Effect Of Polygonum Multiflorum Thunb On Bilirubin Metabolism Mediated By UGT1A1 And Its Hepatotoxicity

Objective1.To establish a high performance liquid chromatography method for simultaneous determination of the polyphenols,tetrahydroxystilbene-glucoside and anthraquinones in Polygonum multiflorum Thunbs,optimize the extraction process in Polygonum multifl-orum Thunbs;to establish a fingerprint for investigating the content difference of the main components in Polygonum multiflorum Thunbs from different origins;To provide the experimental basis for development and revision of quality criteria in Polygonum multifl-orum Thunbs.2.To compare the Polygonum multiflorum Thunbs from different origins induced hepatotoxicity in mice,and to analyze the influence of main components on mice liver toxicity.3.To establish a high performance liquid chromatography method for simultaneous determining the concentration of bilirubin and its metabolites（BGs）in UGT1A1 incuba-tion system;to research the inhibition of Polygonum multiflorum Thunbs and its main components on UGT1A1mediated-bilirubin glucuronidation,and to disclose the reason and mechanism of bilirubin related diseases or adverse drug reaction（e.g jaundice）induced by Polygonum multiflorum Thunbs.Method1.Determination of the main components in Polygonum multiflorum Thunbs by HPLC method.Chromatographic conditions:Diamosil C₁₈ chromatographic column（4.6mm×250 mm,5μm）,mobile phase:acetonitrile-0.1%acetic acid aqueous solution,gradient elution,flow velocity is 1.0 mL/min,the wavelength is 280 nm,column temperature is 30℃,injection volume is 10μL;Polygonum multiflorum Thunbs were extracted by heating reflux method,and influence of the extraction solvent（water,anhydrous ethanol,75%ethanol and 50%ethanol）,extraction time（0.5 h,1 h,2 h and 4 h）,extraction times（once and twice）on its content determination was investigated,respectively.2.Polygonum multiflorum Thunbs induced liver toxicity in mice.Kunming mice were randomly assigned to 13 groups,each group was 6（half male and half female,20±2g）,the experimental groups were fed with the alcohol extracts of Polygonum multiflorum Thunbs from different origins,the control group was fed with normal rat food,the blood via the eyes venous plexus was taken on 0,15,30 days respectively;liver toxicity indexs,such as alanine aminotransferase（ALT）,aspertate aminotransferase（AST）,alkaline phosphatase（ALP）,glutamine transferase（GGT）,lactate dehydrogenase（LDH）,total bilirubin（TBIL）,total bile acid（TBA）,were determinated with the mice serum,and Polygonum multiflorum Thunbs induced liver toxicity was evaluated,and the relationship between differences of main components in Polygonum multiflorum Thunbs and liver toxicity（biochemical indicators）was analyzed.3.Simultaneous determination of bilirubin and its metabolites by the HPLC method.Chromatographic conditions:Diamosil C₁₈ chromatographic column（4.6 mm×200 mm,5μm）,mobile phase:acetonitrile-0.1%formic acid aqueous solution,gradient elution,and the flow rate is 1.0 mL/min,the wavelength is 450 nm,column temperature is 40℃,injection volume is 100μL.The incubation system of human UGT1A1 enzyme mediated-bilirubin glucuronidation and its inhibition was established.The incubation system included potassium phosphate buffer（pH 7.4）,Polygonum multiflorum Thunbs extracts or its components（1¹00μM）,bilirubin（0.2²μM）,UGT1A1（12.5μg/ml）,alamethicin（0.022 mg/ml）,magnesium chloride（0.88 mM）,UDPGA（3.5 mM）,mixed in a certain order,and incubated in 37℃water bath for 15 min.The reaction was stopped by adding cold methanol containing 200 mM ascorbic acid into the incubation system.Result1.The established HPLC method for determination of the main components in Polygonum multiflorum Thunbs showed good isolation,precision and reproducibility（RSD≤2.98%）.Each component in the samples was stable within 48 h in room temperature（RSD≤3.14%）.The HPLC method displayed good linearity（r²≤0.9990）,the recoveries of 9effective components were 99.44¹03.12%with RSD≤4.08%;influence of the extraction solvent,extraction time and extraction times on determination of 9 components exhibited statistically significant（p≤0.05）;Content of main components of Polygonum multiflorum Thunbs from different origins showed significant difference.Of them,the Polygonum multiflorum Thunbs from Guizhou,Sichuan,Shandong,Guangdong and Shanxi meet the quality standards of the"Chinese pharmacopoeia（2015）".2.Compared to the control group,the average serum biochemical indices of the experimental group mice（except for TBA and LDH）on 15 days were significantly increased（p≤0.01）,the average of serum biochemical indicators（except LDH）on 30 days were significantly risen（p≤0.01）;the liver toxicity index among different experimental groups displayed significant difference;Compared to the other experimental groups,the main biochemical indexs from group-1（Guangdong）,group-6（Shanxi）and group-7（Henan）showed increase obviously;it displayed positive correlation between content change of anthraquinones compounds in Polygonum multiflorum Thunbs from different origins and change of the two serum biochemical indices,e.g.TBIL and TBA（p≤0.05）.3.The established HPLC method for determination of bilirubin and its three metabolites showed good isolation,sensitivity,precision and reproducibility（RSD≤2.53%）.Each component in the samples was stable within 24 h in room temperature（RSD≤2.38%）.It exhibited good linearity in the range of 0.01³μM bilirubin（r²≥0.9991）,the extraction recovery was≥92.48%with RSD≤3.65%;IC₅₀ of Polygonum multiflorum Thunbs extraction（emodin as marker）,emodin,tetrahydroxystilbene-glucoside,rhein,epigallocatechin gallate,Resveratrol,Oligomeric proantho cyanidins were 0.055,6.71,27.3,86.5,8.4,20,9.7μM,respectively.Conclusion1.The established HPLC method for simultaneous determination of multiple components in Polygonum multiflorum Thunbs was simple,sensitive,reliable and reproducible;the optimal extraction process for Polygonum multiflorum Thunbs was as followed:reflux extraction twice with 75%ethanol,each time 1 hour;content of the main components in Polygonum multiflorum Thunbs from different origins were different.Polygonum multiflorum Thunbs from several origins did not met the quality standard of Chinese pharmacopoeia;the established"fingerprint"can be used as quality control and evaluation of Polygonum multiflorum Thunbs,and provide an important reference for revision of the pharmacopoeia standard of Polygonum multiflorum Thunbs.2.Polygonum multiflorum Thunbs displayed liver toxicity in mice,and exhibited significant difference from different origins.Anthraquinone compounds in Polygonum multiflorum Thunbs might be the main hepatotoxic ingredient.3.The established incubation system for UGT1A1 enzyme mediated-bilirubin glucuronidation and its inhibition was stable and reliable,can be used to research bilirubin metabolism and its tregulation in vitro;the established HPLC method for determination of bilirubin and its metabolites was simple,accurate,reliable,and can be used to bilirubin related research.Bilirubin glucuronidation can be inhibited by Polygonum multiflorum Thunbs and its components（e.g.emodin）,which resulted in jaundice and hepatotoxicity of Polygonum multiflorum Thunbs.