Comparative Study On The Quality Of Bupleurum In Northwest Hubei Province With Bupleurum Chinese DC

ObjectiveStudy on the quality evaluation and germplasm resources of Bupleurum in Northwest of Hubei Province, provided experimental and theoretical basis for the development and utilization of the Bupleurum in Northwest of Hubei ProvinceMethods1. Study on the resource of Bupleurum in Northwest of Hubei Province1.1Investigation on Bupleurum resources in northwest area of Hubei ProvinceThrough investigate the resources of the wild and cultivated bupleurum in northwest area of Hubei Province, acquisited and producted twenty plant specimens of bupleurum in northwest of HuBei province, compared and identified the difference of the root, stem, leaf, flower, fruit, seed and plant, to establish the retrieval table of plant classification, provide the basis for the identi of plant properties.1.2Identification of Bupleurum marginatum Wall. ex DC. in northwest area of Hubei ProvinceCompared the root and seed of Bupleurum marginatum Wall. ex DC., Bupleurum chinese DC. and Bupleurum Falcatum Linne.by paraffin method. Observed the microstructure of the roots and identif icated the microscopic characteristics. Identificated the species of Bupleurum margina tum Wall. ex DC. and Bupleurum chinese DC. by foam reaction and TLC.1.3Study on the kinship of Bupleurum marginatum Wall. ex DC. in northwest of HuBei province by ISSR molecular marker technology Analysised genetic diversity of Bupleurum marginatum Wall. ex DC. in northwest of HuBei by ISSR molecular marker technology and established the genetic diversity fingerprint, analysis the main difference and genetic relationship of Bupleurum marginatum Wall. ex DC. in northwest of HuBei at molecular biology1.4Study on the key technology of home-grown research of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province Research on the growth phenology of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province, formulated the quality standards of the Seed of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province; and observed the relationship between the temperature, culture media and seed germination rate. Study the best harvest time of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province2. The quality evaluation of Bupleurum marginatum Wall. ex DC. in Northwest of Hubei Province2.1Compared the Chemical compositions between Bupleurum marginatum Wall. ex DC. in northwest of Hubei province and Bupleurum chinese DC, establish the characteristic spectrum of Bupleurum marginatum Wall. ex DC. in northwest of Hubei provinceDetected the content of the SSa、SSd in Bupleurum by HPLC, Compared the content of the SSt between Bupleurum margina tum Wall. ex DC. and Bupleurum chinese DC.. Detected soluble polysaccharide and polysaccharides of Bupleurum through the Anthrone-sulfuric acid colorimetric,compared the total polysaccharide content. Measured the absorbance by visible spectrophotometry, calculated the content of total flavonoid. Measured the content of the volatile oil of the Bupleurum by UV-visible spectrophotometry, and determinated the ash. Detected the volatile oil and total saponins of the Bupleurum by UV-visible spectrophotometry,which were made with Honey wine vinegar before and after. Compared the active ingredient of Bupleurum by the different concoction methods. Established the fingerprint of Bupleurum margina tum Wall. ex DC. in northwest of Hubei province by HPLC. Isolated and identif icated the constituents of Bupleurum compounds by UPLC-MS, established the characteristic spectrum of Bupleurum marginatum Wall. ex DC. in the northwest of Hubei province,and comparing the characteristic spectrum peaks relative peak area.2.2Compared the pharmacological activity between between Bupleurum margina tum Wall. ex DC. in the northwest of Hubei province and Bupleurum chinenseThe antipyretic effects of Bupleurum decoction were monitored at the2,4-dinitrophenol-induced rat pyrexia. The analgesic effect was verified by the test of HAc-induced writhing response in mice and Hot-Plate. The anti-inflammatory of Bupleurum decoction were monitored by the test of ear and foot-swelling in rats. Induced Liver injury in rats with peanut oil of tetrachloride nitrogen, detected ALT, AST, ALP of Serum, and SOD, MDA, GSH of liver tissue, observed the Histopathology and immunohistochemical indicators of liver tissue, compared the anti-inflammatory and hepatoprotective effects between Bupleurum marginatum Wall. ex DC. in northwest of Hubei province and Bupleurum chinese DC..Result1. Study on resource of Bupleurum in Northwest of Hubei Province1.1Investigation on Bupleurum resources in northwest area of Hubei ProvinceThe result show that:the Bupleurum around the region,known as the Bupleurum chinense DC,had been identified as the dried roots of Bupleurum margina tum Wall. ex DC. of Umbelliferae, a small amount of Bupleurum had been identified as the dried roots of Silene fortunei Vis.1.2Identification of Bupleurum marginatum Wall. ex DC. in northwest area of Hubei ProvinceThe characteristic of Bupleurum marginatum Wall. ex DC. and Bupleurum chinense DC. as follows:Bupleurum margina tum Wall. ex Dc.: The small bracts narrow and small, lanceolate; Leaf blade thick, nearly leathery, margin conspicuously white-cartilaginous; Root thick, spindle, the surface usually brown or dark brown; Stem base distortion, few branched, upper Leaves long-lanceolate to linear, green. Bupleurum chinese DC.:Leaves thin, no margin conspicuously white-cartilaginous; Root stout, elongate, brown, woody, usually branched; Stem solitary or several, dichotomously much-branched bove, rod abends into zigzag. Small total bracts thick, three veins, margin not transparent; Small umbellules much and small. Under the ultraviolet visible light, the result of TLC showed that different kinds of bupleurum under different growth conditions have the same color spots in the same position at TLC chromatogram controled with control medicinal materials and Bupleurum saponins reference substance.1.3Study on the kinship of Bupleuruw marginatum Wall. ex DC. in northwest of HuBei province by ISSR molecular marker technology The results showed that7ISSR primers amplified115ISSR stripes,12samples of Bupleurum margina tum Wall, ex DC. amplified81ISSR bands, there were42polymorphic bands, the rate of polymorphic was51.9%. Twenty samples of Bupleurum marginatum Wall. ex DC. were clustered into one group, two samples of Bupleurum chinese DC. were clustered into another group, the coefficient of similarity was0.55, with close phylogenetic relationship.1.4Study on the key technology of home-grown research of Bupleurum margina tum Wall. ex DC. in northwest of Hubei provinceThe germination rate of Bupleurum marginatum Wall. ex DC. could increase to64.8%at40℃water temperatrue; the germination medium have no significant influence on the germination rate of Bupleurum margina tum Wall. ex DC., but the germination rate of Bupleurum margina tum Wall. ex DC. significantly descended above30℃. The content of the saikosaponin show an increasing trend from May to July, arrived the peak at july, then show an downward trend, meet the minimum at October, the result show that Bupleurum marginatum Wall, ex DC. should be harvested before it wither up.2. The quality evaluation of Bupleurum marginatum Wall. ex DC. in Northwest of Hubei Province2.1Compared the Chemical compositions between Bupleurum marginatum Wall. ex DC. in northwest of Hubei province and Bupleurum chinese DC., establish the characteristic spectrum of Bupleurum marginatum Wall. ex DC. in northwest of Hubei provinceThe content of SSt are similar between wild and, less than wild bupleurum in Henan and Gansu, much higher than Bupleurum chinese DC. of Shanxi and Mishima Shibako of Hubei, the results show that the quality of Bupleurum marginatum Wall. ex DC. of the northwest of Hubei province is good.Bupleurum marginatum Wall. ex DC. in the Neixiang County of Henan province and zhigui County of Hebei province have more total saponins, the content of total saponins is similar between Bupleurum chinese DC. in Yunchen County of Shanxi province and Bupleurum margina tum Wall. ex DC. in Fangxian and Baokang County of Hubei province, the results show that growth environment is the important factor for the content of total saponins.The results show that the content of polysaccharide of the cultivated Bupleurum marginatum Wall. ex DC. in northwest of Hubei province is slightly lower than the wild Bupleurum, the content of polysaccharide of Bupleurum overground part also contains a certain amount of polysaccharide, polysaccharide content is high in the Mishima Shibako of Nanzhang County of Hubei province and cultivated Bupleurum of Yunchen County of Shanxi. the results show that the content of polysaccharide were great difference in the different growth conditions, different varieties and different position of Bupleurum.The content of flavonoids in the root of wild Bupleurum marginatum Wall. ex DC. in northwest of Hubei is higher than the cultivated Bupleurum. aboveground parts is higher than is higher than that of underground parts.The content of flavonoids of Bupleurum in the savage Valley town of Nanzhang County of Hubei province is higher than Yunchen County of Shanxi province, Ganshu province and Nanzhang County of Hubei province. The results showed that the content of total flavonoid of the ground part of Bupleurum was significantly higher than that of root, so as to choose the right medicinal parts according to the selection of appropriate pharmacological effects.The content of volatile oil of Bupleurum marginaturn Wall, ex DC. is higher than Bupleurum Chinese DC. and Mishima Shibako, the content of volatile oil of Bupleurum margina turn Wall, ex DC. in northwest of Hubei province is higher than the wild Bupleurum. The results show that it is suitable for implementation of wild Bupleurum sibiricum redoute.The content of volatile oil was higher after processing, from high to low is honey, wine, vinegar. The contents of total saponin of Bupleurum from high to low is honey, Radix. vinegar, wine. The single factor variance analysis by SPSS16.0showed that, Radix Bupleuri and vinegar products, The contents of total saponin of radix Bupleuri, vinegar products and wine products have significant difference (P<0.05), other groups had extremely significant difference (P<0.01). The results show that, before and after processing. the effectiveness of taste changed is due to the conversion of chemical composition changed, that is consistent with the theory of traditional Chinese medicine processing purpose. There were8good separation of common peak in fingerprints spectrum, the difference of the fingerprint similarity was small between Bupleurum Chinese DC. and Bupleurum scorzonerifolium willd. the difference of the fingerprint similarity was big between aboveground parts of Bupleurum marginatum Wall, ex DC. and Bupleurum falea tum L.Separated and identified7saponins according to the information by Mass, and as the fingerprint peak of bupleurum, there are significant differences between the relative peak area of characteristic peak of each sample, the content of saikosaponin A, D are also different.2.2Compared the pharmacological activity between between Bupleurum margina tum Wall. ex DC. in northwest of Hubei province and Bupleurum chinenseThe result Show that the decoction of Bupleurum chinese DC. and Bupleurum marginatum Wall. ex DC. all showed significant antipyretic effect, and there were no significant difference between them. Both the decoction and alcohol extract of Bupleurum chinense DC. and Bupleurum marginatum Wall. ex DC. possessed the analgesic effect in the test of HAc-induced writhing response, and there were no significant differences. The decoction of Bupleurum chinense DC. and Bupleurum marginatum Wall. ex DC. all showed the analgesic effect in the test of Hot-Plate, and there were no significant differences, but alcohol extract of them had no significant analgesic effect.Bupleurum chinese DC. and Bupleurum marginatum Wall. ex DC. all showed significant anti-inflammatory effect in the experiment of ear swelling,and there were no significant difference between them. Bupleurum chinese DC. and Bupleurum marginatum Wall. ex DC. all showed significant anti-inflammatory function at2h,4h,6h in the test of foot swelling experiment, and there were no significant difference between them; Bupleurum marginatum Wall. ex DC.5.0g/kg dose group and Bupleurum chinese DC.10.0g/kg dose groups had significant difference compared with model group at the test of ALT,AST. All the treatment group had no significant difference compared with model group at the test of ALP. The decoction of Bupleurum chinese DC. and Bupleurum marginatum Wall, ex DC. all groups had significant difference compared with model group at the test of SOD. Bupleurum marginatum Wall, ex DC.10. Og/kg dose group and Bupleurum chinese DC.10.0g/kg dose groups had significant difference compared with model group at the test of MDA,GSH. The result of the Histopathology and immunohistochemical indicators of liver tissue Showed that the decoction of Bupleurum chinese DC. and Bupleurum marginatum all showed significant hepatoprotective effect, and there were no significant difference between them.Conclusion(1) Bupleurum marginatum Wall, ex DC. was the main kind of bupleurum in northwest of HuBei province, bupleurum in northwest of HuBei province, known as the " Bupleurum chinense DC.", and sold all over the country were the dry root of bupleurum marginatum in fact.(2) The biodiversity and germplasm resources of Bupleurum marginatum Wall, ex DC. in northwest of Hubei Province is abundant, it is conducive to the development of production of Bupleurum marginatum Wall. ex DC.. The ISSR molecules technology can significantly distinguish Bupleurum marginatum Wall. ex DC. from its sibling species, it also can be used as the molecular characteristics of the species identification.(3) Seeds contained germination inhibitors and embryo after-ripening are the important factors to affect the germination rate of Bupleurum seeds.(4) Establish the fingerprint spectrum and quality evaluation system of Bupleurum marginatum Wall. ex DC. in northwest of Hubei province. The fingerprint of Bupleurum marginatum Wall. ex DC. can be used as the basis of the overall quality establish system. and establish the "identity card" for bupleurum, to ensure safety and controllable of clinical medication, provide theoretical basis for the modernization of traditional Chinese medicine.(5) Analysis a large number of wild and cultivated Bupleurum marginatum Wall. ex DC., compared with other samples of bupleurum chinense DC, which is recorded in the Chinese Pharmacopoeia, founded that the content of the effective components were similar or more than the bupleurum chinenseDC., which meet the standard of the Chinese Pharmacopoeia, or higher than that. The activity effect of antipyretic, analgesic, anti-inflammatory, and hepatoprotective were also close to or more-than the bupleurum chinense DC..Furthely, it was confirmed by ISSR that Bupleurum marginatum Wall, ex DC. and bupleurum chinense DC. were two species separately,but the relationship of them was closely. The root of Bupleurum marginatum Wall, ex DC. was used as bupleurum chinense in the history for long time,which was one of the mainstream commodity of three major bupleurum, Bupleurum marginatum Wall. ex DC. in northwest of Hubei Province has been sold all the country considered as the high quality of bupleurum chinense DC.. On the basis of the above, bupleurum chinense DC. is one of the medicinal plant resources of the high quality of bupleurum. the root of Bupleurum was specified as the original plant in Chinese Pharmacopoeia, therefore, we suggested to restore the original plant status of Bupleurum marginatum Wall. ex DC. in Chinese Pharmacopoeia, or add the Bupleurum marginatum Wall. ex DC. into Pharmacopoeia independently, in order to make full use of this valuable resource.