Stevia is a source for alternative sweeteners: potential medicinal effects MARÍA HERRANZ-LÓPEZ1, ENRIQUE BARRAJÓN-CATALÁN1, RAÚL BELTRÁN-DEBÓN2, JORGE JOVEN2, VICENTE MICOL1* Vicente Micol *Corresponding author 1. Universidad Miguel Hernández, Instituto de Biologia Molecular y Celular Avda. de la Universidad s/n., Elche (Alicante), E-03202, Spain 2. Universitat Rovira i Virgili, Centre de Recerca Biomèdica, Hospital Universitari de Sant Joan, IISPV C/Sant Joan s/n, Reus, 43201, Spain

ABSTRACT: The leaves from Stevia rebaudiana have been used as a source for natural sweeteners for decades in several Asian countries. Stevioside, the major sweet compound from this plant, is 300 times sweeter than sucrose, but has a little bitter aftertaste. This may be overcome via a simple enzymatic modification or via the use of another less abundant diterpenoid glycoside, rebaudioside A, which is devoid of such effect. Stevia refined extracts have been used in the US since 1995 only in the dietary supplements market. In 2008, American FDA finally gave the approval for Generally Recognized as Safe (GRAS) status to highly purified rebaudioside A. France also approved rebaudioside A for food and beverages in 2009. Most of the European Union countries are awaiting the EU approval on stevia products by 2011. Pharmacological studies show that all the glycosylated diterpenoids are degraded in the intestinal tract into their aglycone steviol, therefore this compound is considered the pharmacologically active form. Global authorities (JECFA) have set 4 mg/kg body weight as acceptable daily intake (ADI), expressed as steviol compound. Approval in other countries, however, will require further research on safety and toxicology of stevioside and related compounds. Unexpectedly, recent research suggests that stevioside and rebaudioside A may bear some therapeutic properties. Particularly, a decrease of postprandial glucose and glucagon levels as well as insulinotropic effects have been shown in several cellular and animal models. In human and animals, anti-hypertensive and immunomodulatory properties are also plausible. More recently, the antioxidant and anti-inflammatory effect of stevia-related compounds have significantly reduced the size of the Sweeteners atherosclerotic lesion in an animal model of metabolic syndrome. The increase of obesity in modern societies is driving consumer trends towards the use of non-caloric and natural sweeteners. Hence, a dramatic increase of the use of stevia- related sweeteners is expected in substitution of synthetic sweeteners such as aspartame or saccharine. Additionally, stevioside and rebaudioside A, may provide medicinal properties to reduce obesity and cardiovascular risk.

KEYWORDS: alternative sweeteners, Stevia sp., stevioside, rebaudioside A, metabolic syndrome, obesity, atherosclerosis. vol 21 n 3 -

INTRODUCTION SWEET COMPOUNDS FROM STEVIA SP. AND THEIR CHEMICAL PROPERTIES Stevia rebaudiana (Bertoni) is an herb from the

May/June 2010 Asteraceae family commonly known as sweetleaf, The main compounds from stevia leaves are stevioside (5-10 - sugarleaf or simply stevia, which is originally native to percent dry weight) and rebaudioside A (2-4 South America. The leaves of this plant have been percentiterpenoid glycosides (1). Stevioside has glucosyl and traditionally used by native people in sophorosyl (diglucose with β, 1-2 South America as sweetener and in substitution) beta residues attached to

industry hi-tech The increase of obesity in traditional medicine for centuries. In the aglycone steviol. Rebaudioside A the 1970s, Japan was the first country modern societies is driving contains an additional glucose bound OOD which marketed stevioside consumer trends towards to the sophorosyl moiety through b, 1-3

gro F containing additives as a food and glycosidic bond (Figure 1). Other minor

A the use of non-caloric and pharmaceutical sweetener, followed diterpenoids in stevia are dulcoside A, by many other Asian countries such natural sweeteners stiviolbioside, and rebaudiosides B, C, D as China, Malaysia, Singapore, and E (2). These compounds have a Korea, Taiwan and Thailand. In the last decade USA, relatively high molecular weight (>800) and it is unlikely that Canada, Australia and New Zealand started allowing the may be absorbed in the intestine. However, bacterial use of Stevia sp. for nutritional supplements. Most of these intestinal flora of mammals, including humans, are able to countries together with some convert them into its aglycone, steviol European countries recently A dramatic increase of the (1, 3, 4). Whether this compound approved some highly purified represents the pharmacologically extracts from stevia for the food use of stevia-related active form is probable although category. Further unrestricted sweeteners is expected in debatable (5). Rebaudioside A, B, and approval as a food additive will substitution of synthetic D and stevioside show similar sweetness require comprehensive clinical sweeteners such as potency (300-350 fold sucrose), but studies in order to establish medicinal rebaudioside E and dulcoside are properties, to assess pharmacology aspartame or saccharine significantly less sweet. Stevioside, of pure active compounds and to dulcoside A, and rebaudiosides B and clarify some uncertainties about safety, with particular C have a slight bitter aftertaste (6), difficulting their use in attention to reproductive effects. some food applications but this has been solved for

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Truvia (codeveloped by PepsiCo), and (codeveloped by PepsiCo), and , Cargill with Cargill , Success Sweet European advisors are still concerned European advisors are still concerned which are safety aspects several on limiting the development of stevia- used as a widely compounds related sweetener. Massive use in other suggests that this however, countries, The future. will change in the near is Agency Safety Food European on steviol expected to release a report glycosides in 2010, which may drastically change the use of stevia- RebA-Sweet and ETS-Sweet. saccharin and other synthetic sweeteners, from where its use where from sweeteners, synthetic other and saccharin Stevia especially Korea. countries, Asian other to was spread use its but 1980’s, the in market USA the in introduced was safety for years while to dietary supplements was limited was A rebaudioside Finally, investigated. further were issues Generally 2008 and rated by FDA in December approved as a general-purpose its use for as Safe (GRAS) Recognized products. poultry and meat excluding foods, in sweetener Switzerland, Russia, in released also been has Approval Zealand. It is therefore predictable that Australia and New changes in technology and there will be considerable should suppliers Particularly, material. raw the of distribution to technology production best the select to forced be compound high and batches among consistency achieve be a key step in seems to granulation instance, purities. For has a form granulated process since the manufacturing the on beverages but that than fine powder better performance investments in production technology. implies considerable are suppliers and providers global hand, other the On already delineating a clear trend in of natural the market of intention manifested the by exemplified sweeteners favouring sucralose, abandon to Coca-Cola and PepsiCo distributors main the Currently, A. rebaudioside of use the with RebA97, and brands for this compound are: PureCircle with Corp. Tech GLG are products stevia-derived for applications possible The sauces, table top dairy products, bakery, beverages, broad: supplements, sweeteners, confectionery, food feeding. Companies pharmaceutical products and animal have etc., Life), (SoBe PepsiCo (Truvia), Coca-Cola like European the Within products. different launched already containing especially beverages, products, the first Union, being launched in are A) rebaudioside stevia (97 percent like Switzerland and non- countries European In other France. is t produc the Australia and Ukraine Russia, as European 2008. already approved for food purposes since already approved has France Meanwhile, products. related Rebaudioside A (97 percent) for food and beverages via a transitory directive. SAFETY PHARMACOKINETICS. AND ABSORPTION STEVIOSIDE ISSUES Several studies performed in animals and humans proposed are glycosylated diterpenoids other and stevioside that aglycone their into intestinal flora bacterial by the degraded of metabolite major the be to 4). Steviol appears 3, steviol (1, these compounds into the blood circulation appearing In the liver steviol may yield oxidation following oral ingestion. by steviol of metabolism I phase a and metabolites (phase II conjugation Further P450 is suggested. cytochrome as occur may also metabolites steviol of metabolism) suggested by Geuns et al. (10, 11), since they found steviol glucuronide as the only detectable metabolite in blood and (codeveloped with Coca-cola), Prodalsya with “Ultra Pure”, (codeveloped with Coca-cola), Prodalsya Merisant with PureVia Monteloeder with concerns Stevia-derived products Stevia-derived products established some safety established some safety USA will disappear once USA will disappear once limitations in Europe and limitations in Europe and decades and presumably decades and presumably have been used in Asia for have been used in Asia for is constantly growing in USA and sales sales and USA in growing constantly is sp. Stevia Figure 1. Chemical structure of the diterpenoid glycoside rebaudioside A A rebaudioside glycoside diterpenoid the of structure Chemical 1. Figure steviol. containing the glycoside stevioside and the aglycone Aspartame and saccharine are the main substitution targets Aspartame and saccharine are the main Facts Packaged According compounds. stevia-derived for weight-loss the firm, research market US-based an (PF), increasing are factors determining current and other efforts predicted $3.2 a market to the global artificial sweeteners & Sullivan), such market Frost billion by 2012. In 2008 (source $1.3 to close be to was estimated position leading a has billion. Sucralose with approximately a 36 percent of share, by value, in food applications. Data provided by Intelligence 52 weeks for Inc. Resources Information between March 2008 March 2009, and were substitutes sugar of sales US of sucralose those $372m, and roughly period same the $217m.In represented and saccharin for sales time, of market The respectively. $37m, and $48m were aspartame for a 287.5 underwent percent increase within the period 2003- 2007, whereas those for sucralose were less notorious (90 and aspartame for sales which in scenario an in percent), PureCircle saccharin decreased considerably (8). Moreover, reported sales of rebaudioside A in USA of $34.1m in 2008 growing towards a trend indicating and $60m in 2009, This is probably due use. human for in this compound interest natural it is a 100 percent several reasons, including that to and temperatures high is stable at it sweetener, non-caloric processing food of variety a with compatible consequently methods and it is sold at competitive prices as compared with natural sweeteners like thaumatin. Therefore, it may industry searching for in an perfect choice the represent synthetic chemical or of further need the without sweeteners process (9). Stevia-derived compounds were first used in the food of use the banned Japan when 1970’s early the in industry DISTRIBUTION, LEGISLATION AND PROSPECTIVE ECONOMIC ECONOMIC PROSPECTIVE AND LEGISLATION DISTRIBUTION, VALUE FOR STEVIA-DERIVED PRODUCTS stevioside through enzymatic processing (7). Rebaudioside Rebaudioside (7). processing enzymatic through stevioside effect. unwanted of such devoid is naturally A, however urine in subjects receiving stevioside orally at a dose of 750 effect seems to be mediated via an incompletely mg/day for 3 days. A similar metabolic pathway has been understood action on the enzyme phosphoenol pyruvate found for rebaudioside A. For both compounds, mean carboxy kinase, which is a key step in hepatic maximum concentration of steviol glucuronide appears to gluconeogenesis (28). The same effects have been recently be between 3 and 3.6 µM with a median time to peak described in non-diabetic rats after the oral consumption of concentration of 8 h (12). Biliary and 20 mg/Kg of stevia leaves although this urinary tracts are the major routes for In USA and some European was not appreciated with regular steviol glucuronide excretion but, in consumption of 5.5 mg/Kg stevioside, humans, the urinary excretion prevails countries, rebaudioside A indicating that other steviol glycosides probably involving renal organic anion (97 percent) purified from should be considered (29). transporters. Due to their popular use as Stevia sp. has been Other reports are focused on the sugar substitutes and in remedies, already considered safe for capacity of stevia-related compounds toxicological properties of stevioside to modulate insulin secretion and and rebaudioside A have been its use in food processing glucose tolerance by peripheral extensively studied. In addition, tissues. Steviol and stevioside enhance carcinogenic and teratogenic potentials as well as insulin secretion in isolated mouse pancreatic islets (30). This reproductive effects have also been evaluated. Taken in vitro predicted insulinotropic action of stevioside was together, most studies agree that oral stevioside at an further confirmed in glucose tolerance tests performed in a acceptable daily intake (ADI) of 5 mg/kg body weight is type 2 diabetes rat model using either an intravenous safe and not carcinogenic or teratogenic (1). Certainly, injection of stevioside 0.2 g/Kg body weight (19), or after JECFA has set and ADI value of 4 mg/kg body weight the oral consumption of 25 mg/kg/day for six weeks (31). expressed as steviol, or 11 mg/kg as stevioside although Stevioside (5 mg/kg) was also administered to rats with these estimates are considered highly conservative (13). insulin resistance induced via a high-fructose diet and anti- hyperglycemic, insulinotropic and glucagonosuppresive effects were observed (32). In humans, however, the THERAPEUTICAL POSSIBILITIES OF STEVIA SP. PRODUCTS IN THE insulinotropic effect of stevioside is only observed in MANAGEMENT OF METABOLIC SYNDROME diabetic patients but not in normal individuals. This is important for general use as well as the potential to induce The prevalence of obesity continues to increase with more hypoglucemia (19, 30). Interestingly, the effects in than 50 percent of Europeans currently classified as stimulating insulin secretion and increasing insulin sensitivity overweight and up to 30 percent as are more apparent in skeletal muscle,

Sweeteners clinically obese. The risk of developing Stevia-derived compounds the major site of glucose disposal. coronary heart disease is directly Experiments performed with related to the concomitant burden of have exhibited the rebaudioside A also showed an obesity-related cardiovascular risk capacity to control increase of insulin release in mouse factors clustered in the metabolic glucose levels through its islets under a high glucose syndrome (MS): dyslipidemia, insulin insulinotropic and environment (33), but failed to prove a

vol 21 n 3 resistance, diabetes and hypertension. similar effect in type 2 diabetic rats - The recent rapid increase in childhood glucagon suppression (34). overweight and obesity will lead to a effects in cellular and Diabetes is clinically associated to further increase in prevalence of type 2 animal models hypertension and dyslipidemia, the diabetes and cardiovascular diseases main components of MS. In a dietary

May/June 2010 (14, 15). Health concerns are, therefore, changing some approach, the combination of stevioside and soy proteins - dietary habits and the search for alternative, natural and has been successfully assayed in type 2 diabetic rats with non-caloric sweeteners may represent a major issue in the significant reductions in plasma glucose, blood pressure as clinical management of this condition (16). Current initiatives well as in plasma cholesterol and triglycerides (35). Similar are aimed to change our approach to eating, nutrition and beneficial effects have been also observed in humans industry hi-tech physical activity as a multifactorial approach in which following the ingestion of 1 g stevioside (36), indicating that natural sweeteners may play a substantial role. the search for the active compounds and the uncovering OOD Stevia-related compounds have been suggested to exert a of mechanisms of action may help to develop a drugable

gro F broad spectrum of therapeutic effects on human health. It compound of potential use in humans. Apparently A has been found to be antihypertensive (17, 18), contradictory results may also be found in the literature antihyperglycemic (19, 20), antioxidant (21), effective using doses of < 1 g/day stevioside; long-term consumption against some viral infections (22), and has shown of typical amounts of stevioside as sweetener has no effect antiinflammatory and antitumor properties (23, 24). neither in diabetic and healthy Stevioside has also been reported to influence glucose subjects nor in hypertensive metabolism (25, 26) and renal function (27).

Effects on hyperglycemia and glucose homeostasis Some observations support the notion that stevioside and stevia extract may be used to ameliorate the manifestations of diabetes and consequently considerable effects in both hepatic glucose production and in peripheral glucose disposal may be anticipated. Steviol but no stevioside inhibits intestinal glucose absorption by 40 percent and may alter the morphology of intestinal absorptive cells (25). Stevioside, at 10 mg/Kg (15 days) lowers the high plasma glucose concentrations found in type 1 and type 2 diabetes experimental models. This hypoglycemic

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and NF-kB signalling signalling and NF-kB monocytic THP-1 cells by interfering monocytic THP-1 cells by interfering IKKb with the steviol and Stevioside (23). pathway also exert anti-inflammatory activity on represent which cells, epithelial colonic injury pathogen of sensor immune an that importance particular is of It (45). non- healthy, in induce may stevioside infectedindividuals additional an benefit, favouring the immune-related . For instance, stevioside, rebaudioside A and C rebaudioside A and C in vitro. For instance, stevioside, treatment may be also useful in limiting fatty liver disease, a a disease, liver fatty limiting in useful also be may treatment of MS. component further inflammation Effects on an or inflammation low-grade that documented well is It or worsen may cause inflammatory response excessive those diseases, especially of frequent clinical manifestations and atherosclerosis metabolic derangements, related with anti- that suggest also evidences Cumulative cancer. may have a relevant role in the inflammatory drugs treatment of such conditions but available molecules effects. adverse by severe accompanied usually are Treatment with natural flavonoids is currently under intense research. as demonstrated both in vivo Stevioside is anti-inflammatory and and dulcoside A inhibit 12-O-tetradecanoylphorbol-13- Stevioside (24). skin mouse in inflammation induced acetate may also suppress lipopolysacharide- (1 mM) but no steviol TNF-a and IL-1b (proinflammatory induced release of the in participating both oxide release, nitric cytokines) and in human development of inflammatory disorders, mice, In infections. future against monocytes of activity function several doses of stevioside promote phagocytic and increased humoural immune response. Similar effects have been observed in vitro (46). CONCLUSIONS will sweeteners alternative for market It is expected that the use the that years and following the in considerably increase will replace classic synthetic non- products stevia-related of and beverages. of food processing in the caloric sweeteners from derived products purified some of advantages The multiple are alternative sweeteners of as a source Stevia sp. caloric absence of the thermostability, high and include their will probably and processing, further value and need of provide medicinal effects that may be used in diabetic, in is well documented It subjects. obese and phenilketonuric these of capacity the models animal and cellular compounds to control glucose levels through its insulinotropic and glucagon suppression effects. Moreover improve to capacity its towards also point studies some of animal models in defense antioxidant insulin signalling and widely be can rebaudioside A Stevioside and atherosclerosis. used as sweeteners in many foods and beverages, and also may compounds these of consumption therefore, against inflammatory protect and immunity innate enhance diseases. stevia–related products may be useful to Whether againstdrugs manifestationsmetabolic develop of syndrome, remains an interesting possibility. These compounds have been used in Asia for decades and will disappear USA and Europe in limitations presumably once established some safety concerns in ongoing some and USA In studies. human clinical comprehensive European countries, products such as rebaudioside A with purity >97 percent have been already considered safe for its use in food processing. was was

obesity and obesity and Stevioside and Stevioside and provide medicinal provide medicinal cardiovascular risk properties to reduce properties to reduce rebaudioside A may rebaudioside A may and Cat, . Also in adipose tissue the the tissue adipose in Also . Glut4 and Fabp4 , , alpha Lxr safe and consequently blaze new trails in research oriented safe and consequently blaze new trails in research oriented with conditions in prevention atherosclerosis towards unpublished Preliminary, syndrome. metabolic associated stevioside that indicate laboratory our in obtained results data were obtained at a daily dosage which is considered considered is which dosage daily a at obtained were data restored as an action of stevioside, indicating an restored as an action of stevioside, indicating an in the in antioxidant defence. Importantly, improvement there was a aortic arch of stevioside-treated animals, reduction of macrophages, lipids in the total amount and oxidized lipoproteins that were associated with a significant reduction in the size the atherosclerotic of lesion. These results were interpreted as a significant reduction of oxidative stress and inflammation caused by stevioside via these All genes. involved importantly of modulation the considered is which dosage daily a at obtained were data safe and consequently blaze new trails in research oriented with conditions in prevention atherosclerosis towards unpublished Preliminary, syndrome. metabolic associated stevioside that indicate laboratory our in obtained results expression antioxidant of enzymes, Sod3 Effects on atherosclerosis More recently (44), the effects of stevioside on adipose tissue and obesity-associated insulin resistance, inflammation, oxidative stress and atherosclerosis have been investigated combined which model animal an in leptin and LDL-receptor deficiency, i.e. and hyperlipidemia. massive obesity The authors found no effect of a significant with weight body on treatment stevioside in increase significant a and stress oxidative in reduction blood lowered also It concentration. adiponectin plasma treatment glucose, insulin and cholesterol. Stevioside visceral in uptake glucose and adipogenesis improved of key genes such adipose tissue increasing the expression as Effects on hypertension Effects on that suggests also date to collected data Experimental extract may decrease blood pressure, stevioside and stevia dose-dependent and an effect that is time-dependent, is consumption (31, 39-43). In humans, it requires prolonged that long-term oral stevioside intake is generally considered be considered as an alternative or well-tolerated and may for hypertensive patients. The anti- supplementary therapy may extracts stevia and stevioside of effects hypertensive (increasing volume plasma both on actions represent and vascular resistance (increasing diuresis and natriuresis) importantly, these effects are not vasodilation). More either normal or low blood pressure present in humans with these compounds the amounts of they are unlikely at and provided via a normal diet. and normotensive subjects (11, 37). 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