| Tetrastigma hemsleyanum Diels et Gilg (Sanyeqing), belongs to the grape family Vitaceae, mainly distributed in many provinces of China. The modern pharmacological tests revealed that its root tubers possess various activities, especially excellent anti-tumor capability. It is a very valuable herb medicine. Up to now, there is little study on antioxidant, antimicroorgnism and apoptotic mechanism of Sanyeqing.Therefore, in this study we aimed to investigate biological activities, apoptosis inducing mechanisms of Sanyeqing extracts, by which the application of Sanyeqing will been broaden.1. The dried powder of root tuber of Sanyeqing was refluxed in 80% ethanol. The extract was suspended in water after removing the solvents under reduced pressure and sequentially partitioned with different solvents. Then, I petroleum ether fraction (PEF), II chloroform fraction (CFF), Ⅲ ethyl acetate fraction (EAF), Ⅳ n-butanol fraction (BAF) and V aqueous fraction (AF) of Sanyeqing were obtained.2. The antioxidant activities were evaluated by DPPH, ABTS, FRAP assay. EAF showed the most efficient antioxidant effects, which suggested that it is a promising resource for research and development of antioxidant agents. After repeated separation by various chromatographic methods, two known compounds (β-sitosterol and Quercetin) were obtained. Their structures were elucidated by HPLC-MS/MS,1H NMR,13C-NMR spectra and compared with literatures.3. The following microorganisms are used as tested species:S. aureus LMA 1213 and B. cereus LMA 0106, E. coli LMA 1226, S. typhi LMA 0217, K. pneumoniae LMA 0725, M. racemosus LMA 3221, P. citrinum LMA 7126, A. flavus LMA 0816, A. niger LMA 3601, R. nigricans LMA 2429. Inhibitory diameter, minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) of Sanyeqing extracts were evaluated using oxford cup method and broth micro-dilution method. CFF showed the strongest activity against S. aureus and B. cereus, MIC value is 62.5 μg/ml. EAF exhibited the strongest inhibitory activity against E. coli, S. typhi and K. pneumonia, MIC value ranges from 125 μg/ml to 250 μg/ml. while CFF showed good activity against P. citrinum, A. flavus, A. niger and R. nigricans, MIC value ranges from 31.3 μg/ml to 125 μg/ml.4. MTT assay was used to evaluate cytotoxicity activities of Sanyeqing extracts. CFF showed the strongest activity against HeLa cells. The IC50 values of CFF, PEF and EAF against HeLa cells were 8.59 ± 0.98,121.40 ± 1.27 and 148.54 ± 1.78 μg/ml, respectively. The IC50 values of BAF and AF were above 160 μg/ml. And no obvious toxicity of five fractions was observed in HEK-293 cell line.5. The percentage of LDH release increased greatly (p<0.01) after treatment with PEF and CFF, which indicated that the repture of HeLa cell membrane. No obvious LDH release was observed at low concentration of EAF. But an increase of LDH release was observed at 160 μg/ml of EAF, much lower than that of PEF and CFF.6. The mechanism of apoptosis induction on HeLa cells induced by PEF and CFF was investigated. The effects of apoptosis inducing activities on HeLa cells, disruption of Δψm and augments the activation of Caspase-3, Caspase-8, Caspase-9 were observed in this study. The result demonstrated that both intrinsic and extrinsic routes were involved in the apoptosis of HeLa cell induced by PEF and CFF. Furthermore, PEF and CFF induce cell cycle arrest in G0/G1, G2/M phase.The decreases in CAT, SOD, GSH-pX and increases in MDA were observed, which indicated that the oxidative stress appears to be one of the major mechanisms by which Sanyeqing promotes the apoptosis of HeLa cells.7. Total 30 cositituents were identified from PEF extracts by GC-MS, representing 93.33% of the components. The major compounds in PEF extracts were hexadecanoic acid (15.67%), oleic acid (17.72%), linoleic acid (16.54%), campesterol (1.58%), and β-sitosterol (10.29%). |
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