Studies On The Preparation Technology, Quality Standard Of Total Flavonoids From Bidens Bipinnata L. And Its Protective Effect Against Carbon Tetrachloride-induced Liver Injury In Mice

Acute liver injury has a very high morbidity and mortality and is a common pathology.It is mostly induced by viral hepatitis,alcoholism,iron overload,or drug toxicity.So it has been very important clinical significant to control the development and process of liver injury in time.The incidence of virus hepatitis of our country is the highest in the world.Many drugs have been used in the treatment of liver damage. However,it is still difficult to achieve satisfied therapeutic effect.Whereas,the therapeutic strategy of modem medicine has adjusted from allotherapy to regulate therapeusis,from antiviral research to promote the ability of the organism to defend disease.The diseases of liver can be theraped better using the treatment of traditional Chinese medicine.The treatment of traditional Chinese medicine not only can improve the function,but also can revive the structure.Bidens bipinnata L.,a species of the genus Bidens(Compositae),commonly known as "po-po-zhen" in China,was first recorded on the "Supplement to the Herbal" written by a famous physician in the Tang Dynasty one thousand years ago.It is widely distributed in majority province in China.Bidens bipinnata L.had been traditionally used for many years.It is bitter in taste,neutral in nature and innocuity, and has the effect of heat-clearing,detoxicating and eliminating stasis to subdue swelling.It contains many chemical compositions,such as flavonoid glycoside, saponin,alkaloid,aetherolea.It was largely used to treat hepatitis,inflammation, malaria,hypertension,diabetes,peptic ulcer,snake bite,smallpox.Flavonoids are rich in Bidens bipinnata L.,in which the content is ad high as 5%in the leave.But so far, the total flavonoids of Bidens bipinnata L.are not fully studied.In addition,in the clinical practice,Bidens bipinnata L.and compound preparation containing Bidens bipinnata L.had obvious therapeutic effect on virus hepatitis,which can significantly degrade the ALT and AST of hepatopath with no obvious toxicity and side effects.In order to develop a new use of Bidens bipinnata L.,a flavonoid-rich extract of Bidens bipinnata L.was prepared and its hepatoprotective activity were studied in this paper.The main contents were divided into some sections as follows:1.Studies on the preparation technology of total flavonoids from Bidens Bipinnata L.We applied orthogonal design L₉(3⁴) to optimize the extraction technique of total flavonoids of Bidens bipinnata L.The best extraction technology was that 12-fold and 80%ethanol,1 hour of reflux,and 2 times of extraction.In addition,to screen macroporous resins for isolation and purification of total flavonoids of Bidens bipinnata L.(TFB) through investing the property of adsorption and desorption of 7 kinds of macroporous adsorption resins for TFB.The static and dynamic adsorption-desorption methods were applyed to compare the adsorption quantity,the rate of desorption and adsorption kinetics of different macroporous adsorption resins. The HPD100 resin was chosen as the suitable medium to prepare TFB.And the conditions for preparation of TFB were studied.The technology conditions were as follow,the concentration of the extraction sample was 1～1.5mg·mL^-1,the maximum adsorbing capacity for the total flavonoids of Bidens bipinnata L.was 4.99 mg·ml^-1 with the adsorption velocity 2BV·h^-1,decontamination with 4BV distilled water then 70%ethanol was used to elute the total flavonoids,the eluting volume was 4BV and the eluting velocity was 4 BV·h^-1.On this condition to adsorption and elution,the HPD100 resin could be used 5 times repeatedly.After adsorption on macroporous resin and desorption by ethanol,the total content of flavonoidds in Bidens bipinnata L. was 72.1%,and the yield was 4.7%. 2.Quality standard of total flavonoids from Bidens Bipinnata L.The quantitative control(QC) is the critical of the whole work.We utilized chemical differential method and thin layer chromatography(TLC) to identify TFB. And applying ultraviolet spectrophotometry to determine the content of TFB using NaNO₂-Al(NO₃)-NaOH chromogenic system.The content of TFB was 73.3%,and the average recovery was 96.51%(RSD:2.61%).This method wad convenient,rapid and has good reproducibility.The HPLC method was used to determine the content of hyperoside.The analysis of hyperoside was carried out on Hypersil ODS2 5μm column (4.6mm×250mm).The mobile phase was ACN-3%HAC(13:87).The folw-rate was 1.0ml·min^-1.The detection wavelength was 363nm.And the column temperature was 30℃.We applied external reference method to quantitate the hyperoside of TFB. The liner range of hyperoside was 1.2526～34.1μg·mL^-1,and the correlation coefficient(r) was 0.9998.The recovery was 99.62%and RSD was 1.75%.This method can separate flavonoids well,and also can be used in the quality control of TFB.This work will be basis of the next drug development in the future.3.Protective effect of total flavonoids from Bidens bipinnata L.against carbon tetrachloride-induced liver injury in mice and its partly mechanisms of actionUnder the optimized preparation technology of total flavonoids of Bidens bipinnata L.(TFB),we applied the model of carbon tetrachloride(CCl₄)-induced acute liver injury in mice to investigate hepatoprotective activity of TFB.The results suggested that oral administration of TFB at doses of 50,100 or 200 mg·kg^-1 for 7 days,significantly reduced the elevated of liver index,serum transaminases[alanine aminotransferase(ALT) and aspartate aminotransferase(AST)].Liver histopathology showed that pretreated with TFB could obviously abatement the degree of necrosis and inflammation of hepatic tissues.These results suggest that TFB has significant hepatoprotective activities.In addition,TFB markedly inhibited CCl₄-induced lipid peroxidation,and enhanced the activities of antioxidant enzymes[superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px)].Moreover,pretreatment with TFB suppressed the nitric oxide production and the nuclear factor-kappa B activation in CCl₄ treated mice. Ultrastructural morphology showed that mice pretreated with TFB had a significant improvement in the degree of damages of the organelles of hepatocytes.These results suggest that the mechanism of hepatoprotective activities of TFB is related to,at least in part,the antioxidant properties,inhibition of the production of NO and the expression of NF-κB.