| Inconsistent quality of Chinese medicinal materials is currently one of the major problems in the development of traditional Chinese medicine. A major cause of this problem is the same medicine has varied sources. For a long time, though several kinds of plants from the same genus had been used as one kind of crude drug, its identification and quality control are not satisfactory.As a traditional Chinese medicine, chaihu (Radix Bupleuri), at the earliest, was ranked the top grade in the "Shen Nong’s Herbal Classic". It was a medicine of deficient heat clearing which was used for influenza, fever, alternate chills and fever, malaria, stagnation of liver-Qi, chest rib swelling, anal prolapse, uterus prolapse, menoxenia, and so on. Its officially authorized origins in2010Chinese Pharmacopoeia are Bupleurum chinense and B.scorzonerifolium. According to the past statistical records, the genus Bupleurum represents42species,17varieties and7forma in China. But most of the plants since ancient times had been utilized as Radix bupleuri. In addition, citizens in some regions sell the plants of Bupleurum theirselves according to the offerings and historical usage habits. However, the phytomorph and microscopic characteristics of Bupleurum medicinal plants are fairly approximate, and the chemical constituents contained in plants would change a lot in the process of growth and harvesting. So, there is a considerable difficulty in distinguishing these species with traditional identification methods. At present,5or6kinds of local Bupleurum plants were jumbled together to be used, even including toxic (B.longiradiatum) or little active constituents contained materials (B.tenue). This lead to confusion in the market and uneven quality of the drug. Therefore, it is necessary to establish a reliable and effective identification method for improving the quality standard for Radix Bupleuri.Metabolomics, a new science and technology developed in the mid of1990s (after genomics, transcriptomics and proteomics) was an indispensable basic disciplines for systems biology research. It refers to a holistic analytical approach to all the low molecular weight metabolites in an organism or cells. As an important approach of metabolomics,(ultra) high performance liquid chromatography tandem mass spectrometry provide an analysis platform on small molecules with strong separation and analysis of chemical composition in organism. Especially with the help of electrospray ion source chromatography mass spectrometry analyzer, the molecular ion fragments can be accurately detected and identified. The generated metabolic spectrum would more accurately reflect the biological system state compare to other analytical methods. There will benefit for quality identification of crude drug, as well as establishing a systematic standardized chemical composition database (like Radix Bupleuri).Principal component analysis is a kind of non-supervised multivariate statistical method which was designed to transform multi-index into several comprehensive index using dimension reduction. As a high-dimensional data analysis method, PCA focuses on discovering the inner relationship between exterior characterization of chemical data and internal composition of materials. It is a commonly used and important analysis method for metabolomics which can be dealt with abundant organisms metabolic information (Bupleurum or other multi-source herbs) from mass spectrometry analytical instruments.Objective:To make a detailed investigation of natural resource and medicinal situation of Bupleurum and to raise a strategy which the crude drugs of Radix Bupleuri cultivars would attain an accurate identification and reasonable exploitation. To establish an accurate, fast and reliable identification and quality evaluation system by means of principal component analysis on LC-MS data of Bupleurum so as to provide a reasonable study basis for expansion of new drug source from different Bupleurum species.Methods:1. Geographical distribution of Bupleurum medicinal plants were confirmed based on the documentary guidance. Several-10groups of specimens of more than10origins were intended to collect for widely distributed species such as Bupleurum chinense. But species of limited distribution were intended to collect as many groups as possible. In addition, take full advantage of collections from various herbaria to ensure those Bupleurum samples would be comprehensive and representative. Along with accurate classification and identification, samples information of origins and species should be summed up before unified store in a cool dry place.2. Metabolic spectrum analysis of Bupleurum plants were carried out by means of ultra-high performance liquid chromatography tandem electrospray ionization mass spectrometry (UPLC-ESI-MS). Device parameters such as chromatographic column temperature, injection quantity, mobile phase, velocity, sample preparation would be successively inspected.3. Metabolic spectrum data of both above and below ground parts of Bupleurum plants were acquired under the best sifted experimental conditions. The content of saikosaponin A, C, D was respectively assayed. The samples quality of different species and origins were exclusive determined by PCA with SIMCA-P.4. Metabolic spectrum data of the samples were collected and summed up to establish a metabolomics database of Bupleurum medicianl plants by software of VC++and SQL Server2005.Results:1. There were a total of213specimens which belonging to19species,7varieties and4forma were collected during the resources survey. Survey result shows that over60%of the whole Bupleurum resources reserves were mainly distributed in china’s northeast, north and the northwest, and most of them were Bupleurum chinense, B.scorzonerifolium and B.yinchowense.2. Due to the different customs, Bupleurum medicinal plants were mostly conventional varieties. Because of impure provenance or mixed varieties planted in different region, there were still several cultivars grow in cultivated land. At present, the yield of cultivation production has far more than the wild products. Coupled with a concentrated single species planting for a long time, germplasm degradation and resistance reduction came out. As a result, most of the quality of the materials from different regions was uneven.3. As a commodity, no matter the source was wild or cultivated, different Bupleurum medicinal plants using a same name during buying and selling process. In addition, Bupleurum herbs of the same species often have several different trade names in some regions. So, there was a homonym or synonym confusion which the same base source of plants classified in different parts of goods and the same product name may include a different source plants. As a result, the name confuses the variety.4. The sample extraction condition was20times5%ammonia methanol extract for five times,30minutes each time. The total extraction rate of saikosaponin A and D in demonstration test with five parallel samples were0.5176%,0.5144%,0.5181%,0.5152%,0.5167%, an average of0.5168%, RSD is0.409%. The result shows that the optimized extraction condition is feasibility and reproducibility.5. Assay of saikosaponin A, C, D, and other ingredients contained in Bupleurum medicinal plants by the analysis method of UPLC-ESI-Q-TOF-MS is rapid, accurate, sensitive, reliable and specific. Linear regression equation of peak area corresponding the injection volume of saikosaponin A, saikosaponin C, saikosaponin D, respectively was Y=3.144X+37.08, r=0.9994; Y=18.40X+39.18, r=0.9989; Y=3.225X+161.8, r=0.9989. The result shows that saikosaponin A in the range of4.860μg-14.580μg, saikosaponin C in the range of0.3135-3.1350μg, saikosaponin D in the range of2.170μg-10.850μg respectively has a good linear relationship. The content of saikosaponin A in the samples was ranged from0.0452%-0.3963%, the content of saikosaponin C was ranged from0.0096%-0.1945%, the content of saikosaponin D was ranged from0.1082%-0.3673%.6. Two principal components were determined by the software of SIMCA-P during the analysis. By the help of PCA, similarities and differences of chemical composition in samples can be discriminated. Samples were able to classify according to the difference of chemical composition and species. A correlated relationship was initially performed among Bupleurum medicinal materials quality with variety and origin.7. A proprietary database of chemical composition of Bupleurum medicinal plants with over300spectrums was established to provide a reference for future research work.Conclusion:1. As a kind of traditional herbs, Bupleurum medicinal plants were widely distributed in the north, northwest northeast of China. Although developments of Bupleurum medicinal plants cultivation, to a certain extent alleviate the inadequate supply of Radix Bupleuri in the market. However, wild resources of medicinal plants had live through a variety of disasters and adverse environmental choice. It is a valuable species gene pool should be protected which the cultivars didn’t possess or had disappeared.2. According to a comprehensive consider premise on the yield and quality of Radix Bupleuri, a standardized management and efficient planting technology of medicinal materials cultivation should be put into practice as early as possible for suitable Bupleurum medicinal plants, so as to improve the quality of the crude drug from the beginning.3. UPLC-ESI-Q-TOF-MS offers a more comprehensive approach for chemical composition of multi-source medicinal plants of Bupleurum. Meanwhile, the analysis approach also is an alternative method of medicinal materials quality evaluation and identification, which pays more attention to the quality differences related to different varieties and origins.4. With the help of chemical composition database of Bupleurum medicinal plants, research data would be fully utilized to promote a standardized management on cultivation, processing, sales and modernization process of Radix Bupleuri. |
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