Review Paper Chemistry and in Vivo Profile of Ent-Kaurene Glycosides Of

Natural Product Radiance, Vol. 8(2), 2009, pp.181-189 Review Paper

Chemistry and in vivo profile of ent-kaurene glycosides of Stevia rebaudiana Bertoni—An overview Manishika Sharma1, Naveen Kr Thakral2 and Seema Thakral3* 1G.V.M. College of Pharmacy, Sonipat-131 001, Haryana, India 2Laborate Pharmaceutical India Ltd., Poanta Sahib-173 025, Himachal Pradesh, India 3PDM College of Pharmacy, Bahadurgarh-124 507, Haryana *Correspondent author, E-mail: [email protected] Received 17 January 2008; Accepted 16 December 2008 structures are constituted by a Abstract The ent-kaurene type of diterpenoid glycosides are typically the characteristics of leaves perhydrophenanthrene unit (A, B and C of Stevia rebaudiana Bertoni, the plant with sweet leaves. Relative to sucrose, the potent rings) fused with a cyclopentane unit (D sweetness intensities of these glycosides have projected them as cost effective sucrose substitute. In ring) formed by a bridge of two carbons the present paper, the structural and physicochemical features of ent-kaurene glycosides of Stevia between C-8 and C-13 (Fig. 1 a). Various along with an insight into the structure-sweetness relationship are presented. Despite their age-old criteria are employed for the widespread use in several parts of the world, there still remains certain concern regarding safety profile of these glycosides. Henceforth, the pharmacokinetic, pharmacological and toxicological nomenclature of the kaurene diterpenes, evaluation of ent-kaurene glycosides is reviewed. the most prevalent being the inversion of Keywords: ent-kaurene glycosides, Stevia, Stevia rebaudiana, Structure-sweetness relationship, the conventional description of Sweeteners. stereochemistry when the name is IPC code; Int. cl.8 — A23L 1/09, A61K 36/28, A61K 127/00 preceded by the prefix “ent-”. The nomenclature, numbering and stereochemistry of the ent-kaurene concentrated liquid, crushed leaf or Introduction skeleton (Fig. 1a) have already been ent concentrated white powder. The greenish- The -kaurene type of established by IUPAC recommendations 4. black concentrated liquid is 70 times diterpenoid glycosides are typically the All ent-kaurenoic glycosides are Stevia while the crushed leaf form is about 30 characteristics of leaves of derived from a common moiety, rebaudiana times as sweet as sugar. With reference to Bertoni (Family— kaurenoic acid (Fig. 1b). Kaurenoic acid its sweetening power, it is estimated that Asteraceae), the plant with sweet leaves. is one of the intermediate compounds 30 ml (1 oz) of Stevia extract is equivalent Owing to presence of these sweet-tasting involved in the biosynthesis of diverse to 3 Kg (6.6 lbs) of sucrose3. glycosides, the plant is also known as sweet kaurene diterpenes, including Caa-he-é The structural and herb, honey leaf, or sweet gibberellins, which represent the chrysanthemum. It is also regarded as the physicochemical features of ent-kaurene glycosides of Stevia along with an insight important group of growth “sweet herb of Paraguay”, as its leaves have phytohormones. However, in plant, this been traditionally used for hundreds of into the structure-sweetness relationship and pharmacokinetic, pharmacological kaurenoic acid moiety diverts toward the years by natives of Paraguay and Brazil to ent- and toxicological evaluation of ent- formation of kaurene glycosides sweeten local teas, medicines and as a through steviol, which is the aglycone for ‘sweet treat’. In addition, the leaves are kaurene glycosides are reviewed in the present paper. all the Stevia glycosides. also used for their medicinal benefits in The ent-kaurene type of high blood pressure, obesity, topical glycosides present in Stevia are referred dressing for wounds and other skin Chemistry of ent-kaurene to as steviol glycosides, though steviol is 1-2. disorders Glycosides not the glycoside but the aglycone moiety As an alternative to the Kaurenes represent an important of all these glycosides. Chemically steviol conventional sugar, it is available as a group of tetracyclic diterpenes. Their is ent-13-hydroxy kaur-16-en-19-oic acid Vol 8(2) March-April 2009 181 Review Paper

12 12 11 13 CH 20 2 13 17 20 11 CH C 16 2 14 C 16 D 14 1 1 D 2 10 8 15 2 10 8 15 A H A B H 3 5 B 7 3 5 7 4 6 4 6

H H 18 18 19 (a)COOH (b) Fig. 1 (a-b): Structure of ent-kaurene (a) and ent-kaurenoic acid (b)

glucose in total, with the middle glucose rebaudioside A present in Stevia is the of the triplet connected to the central main determinant of taste quality. When steviol structure. The aqueous extract from steviol glycosides are more than 50% of Stevia rebaudiana leaves leaves is known to contain about ~ the total glycosides, the taste is common/ 10-70% of stevioside and ~ 20-70% of traditional, with a metallic or liquorice (Table 1), a diterpenoidic carboxylic rebaudioside A, with a total glycoside after-taste, whereas when rebaudioside A alcohol5. contents of more than 90%. The remaining makes up more than 50% of the glycosidal A total of eight ent-kaurene glycosides are present in lesser amounts11, 12. contents, the taste is improved with a diterpene glycosides have been isolated In addition to these glycosides, it also reduced after-taste15. from S. rebaudiana namely: stevioside, contains a diverse mixture of labdane steviolbioside, dulcoside A and diterpenes, triterpenes, stigmasterol, Aqueous solubility: All the ent-kaurene rebaudiosides A, B, C, D and E6. tannins as well as volatile oils13. glycosides possess good aqueous solubility S. rebaudiana is an anomaly in the and can be easily extracted with aqueous genus Stevia, as it has been reported that General features solvents16. none of the other 230 species belonging All the ent-kaurene glycosides of to this genus has ever been found to Stevia have been demonstrated to possess Thermostability: Contrary to sugar produce these compounds at high certain common features; some of the which starts to caramalize at about 150- concentration levels7. The yield of important ones are as follows: 160°C, ent-kaurene glycosides can withstand temperatures up to 200°C. The stevioside is the highest (2-10%), Sweetness: These glycosides are about 200 sweetness property of aqueous solution of rebaudioside follows next (about 1%) and times sweeter than sucrose. In addition stevioside does not change when heated the other constituents are the minor quality is superior to sugar in mildness 8-10 to 95°C for two hours17. components . The basic chemical and refreshment2. These tend to produce structures along with important a sweet taste less instantly than sucrose Stability towards extreme pH: These physicochemical properties of these but the taste lasts for a longer period. glycosides are exemplified in Table 1. glycosides are exceptionally stable towards As evident from Table 1, ent- Relative sweetness: Rebaudioside A is pH ranging between 2 to 10. A 0.02% kaurene glycosides are formed by sweeter than other steviol glycosides (300 stevioside solution at pH 3 maintained at times compared with sucrose) and also 95°C for 1 h shows no significant replacing the substituents R1 with glucose has a more palatable taste profile, having degradation while a 12% solution of and R2 with combinations of glucose and rhamnose. Glycoside stevioside differs less of the metallic/liquorice taste often stevioside at pH 2 under these conditions from rebaudioside in the feature that the associated with steviol glycosides14. decomposes17. former is composed of glucose, sophorose Index of taste: Ratio of Stevioside and and steviol while, the latter has four No calorie sweeteners: These

182 Natural Product Radiance Review Paper glycosides have zero glycemic index, zero Flavour enhancement: In conjugation of providing oral health benefits by glycemic load and no calories effects8. with other flavours, ent-kaurene interference with cell surface functions of glycosides act as a flavour enhancer cariogenic bacteria. Taste enhancement: These glycosides producing a stronger flavour, allowing a As these glycosides are heat and produce synergistic effect on taste when smaller quantity of the flavour to be pH stable, non-fermentable and do not used in conjunction with other incorporated. darken upon cooking, these have been sweeteners, e.g. glycerrhizin, aspartame, exploited in a wide range of applications acesulfame and cyclamate (but not with Oral hygiene: Steviol, isosteviol and in food products since ages. saccharin)18. It was found that sweetness rebaudioside B, C and E have been found property of stevioside gets improved along to inhibit the glucan–induced aggregation Synthesis of ent-kaurene glycosides with reduction in aftertaste on of cariogenic organism, Streptococcus The in vivo biosynthetic combination with sucrose and fructose14. mutans19. Thus, they have the potential pathway for ent-kaurene glycosides has

Table 1: Structure and properties of eight ent-kaurene glycosides of Stevia

OR2

12 13 20 11 CH2 C 16 14 D 1 2 10 8 15 A H B 3 5 7 4 6

H 18 COOR1

Steviol: the aglycone of ent-kaurene glycosides (R1 and R2=H)

Compound R R Mol.Wt. M.P. (°C) Solubility (%) Sweetness RS QT 1 2 Stevioside β-Glc β-Glc-β-Glc(2→1) 804 196-198 0.13 100-270 143 0 Steviolbioside H β-Glc-β-Glc(2→1) 642 188-192 0.03 10-15 NA NA Rebaudioside A β-Glc β-Glc-β-Glc(2→1) 966 242-244 0.80 150-320 242 +2 β-Glc(3→1) Rebaudioside B H β-Glc-β-Glc(2→1) 804 193-195 0.10 10-15 NA NA β-Glc(3→1) Rebaudioside C β-Glc β-Glc-α-Rha(2→1) 958 215-217 0.21 40-60 NA -1 (Dulcoside B) β-Glc(3→1) Rebaudioside D β-Glc- β-Glc-β-Glc(2→1) 1128 283-286 1.00 200-250 221 +3 β-Glc(2→1) β-Glc(3→1) Rebaudioside E β-Glc-β- β-Glc-α-Glc(2→1) 966 205-207 1.70 150-200 174 +1 Glc(2→1) Dulcoside A β-Glc β-Glc-α-Rha(2→1) 788 193-195 0.58 40-60 NA -2

RS : Relative sweetness to glucose; QT : Quality of taste ; +better,-worse; NA: Data not available

Vol 8(2) March-April 2009 183 Review Paper been identified and it has been found that Structure property relationship of Rubusoside was also treated with these are derived from the diterpene ent-kaurene glycosides the CGTase-starch system to undergo trans- steviol, which is produced via The sweetness of steviol α-1, 4-glucosylation and a large number ent-kaurenoic acid from a branch of the glycosides differ substantially reflecting of products were obtained. Strong gibberellic acid biosynthetic pathway20, 21. relatively small variation in their structure. enhancement of the sweetness intensity was Numerous attempts have also been Numerous ent-kaurene glycosides isolated observed for the products that were di- or made to chemically synthesize the from S. rebaudiana and Rubus tri- glucosylated at the 13-O-glucosyl aglycone steviol and then to convert it to suavissimus S.K. Lee supplied good moiety. On the other hand, the sweet glycosides. The bicyclo [3.2.1] lead compounds to study structure- tetraglucosylation at the 13-O-glucosyl octane ring unit has been the hot spot of sweetness relationship, since the former moiety as well as glucosylation at the contains several glycosides of same 19-O-glucosyl moiety led to decrease in synthesis for number of chemists. Different aglycone steviol, and the latter contains sweetness28. These results were similar to synthetic approaches utilized for the many glycosides, most of which have the case of stevioside as the starting synthesis of steviol can be summarized as common sugar moieties (13- and 19-bis- material, and strongly suggested that for follows: β-D-glucoside) with slightly different enhancement of intensity of sweetness of 1. Mori’s approach for synthesis of aglycones. Two of the sweetest of ent- steviol glycosides, the elongation of steviol using 6-methoxy-1-methyl- kaurene glycosides i.e. stevioside and 13-O-glucosyl moiety up to a total of tetralone as starting material22,23. rubusoside (a sweet glycoside with steviol four glucosyl units, accompanied by as aglycone i.e. steviol 13, 19-di-O-β- suppression of glucosylation at 19-O- 2. Ziegler and Kloek’s approach for glucoside, isolated from R. glucosyl moiety, is desirable. These synthesis of steviol involving stereo suavissimus) taste somehow bitter and fundamental findings have been used to controlled photoaddition of allene to show aftertaste. In order to improve the obtain better sweeteners using more cyclopent-1-ene-1-carboxaldehyde as sweetness and the taste, modifications of specific transglucosylation systems. These key synthetic step24. sugar moieties of both the glycosides were methods have been used in Japanese food 3. Cock’s approach towards partial performed by enzymatic glycosylations industry8. synthesis of steviol involving ent-kaur- and/or enzymatic trimming33. Thus, In addition to above, attempts 16-en-19-oic acid as starting treatment of stevioside with soluble starch have been made to improve intensity of material25. as donor in presence of cyclomaltodextrin- sweetness of these glycosides glucanotransferase yielded a complex through trans-α-1,4-glucosylation of The steviol hence produced can mixture of products, which were mono-, steviolmonoside and steviolbioside29, be glycosylated to afford stevioside. This di-, tri-, and polyglucosylated derivatives through trans-α-1,4-glucosylation of can further be synthetically converted to on both existing glucose moieties (Table 2). 19-O-β-galactosyl esters30, 31 and through rebaudioside A by selective enzymatic Significant improvement of the protection of 19-O-glucosyl group against removal of glucose unit for stevioside at quality of taste and intensity of sweetness trans-glucosylation32, 33. C-13 position and systematic was observed for most of the glucosylated reintroduction of two glucose units26. products particularly for S1a and S2a, In vivo profile Although a great amount of which are mono- and di- glucosylated at Stevioside, in the form of pure work has been done to chemically the terminal glucosyl moiety at the compound or S. rebaudiana leaf extract synthesize these glycosides, but the 13-O-sophorosyl group, respectively. has been widely used as food additive, production of ent-kaurene class of However, glucosylation of 19-O-glucosyl particularly in Japan, Brazil and Korea. sweet glycosides through chemical group i.e. compound S1b, S2c, and S3c However, studies relevant to pharmacology synthesis for commercial purpose is quite showed a decrease in the intensity and/or and toxicology are relatively jejune. Much expensive. quality of sweetness27. of the literature is published as reports

184 Natural Product Radiance Review Paper or abstracts in hard to obtain journals, with 50% inhibition observed at 1.2mM39. Effect on renal functions: In rats, oral though some earlier reviews on Stevia and High levels of stevioside can cause severe administration of its extract as well as its glycosides have appeared in some disruption of mitochondrial cristae in stevioside for 40-60 days induced diuresis books18, 34-36. kidney tubules40. However, the by reducing vascular resistance and mitochondrial actions of stevioside are increases fractional excretion of sodium ADME of ent-kaurene glycosides observed only in isolated organelles, and and potassium ions47, 48. Stevioside itself ADME (Absorption, Distribution, are not observed in intact cells. This had a clearance rate less than that of Metabolism, and Excretion) studies of suggests that stevioside does not permeate p-amino hippuric acid but greater than steviosides had been performed in rats. the cell membrane41. that of inulin, which suggests that Stevioside is not readily absorbed from glycoside is actively secreted by renal the upper small intestine owing to its high Effect on carbohydrate metabolism: tubular epithelium49. molecular weight. However, most of the In isolated perfused rat liver, stevioside stevioside administered is degraded by inhibits transport of monosaccharides i.e. Effects on reproductive systems: bacteria of the colon, resulting in free glucose, fructose and galactose in both There has been some controversy over steviol which is easily absorbed. In the the directions42. It halves transport rate whether the plant and its extract have any human colon, only the groups of of glucose into liver and also inhibits contraceptive effects50. It has been claimed bacteroidaceae were efficient in hepatic release of glucose. In Hamsters that Paraguay Indians used the herb as a hydrolyzing stevioside into steviol and fed stevioside for 12 weeks, glucose male contraceptive beverage. Subsequently, steviol-16,17α-epoxide. After degradation absorption was found to be inhibited several research groups duplicated these of stevioside to steviol by bacteria of the leading to decrease in body weight of experiments and found no evidence of colon, part of the steviol is absorbed by hamsters43. Oral administration of contraceptive or antifertility effects of its the colon and transported to the liver by steviosides (7% of diet) in rats for 56 days leaves in rats37. Thus, when fed to male portal blood and part is excreted in feces. caused no change in blood sugar and female rats, for 60 days prior to mating 8 In the liver, the steviol glucuronide is level . However an increase in plasma to both and for first seven days of formed; it is released into the blood and glucose levels was found when stevioside gestation to female rats, no effect were 44 filtered out by the kidneys into the urine. was given by intravenous infusion . The seen on either mating performance or The high levels of steviol glucuronide in magnitude of effect of intake of fertility51. However, some recent reports the urine suggest that there is no steviosides on blood glucose levels in have indicated decrease in the epididymal accumulation of steviol derivatives in the vivo is still under investigations and there sperm count, plasma testosterone 37 human body . Intravenous administration are reports indicating decrease in the concentration and decrease in organ body of radiolabelled stevioside in rats blood glucose and hepatic glucose levels weight ratio of testis and cauda exhibited fast accumulation in small in rats after four weeks and in human epididymis upon administration of higher intestine and then in liver. Within two volunteers eight hours after consumption doses of aqueous extract of leaves to adult hours, 52% of the radioactivity of an extract45. male Wistar albino rats for a period of 65 administered appeared in the bile38. Effect on cardiovascular system: The days52. extract has been found to reduce heart Pharmacological effects rate and mean arterial blood pressure. Toxicological evaluation The ent-kaurene glycosides are Similarly, stevioside also lowers mean Mutagenicity studies: Studies on diverse in their actions. Some of the arterial blood pressure, an effect that is stevioside crude crystals and rebaudioside important ones are discussed below: blocked by Indomethacin. This suggests A indicate that the compounds are not Effect on energy metabolism: that cardiovascular action of stevioside is mutagenic. In 1999 the Joint FAO/WHO Stevioside interfere with oxidative mediated via a prostaglandin-dependent Expert Committee on Food Additives phosphorylation in isolated mitochondria, mechanism46. (JECFA) clearly stated that there was no

Vol 8(2) March-April 2009 185 Review Paper indication of carcinogenic potential of added safety factor of 100. The major extract62. However, almost all male rats stevioside53. This has been shown in findings were reduced spermatogenesis, including controls developed interstitial several bacterial assays, e.g. the Ames test decreased seminal vesicle weight, cell tumours in the testis and effect on in Salmonella typhimurium, forward interstitial cell proliferation in the testes, male reproductive system was a major mutation test in Bacillus subtilis, spore medullary cell proliferation in the adrenal concern. rec-assay and tests for chromosomal glands, atrophy of the thymus, Fertility and teratogenicity: In one of aberration in mammalian cells in CHL and inflammatory lesions in the trachea and the rat studies63, Wister rats were given human lymphocytes in vitro54. lungs, age-related changes of the kidneys stevioside in the diet (0.15, 0.75 and 3%, Chromosomal abnormalities manifested (such as degeneration of tubular equivalent to 150, 750 and 3000 mg/kg as increased in number of gaps and epithelium, hyaline casts and glomerular bw/day, 96% purity). Males were treated interchanges are seen only with stevioside sclerosis) and pigmentation and increased for 60 days before and during the mating at high concentrations8, 55. However, the haematogenesis of the spleen. Similar period and females for 14 days before the stevioside metabolite steviol shows results were observed in another study on mating period and for 7 days during mutagenic activity in the forward mutation F344 rats employing purified stevioside gestation. Results showed no treatment assay although not in reverse Ames assay56. Steviol is believed to be mutagenic as a Table 2: Relative sweetness and quality of taste of result of bioactivation, the nature of different glucosyl derivates of Stevioside bioactivated mutagenic metabolites being 57 still unknown . Thus, steviol is mutagenic OR2 in S. typhimurium TM677 in the 12 11 13 20 CH presence of a metabolic activation system, 2 C 16 as well as in CHL cells in vitro (gene 14 D mutations and chromosomal 1 2 10 8 58 15 aberrations) . In S. typhimurium, it A H B induces mutations of the guanine 3 5 7 phosphoribosyltransferase (gpt) gene59. 4 6

H 18 Acute toxicity: Stevioside and steviol COOR1 have very low acute oral toxicity in the mouse, rat and hamster. The acute LD50 of stevioside has been reported as > 1700 Compound N–R1 N-R2 RS QT mg/kg body weight in rats; > 15 g/kg 60, 61 Stevioside 1 2 160 0 (orally) in mice . S1a 1 3 180 +4 S2a 1 4 205 +4 Chronic toxicity: A purified hot water S3a 1 5 117 +3 extract of S. rebaudiana containing the S1b 2 2 133 +2 equivalent of about 95% total sweet S2b 2 3 136 +1 S3b 2 4 146 0 glycosides (74.5% stevioside and 16.3% S2c 3 2 136 0 rebaudioside) was administered to 70 S3c 3 3 150 +1 F344 male rats for 22 months and 70 S3d 4 2 121 +3 female rats for 24 months. The high dose Rubusoside 1 1 134 -2 selection was based on an estimated N–R1: No. of glucosyl residues at R1;N-R2 : No. of glucosyl residues at R2; human intake of 4 mg/kg BW and an RS : Relative sweetness to glucose; QT : Quality of taste ; +better ;-worse

186 Natural Product Radiance Review Paper related effects on fertility or mating liver. Some of the mitochondrial Conclusion performance and the foetuses did not parameters that have been investigated for It is remarkable that the sweet develop any malformations. In the other different ent-kaurene moieties of it are ent-kaurene glycosides are accumulated rat study62 stevioside (95.6% purity), coupled and uncoupled respiration, at high levels in the leaves of S. given at doses of 250, 500 and 1000 mg/ L-glutamate dehydrogenase, ATP rebaudiana, given that the plant leaves kg bw/day from day 6 through day 15 of dependent swelling, NADH oxidase are not generally regarded as storage pregnancy induced no teratogenic effects. activity, DNP stimulated ATPase succinate organs for secondary metabolites. Relative However, steviol, the metabolite of dependent swelling, succinate to sucrose, the potent sweetness stevioside, is toxic to pregnant hamsters dehydrogenase and succinate oxidase intensities of these glycosides have (dose 0.75g/kg bw/day) and their activity. It has been shown that stevioside projected them as cost effective sucrose foetuses when administered on day have inhibitory effects on these enzymatic substitute. Despite their widespread use 6 through 10 of gestation64. It produces activities at quite high concentration as in several parts of world, no evidence of decreased maternal weight gain and compared to other ent-kaurene analogs adverse reactions due to the ingestion of high maternal mortality. The number of of Stevia8. its extracts or stevioside by humans has live births per litter and mean fetal appeared in the biomedical literature. weight decreased. The maternal kidneys Clinical trials However, the famous Phillip’s comment showed a dose-dependent increase in Results from several clinical ‘that Stevia has been in use for centuries severity of convoluted tubules in the studies support the findings that no does not necessarily make it safe’ is quite kidneys. significant effects on blood pressure and relevant. Steviol, a metabolite of blood sugar occur with high steviol stevioside, produced in the human Nephrotoxicity: Nephrotoxic effects of glycoside doses delivered over several intestinal microflora is known to be stevioside have been observed in rats months. However, two clinical trials on genotoxic and to induce developmental following single subcutaneous injection patients with high blood pressure have toxicity. Hence, additional efforts are (1.5 g/kg, SC; dose equivalent to about reported blood pressure reductions after warranted to resolve issues pertaining to 250 times the average daily intake of long-term treatment with stevioside. One the safety concerns. human consumers). Blood urea levels clinical trial using stevioside found a began to rise 3 h later, an indication of beneficial effect on postprandial glucose References inability to excrete nitrogen. Other effects homeostasis in type 2 diabetic patients. 1. Elkins R, Stevia: Nature’s Sweetener, include elevated plasma creatinine and These raised concerns about potential Woodland Publishing, Orem, 1997. increased urinary glucose and increase in risks for normotensive and diabetic 2. Kinghorn AD and Soejarto DD, Intensely sweet urinary levels of two enzymes: γ- consumers. Two randomized, placebo- compounds of natural origin, Med Res Rev, glutamyltranspeptidase and alkaline controlled, double-blind clinical trials 1989, 9, 91-115. phosphatase. In addition there was also were conducted to examine the effects of 3. www.dweckdata.com/Published_papers/ an indication of degeneration of kidney rebaudioside A on the blood pressure of Rainforest_Plants.pdf tubules and disruption of mitochondrial healthy subjects and on glucose 4. García PA, Oliveira AB and Batista R, cristae on long-term usage. Such effects homeostasis in type 2 diabetics. The Occurrence, biological activities and synthesis have also been demonstrated in studies concluded that 1,000 mg/day of of kaurene diterpenes and their glycosides, hamster65, 66. rebaudioside A had no clinically Molecules, 2007, 12, 455-483. significant effects on blood pressure in 5. Mosettig E, Beglinger U, Dolder F, Lichti H, Hepatotoxicity: Stevioside, at healthy subjects or on glucose Quitt P and Waters JA, The absolute configuration of steviol and isosteviol, J Am concentration level of ~1.5mM, has been homeostasis or blood lipids in type 2 Chem Soc, 1963, 83, 3163-3164. displayed to have no effect on activity of diabetic patients. No adverse effects were 67, 68 6. Pasquel A, Meireles MAA, Marques MOM glutamate dehydrogenase of rat or bovine observed in either study . and Petenate AJ, Extraction of glycosides with

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