| Cirsium japonicum DC is a kind of traditional C hinese medicine and food homology variaty, which is frequently cited in ancient Chinese medical texts such as Bencao Gangmu(Compendium of Materia Medica). It lives wild on the hillside, roadside, distributes in most parts of China, the efficacy part of which is the aerial parts or roots. Cirsium japonicum DC contains plenty of chemical ingredients, such as triterpene steroids, volatile oils, flavonoids and flavonoid glycosides. Cirsium japonicum DC tastes sweet with a little bitter, and can be used to treat a variety of clinical tuberculosis, hypertension, urinary tract stones, hematuria, and carbuncle boils early lung caused by wind- heat rhinitis. Modern pharmacological studies have found that it has preventing bleeding, anti-bacterial, and anti-tumor effect.The total extract was obtained by the extractio n of heating reflux with 70% ethanol and then portioned with petroleum ether, ethyl acetate, n-butanol and water, which yielded petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and water fraction, respectively. Different models, antioxidant(ferric reducing/antioxidant power assay, DPPH radical scavenging assay and ABTS scavenging assay), antitumor and protecting liver cells from chemical injury properties were use to evaluate the activity of the total extract(70% ethanol) and four fractions extracted from Cirsium japonicum DC. Results showed that petroleum ether and ethyl acetate fraction were found to have significant inhibitory effect against human breast cancer cells MCF and human hepatoma cell line, thus these two fraction were choose to be separated for anticancer compounds. As for the activity against liver damage, the n-butanol fraction, and water fractio n showed significant activity. Consequently, n-butanol fraction was selected for further separation and purification.Three fractions were separated and purified by various column chromatographies(including silica gel column, Sephadex LH-20 gel column and ODS column) and the prepared HPLC. Six compounds were obtained and four of them were identified by mass spectrometry and nuclear magnetic resonance as 1- nonadecanol; β-sitosterol; β-daucosterol; apigenin-7-O-β-D-glucopyranoside, respectively.Antioxidant, antitumor and anti- inflammatory activities of the six compounds isolated from three fractions was investigated. Results showed that compound 4 and 6 showed strong overall reduction force, compound 6 showed strong DPPH radical scavenging and ABTS scavenging activity. In the evaluation of cytotoxicity against tumor cell lines, compound 1, 2 and 3 exhibited obviously action on the MCF cell lines, compound 3 and 6 exhibited obviously action on the Hep G2 cell lines. All the compounds had little cytotoxicity to the liver cell HL7702. In the evaluation of protecting liver activity, compound 6 showed a similar trend to the positive control silymarin, the best liver protecting concentration of compound 6 was 25μg / m L, hepatoprotective effect of the other five compounds are not so significant.The flavonoids of Cirsium japonicum DC was purified using three macroporous resin(AB-8, DM301 and HPD100), to maximize the adsorption and desorption of Cirsium japonicum DC, effect on the adsorption amount of resin of the adsorption temperature and sample concentration were detected, and then the most suitable type of resin and purified conditions: AB-8, at room temperature, adsorption 220 minutes or more continously, 55% ethanol for desorption. Under this condition, total flavonoids of the purified dry sample were determined as 203.33mg/g.In addition, the total flavonoids sample was subjected to in vitro antioxidant, anti-cancer and hepatoprotective activity, the results showed that the reducing power and the DPPH radical total flavonoids were weak than the positive control and silymarin, but in terms of clearing ABTS radical, C irsium japonicum DC total flavonoids showed stronger scavenging effect than silymarin, and is close to VC positive control group. T he anticancer activity of Cirsium japonicum DC total flavonoids was lower than the five- fluorouracil control at low concentrations significantly, but with the increase of the concentration, its anticancer activity increases faster than that of positive control group, with good anti-cancer effect. The hepatoprotective activity of Cirsium japonicum DC total flavonoids showed that the total flavonoids have little damage to the normal liver cell HL-7702, at the highest concentration of 400μg/ ml, the cytotoxicity to normal liver cell is about 30 percent, the study of anti-CC l4 liver injury showed the hepatoprotective effect of the total flavonoids is consistent with the trend of the positive control, and there is no significant difference with the positive control, thus the Cirsium japonicum DC flavonoids can be further developed for the hepatoprotective products. |
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