Preliminary Study On The Chemical Constituents Of Flavonoids From Southern Scutellaria And Their Liver-protecting Effects Before And After Processing

Semen Cuscutae(Tu-Si-Zi)is a well-known traditional chinese medicine first recorded in the famous Shen Nong's Herbal as an upper grade drug.Tropism of taste:Gan Wen.It could be used for medicine,dietary supplement and cosmetic due to its biological and pharmacological activities.It has been used to nourish liver and kidney,prevent miscarriage and improve eyesight thousands of years.Recently,many studies have revealed that flavonoids which are the main constituents of Tu-Si-Zi could enhance reproductive system,improve immunesys temand have potent antioxidant and anti-aging benefits.Besides,flavonoids could nourish liver and improve eyesight,improve the function of cardiovascular system and anticancer,Processed Tu-Si-Zi has a long history.In ancient times,the most commonly used method was wine processing method(jiujin,jiuzheng,jiuchao,jiuzhu,jiuwei).Second,the method was salt processing method.Besides,there were frying method,huangjing juice and migan juice processing method.The species recorded on the "Tu-Si-Zi" item of China pharmacopoeia(2010)is salt processed Tu-Si-Zi.In Qing Dynasty Yan-Xiting concluded that different processed Tu-Si-Zi had different activities:Processed by stir-heating with salt to fill kedney;Processed by cooking in Huangjing juice to warm spleen and stomach;Processed by stir-heating with wine to warm muscle;Processed by stir-heating with wine and rice to discharge diarrhea.This study is a extension part of national natural fund project " the material foundation and principle of the research which Cuscuta australis processed by salt enhance the efficacy of protecting kidney".This study is from the change of flavonoids' concent to find the different effect of raw product and various processed products for liver protection.It may help to explain why they have traditionally been used to treat different clinical symptoms and expatiate upon the process principium.In the research there were four kinds of samples(crude Tu-Si-Zi,Yanzhi-Tu-Si-Zi,Jiuzhi-Tu-Si-Zi and Qingchao-Tu-Si-Zi).Objective:The flavonoids of Cuscuta australis are its main active ingredients.By comparison the flavonoids of raw products and processed products,we find the flavonoids content change.Raw product(Raw Tu-Si-Zi)and processed products(Yanzhi-Tu-Si-Zi,Jiuzhi-Tu-Si-Zi and Qingchao-Tu-Si-Zi)are used to acute liver injury model and oxidative damage to liver cells model.We observe the protection of raw product and processed products.We attempt to clarify the different role of raw product and processed products on hepatoprotective effect.Methods:Through using macroporous resin column,polyamide column and so on,we separate the ethyl acetate part and n-butanol part of Cuscuta australis;Respectively we made and processed products(Yanzhi-Tu-Si-Zi,Jiuzhi-Tu-Si-Zi and Qingchao-Tu-Si-Zi),using HPLC to determine the five flavonoids(Hyperoside?Astragalin?Quercetin?Kaempferol?Isorhamnetin)content of the raw product and various processed products;In vitro experiment(Cells experiment):We have the alcohol extraction of raw products and processed products effect on normal liver cells(L02),and observe the growth of cells to determine the appropriate concentrations of experiment;We study the concentration of H2O2 on L02 cell oxidative damage;We use the alcohol extraction of raw product and various processed products act on L02 cells which were oxidized by H2O2,and investigate the growth of cells,at the same time,using Elisa kits to detect the content of MDA,GSH,TNF-a,and IL-6 in cell supernatant;Animal experiment:Adopting preventive medication,the alcohol extraction of raw product and various processed products were filled the mice's stomach for seven days,after the last dose,D-galactosamine were injected to mice,which caused acute liver injury in mice model.blood is used to detect the serum indexes,get used to make pathological liver.Results:We separate and identify six flavonoids compounds,respectively Hyperoside?Quercetin-3-O-?-D-galactopyranosyl(2?1)-?-D-apiopyranoside?Astragalin?Quercetin?Kaempferol?Isorhamnetin.The content of hyperoside?astragal in?quercetin?kaempferol in raw product and various processed products have a significant difference:The content of hyperoside?astragalin in raw Tu-Si-Zi is high,and the proportion of content is 1:1;After processing,the content of astragalin present downward trend,Jiuzhi-Tu-Si-Zi drop to the lowest levels,followed by Yanzhi-Tu-Si-Zi and Qingchao-Tu-Si-Zi;the content of hyperoside present upward trend,the content of Yanzhi-Tu-Si-Zi is the highest,followed by Qingchao-Tu-Si-Zi,and the content of Jiuzhi-Tu-Si-Zi fall slightly;the content of kaempferol present downward trend,Jiuzhi-Tu-Si-Zi drop to the lowest levels,followed by Yanzhi-Tu-Si-Zi and Qingchao-Tu-Si-Zi;the content of quercetin present upward trend,the content of Jiuzhi-Tu-Si-Zi is the highest,followed by Qingchao-Tu-Si-Zi and Yanzhi-Tu-Si-Zi;The result of in vitro experiment(Cells experiment):The raw product and various processed products have a promoting effect on L02 cells growth,and in 25 mg/L-200 mg/L range of concentration,with the increase of concentration,promoting effect of reinforcement;The alcohol extraction of the raw and processed products have a influence on L02 cells whinch H2O2 oxidation damaged,by comparison cells rate,the alcohol extraction of raw and processed products group and model group were significantly different;By comparison the content of malondialdehyde(MDA)and glutathione(GSH)in cells supernatant,we found malondialdehyde content in the model group increased significantly,the reduced material GSH content decreased obviously;the content of malondialdehyde and glutathione in Qingchao-Tu-Si-Zi100 group,Jingzhi-Tu-Si-Zi100 group and Yanzhi-Tu-Si-Zi100 group had significant difference with the model group,antioxidant capacity:the alcohol extraction of Jiuzhi-Tu-Si-Zi>the alcohol extraction of Yanzhi-Tu-Si-Zi>the alcohol extraction of Qingchao-Tu-Si-Zi>the alcohol extraction of Tu-Si-Zi.the content of tumor necrosis factor(TNF-a)and interleukin 6(IL-6)in cells supernatant which of Tu-Si-Zi50 group,Tu-Si-Zi100 group and Qingchao-Tu-Si-Zi50 group were significantly difference from model group,anti-inflammatory effects:the alcohol extraction of Tu-Si-Zi>the alcohol extraction of Qingchao-Tu-Si-Zi>the alcohol extraction of Jiuzhi-Tu-Si-Zi>the alcohol extraction of Yanzhi-Tu-Si-ZiThe results of animal experiment:In the model rat serum,AST,ALT were significantly elevated.The level of AST in Tu-Si-Zi and Yanzhi-Tu-Si-Zi were significantly different from model group.This showed Tu-Si-Zi and Yanzhi-Tu-Si-Zi have better protection on liver which D-GalN induced acute liver injury model,including the protection of raw Tu-Si-Zi stronger than Yanzhi-Tu-Si-Zi group;Compared with model group,the content of malondialdehyde and glutathione in raw product group and various processed product groups had no significant difference.The results of mice liver tissue pathological slices:The liver cells of model rat liver tissue became swelling,eosinophilic granular cytoplasm increased,nuclear chromatin significantly reduced,the Kupffer cells and neutrophils of hepatic blood sinus increased,and there were many infiltration of inflammatory cells in portal area and perivascular.Compared with model group,Tu-Si-Zi and positive drug treatment group can mitigate lesions to liver tissue.Conclusions:The alcohol extraction of raw and processed products have a promoting role on the growth of L02 cells,the theory of traditional Chinese medicine Cuscuta australis"nourish liver and protect liver " are consistent;the alcohol extraction of raw and processed products group can decrease the H2O2 oxidation damage degree,have a protective effect on L02 cells whinch H2O2 oxidation damage;the alcohol extraction of Jiuzhi-Tu-Si-Zi had the strongest antioxidant capacity,the anti-inflammatory effect of the alcohol extraction of Yanzhi-Tu-Si-Zi are strongestRaw Tu-Si-Zi and Yanzhi-Tu-Si-Zi had protection which D-GalN induced acute liver injury,the protection of raw Tu-Si-Zi stronger than Yanzhi-Tu-Si-ZiCombined with the results of animals,pathology and cell experiment,we draw the conclusion that raw Tu-Si-Zi can protect liver best.From the cell experiment,we knew the antioxidant effect of Jiuzhi-Tu-Si-Zi was best,but experiments on animals showed no statistical significance,we need to be further in-depth study.