Study On The Chemical Constituents Of Heterosmilacis Japonicae Rhizoma And Authentication With Its Two Kinds Of Closely Related Species Crude Drugs

Tubixie(Heterosmilacis Japonicae Rhizoma)originates from the rhizoma of Heterosmilax japonica Kunth.(Liliaceae)which has the effects of “disintoxicating” and “dispelling wind-damp”.And its closely related species crude drugs including Tufuling and Baqia.Tufuling(Smilacis Glabrae Rhizoma)originates from the rhizoma of Smilax glabra Roxb.Baqia(Smilacis Chinae Rhizoma)originates from the rhizoma of Smilax china L.Tubixie has been widely used in South China area.But Tubixie is easy confused with Tufuling and Baqia.As we all known,there have been a lot of researches about Tufuling and Baqia,the research of Tubixie was more less than them,so the medicinal value needs further study.Sometimes Tubixie is also called Tufuling in the folklore for the character of Tubixie is similar with to Tufuling.People take Baqia act as Tubixie and Tufuling because it is easy to be got.In the above case,studies on chemical constituents of Tubixie is particularly important.At the same time,it is difficult to solve the practical problems in the production of Chinese herbal medicines if the lack of stronger specific comparative authentication aimed to active and index component.The paper shows the n-butanol fractions of Tubixie were studied firstly,and then the comparative authentication was carried out in Tubixie and other two by using UV Spectrophotometry,TLC,HPLC and DNA barcoding.The results are as follow:(1)Four compounds were separated from the n-butanol fractions of Tubixie through various column chromatographic technique.The four compounds respectively are(R)-Methyl-4-(2,4-dihydroxy-3,3-dimethylbutanamido)butanoate(1),(3S)-1,2,3,4-tetrahydro-?-carboline-3-carboxylic acid(2),Ethyl-?-D-xylobioside(3)and daucosterol(4).Compound 1 is a new natural product,compound 2 and compound 3 were isolated from the plant for the first time.(2)Using rutin as standard substance,total flavonoids content of Tufuling,Baqia and Tubixie were determined to be 54.57 mg/g,30.52 mg/g and 3.75 mg/g respectively by spectrophotometric method.Using dioscin as standard substance,total saponins content of Baqia,Tufuling and Tubixie were determined to be 1.97 mg/g,1.00 mg/g and 0.54 mg/g respectively by spectrophotometric method.In general,compared with the other two kinds of crude drugs,the content of Tubixie in both total flavonoids and total saponins was the lowest.This study provides an effective and accurate method for identification of Tubixie,Tufuling and Baqia.Tubixie,Tufuling and Baqia were respectively identified qualitatively by TLC,and the content of astilbin and resveratrol were determined by HPLC.Three kinds of Chinese herbal medicines can be identified by TLC.Astilbin was found in Tufuling.Resveratrol was detected in Tufuling and Baqia.However,the above of two chemical components were not detected in Tubixie.The contents of astilbin and resveratrol come from the three kinds of Chinese herbal medicines were quantitied by HPLC further.The average contents of astilbin and resveratrol in Tufuling were 13.28 mg/g and 0.17 mg/g respectively,The average contents of astilbin and resveratrol in Baqia were 1.62 mg/g and 0.64 mg/g respectively,same as above,Tubixie was not detected these two compounds.According to the above conditions,the three kinds of crude drugs can be distinguished correctly.(3)DNA barcording based on ITS2 sequence was established in order to identify Tubixie,Tufuling and Baqia.NJ tree shows that they can be distinguished from molecular level obviously.Besides,this molecular biological methods provided an effective method for authentication of Tubixie and its adulterants,which is proved to be an effective complement method for traditional pharmacognostic authentication.In order to solve the authenticity of Tubixie in actual purchase,production and use,the method should be explored to practice.