Identification Of Steroidal Saponins From Tribulus Terrestri L. And Its Protective Effects On Cerebral Ischemia Injury

Tribulus terrestris L. is a common and well known traditional Chinese medicine which is used to calming the liver, relieving depression, promoting blood flow, eliminating Wind, improving vision, relieving itching. Studies on the chemical constituents in Herba tribuli (Tribulus terrestris L.) initiated in the 1960s, many papers have been published on its chemical constituents and pharmacological activities. Crude preparation of tatol saponins from stem and leaf exhibited its abilities in antagonizing myocardial ischemia, reducing blood pressure, reducing cholesterol, increasing ratio of lecithin to cholesterol, and preventing the deposition of lipid in arteries, myocardium and liver. Since the 1980s, the research on tribulus terrestris L. started in China, several medicine have been developed, one of them is xin nao shu tong which can enlarge the coronary vessel and improve the coronary circulation and result in alleviate coronarism and myocardial ischemia. The main components of tribulus terrestris L are saponins, flavonoids, alkaloids, polysaccharides, sterols, amino acids, terpenes, fatty acids and inorganic salts etc.The interest in the fruit of Tribulus terrestris L. started in the 1990s and was concentrated on the isolation and identification of its steroidal saponins, but concerning the mechanisms on how the steroidal saponins perform its function in anti-cerebral ischemia remain unclear. The total sapoinis from the fruit of Tribulus terrestris L,(content of steroidal saponins was higher than 60%) was isolated. The results from pharmacological experiments showed that the total saponins could diminish the areas of focal-focal type brain ischemic preconditioning in rats, inhibit the elevation of acute incomplet brainischemia vascular permeability in rats, and prevent the brain edema; in addition, the total saponins could reduce cerebral thromboxane A₂ (TXA₂) level and produce no effects on prostacyclin (PGI₂), thus, it could also change the ratio of PGI₂ to TXA₂, inhibit the formation of thrombus, decrease endothelin(et) content during cerebral ischemia reperfusion injury of the rat brain, increase the ability of GSH in removing free radicals, abase blood viscosity blood stasis in rats model, inhibit platelet aggregation, improve hemorrheology,accelerate fibrinolysis, enhance the ability of mouse hypoxia tolerance, and produce significantly protective effects on experimental brain ischemia. In order to identify the effective constituent of anti-brain infarction in total saponins, the chemical constituents from s total saponins was purified and studied, the results showed that purity of steroidal saponins is more than 95%, the content of furostanol saponins is more 90%.On the review of chemical constituents of Tribulus terrestris L. and its pharmacological activities, we investigated constituents of saponin of tribulus terrestris L.14 compounds were isolated from it by using open-column(silica gel, macroreticular resin, Sephadex LH-20, ODS), preparative HPLC methods et al. On the basis of spectroscopic analysis, including ¹H NMR, ¹³C NMR, ESIMS, HMBC, HMQC, ¹H^-1HCOSY, TOCSY and chemical methods, we identified 14 steroidal saponins, in which five is new. Five new compounds were identificated as 26-O-β-D-glucopyranosyl-(25R)-5α-furost-3β,22α,26-triol-3-O-[β-D-xylopyraose(l-^ 3)]-[β-D-xylopyraose( 1→2)]-β-D-g]ucopyranosyl(1→4)-[α-L-rhamnopyranosyl( 1→2) ]-β-D-galactopyranoside (1); 26-O-β-D-glucopyranosyl-(25R)-5a-fiirost-A20(22)-ene -3β,2α,26-triol-3-O-β-D-galactopyranoside(1→2)-β-D-glucopyranosyl(1→2)-β-D- ga lactopyranoside (2) ;25R-5α-furost-Δ20(22)-ene-12-ketone-3β,26-diol-26-O-β-D-glu copyranoside; (3); 26-O-β-D-glucopyranosyl-5α-furost-Δ20(22)-ene-3β,26-diol-3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranosyl( 1→4)]-β-D-galactopyranoside(4); 26-O-β-D-glucopyranosyl-5α-furost-12-ketone-2α,3β,22α,26-tetrol-3-O-α-L-gluco pyranosyl(1→2)-β-D- glucopyranosyl(1→4)-β-D-galactopyranoside (5).To investigate the protective effects of Saponins of tribulus Terrestris on the focal cerebral ischemia in rat, the rat model of focal cerebral ischemia was established by common carotid arteries occlusion (CCAO) operation. The results from the experiments of the saponins with dose of 10, 5mg/kg showed that saponins could significantly decrease the infarction area and improve behavior score (p<0.01) and increase the activities of GSH-PX, SOD and decrease the content of MDA, NO, FFA in serum of focal cerebral ischemia mice. The saponins of tribulus Terrestris could reduce apoptosis percentage of neuron and could stabilize the mitochondria membrane potential, upregulate Bcl-2 protein expression, downregulate Bax protein expression, enhance Bcl-2 mRNA expression level and could depresse Bax mRNA expression level in the brain tissue of focal cerebral ischemia in rats. The conclusion is that steroidal saponins from Tribulus terrestris can produce the protective effects in rat via antioxidant and antiapoptosis pathway.