Studies On The Phytochemical Constituents And Pharmacological Activities Of Gymnema Sylvestre (Retz) Schult

Gymnema sylvestre(Retz) Schult is a liana plant of the Asclepiadaceae family that grows in tropical and subtropical regions of the World. In China, it is distributed mainly in the provinces of Guangdong, Guangxi and Yunnan and is traditionally used by local people as an anti-inflammatory and analgesic herbal medicine. It is also a folklore medicine in India used for the treatment of malaria, hyperglycemia, mosquito and snake bites. Pharmacological studies showed that G. sylvestre has anti-caries, hypoglycemic and weight reducing effects.During the accomplishment of this dissertation, twenty five chemical constituents were isolated and identified from the chloroform and n-butanol extracts of the plant stems. Those extracts had been evaluated for hypoglycemic activity using two pharmacological models in mice. A part of the chemical constituents were investigated for α-glucosidase inhibitory and anti-tumor activities using in vitro experiments, and anti-inflammatory effect by in vivo experiment. 1. Studies on the chemical constituents of Gymnema sylvestre(Gs)By repeated column chromatography and final purification on preparative HPLC, thirty compounds were isolated from the chloroform and butanol soluble extracts of Gs and the structures of twenty five compounds were determined, including eight new compounds, namely gymsylvestroside A(1), gymsylvestroside E(2), gymsylvestroside B(3), gymsylvestroside C(4), gymsylvestroside D(5), gymsylvestroside F(6), gymsylvestroside G(7) and gymsylvestroside H(8). The known compounds were stephanoside K(9), gymnpregoside 8(10), gymnpregoside L(11), gymnpregoside I(12), gymnpregoside I(13), gymnpregoside D(14), gymnpregoside C(15), sarcostin(16), prosapogenin 8(17), isoimperatorin(18), aviprin(19), Eryciboside A(20), Stigmasterol(21), kaempferol-7-ether-3-O-β-D-glucopyranoside(22), n-Heneicosane(23), heptadecanol(24) and β-sitosterol(25). Their structures were determined by spectroscopic methods and their physico-chemical characters. Compounds 17-22 were found in this plant for the first time and compounds 18-20 were isolated from the Gymnema genus for the first time. 2. Primary observation on the hypoglycemic activity of Gs extractAdrenaline-induced hyperglycemia in mice Hyperglycemia was induced in mice by intraperitoneal injection of epinephrine and the blood glucose levels of the mice were monitored at 0.5, 1 and 2 h after administration of Gs samples in order to evaluate their hypoglycemic effects. The crude extract was given at doses of 10, 20 and 40 g/kg and the chloroform, n-butanol and water soluble fractions were given at dose of 40 g/kg. The crude extract and the n-butanol fraction exhibited hypoglycemic effects at dose of 40 g/kg in 1 h after the dosing. The chloroform and water fractions were not so active.Glucose-induced hyperglycemia in mice Hyperglycemia mice made by oral administration of glucose solution were used in this experiment. The crude extract was given at doses of 10, 20 and 40 g/kg and the fractions were administered at 40 g/kg dose Blood sugar levels of the mice were measured at 0.5, 1 and 2 h after the dosing.The crude extract and the n-butanol fraction exhibited evident hypoglycemic effects at dose of 40 g/kg in 1 h after the dosing, better than the chloroform and water fractions. 3、Pharmacological activities of some isolated compounds 3.1 α-Glucosidase Inhibitory EffectThe Saccharomces cerevisiae α-glucosidase inhibition activities of compounds gymsylvestroside A(1), B(3), C(4), D(5) and E(2) were assayed using p-nitrophenyl-α-D-glucopyranoside(p NPG) as substrate and acarbose as positive control. Gymsylvestroside A-E showed inhibition rates of 4.9%、3.8%、6.6%、3.6% and 9.5% at consentrations of 1.02, 1.07, 1.11, 1.16 and 1.12 mg/ m L, respectively. The inhibition rate of acarbose at 0.50 mg/m L was 26.4%. 3.2 TPA-induced mouse ear edemaThe TPA-induced mouse ear edema experiment was used to evaluate the anti-inflammatory activities of compounds 1–4. Compounds 1–4 did not show significant anti-inflammatory effect in this experiment. 3.3 Anti-tumor experimentsThe anti-tumor activities of compounds gymsylvestroside A, B, C and D were determined by in vitro cell experiments on leukemic cancer cells K562. Inhibition rates of 23%-26% were observed. SummaryIt has been reported in the literatures that the hypoglycemic activity of G. sylvestre was the combined effect of diverse saponin compounds named as Gymnema sylvestre acid. Besides conduritol A, other hypoglycemic compounds from G. sylvestre have rarely been reported so far. In this study the hypoglycemic effects of different solvent fractions were investigated using the adrenaline and glucose induced hyperglycemia models, and the n-butanol fraction, which showed positive results in the hypoglycemic tests, was selected as the object of phyrochemical study. New C21 steroidal saponins were isolated from this fraction. C21 steroidal saponins have been found in the Asclepiadaceae family previousely, but were isolated from this plant for the first time. This kind of compounds possesses diverse pharmacological effects, especially anti-tumor activity. The C21 steroidal saponins obtained in this experiment were preliminary evaluated for α-glucosidase inhibitory and anti-inflammatory effects, as well as anti-cancer activity on K562 cells. The results may be helpful to further studies on this plant.