- insulin. Holvoet et al. (2015) published a research pendent manner. Thus, from the experimental findings article which supports the Stevia products that help it was specified that rebaudioside A may reduce the in the reduction of insulin resistance by improving risk of hypoglycaemia and can be recommended over the glucose metabolism and enhancing the insulin sulphonylureas. The purified forms of rebaudioside A secretion by the breakdown of fat and bile acids, were granted the GRAS (Generally Recognised as Safe) which aids in the control of weight. status by the US FDA. The main steviol glycoside stevioside is an effec- Consequently, for supporting the positive energy tive antihyperglycaemic agent. It directly acts on the balance, healthy glucoregulation and for pancreatic β-cells and enhances the insulin secretion without gland rejuvenation the table sugars can be substituted altering the K+-ATP channel activity and cAMP with low-calorie sweet tasting, excellent natural sugar level in the islets (Jeppesen et al. 2000). Stevioside alternatives stevioside and rebaudioside A of Stevia. possesses a hypoglycaemic effect by suppressing Saravanan and Ramachandran (2013) studied the secretion of glucagon from the α-cells of the the modulating efficacy and protective effects of a pancreas (Shibata et al. 1995). The direct effect of diterpenoid rebaudioside A on the antioxidant sta- stevioside on glucose transport activity in skeletal tus and lipid profile in experimental streptozotocin muscle was studied by Lailerd et al. (2004). It was (STZ)-induced diabetic rats. Wistar rats were induced reported that the low concentration of stevioside has diabetes by a single intraperitoneal administration of an immense action in glucose transport in skeletal STZ (40 mg/kg BW). The diabetic Wistar rats showed muscle. Chen et al. (2005) suggested that stevio- decreased levels of insulin and elevated levels of plas- side enhances the insulin secretion and also helps ma glucose, thiobarbituric acid reactive substances, in insulin utilisation by regulating blood glucose and hydroperoxides. There was a decreased activity levels in insulin-deficient rats. It was due to the of enzymatic antioxidants (superoxide dismutase, stevioside action of slowing down gluconeogenesis catalase, and glutathione peroxidase) and the levels which decreases the phosphoenolpyruvate carboxy- of non-enzymatic antioxidants (vitamin C, vitamin E, kinase (PEPCK) gene expression in rats’ liver. The and reduced glutathione) in diabetic rats. The lipid long-term exposure of fatty acids in pancreas halts profile levels were significantly increased in plasma the activity of α- and β-cells causing diabetes. In such as triglycerides, total cholesterol, phospholipids, this view the function of stevioside has also been free fatty acids, low density lipoproteins (LDL-cho- studied by Hong et al. (2006) and they concluded lesterol), and very low-density lipoproteins while that stevioside is a strong antidiabetic agent as it can plasma high-density lipoproteins were significantly upregulate the activity of genes responsible for fatty decreased in diabetic rats. The administration of acid metabolism, namely oxidation, conversion, and rebaudioside A (200 mg/kg BW) orally reversed the gene disposal by counterbalancing the hypersecre- lipid peroxidation products, insulin, plasma glucose, tion of α-cells caused by fatty acids. enzymatic, non-enzymatic antioxidants, and lipid Gregersen et al. (2004) conducted an experiment profile levels close to normal. The outcome of this in diabetic humans for insulin sensitivity, where study suggests that the natural low-calorie sweet- 286 Czech J. Food Sci., 34, 2016 (4): 281–299 Review doi: 10.17221/335/2015-CJFS ener rebaudioside A exhibits antilipid peroxidative, tests for chromosomal aberrations in mammalian cells antihyperlipidaemic, and antioxidant properties. in Classical Hodgkin Lymphoma (CHL) and human lymphocytes (Joint FAO/WHO Expert Committee on Food Additives 1999). Genotoxicity Stevioside of 99% purity showed positive results in Salmonella typhimurium (S. typhimurium) strain Intense in vitro and in vivo studies have been done TA98 at 50 mg/plate (Suttajit et al. 1993). The on genotoxicity of steviol glycosides (stevioside and results of data analysis showed a 4-fold increase in rebaudioside A). Twenty expert panels along with revertants without S9 extract and a 2-fold increase international food safety agencies have concluded with bacterial strain S9. The study used pre-incubated that the extensively used sweet steviol glycosides, stevioside with and without β-glucosidase. The same stevioside, and rebaudioside A, are not genotoxic. mutagenic results are shown by both treated and In the latest publications a major concern has been untreated samples which reveal that at 50 mg/plate, expressed on the basis of selected studies and over- stevioside (without β-glucosidase or S9), stevioside all database suggests that steviol glycosides may be metabolite(s) (stevioside + S9), steviol (stevioside + mutagenic, but it has been recommended that ad- β-glucosidase), and steviol metabolite (s) (stevioside + ditional in vivo genotoxicity studies are required to β-glucosidase + S9) are all mutagenic in TA98. Sut- complete their safety profiles (Urban et al. 2013). tajit et al. (1993) also stated that human lympho- A statement from the Joint Expert Committee for cytes incubated with 1, 5, and 10 mg/ml stevioside Food Additives in 2005 concluded that stevioside for 24 h did not cause any chromosomal aberrations. and rebaudioside A did not show any evidence of However, Klongpanichpak et al. (1997) concluded genotoxicity either in vitro or in vivo (JECFA 2005). on the basis of results that stevioside is not mutagenic Brusick (2008) reviewed many literature sources in S. typhimurium strain TA98 at a concentration on the genotoxicity of stevioside, two out of 16 studies of 50 mg/plate though S9 extract is used from mice, confirmed genotoxic activity for stevioside. In con- hamsters, rats, and guinea pigs where Suttajit et trast, Awney et al. (2011) presented the data which al. (1993) showed the strongest outcome or results is well-designed and well-conducted on subchronic without S9 extract. toxicity studies showing that no adverse effects are Matsui et al. (1996) reported that doses of stevio- noticed after the consumption of rebaudioside A. side up to 5 mg/plate were not mutagenic in strains The steviol glycoside, chiefly stevioside, is subjected of S. typhimurium TA97, TA98, TA100, TA102, and to both in vitro and in vivo assays so as to detect TA104 with or without S9 or in S. typhimurium damage to DNA (Brusick 2008). The tests consist strains TA1535 and TA1537, and E. coli WP2 uvrA/ of measuring chromosome alterations, mutations, pKM101 with S9. This compound was not mutagenic and simple breakage of DNA. The exception is with in S. typhimurium strain TM677 with or without S9 a single positive trial in strain TA98 of the Ames test at 10 mg/ml, either. The negative results were also (stevioside at 50 mg/plate which exceeded the recom- given by stevioside in the umu test with or without mended upper concentration limits for that assay), S9 and were negative in the spore and streak rec- for steviol glycosides including rebaudioside A, all in assays with or without S9 at 10 mg/disk of stevioside. vitro test results, do not produce any evidence that Williams and Burdock (2009) investigated the DNA damage is induced by steviol glycosides. The potential of rebaudioside A in inducing genotoxicity in vivo study is based on assessing the capability of in three in vitro and two in vivo assays [conducted ac- stevioside to induce the breakage of DNA strand in cording to the Organisation for Economic Cooperation rats and mice and a micronucleus test for chromo- and Development (OECD) guidelines]. It was found some damage in the mouse. No genotoxicity was that this compound at concentrations up to 5000 µg/ml seen by in vivo assays conducted in rats and mice was not mutagenic in a chromosomal aberration test when given a dose up to 2000 mg/kg BW. In another using Chinese Hamster V79 cells, in the Ames test study on crude crystals of stevioside it was specified using Escherichia coli and S. typhimurium, and in a that the compound is not mutagenic. This has been mouse lymphoma assay using L5178Y+/– cells, with shown again in numerous bacterial assays, specifi- and without metabolic activation. Moreover, rebaudio- cally the forward mutation test in B. subtilis, spore side A at doses up 750 mg/kg BW was non-genotoxic rec assay, Ames test in S. typhimurium and in vitro in a bone marrow micronucleus test in mice and in 287 Review Czech J. Food Sci., 34, 2016 (4): 281–299

doi: 10.17221/335/2015-CJFS rats at 2000 mg/kg BW in an unscheduled DNA syn- reduce the tumour formation and tumour promotion by thesis test. Thus, this study supports the generally blocking the Epstein-Barr virus early antigen (EBV-EA) recognised as safe determination of rebaudioside A induction (Konoshima & Takasaki 2002; Yasukawa and provides enough evidence that at tested doses et al. 2002; Takasaki et al. 2009). rebaudioside A is not genotoxic. The mutagenicity of stevioside was tested for chro- Rebaudioside A does not cause any chromosome mosomal effects on cultured human lymphocytes damage, mutations, or DNA strand breakage in quite and in S. typhimurium strains TA98 and TA100; a few in vitro and in vivo studies (Pezzuto et al. results revealed that stevioside does not show any 1985; Nakajima 2000a, b; Sekihashi et al. 2002). significant chromosomal effect in healthy donors’ cultured blood lymphocytes although it was not mutagenic at concentrations up to 25 mg/plate but Carcinogenicity showed the direct mutagenicity to only TA98 at 50 mg/plate (Suttajit et al. 1993). The stevioside Stevioside and rebaudioside A are the most im- effect against tumour was studied and results showed portant secondary metabolites in leaves of Stevia that stevioside slowed the tumour promoting agent rebaudiana, which is approved in non-toxic category (TPA) induced tumour promotion in a mice skin whereas toxicity was studied under acute oral condi- carcinogenesis (Nakamura et al. 1995). tions due to its structural behaviours and historical The highlighting novel study by Paul et al. (2012) uses (Medon et al. 1982). Studies suggest that crude reported that the consumption of stevioside re- crystals of the compounds stevioside and rebaudio- duces a risk of breast cancer. It was observed that side A are not mutagenic. The anti-inflammatory stevioside decreases certain stress pathways in the and cancer protecting property of S. rebaudiana is body that contribute to the cancer cell growth and due to the presence of water soluble and bioactive enhances cancer apoptosis (cell death). The low compounds like chlorophylls, xanthophylls, stevio- concentration of stevioside was studied for toxi- side, and rebaudioside A (Koubaa et al. 2015) which cological effects on apoptosis induced by serum help in the phagocytic function of cells by completely deprivation using the PC12 cell system by means engulfing and processing the particles, and protecting of DNA electrophoresis and TUNEL signal assays. the body from potential threats by means of boosting On the basis of analysed data it was established that the immune system (Salvador 2014). stevioside enhanced apoptosis induced by serum The Joint FAO/WHO Expert Committee on Food deprivation and this is due to increased expression Additives (JECFA) in 1999 clearly declared that ste- of Bax and of cytochrome c released into the cyto- vioside does not acquire a carcinogenic potential. sol, suggesting that stevioside affects the regulation The carcinogenic effects of stevioside were studied of the normal apoptotic condition (Takahashi et on urinary bladder initiation and promotion. Findings al. 2012). The sweet glycoside stevioside extracted showed that no development of neoplastic or preneo- by methanol and ethanol solvent showed antican- plastic lesions was observed in the urinary bladder cer activity against Caco cell line with IC50 value by stevioside (Hagiwara et al. 1984). The combined 10 and 12 μg/ml and cytotoxic property against outcome of an oral 24-month carcinogenicity and CaSki cell line with IC50 value of 20 and 5 μg/ml, chronic toxicity study of stevioside with 85% purity in respectively (Deshmukh & Kedari 2014). Wistar rats showed that no preneoplastic or neoplastic Administration of rebaudioside A in a single dose lesions were seen in any rat tissue. On the other hand, of 2000 mg/kg BW to male Wistar rats does not show the lack of toxicity was also observed in subchronic any signs of toxicity after observation for 16 h post studies after giving the maximum dose of 600 mg/kg dosing (Williams & Burdock 2009). BW/day (Xili et al. 1992). The stevioside hydrolysed The bacterial reverse mutation test (Ames test) product isosteviol inhibits the human cancer cell growth was used to study the toxicity of rebaudioside A us- in vitro (with LD50 values of 84 to 167 μmol) and DNA ing standard S. typhimurium and Escherichia coli; replication (Mizushina et al. 2005). results showed no significant increase in the number In a two-stage model of mouse skin carcinogenesis with of relevant colonies exposed to rebaudioside A at subsequent sequential exposure to 12-O-tetradecanoyl- concentrations up to 5000 µg/plate. Rebaudioside A in phorbol-13-acetate (TPA) and 7,12-dimethylbenz[a] the Ames test was found to be non-mutagenic in these anthracene (DMBA) have been reported to inhibit and two bacterial strains (Williams & Burdock 2009). 288 Czech J. Food Sci., 34, 2016 (4): 281–299 Review doi: 10.17221/335/2015-CJFS

The mutagenic potential of rebaudioside A was cular therapeutic agents. Leaves of S. rebaudiana evaluated in cultured human lymphocytes; the analysed possess valuable biological properties due to the data showed no significant increase in the incidence presence of the important glycosides stevioside and of chromosomal aberrations or polyploidy in cultured rebaudioside A. An alternative therapy was offered Chinese Hamster V79 cells following 4 and 20 h treat- by these isolated glycosides, non-caloric sweeteners ments at any of the doses tested with or without S9 whose consumption may possibly exert valuable ef- metabolic activation (Williams & Burdock 2009). fects on human health by functioning as heart tonic Rebaudioside A was tested for the mutagenic potential for lowering the elevated blood pressure, regulating on mouse bone marrow cells by means of the mam- the heartbeat, relaxing the arteries, inhibiting vas- malian erythrocyte micronucleus. The results of the cular contractility and diuretic action (Gardana et test showed that the maximum dose of 5000 µg/ml al. 2010). Usual consumption of these compounds did not produce any statistically significant increase decreases the content of blood cholesterol by main- in the incidence of polychromatic immature eryth- taining the lipid profile levels (Atteh et al. 2008). It rocytes (Williams & Burdock 2009). Additionally, also improves blood coagulation and cell regenera- rebaudioside A was shown not to cause any signs of tion, suppresses neoplastic growth and strengthens toxicity when administered to the mice at doses of blood vessels (Wingard et al. 1980; Jeppesen et al. 150, 275, or 750 mg/kg of BW compared to the plain 2003; Barriocanal et al. 2008; Maki et al. 2008). saline control (Williams & Burdock 2009). The role of stevioside in inhibiting the athero- sclerosis by improving insulin signalling and anti- oxidant defence in obese insulin-resistant mice was High blood pressure and cardiovascular effects studied by Geeraert et al. (2010). Results showed that stevioside action in obese insulin-resistant mice A lot of reports have broadly shown that various enhanced adipose tissue maturation and increased classes of plant-derived substances like diterpenoids glucose transport, insulin signalling, and antioxidant exert significant cardiovascular and antihyperten- defence in white visceral adipose tissues. Additionally, sive effects (Melis 1991; Tirapelli 2008). The di- stevioside also helps in reduction and inhibition of terpenoids stevioside and rebaudioside A show a the plaque volume in the aortic arch by decreasing great potential source of the latest prototypes for the number of macrophages, lipid, and oxidised low- the discovery and development of new cardiovas- density lipoprotein (ox-LDL) content of the plaque.

Figure 3. Mechanism of stevioside action on the cardiovascular system. Stevioside decreases the vascular resistance which promotes the blood pressure reduction by means of the inhibition of extracellular Ca2+ influx which stimulates the release of a vasodilator prostaglandin. Stevioside promotes diuresis, natriuresis, and reduction of Na+ reabsorp- tion ensuing in a reduction of the extracellular fluid volume (Tirapelli et al. 2010)

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A group of patients treated with stevioside was primarily by inhibiting the extracellular Ca2+ influx. well-tolerated with no side-effects and reported to Additionally, in vitro effects of stevioside showed have significantly superior quality of life scores than that stevioside induces hypotension in conscious the placebo group. However, more patients in the hypertensive rats (Lee et al. 2001). placebo group developed left ventricular hypertro- The precise fundamental mechanism of stevioside phy (Hsieh 2003), high blood pressure may often underlying the cardiovascular actions is summarised cause the abnormal thickening of the heart muscle. in Figure 2. Stevioside affects vascular resistance by A placebo-controlled double-blind study established inhibiting the extracellular Ca2+ influx and the release that stevioside taken orally at doses of 250 mg three of vasodilator prostaglandin which in general helps times a day in patients with mild to moderate hyperten- in a reduction of blood pressure. This compound sion for one year resulted in a significant, long-term also results in a reduction of the extracellular fluid decrease in both the systolic and diastolic blood pres- volume and produces diuresis and natriuresis. Tox- sure (Chan et al. 2000). Following the initial study icity of stevioside in rodents was investigated and by a similar research team by means of an increased results showed that the intake of stevioside as high as dose of stevioside (1.5 mg) stimulated the decrease in 7.9 mg/kg BW produces no acute toxicity (Xili et al. blood pressure (Hsieh et al. 2003). Studies revealed 1992). These major findings might provide significant that purified stevioside produces changes in prosta- information about the possible use of stevioside in glandin activity and induces diuresis, natriuresis, and the treatment of arterial hypertension. A randomised, hypotension in rats (Melis et al. 1985). double-blind 4-week study by Maki et al. (2008) The first study on stevioside was conducted in 1977 evaluated hemodynamic effects of the consumption to investigate the cardiovascular effects of stevioside of 1000 mg/day rebaudioside A against placebo in in rats (Humboldt & Boech 1977). It was estab- 100 individuals with normal and low-normal systolic lished that stevioside induces diuresis and a marked blood pressure (SBP) and diastolic blood pressure decrease in the mean arterial pressure and heart (DBP). The study groups were mostly female (76% rate. The mechanism behind the role of stevioside in rebaudioside A and 82% placebo) with an average age cardiovascular effects was investigated initially and it of around 41 (range 18–73) years. The mean resting was found that an intravenous infusion of stevioside seated systolic blood pressure/diastolic blood pres- (8 and 16 mg/kg/h) in normotensive rats produced a sure was 110.0/70.3 and 110.7/71.2 mm Hg for the noticeable dose-dependent hypotensive effect with rebaudioside A and placebo groups, respectively. On diuresis and natriuresis (Melis & Sainati 1991). comparing with the placebo, rebaudioside A does More recently, studies on stevioside suggested that not significantly change the resting seated systolic it induces a reduction in the mean arterial pressure blood pressure/diastolic blood pressure, mean arte- and lower renal vascular resistance by promoting rial pressure, heart rate, or 24-h ambulatory blood renal vasodilatation. This vasodilator effect is due to pressure responses in patients with low-normal to the blockage of Ca2+ channels because verapamil, a normal blood pressure. The results of the present Ca2+ channel blocker, improved the systemic effect of study suggest that 1000 mg/day consumption of stevioside, while CaCl2 infusion reduced the vasodila- rebaudioside A does not produce any clinically vital tor response of stevioside (Melis & Sainati 1991). changes in the blood pressure in healthy adults with The stevioside in a dose-dependent manner relaxed normal and low-normal blood pressure. the endothelium-intact and endothelium-denuded arteries in rat isolated aortic rings, contracted with vasopressin (Lee et al. 2001). The stevioside-induced Renal function relaxation was not influenced by methylene blue, a guanylate cyclase inhibitor, presenting that the Abundance of information was offered by tra- relaxation was not mediated by the cyclic guano- ditional herbalism regarding the treatment of the sine monophosphate (cGMP)-NO pathway. The kidney disease. The presence of diterpene glycosides authors used the cultured aortic smooth muscle cells in the herb S. rebaudiana exhibits a high degree (A7r5) and found that the Ca2+ influx was blocked of natural antioxidant activity and is used as high by stevioside but it was ineffective in inhibiting the potency sweeteners. intracellular Ca2+ release. Therefore, the resulting Toskulkao et al. (1994) studied the interac- data indicate that stevioside induces vasorelaxation tion between urinary enzyme levels and changes in 290 Czech J. Food Sci., 34, 2016 (4): 281–299 Review doi: 10.17221/335/2015-CJFS plasma creatinine, and blood urea nitrogen levels the transepithelial transport of p-aminohippurate on in rats treated with stevioside by means of concur- the luminal side and its reversible inhibitory effect rent changes of the kidney. There is an increase in on the basolateral side indicate that stevioside does blood urea nitrogen after subcutaneous injection not permanently change the p-aminohippurate trans- with stevioside (1.5 g/kg BW) at 3 h onward. The port and does not harm the renal tubular function at blood urea nitrogen and creatinine level increases normal human intake levels (Jutabha et al. 2000). to a maximum after stevioside injection with ap- In a recent study, Hashemi et al. (2014) investigated proximately 180 and 132% at 9 hours. At this point the feasible protective effects of rebaudioside A on in time stevioside causes a significant increase in acetaminophen (APAP)-induced oxidative stress in alkaline phosphatase (AP), glucosuria and glutamyl the kidney of mice. The oxidative stress was induced transpeptidase (GTP), however, no significant changes in the kidney of BALB/c mice by the intraperitoneal in proteinuria, N-acetyl-d-glucuronidase (NAG), or (i.p.) administration of a single dose of 300 mg/kg glutathione-S-transferase (GSH-S-TF). Degeneration acetaminophen. Thirty minutes after acetaminophen of the proximal convoluted tubule cells was detected injection a number of these mice were treated with after histopathological examination of the kidney rebaudioside A (700 mg/kg) (i.p.). Later, after two induced by stevioside but no lipid peroxidation was and six hours of acetaminophen injection all BALB/c found. Results revealed that stevioside causes nephro- mice were sacrificed and glutathione (GSH), malon- toxicity at the proximal convoluted tubules rather dialdehyde (MDA), free acetaminophen, and glu- than at the other tubules or glomeruli. It was most tathione conjugated acetaminophen (APAP-GSH) probably by a defect of the cell volume regulation were determined in the kidney tissues of sacrificed due to depletion of intracellular ATP and disruption mice. Thus, findings suggest that though rebaudio- of microvilli, and nuclear dysfunction. side A was not successful in preventing the initia- In another study by Melis et al. (1992) the effect tion of acetaminophen-induced oxidative stress, as of stevioside from S. rebaudiana leaves on the renal indicated by GSH depletion and lipid peroxidation function of normal and hypertensive rats was studied. in kidneys of mice, but afterwards it attenuated lipid The examined stevioside functions as a systemic peroxidation by reducing acetaminophen conversion vasodilator which aggravates hypotension, diuresis, to its activated metabolite, specifically N-acetyl- and natriuresis in both the normal and hypertensive p-benzoquinone imine (NAPQI), which produced rats. The administration of stevioside continuously APAP-GSH conjugate in kidneys of mice. Thus, to both normal and hypertensive rats increased the rebaudioside A acts as a principal compound in the renal plasma flow and glomerular filtration rate, alleviation of acetaminophen induced oxidative stress which was due to the vasodilation of both the afferent in kidneys of mice after acetaminophen overdoses. and efferent arterioles. A similar study shows that long-term oral intake or acute intravenous admin- istration of stevioside lead to a decreased plasma Fertility and teratogenicity volume producing diuresis and natriuresis. On the other hand, the infusion of stevioside directly into It is controversial whether the plant S. rebaudiana rats’ artery induces diuresis. This reaction was due or their extracts possess some contraceptive effect to decreased proximal tubular reabsorption as in- (Planas & Kuc 1968). Paraguayan Matto Grosso dicated by lithium clearance (Chatsudthipong & Indian tribes used the leaves of this herb S. rebau- Thongouppakarn 1995), signifying that stevioside diana in tea or beverages as a male or female oral targets at the proximal tubule of the kidneys. contraceptive. This research was designed to investigate the ef- The legislative body mentioned in the application fect of stevioside on the transepithelial transport of that accessible results do not explain that stevioside p-aminohippurate in isolated S2 segments of rabbit induces teratogenic or embryotoxic effects and this proximal renal tubules using in vitro micro-perfusion. report is scientifically supported by three studies, The result shows that stevioside, at a concentration one in the hamster (Yodyingyuad & Bunyawong of 0.70 mM, inhibits the transepithelial transport of 1991) and two in the rat (Mori et al. 1981; Usami p-aminohippurate by interfering with the basolateral et al. 1995). The effect of stevioside on growth and entry step, the rate-limiting step for transepithelial reproduction was studied. Stevioside at different doses transport. The absence of the effect of stevioside on (500, 1000, and 2500 mg/kg BW/day, 90% purity) 291 Review Czech J. Food Sci., 34, 2016 (4): 281–299

doi: 10.17221/335/2015-CJFS was fed to hamsters during the mating time and they histopathological examination of the testes in high- were permitted to bear three litters. In the course of dose males from both 28 and 90 day feeding studies treatment mating performance, fertility, pregnancy, (Curry et al. 2008). The results were unexceptional number of foetuses, in addition growth and fertility after macroscopic and microscopic examinations of the offspring were not affected. On the other hand, of the testes from the 28-day study in 100 000 ppm no teratogenic effects were studied (Yodyingyuad group and from the 90-day study of all the male re- & Bunyawong 1991). In another study stevioside productive organs in the 50 000 ppm group. It was was applied in a diet of Wistar rats (0.15, 0.75, and concluded from both studies that in both male and 3%, equivalent to 150, 750, and 3000 mg/kg BW/day, female reproductive systems no treatment-related 96% purity). Before and during the mating period adverse effects were observed. males were treated for 60 days while females for Sloter (2008) tested the rebaudioside A via gavage 14 days before the mating period and for 7 days during in an embryo/foetal developmental toxicity study in gestation. The analysed result shows no treatment pregnant rats. Intrauterine growth and survival were related effects on fertility or mating performance, not affected by the administration of rebaudioside and no developmental malformations were seen in A and there were no developmental variations or the foetuses (Mori et al. 1981). foetal malformations at any dosage level. A dose level Stevioside of 95.6% purity, dissolved in distilled ≥ 2000 mg/kg BW/day (highest dose administered) water, was given to four groups of 26 pregnant Wistar was considered to be the NOAEL for maternal and rats once a day at doses of 0, 250, 500, and 1000 mg/kg embryo/foetal developmental toxicity. BW/day from 6th day to 15th day of pregnancy, on 20th day of gestation the rats were sacrificed, and the foetuses of pregnant Wistar rats were examined for Biotechnological approach malformations. At the final stage the points examined were number of live foetuses, number of resorptions Sixty thousand years back in the middle Palaeolithic or dead foetuses, maternal and foetal body weight, age traditional medicinal systems developed by incor- sex distribution, and incidence of malformations. porating plants or herbs as a mode of therapy, which It was concluded that no teratogenic effects were can be tracked from fossil studies (Solecki 1975). induced in pregnant rats upon the administration of In the past few years a number of major advances stevioside orally (Takanaka et al. 1991; Usami et have occurred in molecular techniques mainly focus- al. 1995). Perhaps, in view of the numerous studies, ing on their benefits and limitations; moreover, the it can be concluded that stevioside does not cause latest technologies improve existing techniques or any developmental toxicity at high doses. Although build up new approaches so as to produce informa- some data suggest that stevioside can affect the tion more quickly, accurately, easily, or in a more male reproductive organ system and reproductive straightforward repeatable manner than the existing performance, for example decreased seminal vesicle methods. Accordingly, for Stevia a number of advanced weight, reduced spermatogenesis, and interstitial molecular techniques are applied, for instance nuclear cell proliferation in the testes (Yamada et al. 1985). magnetic resonance (NMR) studies were approved for Furthermore, few researchers reported that no determining the physical and chemical properties and developmental toxicity was evidenced from pub- confirming the structure of natural sweeteners ste- lished chronic toxicity (Xili et al. 1992; Toyoda vioside and rebaudioside A (Steinmetz & Lin 2009). et al. 1997), subchronic toxicity (Aze et al. 1991), Biotechnology plays an important role in acceler- and developmental and reproductive toxicity (Mori ated development of consistent plant based drugs. et al. 1981; Yodyingyuad & Bunyawong 1991; For multiplication and genetic enhancement of the Usami et al. 1995) studies that purified stevioside medicinal plant like S. rebaudiana (Bertoni) biotech- or rebaudioside A have an undesirable influence on nological tools are important which help in adopting the male or female reproductive systems. new techniques, for example in vitro regeneration, On the basis of past database and continuous de- genetic transformations, and plant tissue culture. bate over the reproductive effects of stevia extracts The biotechnologists show enormous interest in the additional investigations were conducted to prove production of valuable secondary products by using the reproductive safety of high-purity rebaudio- the cell culture technique. In the current situation side A. The initial phase of the evaluation included a plant tissue culture is a noble, innovative, effective, 292 Czech J. Food Sci., 34, 2016 (4): 281–299 Review doi: 10.17221/335/2015-CJFS and efficient method for obtaining their substances insect and plant-animal interactions (Pichersky & on a large scale and for converting less medicinally Gershezon 2002). vital plant metabolites to a valuable product. Few scientists performed a small number of mo- An extremely low germination percentage was lecular level investigations from the Stevia leaf cDNA shown by seeds of S. rebaudiana (Felippe & Lucas library. Brandle et al. (2002) one-time sequenced 1971; Monteiro 1980; Toffler & Orio 1981) and a 5548 expressed sequence tags (ESTs) and studied the practice of cutting the plant through vegetative propa- association of the MEP pathway for steviol biosynthesis gation is restricted to a small number of individuals without involving the mevalonic acid (MVA) pathway. (Sakaguchi & Kan 1982). The simple and quick S. rebaudiana has many applications in nano- process for the mass propagation of S. rebaudiana is technology and includes a potential to recommend through tissue culture techniques which include many positive applications for food and health. The dried areas such as cell nutrition, cell division, differentia- stevia leaf extract was used as a shadow in AgNO3 tion, and cell preservation. But at present, bulk cells solution to synthesise silver nanoparticles (Yilmaz are cultivated in vitro or as clone from single cells to et al. 2011). X-ray diffraction and transmission elec- produce whole plants from the isolated meristem, tron microscopy (TEM) specify that nanoparticles are subsequently the callus is induced and complete polydispersed and spherical with diameter ranging plantlets are developed by organogenesis or by em- from 2 nm to 50 nm. bryogenesis. Plant propagation by means of in vitro The “nutraceutical” application of the Stevia extract method holds a remarkable potential for the produc- comprises foods and beverages as pharmaceutical tion of high-quality plant-based medicines (Murch compositions such as protective hydrocolloids (like et al. 2000). The micropropagation of S. rebaudiana proteins, modified starches), secondary metabolites, through shoot tip culture was reported by Das et al. bioactive compounds, antioxidants, and antibacterial (2011). In support of multiple shoot proliferation agents that may possibly be added to soft drinks, within 35 days of culture, Murashige and Skoog (MS) nutritious bars or candies, with the aim that an adult medium (Murashige & Skoog 1962) supplemented can consume a large amount of preferably 100–500 mg with 2 mg/l kinetin was found to be the most excel- of Stevia extract. lent option producing more than 11 shoots from a The diverse applications of Stevia have brought single shoot tip explant. MS media without growth the interest of researchers to start research in a regulators worked vigorously for root induction variety of fields for understanding the chemistry, but when supplemented with indole-3-acetic acid biochemistry, structure, behaviour, and role of these (IAA) and benzyl adenine (BA), they had an adverse steviol glycosides. Researchers have been working effect on root induction. The peroxidase assay was for a long time in the field of immunology to clarify performed along with inter-simple sequence repeat the biological nature and safety aspects of steviol (ISSR) fingerprinting to authenticate the genetic glycosides (Ahmed et al. 2011). clonality of in vitro generated propagules. Thus, an overall study shows that the shoot tip culture in agar medium containing a high concentration of kinetin CONCLUSION helps in the micropropagation of S. rebaudiana. The practice of metabolic engineering experi- The leading food scientists of the world consider ments may disclose undiscovered branches to al- bioactive constituents of S. rebaudiana as the “sweet- ready known metabolic pathways. The achievements eners of the future or millennium”. Stevia has diverse in metabolic engineering of diterpenoids help in applications in various fields such as in global food determination of biological activities of transgenic industry and medicinal world. Traditionally, the leaves plants engineered for a diterpenoid pathway. The of S. rebaudiana are acknowledged as producer of presence of diterpene steviol glycosides (stevioside diterpenoid steviol glycosides which are low in calo- and rebaudioside A) in leaves of S. rebaudiana pro- ries, non-hazardous, anti-microbial, and effective vides significant applications in industrial products sugar substitute of commercial value in a number for instance flavouring agents, antimicrobial agents, of countries. The chemistry, biochemistry, and bio- commercial sweeteners, and pharmaceuticals. The technology have played a vital role in understanding essential role of these glycosides is plant-plant com- the structure, behaviour, and role of these steviol munication, plant-environment interaction, plant- glycosides. The main active principal metabolites of 293 Review Czech J. Food Sci., 34, 2016 (4): 281–299

doi: 10.17221/335/2015-CJFS these glycosides are stevioside and rebaudioside A, Bertoni M. (1905): Le kaa He-e-Sa nature et ses proprieties. which have a huge impact on human physiology and Anales Cientificos Paraguayos. Serie I, 5: 1–14. are reported to possess a potential therapeutic mode of Brandle B.E., Telmer P.G. (2007): Steviol glycoside biosyn- applications in diabetes, cardiovascular, cancer, renal, thesis. Phytochemistry, 68: 1855–1863. and reproductive system. There is a requirement for Brandle J.E., Richman A., Swanson A.K., Chapman B.P. much skill for the toxicological evaluation of these (2002): Leaf ESTs from Stevia rebaudiana: a resource for glycosides to settle issues related to safety concerns. gene discovery in diterpene synthesis. Plant Molecular Therefore, some biotechnological techniques must be Biology, 50: 613–622. implemented to cover a broad spectrum of scientific Brusick D. (2008): A critical review of the genotoxicity of applications for understanding the potential benefits steviol and steviol glycosides. Food and Chemical Toxi- offered by the remarkable plant S. rebaudiana. cology, 7: S83-S91. Carakostas M.C., Curry L.L., Boilea A.C., Brusick D. (2008): References Overview: the history, technical function and safety of rebaudioside A, a naturally occurring steviol glycoside, Abudula R., Matchkov V.V., Jeppesen P.B., Nilsson H., Aal- for use in food and beverages. Food and Chemical Toxi- kjaer C., Hermansen K. (2008): Rebaudioside A directly cology, 46: S1–S10. stimulates insulin secretion from pancreatic beta cells: a Chan P., Tomlinson B., Chen Y.J., Liu J.C., Hsieh M.H., glucose dependent action via inhibition of ATP-sensitive Cheng J.T. (2000): A double-blind placebo-controlled K+-channels. Diabetes, Obesity and Metabolism, 10: study of the effectiveness and tolerability of oral stevio- 1074–1085. side in human hypertension. 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Corresponding author: Dr Ena Gupta, University of Allahabad, Centre for Biotechnology, Uttar Pradesh, India; E-mail: [email protected]

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