Quality Evaluation Study Of Stemona Tuberosa Lour.

Derived from Stemona tuberosa Lour. (Genus:Stemona; Family:Stemonaceae), which is mainly distributed in Guangxi Zhuang Autonomous Region, Yunnan, Guizhou, Hunan and Guangdong provinces, Stemona Tuberosae Radix is one of the three sources of Stemonae Radix as recorded in the Pharmacopoeia of the People's Republic of China (Edition 2005 and 2010). It is often used clinically; it can relieve cough orally and kill parasites extenally and has a wide range of application. This paper focused on the following aspects:herbalogical study of Stemonae Radix; resource and commodity investigation, pharmacognostical identification and quality evaluation as well as corelation between fingerprint spectrum characteristics and cough relieving index of total alkaloids from S. tuberosa.1. Herbalogical study Stemona Radix was originally written down by the name of "Baibugen". Historically, "Baibu" and "Baibugen" were often used identically or almost identically. The two names were based on the quantity and arrangement of tuberous roots, and this was the same in all related books; other names were all recorded as aliases; "Baibing" is not the alias of "Baibu". In herbal medicine books, the source of Stemona Radix was chiefly from Stemona sessilifolia Miq., Stemona tuberosa Lour., Stemona japonica (Bl.) Miq and plants in the genus Asparagus of the family Liliaceae. Now, the crude drug is primarily derived from S.sessilifolia, S. tuberosa, S. japonica. Stemonae Radix is produced in areas located in the south of Qinling Mountains, Stemona Tuberosae Radix has a production area more southward than that of Stemona Sessilifoliae Radix, and Guangxi and its neighbouring provinces are the main production areas of Stemona Tuberosae Radix.2. Resource and commodity investigation By looking up relevant documents and specimens, consulting relevant specialists and conducting field investigation, we acquired the information about the distribution and the chief production areas of S. tuberosa in China and Guangxi. All wild, Generally it was found in remote mountains and the species is primarily distributed in the area on the south of the Yangtze River valley. Global positioning system (GPS) showed that it grows between 35～1577 meters above see level. By and large Stemona Tuberosae Radix is the commercial crude drug of Stemonae Radix in Guangxi and its neighbouring provinces and has a high production in these areas; in Guangxi, Stemona Tuberosae Radix is chiefly produced in Baise, Hechi and Guilin, with some imported from Viet Nam. Stemonae Radix was sold in all big crude drug markets, Stemona Tuberosae Radix being in the majority.3. Pharmacognostical identification (1) Character identification Possessing a relatively big stele, the root exhibited shallow longitudinal wrinkles or irregular longitudinal grooves. With slightly expanded nodes, the subround stem was sunken on both sides. The broadly ovate leaf comprised 7-15 veins.(2) Microscopic identification The velamen consisted of 3 layers of cells, the first layer being thickened; in the stele, there were 25-30 strands of xylems and phloems arranged alternately; The pith was relatively wide. The stem comprised amphivasal vascular bundles which alternated with each other and formed a ring. The leaf contained in the main veins nearly elliptical vascular bundles, which were surrounded by annular sheaths; cylindrical crystals were scattered in the mesophyll; in the leaf powder, stomata were anomocytic, vessels were mostly spiral, and fibers were long-spindle-shaped.(3) Physicochemical identification In the bismuth potassium iodide and silicotungstic acid tests, all the root, stem and leaf of the crude drug showed a positive response.All the results above provided a basis for identifying the root, stem and leaf of S. tuberosa. Moreover, the vascular bundles in the stem of S. tuberosa were amphivasal.4. Systematic preliminary test and preparation of oxotuberostemonine(1) Systematic preliminary test The result showed that the root, stem and leaf contained almost the same chemical composition, including amino acids, polypeptides, proteins, saccharides, polysaccharides, glycosides, organic acids, phenols, coumarins, lactones, alkaloid, etc.(2) Preparation of oxotuberostemonine No tuberostemonine was detected from Stemona Tuberosae Radix collected from Guangxi and its neighbouring provinces. With high performance liquid chromatography (HPLC), we isolated from the total alkaloids one common compound with high content, which was deduced to be oxotuberostemonine using EI-MS,13C NMR,1H NMR and other methods.5. Quality evaluation(1) Content determination of total alkaloids Using acid dye colorimetry, the content of total alkaloids in 16 batches of Stemona Tuberosae Radix samples was determined; it was 6.56～23.56 mg/g, with an average value of 15.41 mg/g, indicating that there was much variation among the quality of Stemona Tuberosae Radix collected from different production areas. Among all the crude drugs, those from Shangsi and Fengshan of Guangxi and Xinyi of Guangdong had a relatively high content of total alkaloids. This method was simple, rapid and sensitive.(2) Content determination of oxotuberostemonine With ultrasonical extraction by methanol and HPLC, the content of oxotuberostemoninein 16 batches of crude drug samples was determined; it varied much, ranging from 0.69～5.83 mg/g (that of Daxin Guangxi was 5.83 mg/g, Jingxi Guangxi 4.89 mg/g and Gongcheng Guangxi 4.24 mg/g) and the average being 2.40mg/g. This method was simple and accurate and should be considered to be the approach of evaluating the quality of Stemona Tuberosae Radix.(3) TLC A initial TLC method for identifying Stemona Tuberosae Radix using oxotuberostemonine as the reference substance was established. In this method, total alkaloids were isolated. The optimal development system was chloroform:methanol (20:1), the colour developing reagent was modified bismuth potassium iodide reagent. As a result of the TLC test, red brown spots in the chromatogram obtained with the test solution of 16 batches of crude drug samples corresponded in position and colour to the spots in the chromatogram obtained with the reference solution. This method was simple and rapid and had a relatively high specificity for Stemona Tuberosae Radix. In addition, TLC results revealed that there were to a certain degree differences in the spots among samples and in the intensity of corresponding spots, suggesting that obvious variation existed in the chemical composition of different individuals of Stemona Tuberosae Radix.(4) Tests①The average content of water-soluble extracts of individual samples showed a maximum of 80.89% and a minimum of 53.41%, both of which were higher than that in the Pharmacopoeia of the People's Republic of China which stipulates that the content of water-soluble extracts in Stemona Tuberosae Radix is not lower than 50.0%; the total average content was 68.51%.②The total averages of water content, total ash content and acid-insoluble ash content are respectively 7.85%,5.82% and 0.92%. For the maximum average and the minimum average of the individual samples, water content was 11.25% and 5.58%; the total ash content 10.55% and 3.61%; acid-insoluble ash content 2.48% and 0.36%. From above, we could see that for individual samples, the maximum average content was at least one time higher than the minimum average content, suggesting that there was much variation in the quality of Stemona Tuberosae Radix from different production areas.6. Corelation between fingerprint spectrum characteristics and cough relieving index of total alkaloids(1) Establishment of fingerprint spectra HPLC fingerprint spectra of the total alkaloids of Stemona Tuberosae Radix from 16 production areas were established using the chromatographic peak of oxotuberostemonine as the reference peak:The detection wavelength was 230 nm, the eluant was acetonitrile:0.1% water solution of triethylamine, the spectrum was recorded within 85 minutes. Fingerprint spectra varied significantly among different individuals of Stemona Tuberosae Radix which were collected from 16 areas, and only 6 common characteristic peaks were marked,6 out of 16 production areas had a similarity of 0.8 above.(2) Cluster analysis By the use of cluster analysis in SPSS 16.0, we classified the HPLC fingerprint spectra of the total alkaloids of Stemona Tuberosae Radix total alkaloids with peak area as the measure. As a result,16 batches of samples were classified into 4 classes, the differences of which reflected the differences of their distances and habitats.(3) Cough relieving effect Adopting the sequential method, we studied the relieving effect of the total alkaloids on cough induced by ammonia water. The results indicated that total alkaloids of Stemona Tuberosae Radix from different production areas could prolong the time after which ammonia water-induced cough occurred (R>100%); such effects differred notably among them.(4) Corelation between of fingerprint spectrum characteristics and cough relieving effects After getting fingerprint spectra and quantitive data of cough relieving effects of total alkaloids from Stemona Tuberosae Radix, we adopted gray correlation analysis as the method and began to determine the contribution of every chemical ingredient represented by the fingerprint spectrum peak to cough relieving effects. As a result, the relational degree ranged at 0.53～0.84, indicating that all chemical ingredients contributed almost the same to the effects. We deduced that the cough relieving effects were due to the "joint-effort" of every chemical ingredient of total alkaloids; the contribution of common characteristic peaks including oxotuberostemonine to cough relieving effects is very prominent.7. Correlation analysis of quality assessment indexes We studied the correlation of some quality assessment indexes and found that there were differences among the crude drug samples in the variation trend (ascending or descending) of each quality assessment index. We thought that it was not the case that the medicinal effect of the crude drugs became greater as the content of their water-soluble extracts, total alkaloids or oxotuberostemonine got higher; instead, it should be that the medicinal effect was due to the "joint effort" of many ingredients and some unknown highly active component.8. Quality standard draft and drafting instructions Based on the results of this study, combined the stipulation under Stemonae Radix in the Pharmacopoeia of the People's Republic of China (Edition 2010), the initial quality standard draft and the drafting instructions were established.