A Novel Insulin-releasing Substance, Phanoside, from the Plant Gynostemma pentaphyllum

Abstract

Extracts from Gynostemma pentaphyllum Makino (Cucurbitaceae), a Southeast Asian herb, has been reported to affect numerous activities resulting in antitumor, cholesterol-lowering, immunopotentiating, antioxidant, and hypoglycemic effects. We have isolated one active compound by ethanol extraction, distribution in n-butyl alcohol/water, solid phase extraction/separation, and several rounds of reverse phase high pressure liquid chromatography. We have shown by NMR and mass spectrometry that this active compound is a novel saponin, a gypenoside, which we have named phanoside (21-,23-epoxy-,3β-,20-,21-trihydroxydammar-24-ene-3-O-([α-d-rhamnopyranosyl(1→2)]-[β-d-glycopyranosyl(1→3)]-β-d-lyxopyranoside)), with a molecular mass of 914.5 Da. Phanoside is a dammarane-type saponin, and four stereoisomers differing in configurations at positions 21 and 23 were identified, each of which were found to stimulate insulin release from isolated rat pancreatic islets. We have also found that the stereoisomers are interconvertible. Dose-dependent insulin-releasing activities at 3.3 and 16.7 mm glucose levels were determined for the racemic mixture containing all four stereoisomers. Phanoside at 500 μm stimulates insulin release in vitro 10-fold at 3.3 mm glucose and potentiates the release almost 4-fold at 16.7 mm glucose. At these glucose levels, 2 μm glibenclamide stimulates insulin release only 2-fold. Interestingly, β-cell sensitivity to phanoside is higher at 16.7 mm than at 3.3 mm glucose, although insulin responses were significantly increased by phanoside below 125 μm only at high glucose levels. Also when given orally to rats, phanoside (40 and 80 mg/ml) improved glucose tolerance and enhanced plasma insulin levels at hyperglycemia. Extracts from Gynostemma pentaphyllum Makino (Cucurbitaceae), a Southeast Asian herb, has been reported to affect numerous activities resulting in antitumor, cholesterol-lowering, immunopotentiating, antioxidant, and hypoglycemic effects. We have isolated one active compound by ethanol extraction, distribution in n-butyl alcohol/water, solid phase extraction/separation, and several rounds of reverse phase high pressure liquid chromatography. We have shown by NMR and mass spectrometry that this active compound is a novel saponin, a gypenoside, which we have named phanoside (21-,23-epoxy-,3β-,20-,21-trihydroxydammar-24-ene-3-O-([α-d-rhamnopyranosyl(1→2)]-[β-d-glycopyranosyl(1→3)]-β-d-lyxopyranoside)), with a molecular mass of 914.5 Da. Phanoside is a dammarane-type saponin, and four stereoisomers differing in configurations at positions 21 and 23 were identified, each of which were found to stimulate insulin release from isolated rat pancreatic islets. We have also found that the stereoisomers are interconvertible. Dose-dependent insulin-releasing activities at 3.3 and 16.7 mm glucose levels were determined for the racemic mixture containing all four stereoisomers. Phanoside at 500 μm stimulates insulin release in vitro 10-fold at 3.3 mm glucose and potentiates the release almost 4-fold at 16.7 mm glucose. At these glucose levels, 2 μm glibenclamide stimulates insulin release only 2-fold. Interestingly, β-cell sensitivity to phanoside is higher at 16.7 mm than at 3.3 mm glucose, although insulin responses were significantly increased by phanoside below 125 μm only at high glucose levels. Also when given orally to rats, phanoside (40 and 80 mg/ml) improved glucose tolerance and enhanced plasma insulin levels at hyperglycemia. Plant extracts have been used in traditional medicine since ancient times for treatment of various diseases including a disease similar to type 2 diabetes. Several hundred plants are known to have antidiabetic properties and a large number of compounds from plant extracts have been reported to have beneficial effects for treatment of diabetes (1Anhäuser M. Drug Discov. Today. 2003; 8: 868-869Crossref PubMed Scopus (9) Google Scholar, 2Atta Ur R. Zaman K. J. Ethnopharmacol. 1989; 26: 1-55Crossref PubMed Scopus (237) Google Scholar). The phytochemicals with hypoglycemic activity can be classified as alkaloids, flavonoids, glycosides/steroids/terpenoids, polysaccharides, proteins, saponins, and miscellaneous other compounds (3Lamba S.S. Buch K.Y. Lewis H.I. Lamba J. Stud. Nat. Prod. Chem. 2000; 21: 457-496Crossref Scopus (38) Google Scholar). A large number of crude plant extracts and purified substances from plants have been tested in clinical trials for treatment of diabetes (4Izzo A.A. Ernst E. Drugs. 2001; 61: 2163-2175Crossref PubMed Scopus (608) Google Scholar, 5Vuksan V. Sievenpiper J.L. Xu Z. Wong E.Y. Jenkins A.L. Beljan-Zdravkovic U. Leiter L.A. Josse R.G. Stavro M.P. J. Am. Coll. Nutr. 2001; 20: 370S-383SCrossref PubMed Scopus (131) Google Scholar). However, effects with crude extracts remain obscure if the active principles in the extracts are not characterized. Gynostemma pentaphyllum Makino (Cucurbitaceae) is a herb that is widely used in Southeast Asian folk medicine and is gaining popularity also in other countries as an herbal medicine. G. pentaphyllum extracts reportedly have many effects, such as lowered cholesterol, immunopotentiation, as well as antitumor, antioxidant, and hypoglycemic effects (6Li L. Jiao L. Lau B.H. Cancer Biother. 1993; 8: 263-272Crossref PubMed Scopus (76) Google Scholar, 7Lin J.M. Lin C.C. Chiu H.F. Yang J.J. Lee S.G. Am. J. Chin. Med. 1993; 21: 59-69Crossref PubMed Scopus (150) Google Scholar, 8Jang Y.-J. Kim J.-K. Lee M.-S. Ham I.-H. Whang W.-K. Kim K.-H. Kim H.-J. Yakhak Hoechi. 2001; 45: 545-556Google Scholar, 9Zhou Z. Wang Y. Zhou Y. Zhang S. Chin. Med. J. (Engl. Ed. 1998; 111: 847-850PubMed Google Scholar). Although there are substances in G. pentaphyllum extract that belong to different compound classes, a large group of substances in these extracts is saponins. Saponins isolated from G. pentaphyllum are also known as gypenosides. Currently, there are about 90 different gypenosides isolated and characterized. Several representatives of gypenosides are also found in extracts of other plants, such as species of ginseng (10Kuwahara M. Kawanishi F. Komiya T. Oshio H. Chem. Pharm. Bull. 1989; 37: 135-139Crossref Scopus (50) Google Scholar, 11Takemoto T. Arihara S. Nakajima T. Okuhira M. Yakugaku Zasshi. 1983; 103: 173-185Crossref Scopus (65) Google Scholar). Ginseng extracts have been widely studied and, similar to G. pentaphyllum extracts, exhibit antidiabetic effects via hypoglycemic properties (12Xie J.T. Zhou Y.P. Dey L. Attele A.S. Wu J.A. Gu M. Polonsky K.S. Yuan C.S. Phytomedicine. 2002; 9: 254-258Abstract Full Text PDF PubMed Scopus (109) Google Scholar). Although several types of compounds have hypoglycemic properties, the mechanisms for achieving this effect are different. There is a growing body of evidence that saponins, although active in several systems, are responsible for modulation of glucose disposal and insulin release (13Attele A.S. Zhou Y.P. Xie J.T. Wu J.A. Zhang L. Dey L. Pugh W. Rue P.A. Polonsky K.S. Yuan C.S. Diabetes. 2002; 51: 1851-1858Crossref PubMed Scopus (477) Google Scholar). For example, crude saponin fractions of G. pentaphyllum extracts have been shown to have hypoglycemic and hypolipidemic effects in rats (8Jang Y.-J. Kim J.-K. Lee M.-S. Ham I.-H. Whang W.-K. Kim K.-H. Kim H.-J. Yakhak Hoechi. 2001; 45: 545-556Google Scholar). However, the active principles responsible for hypoglycemia are not known. In the present study, we demonstrated that ethanol extracts of G. pentaphyllum strongly stimulate insulin secretion from isolated rat pancreatic islets. We have isolated a novel insulinreleasing substance from this extract, determined the structure of four active isomers by NMR and mass spectrometry, and characterized its effects on insulin release both in vitro and in vivo in the rat. Extraction—An ethanol extract of G. pentaphyllum was supplied by the Institute of Material Medica, Hanoi, Vietnam. The parts of G. pentaphyllum were dried in sunlight before pounding into powder. The powder was extracted with 70% ethanol in a Soxhlet extractor for 4 cycles. The ethanol was evaporated under reduced pressure to give a crude extract. 15 g of the crude extract was dissolved in 200 ml of water and centrifuged for 20 min at 3000 × g. The supernatant was collected, diluted with 200 ml of water, and extracted with 400 ml of n-butyl alcohol with a magnetic stirrer for 10 min. Water and n-butyl alcohol (Merck) were allowed to separate for 30 min in a separating funnel. The n-butyl alcohol phase was collected, and the solvent was evaporated until most of the n-butyl alcohol had been removed. The residue from the n-butyl alcohol extraction was diluted with 1000 ml of water. Portions of 500 ml were mixed with 200 g of Amberlite XAD-16 HP (Supelco, Bellefonte, PA) and stirred for 30 min. The unabsorbed material was removed by filtration, and the Amberlite was collected and washed with water. The adsorbed material was eluted from the Amberlite with aqueous acetonitrile (Riedel-de Haën) at different concentrations (25, 60, and 80% acetonitrile). Chromatography—The subsequent two high pressure liquid chromatography (HPLC) 1The abbreviations used are: HPLC, high pressure liquid chromatography; CV, column volumes; DQF, double quantum-filtered; ROESY, rotating Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; HSQC, heteronuclear single quantum correlation; HMBC, heteronuclear multiple bond correlation; KRB, Krebs-Ringer bicarbonate; NOE, nuclear Overhauser effect; iNOS, inducible isoform of nitricoxide synthase. steps were carried out on an ÄKTAexplorer and the last on an ÄKTApurifier HPLC system (Amersham Biosciences AB, Uppsala, Sweden). The bioactivity was located in the 60% acetonitrile fraction (total volume 500 ml). Portions (150 ml) of this fraction were diluted with 400 ml of water, applied to a Vydac C18 column (22 × 250 mm; 218TP152022), and eluted with a linear gradient of 20–57.5% B (eluent A, water; eluent B, 95% acetonitrile) in 4.5 column volumes (CV) at a flow rate of 25 ml/min. The fractions were analyzed for insulin-releasing activity. In the second chromatographic step, the active fraction from the first reverse phase HPLC separation was rechromatographed on a SOURCE 15RPC 16/90 column in a linear gradient of 32–40% B in 9.33 CV at a flow rate of 8 ml/min. In the last chromatographic step, the active fraction was purified on an ÄKTApurifier HPLC system using a C18 column (ODS-AP, 10 × 100 mm, 5 μm, YMC, Germany) and the same solvent system described above employing a gradient of 40–41% B in 4 CV at 6 ml/min. The structure of the pure component was elucidated using 1H-1H and 1H-13C two-dimensional NMR spectroscopy and mass spectrometry. NMR—NMR spectra were recorded with a Bruker DMX-600 spectrometer equipped with a cryoprobe in Me2SO-d6 solution at 25 °C. Chemical shifts are reported in parts/million relative to residual solvent signal (δ(1H), 2.50 parts/million; δ(13C), 39.5 parts/million). Two-dimensional spectra recorded included DQF-COSY, ROESY, TOCSY, sensitivity-enhanced 13C HSQC, and 13C-1H HMBC. Pulsed-field gradients were used for all 13C correlation spectra. 13C HMBC spectra were recorded with a coupling evolution delay for the generation of multiplebond correlations set to 62.5 ms. All two-dimensional spectra were run with a data matrix of 4096 × 1024 points, giving τ2max = 250 ms for the 1H nucleus in the acquisition dimension and τ1max = 200 ms for 1H or τ1max = 50 ms for 13C for the indirect dimension. Prior to Fourier transform, the data matrix was zero-filled twice, and a multiplication by shifted sine-bell window function was applied. For 13C-HMBC, the magnitude spectra were calculated. Mass Spectrometry—Positive ion mass spectra were recorded on an Ettan ESI-ToF electrospray ionization time-of-flight mass spectrometer (Amersham Biosciences AB, Uppsala, Sweden) using a capillary exit voltage of 150 V. Samples of chromatographic fractions were infused at 10–50 μl/min via the electrospray ionization source using a syringe pump. For alkali metal adduct measurements, a 50-μl aliquot of the chromatographic fraction was mixed with 2 μl of 5 μm LiCl, NaCl, or KCl solution in water and sprayed into the mass spectrometer. Insulin Release Assay—Male Wistar rats, weighing 250–280 g, were from a commercial breeder (B & K Universal, Sollentuna, Sweden). The animals were kept at 22 °C with free access to food and water before being sacrificed for isolation of their pancreatic islets. The islets were isolated by collagenase (Roche Applied Science) digestion of the pancreas, followed by hand picking (14Lacy P.E. Kostianovsky M. Diabetes. 1967; 16: 35-39Crossref PubMed Scopus (2492) Google Scholar). The islets were then cultured for 24 h in RPMI 1640 medium (Flow Lab Ltd., Irvine, UK) containing 11 mm glucose, 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 0.1 mg/ml streptomycin. Batch incubations of the islets were performed in Krebs-Ringer bicarbonate (KRB) buffer containing 118.4 mm NaCl, 4.7 mm KCl, 1.9 mm CaCl2, 1.2 mm MgSO4, 1.2 mm KH2PO4, 25 mm NaHCO3 (equilibrated with 5% CO2/95% O2, pH 7.4), 0.2% bovine serum albumin, 10 mm HEPES, and 3.3 or 16.7 mm glucose. Following pre-incubation for 30 min in KRB at 3.3 mm glucose, batches of 3 islets were incubated for 60 min in KRB with 3.3 or 16.7 mm glucose and different concentrations of the crude ethanol extracts or the purified compounds. After the batch incubations, aliquots of the medium were analyzed for insulin content by radioimmunoassay (15Herbert V. Lau K.S. Gottlieb J. PubMed Scopus Google Scholar). of Phanoside in Wistar rats and an glucose tolerance were used to the in vivo effect of and 80 In rats phanoside in g body was given to the animals orally 90 min before the of glucose body The group was given only glucose concentrations were in a at 90 min and before and 60, and min the of glucose. the effect of phanoside (40 and 80 body on insulin secretion in plasma were before of the compound before of glucose and 30 min for of of the novel compound was by the insulin release from the isolated rat pancreatic islets as described under and The crude ethanol extract of G. pentaphyllum was dissolved in water, and the solution was extracted with n-butyl After separation of the alcohol and of an the of the activity was in the n-butyl alcohol In the most of the n-butyl alcohol was removed under reduced pressure and the extract adsorbed to Amberlite The material on the was eluted first with water and then with different containing 60, and 80% The activity was found in the containing 60% fraction was purified by reverse phase After of 3 volumes of water, the fraction was on a C18 column The active fraction was purified on a column The material from the active fraction was used for of the insulin-releasing activity described However, using a high C18 the material was into four as four in All of these four fractions were found to be active in the insulin release at both 3.3 mm and 16.7 mm glucose B and of isomers of the active compound using a high reverse phase high pressure liquid chromatography A, the active fraction from the chromatography shown in was rechromatographed on a column × 10 mm 5 μm, 40–41% B in 4 CV at 6 ml/min. The material into four fractions as B, insulin-releasing activity of separate fractions from the chromatography above at 3.3 mm glucose. the of phanoside are with aliquot of each fraction was and for insulin-releasing activity of separate fractions from the chromatography above at 16.7 mm glucose. the of phanoside are with aliquot of each fraction was and for NMR of all four fractions were on the of two-dimensional NMR 1H-1H and 1H-13C spectra in Me2SO-d6 to the NMR all fractions of two one of which was not and to the other A of spectra at different times allowed of the structure of all of 1H-1H TOCSY, 1H-1H DQF-COSY, 1H-13C HSQC, and 1H-13C HMBC spectra to the that the structure different to which both of the other two are at and glucose, at In is to the at 3 of The is to the at The is has an group and two Chemical the of and the 13C NMR data all at the positions for A and = 3 and for the B All are in The and the of and the were from HMBC The HMBC to the of the and the at In the in 1H-1H spectra and the of the of spectra at different times in a in the isomers in fractions and isomers of fractions 2 and 3 the second of the mixture fractions and fractions the rate was to be under the NMR that were the of we that for all there are the and parts of the and that the had to be to the of the in the The of these were from 1H-1H spectra. In the for all four and as shown in by of the relative to the For fractions the have for fractions that group is located relative to the The to that at positions 20 and 21 for the isomers are located at of the at the same for the isomers at the of in the the The of relative to the in all isomers was by and and from to was by from to be in Mass the mass of a signal was for the compound = A and several for phanoside and its were at to and in to several The at the capillary exit of the mass spectrometer. is known that saponins, being not by In to with alkali which can then be by mass spectrometry. this was to the molecular mass of The compound solution from chromatographic fractions containing water and acetonitrile was mixed with different alkali metal and and the mass spectra recorded and In the with the signal increased about 10 and the metal ion A similar mass was with and the metal adduct shifted to the mass and A similar was with not In these the mass were = = = mass not The mass of the metal of phanoside and its are in with the and the structure of phanoside determined by Insulin Release from ethanol extract of G. pentaphyllum increased insulin secretion from isolated rat islets at both and glucose The activity on the of the extract. 2 and 4 mg/ml not mg/ml) of extract increased insulin release at 3.3 mm glucose from to and = for and at 16.7 mm glucose from to and = The fractions of the purified compound from the fraction in by the were used to the effect on insulin release At 3.3 mm glucose, 125 μm and higher concentrations of the were at 16.7 mm glucose, of insulin release was at the compound of μm At the mm, the compound insulin release 10-fold at 3.3 mm glucose and almost 4-fold at 16.7 mm glucose. effects were than the insulin release by a at a In of the of phanoside and 90 min when glucose was glucose levels were similar in all of rats with or 80 phanoside and in rats min the of glucose, glucose levels were increased to mm in all However, phanoside treatment (40 and not 10 significantly the glucose concentrations with and = 10 in each also at 30 min and at the higher and 80 phanoside improved glucose tolerance in In a number of rats = plasma insulin levels were similar in rats (40 and 80 and rats at min and min min glucose the in plasma insulin levels was in than in effect of phanoside on insulin secretion of Wistar rats of glucose. Wistar rats were given phanoside orally in and the group was given only min glucose was insulin concentrations were at and 30 min glucose = A novel compound with insulin-releasing activity was isolated from an ethanol extract of the Southeast Asian herb G. pentaphyllum and demonstrated to be a gypenoside, which we have named a molecular mass of 914.5 Da. in the herbal extract orally to glucose levels, we that the hypoglycemic be a compound that be via the with isolated novel gypenoside, a substance that we have isolated from the extract. is by the that the crude extract a effect on isolated pancreatic islets and that we were to this activity the A large number of saponins, has been isolated from G. The isolated in this has a structure with the of G. pentaphyllum known. However, a similar compound has been isolated from this plant in Z. Wang Arihara S. 1993; activity has been The compound had the same structure had an group at and different with purified phanoside demonstrated a insulin-releasing activity that is both insulin release at a glucose of 3.3 and the effect of 16.7 mm We these effects with a that is known to insulin secretion also at glucose levels PubMed Scopus Google Scholar). At 2 μm glibenclamide has been shown to stimulate insulin release in vitro to a V. S. 1983; 9: Google Scholar). that phanoside at is a than the In of in isolated purified given significantly improved glucose tolerance in Interestingly, this effect was only in the of hyperglycemia. The in glucose concentrations of phanoside was by insulin that the hypoglycemic effect in vivo is to the of insulin The by which phanoside stimulates insulin secretion to be However, has been shown that extract of G. pentaphyllum containing gypenosides by activity and F. S. 2003; 8: PubMed Scopus Google Scholar). of such as was demonstrated to stimulate insulin secretion in isolated rat islets G. J. PubMed Scopus Google is that phanoside insulin release by the of in the However, this effect is has also been shown that gypenosides from G. pentaphyllum release from which is by the J.A. PubMed Scopus Google although this was with a of all gypenosides from G. the other has been demonstrated that insulin release from pancreatic M. T. 2003; PubMed Scopus Google Scholar). the effect of phanoside be by release of in the Extracts of G. pentaphyllum isolated gypenosides have been reported to a of effects, such as the of J.M. Lin C.C. Chiu H.F. Yang J.J. Lee S.G. Am. J. Chin. Med. 1993; 21: 59-69Crossref PubMed Scopus (150) Google (6Li L. Jiao L. Lau B.H. Cancer Biother. 1993; 8: 263-272Crossref PubMed Scopus (76) Google and effects Z. J. Ethnopharmacol. PubMed Scopus Google as well as Z. Wang Y. Zhou Y. Zhang S. Chin. Med. J. (Engl. Ed. 1998; 111: 847-850PubMed Google and properties C.C. Cancer 2002; PubMed Scopus Google Scholar). In of G. L. Z. Xie Y. J. Nat. Prod. PubMed Scopus Google have isolated dammarane-type to the ginseng saponins. ginseng extract has been shown to glucose and body in (12Xie J.T. Zhou Y.P. Dey L. Attele A.S. Wu J.A. Gu M. Polonsky K.S. Yuan C.S. Phytomedicine. 2002; 9: 254-258Abstract Full Text PDF PubMed Scopus (109) Google Scholar). In saponin fractions of ginseng and G. pentaphyllum were demonstrated to have similar effects on and (8Jang Y.-J. Kim J.-K. Lee M.-S. Ham I.-H. Whang W.-K. Kim K.-H. Kim H.-J. Yakhak Hoechi. 2001; 45: 545-556Google Scholar). In the ginseng extract, an was (13Attele A.S. Zhou Y.P. Xie J.T. Wu J.A. Zhang L. Dey L. Pugh W. Rue P.A. Polonsky K.S. Yuan C.S. Diabetes. 2002; 51: 1851-1858Crossref PubMed Scopus (477) Google Scholar). In from the plant G. pentaphyllum we have isolated a active substance with the molecular mass 914.5 a gypenoside, which we have named Phanoside is a and of insulin secretion both in vivo and in vitro in the rat. with