Studies On Purification Of Gymnemic Acid And Its Effect On ?-Amylase

Gymnema sylvestre(Retz.) schult. is a perennial woody vines which belongs to Asclepiadaceae. It also named bock greenbrier rhizome. Gymnema sylvestre(Retz.) schult. is mainly distributed in Vietnam, India, and China's tropical coastal areas such as Guangxi, Hainan provinces. It is bitter and mild-natured. Gymnema sylvestre has been used to treat malaria, tumidness, inflammation.etc. It is a traditional herbs for treating the diabetes In india. Modern research has shown that Gymnema sylvestre can inhibit the sweet response and regulate blood sugar level. Gymnema acid is its main active ingredient, Which belong to triterpenoid saponin compounds. At present, There are so many methods used to isolate gymnema acid, such as traditional column chromatography and thin layer chromatography (TLC). Which has the shortcoming of inefficiency and tedious. This paper gives a systematic investigation of isolation to gymnema acid and its inhibitory effect on alpha-amylase.1. Optimization of purification technology for gymnema acid from Gymnema sylvestre by extraction and macroporous resin.The dried stems and leaves of Gymnema sylvestre were extracted with 60% ethanol,2 times, every time for 1.5h. the extract was evaporated to suspended in water and successively treated with petroleum ether, chloroform and n-butanol. The n-butanol layer were evaporated to dryness and dissolved to 9.3mg/mL in water. Static and dynamic adsorption methods were selected to estimate the purification ability of 6 macroporous resins, including AB-8?D101?ADS-7?NKA-9?HPD-100?ADS-17, for purifying gymnema acid from Gymnema sylvestre. The results showed that the HPD-100 was the best adsorbate of all. The optimum purification conditions were as follow:the concentration of sample liquid was 5.18 mg/ml, the amount of sample was 4 BV,10% ethanol was used to eluate impurities. the desorption liquid was 5 BV 70% ethanol. At finally,87.75% gymnema acid from Gymnema sylvestre were got.2. Isolation and preparation of gymnema acid ? by high speed counter-current chromatographThe K-value was selected by HPLC. A solvent system of n-hexane-n-butanol- methanol-0.02% trifluoroacetic acid water (0.8:9:1:9)was used to isolate gymnema acid ? from gymnema acid crude extract by HSCCC. The lighter layer serve as fixed phase and heaver layer serve as flowing phase. Isolation was performed under rotator speed of 1500 r/min. Temperature was 28? and detection wavelength was 235 nm. Fraction ? were collected according to uv-spectrogram and then detected by HPLC, which was gymnema acid ? with the purity of 90.3%.3. Inhibition Effects of gymnema acid on a-AmylaseThe inhibition effects of gymnema acid on a-amylase were studied with acarbose as positive control. The results showed the IC50 of gymnema acid, gymnema acid I and acarbose were 0.355mg/mL,0.0095 mg/mL and 0.465 mg/mL. Within the scope of the test concentration, gymnema acid had a remarkable inhibition on a-amylase activity. The kinetic analysis showed that the inhibition of gymnema acid on a-amylase was reversible noncompetitive.