The Anti-Proliferation Effect Of Saponins From Tribulus Terrestris L. On Renal Carcinoma Cell Line 786-O

Tribulus terrestris L. has been recorded in the Pharmacopoeia of China as a traditional Chinese herb. There have been many studies on pharmacology of the fruit so far, but it is still unclear on the pharmacal use of the whole plant as traditional medicine is fruit. Based on our former research about Tribulus terrestris L., the objective of this study is to investigate the effect of sapomns from Tribulus terrestris L (STT) on the renal cancer cell line (786-0) in vitro, which may be caused by diabetic nephropathy, and its mechanisms.At first, the anti-tumor effect of STT was to be evaluated by MTT and SRB assay. By using the two methods we found that the STT showed a significant inhibitory effect on 786-0 which was in dose depentment manner. Then we compared the cytotoxic effect of STT on 786-0 with that on human normal kidney cell line (HEK-293), it was observed that STT had less inhibitory effect on the proliferation of HEK-293.In order to further investigate STT induced-apoptosis on 786-0, several methods of morphological observation were used, such as Wright staining, acridine orange staining and electron microscope, the results showed that the morphological changes were induced by STT with apoptosis on 786-0, but the DNA Ladder wasn't tested because the rate of apoptosis was very low. In addition, the effects of STT on the cell cycle, the expression of Bcl-2 protein, the content of Ca²⁺ and the mitochondrial membrane potential(△ψm) in the 786-0 were determined respectively by flow cytometry(FCM). After the 786-0 were treated by STT, it was found that: 1) The distribution of 786-0 on G₀/G₁ and G₂/M phase was decreased; 2) The expressions of Bcl-2 protein and cycle D1 fell; 3) The△ψm was disrupted; 4) The intracellular Ca²⁺ content was increased. What's more, adding 2 m mol/L Ca²⁺, or 1m mol/L EGTA, or 250κmol/L Zn²⁺ to the medium, the cytotoxicity of STT against 786-0 was enhanced, whereas the DNA fragment induction by STT was affected only with 2 m mol/L Ca²⁺. It was speculated that the effect of STT on 786-0 was possibly catalyzed by Ca²⁺-independent DNase. To sum up, STT can significantly inhibit the growth of 786-O in vitro, partially, by apoptosis, which was related to the change of cell cycle, the Bcl-2 protein expression, the intracellular Ca²⁺ content and the potential of mitochondrial membrane.Keeping the basal effective content stable and setting down a strict quality standard are important for Chinese herb modernizing. The HPLC-fingerprinting of the main saponins from Tribulus terrestris L. stored in different periods and their cytotoxicity on 786-O were tested respectively by RP-HPLC [Kromasil C18 analytical column(250mm×4.6mm, 5um), 100ï¼…methanol as eluent with flow rate: 1mL/min] and MTT method. The discipline of changes is found during storage: 1) The determination of main saponins from Tribulus terrestris L was decreased; 2) The cytotoxic effect of STT on 786-O was weakened. In short, the period of storage can affect the determination of main saponins from Tribulus terrestris L. and its effect on cancer cell; partially, the determination of Hecogenin can influence the effect.It was well known that the trace elements are effective components of Chinese herbs. In this study, we tried to discuss the interrelation among trace elements, organic compounds and the effect on AGEs in several plant extracts with method of blurred classification. To begin with, we prepared AGEs with glyoxal, fructose or glucose, and added the plant extracts to test the effect. Secondly, we used Inductively Coupled Plasma—Atomic Emission Spectrometry to determine the contents of seven trace elements, Zn,Mg,Mn,Mo,Cu,Cr,V. By range analysis, correlation coefficients and relative fuzzy matrix were calculated to combine with Zn/Cu and Zn/Mn. It was found that most of the components can inhibit the formation of AGEs, especially, the effect of FFH was close to FRR, cluster in the two plant extracts was similar when relative coefficient is 0.75. The finding of this study suggested that trace elements and organic compounds of STT had much to do with the formation of AGEs and its inhibitory effect on 786-O.