Determination Of 13 Active Compounds In Huang-lian-jie-du Decoction And Screening Of Effective Fraction By Hplc And Safety Assessment Of The Active Components Combination

Huang-lian-jie-du decoction is a classic prescription for clearing heat and detoxifying.Modern pharmacological studies showed that it has the effects of antiinflammatory,antioxidant,hypoglycemic and neuroprotective.Objective: To establish a method for the determination of 13 active components in Huang-lian-Jie-du Decoction(HLJDD)by HPLC,preparing different polar fractions of HLJDD by system solvent ex-traction method.And screening of active fraction of HLJDD by detecting the content of active compounds in different polar fractions.On this basis,according to the proportion of the content in HLJDD," active components combination of HLJDD " is formulated by the four highest active compounds in HLJDD,and the safety of the active components combination of HLJDD was evaluated through in vitro and in vivo experiments.Methods: 1.The detection of the 13 main active compounds in HLJDD was built based on Agilent 1260 HPLC and the content of 13 main active compounds in HLJDD was determined by this method.2.Dried HLJDD powder was extracted with petroleum ether,ethyl acetate,nbutanol and purified water by systematic solvent extraction,and then obtain the different polar fractions of HLJDD.Use the HPLC method to detect 13 main active compounds in the different polar fractions of HLJDD,and screening out the effective fraction of HLJDD based on the test results.3.The four highest active compounds in HLJDD were used to form the active components combination of HLJDD,according to the HPLC detection ratio.Thiazole blue(MTT)was used to detect the effects of HLJDD,HLJDD n-butanol fraction,geniposide,berberine,palmatine,baicalin and active components combination of HLJDD on the viability of Caco-2 cells to detect the cytotoxicity of each drug.4.Kunming mice were randomly divided into normal control group and four active components combination of HLJDD treatment groups,the normal control group was given distilled water and the experimental group was given respectively 5100 mg/kg,1275 mg/kg,318.75 mg/kg,79.6875 mg/kg of active components combination of HLJDD by gavage at one time,feeding and observation for 7 days after administration,the clinical manifestations,death and diet of the mice in each group were observed and recorded during the experiment.After experiment,the mice were sacrificed and the organ damage was observed,the organ index was calculated To determine the acute toxicity of the active components combination of HLJDD.Results and conclusions: 1.The precision,repeatability,stability and add sample collection experiment proved that the HPLC detection method is stable and reliable,and use it to detect the content of 13 active components in HLJDD.2.According to HLJDD dry powder,the yield of HLJDD-petroleum ether fraction,HLJDD-ethyl acetate fraction,HLJDD-n-butanol fraction,HLJDD-water fraction,and HLJDD-precipitation fraction are 0.718%,5.15%,64.45%,25.76%,and 3.70% respectively.The HPLC test results showed that the content and proportion of active components in the n-butanol fraction are most similar to HLJDD,which shows that HLJDD-NBA fraction has similar effective with HLJDD.The four highest active components in HLJDD and HLJDD n-butanol are geniposide,berberine,palmatine,baicalin.The four active components are combined according to the proportion of HLJDD to form an active components combination(Geniposide 44.21%,berberine 32.42%,palmatine 12.02%,baicalin 11.35%).3.The Caco-2 cells were treated respectively by 162.5 μg/mLHLJDD,100 μg/mL HLJDD-n-butanol fraction,100 μg/mL geniposide,100 μg/mL baicalin,50 μg/mL palmatine,6.25 μg/mL berberine,12.5 μg/mL active components combination of HLJDD,the results of MTT test showed that the survival rate were both more than 90%,which indicate that there was no Caco-2 cytotoxicity when the treate concentration was not more than the above,and the active components combination of HLJDD can be used in the Caco-2 cell test.4.The results of acute toxicity test about the active components combination of HLJDD showed that one male mouse died after gavaged two hours in the 5100 mg/kg dose group.The autopsy results showed that the liver was swollen,brittle,the color was uneven and diffuse white spots on it,slight swelling of the spleen and kidneys,congestion and bleeding in the lungs.And the other mice did not die within 7 days.After administration,the mice in 5100 mg/kg dose group showed laziness,food loss,and bunch together,the symptoms improved the next day,the mice in the other treatment groups had no obvious abnormalities.The changes of autopsy were mainly swollen and brittle in liver of the 5100 mg/kg dose group.The organ indexes of liver and spleen in this group were significantly different from those of the blank group(P < 0.05),The heart organ index were all increased but not significantly different from the blank group(P > 0.05).and all organs in the other treatment groups were normal.The result shows that the active components combination of HLJDD has toxic effects on the liver and spleen at a concentration of 5100 mg/kg.But it has no toxicity when the concentration is not more than 1275 mg/kg.