Induction of apoptosis in human hepatocarcinoma SMMC-7721 cells in vitro by flavonoids from Astragalus complanatus

Aim of the study

Flavonoids extracted from the seeds of Astragalus complanatus R.Br. reduce the proliferation of many cancer cells. The present study was carried out to evaluate the effects of these flavonoids from Astragalus complanatus (FAC) on human hepatocarcinoma cell viability and apoptosis and to investigate its mechanisms of action in SMMC-7721 cells.

Materials and methods

Cell viability was measured using the MTT assay. To detect apoptotic cells, SMMC-7721 cells treated with FAC were stained with Hoechst 33258 and subjected to agarose gel electrophoresis. Quantitative detection of apoptotic cells was performed by flow cytometry. The effects of FAC on apoptosis and cell cycle regulatory genes and proteins in SMMC-7721 cells were examined using an S series apoptosis and cell cycle gene array and Western blot analysis.

Results

The growth of SMMC-7721 and HepG2 cells was inhibited by treatment with FAC. Cell death induced by FAC was characterized by nuclear condensation and DNA fragmentation. Moreover, the cell cycle was arrested in the G0/G1 and S phases in FAC-treated SMMC-7721 cells. A sub-G1 peak with reduced DNA content was also formed. The activity of caspase-3 was significantly increased following FAC treatment. Microarray data indicated that the expression levels of 76 genes were changed in SMMC-7721 cells treated with FAC: 35 genes were up-regulated and 41 were down-regulated. Western blot analysis showed that caspase-3, caspase-8, Bax, P21, and P27 protein levels in SMMC-7721 cells were increased after 48 h of FAC treatment, while cyclinB1, cyclinD1, CDK1, and CDK4 protein levels were decreased.

Conclusions

These results suggest that FAC may play an important role in tumor growth suppression by inducing apoptosis in human hepatocarcinoma cells via mitochondria-dependent and death receptor-dependent apoptotic pathways.     

甘草黄酮的提取分离和含量测定

目的：充分利用甘草资源。方法：从提取过甘草酸的废渣中提取甘草黄酮并进行含量测定。结果：用碱酸处理和用大孔树脂处理黄酮含量高，收率也高。结论：从甘草废渣中提取甘草黄酮具有较好的社会效益和经济效益     

天峨野生大果山楂酒中氨基酸与总黄酮的含量测定

目的：对天峨野生大果山楂（Malus doumeri)酒中氨基酸（游离氨基酸,水解氨基酸）,总黄酮进行含量测量。方法：利用高速氨基酸分析仪和分光光度计分别测定该酒中游离氨基酸,水解氨基酸和总黄酮类的含量。结果：该酒含17种游离氨基酸,含量为30．89％,16种水解氨基酸,含量为99．25％;总黄酮含量为15．43％（单位mg/ml)。结论：该酒富含氨基酸和总黄酮化合物,具有较强的生物活性。     

3味山姜属中药黄酮类成分对大鼠环核苷酸水平及交感神经-肾上腺轴的影响

目的：观察高良姜、大高良姜、红豆蔻黄酮类成分对胃溃疡寒证大鼠环核苷酸水平及交感神经-肾上腺轴的影响,探讨3味山姜属中药温热药性的物质基础。方法：采用灌服冰知母水煎液与15%冰乙酸制备大鼠胃溃疡寒证模型,以干姜姜辣素为阳性对照,采用酶联免疫吸附试验（ELISA）法测定腺苷酸环化酶（AC）、磷酸二酯酶2（PDE2）、环磷酸腺苷（cAMP）、环磷酸鸟苷（cGMP）、促肾上腺皮质激素（ACTH）、多巴胺β羟化酶（D-β-H）含量。结果：与空白组比较,胃溃疡寒证模型组大鼠胃组织AC、cAMP含量及cAMP/cGMP比值显著降低,PDE2含量显著升高（P<0.01）。与模型组比较,高良姜、大高良姜、红豆蔻高低剂量组大鼠胃组织AC含量升高;高良姜、大高良姜、红豆蔻高低剂量组大鼠胃组织PDE2含量显著降低,cAMP含量、cAMP/cGMP比值显著升高（P<0.01或P<0.05）。结论：3味山姜属中药黄酮类成分通过调节胃溃疡寒证大鼠环核苷酸水平从而促进交感神经-肾上腺轴功能活动的作用,也体现出黄酮类成分药性温热。     

加杨雄花序的化学成分研究

首次对加杨雄花序了条统从其撮物的石油醚（６０～９０℃）部分,乙酸乙酯部分分离并鉴定了１５个化合物,本语文报道８个黄酮类成分,乔松酮（ｐｉｎｏｓｔｒｏｂｉｎ,１）,高良姜素－３－甲氧基（ｇａ／ｌａｎｇｉｎ ３ ｍｅｔｈｏｘｙ,２）,乔松索（ｐｉｎｏｃｅｒｍｂｉｎ,２）,白杨素（ｃｈｒｙｓｉｎ,４）槲皮素－３,７－二甲氧基（ｑｕｅｒｃｅｔｉｎ０－３,７－ｄｉｍｅｔｈｏｘｙ,５）,鼠李素（ｒｈａｍｍｅｔ     

Dietary flavonoids protect human colonocyte DNA from oxidative attack in vitro

Summary Background & Aims: Epidemiological studies suggest that antioxidant polyphenols in the human diet may protect against diseases such as cancer. In this study we investigated the cytoprotective potential of the flavonoids, quercetin, myricetin, kaempferol and rutin against oxidative DNA damage in human colonocytes in vitro. Methods: Caco-2 cells, which display specialised enterocyte/colonocyte cell functions, were used as an in vitro model for human colonocytes. Hydrogen peroxide was employed as the oxidant. DNA damage (strand breakage, oxidised purines and oxidised pyrimidines) was determined using the alkaline single cell gel electrophoresis or comet assay. Cell growth and viability were measured. Results: Hydrogen peroxide caused a dose-dependent increase in DNA strand breakage in human colonocytes, presumably via oxygen free radical generation. Quercetin and myricetin protected Caco-2 cells against oxidative attack. In addition, quercetin decreased hydrogen peroxide-mediated inhibition of growth. Neither rutin nor kaempferol was effective. However, quercetin, while inhibiting DNA strand breakage, did not alter the levels of oxidised bases following peroxide treatment. The antifungal agent ketoconazole, prevented quercetin cytoprotection in Caco-2 cells, indicating that P450-mediated metabolism may alter the efficacy of the flavonoids against oxidative DNA damage. Conclusion: Flavonoids, particularly quercetin, the most abundant flavonoid in the human diet, are likely to be important in defending human colonocytes from oxidative attack. Received: 7 October 1998, Accepted: 5 November 1998     

Quercetin inhibits tyrosine phosphorylation by the cyclic nucleotide-independent,transforming protein kinase,pp60src

Summary The bioflavonoid quercetin is a potent inhibitor of a cyclic nucleotide-independent, tumor virus-coded protein kinase which phosphorylates tyrosine residues and acts as a cellular transforming protein. Half-maximal inhibition of the protein kinase occurred at 3–4 M quercetin whereas rutin was much less effective. The finding, that quercetin inhibits a cyclic nucleotide-independent protein kinase activity, may provide clues to the diverse pharmacological effects of the bioflavonoids.     

The effect of naringenin on the intestinal and renal transport of organic solutes

Summary Naringenin (4,5,7-trihydroxy-flavanone) inhibits the accumulation of glycine, -methyl-glucoside, p-amino-hippurate and N¹-methyl-nicotinamide in dog renal cortex slices. It also inhibits oxygen consumption by this tissue. Since the sensitivity of amino-acid uptake to the drug is less than the sensitivity of oxygen consumption, the inhibition of this transport might be secondary to an effect on intermediary metabolism. The inhibition of PAH uptake occurs at a lower concentration of the drug, and so naringenin may affect this process at the membrane level.Naringenin inhibits the transport of sugars and amino-acids by dog, guinea-pig and rat small intestine. Both steady-state accumulation and initial rates of entry are affected. Amino-acid uptake is depressed both in the presence or absence of sodium ions. The inhibition is reversible provided short contact times are employed. According to kinetic analysis, naringenin appeared to be a fully non-competitive inhibitor of phenylalanine influx. Examination of the unidirectional transmural fluxes of phenylalanine across guinea-pig intestine revealed that only the mucosal-serosal flux was affected, and then only if the flavanone was added to the solution bathing the mucosal face of the tissue. Naringenin does not inhibit mucosal Na⁺-K⁺-ATPase, but it does alter the intracellular ion concentrations.Although some of the results can be explained in terms of an effect of naringenin on metabolism, others can not. It is argued that naringenin has a direct action on cell membranes.Some of these results have been presented to the Physiological Society and a short report has appeared in their proceedings (Robinson, 1979)     

银叶树树叶中的黄酮类化合物

目的研究银叶树Heritieralittoralis D.的化学成分.方法利用硅胶柱层析进行分离和纯化,并通过波谱分析鉴定结构.结果从银叶树的叶子中分离鉴定了8个黄酮类化合物,分别为栎皮苷(quercitrin 1),槲皮素(quercetin 2),山柰酚-3-O-(6"-0-E-p-香豆酰基)-β-D-吡喃葡萄糖(kaempferol-3-O-(6"-O-E-p-coumroyl)-β-D-gluco-pyranoside 3),山柰酚(kaempferol 4),山柰苷(kaempferitrin5),杨梅黄酮(myricetin 6),圣草酚(eriodictyol 7)和儿茶素(catechin 8).结论化合物2-8首次从该植物中分离得到.     

甘草中黄酮的超声提取及含量测定

目的：提取甘草中的黄酮．并测定其含量。方法：运用超声技术提取甘草黄酮．用比色法测定黄酮含量。结果：室温下超声提取80min较为合适。此时黄酮的含量为1．311％．平均回收率为99．10％．RSD=0．90％(n=6)。结论：运用超声技术提取甘草黄酮速度较快，实验结果令人满意。