Preliminary Study On The Quality Control Methods Of Traditional Chinese Medicine Bruceae Fructus

Bruceae Fructus?Yadanzi in Chinese?,also named duck egg,old duck gall,is the dry mature fruit of Brucea Javanica L.Merr.The fruits have bitter taste,and is considered cold with small poison.This herb was recorded in the"Compendium of Materia Medica".The main production area includes Guangxi,Guangdong,Yunnan and Hainan provinces.The chemical composition of Bruceae Fructus is complex and diverse.It has been reported to have quassinoid,flavonoids,terpenoids,alkaloids,fatty acids,and other structural types.In terms of pharmacological activities,it was reported to show anti-tumor,anti-malarial,insecticidal and other effects.At present,Bruceae Fructus is widely used in Lingnan area,and products Brucea javanica oil emulsion,Brucea javanica oil preparation,Brucea oil emulsion oral liquid,etc.have been widely used in various diseases such as tumors in the clinic.However,quality control methods of Bruceae Fructus were less studied.All the research progress regarding the chemical components and pharmacogical activities were shown in Chapter 1.In addition,authentication of herbs by DNA technologies was also shown in Chapter 1.In the second chapter of this dissertation,the molecular identification of Bruceae Fructus was first carried out using DNA molecular technology.The genomic DNA of Bruceae Fructus was extracted;the ITS primer was designed using the ITS sequence of Bruceae Fructus as a template.Then,using the Brucea genomic DNA as a template,the high-fidelity DNA polymerase was used to amplify the ITS sequences in the genome,and finally the amplified products were sequenced and compared with the ITS sequences of Brucea javanica in Genebank.The results show that our plant samples are genuine herbs of Bruceae Fructus.In the third chapter,the fingerprint of Bruceae Fructus was established by HPLC.The optimized conditions:Cosmosil?4.6 mm×250 mm,5?m?C₁₈ column,mobile phase:H₂O?A?-CH₃OH?B?,gradient 0¹5 min,15%?30%B;15³0 min,30%?45%B;30⁶0min and 45%?65%B,flow rate 1.0 ml·min^-1,detection wavelength 221nm,column temperature 29?,injection volume 10?l.The results showed that the fruits of Brucea javanica from Guangxi Yulin market had significant difference from other samples.In the sample from Guangxi Yulin,the content of yadanzioside A is much lower;The similarity of the seven batches is larger than 0.924,indicating that they are close similar to each other.In addition,the chemical composition of methyl ester of oil-soluble part of Bruceae Fructus was analyzed by GC-MS.It was found that the highest content of this parts was methyl linoleate?59.59%?,followed by methyl oleate 32.04%.To establish a method for quantitative determination of three quassinoids?bruceoside B?bruceoside A and brusatol?in Bruceae Fructus,we established a HPLC method in chapter four.The determination was carried out on a Cosmosil?4.6 mm×250 mm,5?m?C₁₈ column,with the gradient elution program of mobile phase:H₂O?A?-CH₃OH?B?,gradient 0¹0 min,15%?35%B;10³0 min,35%?45%B;30⁶0 min,45%?60%B,flow rate of 1.0 mL·min^-1,and the detective wavelength was 221 nm.Our results showed that using methanol-water as mobile phase for gradient elution,the three components were separated completely without interference.The calibration curve of bruceoside B was linear in the range of 0.722-2.166?g with the correlation of 0.9999;The calibration curve of bruceoside A was linear in the range of2.074-6.222?g with the correlation of 0.9999 and the calibration curve of brusatol was linear in the range of 0.503-1.509?g with the correlation of 0.9999.The average recovery of bruceoside B?bruceoside A and brusatol were 96.5%,98.4%and 103.2%,with the RSD of 3.1%,2.5%,2.9%?n=5?,respectively.The determination results of twelve batch samples showed that the method was accurate,simple and convenient,and could be used in the quantitative determination of bruceoside B?bruceoside A and brusatol in Bruceae Fructus.In summary,the established DNA identification method can be used for plant source identification of Brucea javanica;the established fingerprint and content determination can provide scientific basis for the authentication and quality control of the of Bruceae Fructus.