Preliminary Study On The Chemical Composition And Quality Standard Of Gualou Peel

Trichosanthes pericarpium.is a common traditional chinese medicine and it is mentioned firstly in "shennongbencaojing".The medicine possesses the effecr of removing phlegm and causing laxation,so it is used to cure dry cough with sticky sputum and constipation.Trichosanthes pericarpium.is the dried ripe peel of Trichosanthes kirilowii Maxim.or Trichosanthes rosthornii Harms.Some pharmacological tests have shown that the extracts of the pericarpium possess dilating coronary arte,protecting ischemic myocardium,anti-tumor and anti-inflammatory activities.Because of the differences in medicinal properties and efficacy betweenTrichosanthis fructus,Trichosanthes Pericarpium and Trichosanthissemen,the "chinese pharmacopoeia"takes them as diffremt medicine respectively.Trichosanthis fructus is an aggregation of pericarpium,semen,pulpa and flesh,it is not equal to the pericarpium plus semen.Although there have many reports about the chemical composition of Trichosanthis fructus and Trichosanthissemen,little reports about Trichosanthes Pericarpium have been studied.The effective material basic of Trichosanthes Pericarpium is not clear until now.Because of the differences in medicinal properties and efficacy betweenTrichosanthis fructus,Trichosanthes Pericarpium and Trichosanthissemen,there is an urgent need to study the effective material basic and pharmacological activity respectively to illuminate the difference between them.The quality control of in the "chinese pharmacopoeia" is only using the control herbs in TLC study,none assaying and other method are recorded.There is no report about quality control by using any characteristic chemical constituent.There is an urgent need to establish a quality control method by using some characteristic chemical constituent in Trichosanthes PericarpiumThis experiment is one part of traditional chinese medicine standard in Hongkong.Because of the shortage in the resources of Trichosanthes rosthornii Harms.,Hongkong Department of Health choose the Trichosanthes kirilowii Maxim.as the source of Trichosanthes pericarpium.This experiment studied the quality control of Trichosanthes kirilowii Maxim.Spinasterol has a good biological activities such as anti-tumor,anti-inflammatory and so on.We choose spinasterol as an indexity constituent in the quality control of Trichosanthes kirolowii Maxim.in the permission of Hongkong Department of Health.We also have compared the difference between Trichosanthes kirilowii Maxim.and Trichosanthes rosthornii Harms in thin-layer,characteristic spectrum and the content of spinasterol in Trichosanthes pericarpium.Objective:Studying the chemical constituents of Trichosanthes kirilowii Maxim,to establish the method of TLC,characteristic spectrum and assaying for quality controlMethod and Result:Sample collection:We have investigated the the cultivation of Trichosanthes kirilowii Maxim.in Anguo Hebei,Bozhou Anhui,Feicheng Shandong and the resource distribution of Trichosanthes rosthornii Harms.in Sichuan.We have collected ten batches of Trichosanthes kirilowii Maxim.and eight batches of Harms.the herbarium has been collected as the same time.To study the chemical constituents of Trichosanthes kirilowii Maxim.:Dried fruits(20.0 kg)of Trichosanthes kirilowii Maxim.were extracted exhaustively with 90%EtOH for 3 h twice.After concentration in vacuo,the residue was partitioned with Petroleum Ether,CHCl3,EtOAc and n-BuOH successively.The dichloromethane extract of the plant was choosed as the study object,because the polarity of n-BuOH was too high.Chromatographic methods such as D101 macroporous resin,silica gel column chromatographic technology,Sephadex LH-20,chromatographic technique were used and nine compounds were isolated from the dichloromethane extract of the plant.By using spectroscopic techniques including MS,UV IR and extensive NMR(1H,13C,1H-1H COSY,DEPT,HSQC,HMBC)these compounds were identified,two fatty acids,palmitic acid(compound 1)and lauric acid(compound 2),two sterols,spinasterol(compound 3)and ?-sitosterol(compound 4),two triterpene,oleanolic acid(compound 5)and betulinic acid(compound 6),two flavonoids,gancaonin G(compound 7)and cirsimaritin(compound 8),a coumarin compound,racemosin(compound 9).To establish a method of qualitative discrimination on thin-layer:We have established a method of qualitative discrimination on thin-layer chromatography(TLC)by using spinasterol as an indexity constituent.Developing solvent system:petroleum ether:ethyl acetate(2:1,v/v),spraying reagent:2%(w/v)Vanillin,the result showed that 18 batches of Trichosanthes pericarpium.were at the same Rf value,which proved the feasibility of this method.This experiment was the first time to choose spinasterol as an indexity constituent.in TLC study.To establish a characteristic spectrum of Trichosanthes pericarpium.by using spinasterol as anindexity constituent:We have determined the method of HPLC through the study of the chromatography elution conditions.Chromatographic condition:Agilent 1100 Series,Eclipse XDB C18 HPLC column,(4.6 x 250 mm,5 ?m),mobile phase:methanol:water=99:1,column temperature:30?,detector wavelength:211 nm,Injection volume:20 ?L.We have studied the characteristic components in Trichosanthes Pericarpium with the method we determined,and we have identified four characteristic peaks including spinasterol in Trichosanthes Kirilowii.We have established the characteristic spectrum through the ten batch of Trichosanthes Kirilowii.This experiment was the first time to choose spinasterol as an indexity constituent.in characteristic spectrum study which meet the requirement of the traditional chinese medicine standard in Hongkong.To establish an HPLC method to determine the constituent of spinasterol in Trichosanthes pericarpium.and to determine the scope of it:We have established a HPLC method with a UV detector to determine the continent of spinasterol.Because of 211nm absorption in ultmviolet of spinasterol.Chromatographic condition:Agilent 1100 Series,Eclipse XDB C18 HPLC column,(4.6 x 250 mm,5?m),mobile phase:methanol:water=99:1,column temperature:30?,detector wavelength:211 nm,Injection volume:20 p.L.Result:the method has been supplied to determine eighteen samples,which comes from over our country.The scope of the content in Trichosanthes kirilowii was not less than 223.42mg/kg according to the formula computing in the traditional chinese medicine standard in Hongkong.This experiment was the first time to choose spinasterol as an indexity constituent.in assaying study.The method is simple and accurate with good reproducibility and can be used for the quantitative analysis of spinasterol in Trichosanthes pericarpium.To compare the difference between Trichosanthes kirilowii Maxim.and Trichosanthes rosthornii Harms.in thin-layer,characteristic spectrum and the constituent of spinasterol in Trichosanthes pericarpium:We have compared the difference between Trichosanthes kirilowii Maxim.and Trichosanthes rosthornii Harms.in TLC study,characteristic spectrum study and assaying study.The difference between Trichosanthes kirilowii Maxim.and Trichosanthes rosthornii Harms.in TLCis not obviously,while the difference in characteristic spectrum and assaying were bothobviously.Conclusion:Oleanolic acid and betulinic acid were firstly found in Trichosanthes pericarpium.while,gancaonin G,cirsimaritin and racemosin were firstly found in Trichosanthes.We have obtained 350mg spinasterol in this experiment,which meet the requirement of standard substance.The study of the dichloromethane extract laid the foundation of the further study of Trichosanthes pericarpium.The method of TLC,characteristic spectrum and assaying for quality control of Trichosanthes pericarpium we have established is the first time to study the quality standard of Trichosanthes pericarpium.This experiment of chemical component and quality standard meet the requirement of the traditional chinese medicine standard in Hongkong strictly.The result we have obtained has been recognized by the scientific committee of Department of Health in Hongkong.It is the first time to study the difference between Trichosanthes kirilowii Maxim.and Trichosanthes rosthornii Harms.in TLC,characteristic spectrum and assaying,but what the difference is need further study.Trichosanthes pericarpium.is a common traditional chinese medicine in clinic,three is an urgent need for us to study the chemical constituents and pharmacological effects of it.At the same time the correlationship between chemical constituents and pharmacological need further study.