| Humulus scandens (Lour.) Merr. is originated from dried aerial parts of H. scandens (Lour.) Merr. in the mulberry family, it has hot alexipharmic effect and dieresis, it can be used to be in the treatment of cough with lung heat、lung cancer、 tuberculosis、deficiency-heat and polydipsia、heat stranguria、edema、urine negative、 wet heat durchfall、hot toxin sore、irritating skin ect. Based on the previous study of Research on quality standards of Herba Humulus Scandens and the active component for the inhibition on Mycobacterium tuberculosisin, the thesis contrasted the quality of Humulus scandens (Lour.) Merr. from different habitats, studied the dynamic change of active ingredient of Humulus scandens (Lour.) Merr. in different growth periods, provided references to improve and perfect further quality standards of Herba Humulus Scandens, as well as choose optimum harvesting time. Main contents and results are as follows:1This is the first detailed studied on dry weight accumulation and the dynamic change of active ingredient of Humulus scandens (Lour.) Merr. in different growth periods, and applied the PCA method, made comprehensively analyzes on the dynamic change of active ingredient of Humulus scandens (Lour.) Merr., provided references to determine optimum harvesting time.Two different regions, different harvest time in a growth cycle accumulation of dry matter of humulus scandens and extract and flavones, mignonette, and apigenin-7-O-β-D-glucosidase were dynamically determined, the results show that, there was greater difference on dry matter of humulus scandens and extract and flavones, mignonette, and apigenin-7-O-β-D-glucosidase content from different districts varied, the differences of contents of four components in the same region of fifferent harvest time was significant, through applied the PCA method, made comprehensively analyzes on the dynamic change of active ingredient of Humulus scandens (Lour.) Merr., we found that the principal component factor score higher in February in Sichuan Chengdu area, the principal component factor score higher in August in Hebei Tangshan area, it showed that effective composition dynamic accumulation in different regions had differences, this may be related to the two regions belong to the south and north,geographical location, environment,climate and other natural conditions differ greatly, prompt its harvest time determine origin factors should be considered.2. For the first time, the comparative of medicinal properties of humulus scandens in different origins were studied, found out its appearance on the similarities and differences.Based on the careful observation of the appearance of humulus scandens from different regions, founed its traits are different, mainly reflected in color, bristles and glandular dots on the surface of the leaf, etc:most of the samples wrapped clumps, a few samples had been cut into pieces; most of the samples are yellow-green, a few are sandy or brown; leaf surface differences are more apparent, mainly are the upper and lower surface bristles, there are differences in the density of glandular dots; take the fruits of samples, the fruits color and markings on the surface are different.3. The mignonette, and apigenin-7-O-β-D-glucosidase TLC identification of humulus scandens in different origins were studied.3.1According to the2010edition of "standard of Chinese medicinal materials in sichuan province" of mignonette thin layer identification method, the mignonette element for32batches samples were identified by TLC. The results show that,N0.3(Zhejiang Xianju)、NO.4(Guangdong Shaoguan)、NO.7(Gansu Dangchang)、 NO.8(Jiangsu Lianyungang)、NO.11(Hunan Changsha)、NO.16(Sichuan Yibin)、 NO.21(Shandong Qingdao)、NO.23(Hebei Tangshan、NO.24(Jiangxi Nanchang)、 NO.25(Jiangxi Ji an)、NO.28(Beijing Fengtai area)、NO.29(Chongqing Nanchuan)、 NO.30(Shanghai Chongmingdao)、NO.31(Anhui Bozhou medicine market).. NO.32(Guangxi Yulin medicine market), a total of15samples, a same yellow-green color spots in the TLC graph of sample existed at the corresponding positions compared with the mignonette control, but the size and the strength of the fluorescent spots are different, other samples not detected mignonette, tip on the existence and quantities of the composition of humulus scandens in different origins are different.3.2Through the quantity of sample pretreatment, system and points, distance, deployment humidity, temperature and the thin layer plate saturated time and so on, for the first time, apigenin-7-O-β-D-glucosidase TLC identification were studied. With chloroform:ethyl acetate:methanol:formic acid (8:6:3:1)for a system, separation effect is better, spots more and clear, the Rf value is moderate. Through thin layer chromatography experiment of32batches samples humulus scandens from different origins and wares.32batches samples TLC graphs, NO.1(Liaoning Fushun)(Zhejiang Xianju)、NO.4(Guangdong Shaoguan)、NO.5(Shanxi Xianyang)、 NO.7(Gandu Dangchang)、NO.8(Jiangsu Lianyungang)、NO.11(Hunan Changsha)、 NO.16(Sichuan Yibin)、NO.20(Shandong Jinan)、NO.21(Shandong Qingdao) NO.23(Heibei Tangshan)、NO.24(Jiangxi Nanchang)、NO.25(Jiangxi Ji an)、 NO.26(Fujian Fuzhou)、NO.28(Beijing Fengtai area)、NO.29(Chongqing Nanchuan)、 NO.30(Shanghai Chongmingdao), a total of17samples, a same yellow-green color spots in the TLC graph of sample existed at the corresponding positions compared with the apigenin-7-O-β-D-glucosidase control, but the size and the strength of the fluorescent spots are different, other samples not detected apigenin-7-O-β-D-glucosidase, tip on the existence and quantities of the composition of humulus scandens in different origins are different.4The HPLC fingerprint of humulus scandens were first developed in the study, set up the HPLC fingerprint analysis method of humulus scandens. and t humulus scandens from different origines has carried on the cluster analysis, to find out the differences between the chemical composition of humulus scandens from different origins provides reference basis.4.1Through the selection of chromatographic conditions、the investigation of gradient elution method and preparation of test sample solution, as well as the methodology validation of precision, stability and repeatability, set up the HPLC fingerprint of humulus scandens. Based on HPLC fingerprint analysis of32batches of samples of humulus scandens,15common peak was determined.4.2The similarity analysis for the ethyl acetate part of humulus scandens HPLC fingerprint was studied, results show that different regions humulus scandens herbs fingerprint similarity is low. Clustering analysis, the results show that, division with15distance, can be divided into two parts, similarity above0.7samples, including NO.5(Shanxi Xianyang)、NO.6(Shanxi Yuncheng)、NO.7(Gansu Dangchang)、 NO.9(Guizhou Anshun、NO.10(Guizhou Guiyang)、NO.13(Chengdu Wenjiang area)、 NO.14(Chengdu Jinjiang area)、NO.15(Sichuan Xichang)、NO.18(Sichuan Pengzhou)、 NO.19(Sichuan Suining) can get for the category, the rest samples together for a class; division with10distance, can be divided into three parts, similarity above0.7samples, can get for the category, similarity between0.3～0.7samples, including NO.2(Henan Nanyang)、NO.4(Guangdong Shaoguan)、NO.11(Hunan Changsha)、N0.12(Guangxi Hechi)、N0.16(Sichuan Yibin)、NO.17(Sichuan Mianyang).NO.3(Zhejiang Xianju)、 NO.24(Jiangxi Nanchang)、NO.25(Jiangxi Ji an)、NO.26(Fujian Fuzhou)、 NO.27(Fujian Zhangzhou)、NO.32(Guangxi Yulin medicine market) can get for the category, the rest samples together for a class.5. For32batches of different origin samples of humulus scandens, we examined the moisture, total ash and acid insoluble ash, determinated the extract.5.1According to the2010version of the method specified in the "standard of Chinese medicinal materials in Sichuan province", moisture determination of32batches of different origin samples of humulus scandens was studied. Measured32batches of different origin samples of humulus scandens moisture content as high as9.7%, the lowest was4.9%. Reference in the2010edition of "standard of Chinese medicinal materials in Sichuan province", the provisions of the water shall not be over10.0%,32batches of samples conform to the prescribed standards.5.2According to the2010version of the method specified in the "standard of Chinese medicinal materials in Sichuan province", total ash and acid insoluble ash of32batches of different origin samples of humulus scandens were studied. Measured32batches of different origin samples of humulus scandens total ash content as high as19.83%, the lowest was10.43%; acid insoluble ash as high as4.56%, the lowest was0.30%. Reference in the2010edition of "standard of Chinese medicinal materials in Sichuan province, the total ash content may not be over19.0%, acid insoluble ash content shall not be over5.0%, except the No.24(Jiangxi Nanchang) samples total ash content does not conform to the standard, the others all conform to the prescribed standards.5.3According to the2010version of the method specified in the "standard of Chinese medicinal materials in Sichuan province", extract of32batches of different origin samples of humulus scandens were determinated. Measured32batches of different origin samples of humulus scandens extract as high as21.88%, the lowest was8.73%. Reference in the2010edition of "standard of Chinese medicinal materials in Sichuan province, the provisions of extract not be over12.0%, except theNo.22(Chongqing Shapingba、No.30(Shanghai Chongmingdao)、No.31(Anhui Bozhou medicine market)、No.32(Guangxi Yulin medicine market), the others all conform to the prescribed standards. 6. First established the ultraviolet spectrophotometry method of total flavonoids of humulus scandens and apigenin-7-7-O-D-glucosidase content of HPLC method, flavonoids, mignonette element and apigenin-7-O-β-D-glucosidase contents of32batches of different origin samples of humulus scandens were determinate, to provide reference basis for perfect qualify evaluation standards and the comparative analysis of humulus scandens humulus scandens quality differences between different regions.6.1The uv-spectrophotometry determination of total flavonoids of Humulus scandens was determinated. Through the methodology of investigation,inclouding the preparation of test sample solution, precision, stability, repeatability, recovery, etc. established mignonette element as reference substance of total flavonoids of humulus scandens. Uv-spectrophotometry method. Using this method to samples of different regions to determine the flavonoids content, results showed that the flavonoids content in different origin samples of humulus scandens difference is bigger, NO.3(Zhejiang Xianju)、NO.4(Guangdong Shaoguan)、NO.5(Shanxi Xianyang)、 NO.12(Guangxi Hechi)、NO.16(Sichuan Yibin)、NO.19(Sichuan Suining)、 NO.26(Fujian Fuzhou) and NO.28(Beijing Fengtai area), eight sample content is higher,No.22(Chongqing Shapingba)、No.24(Jiangxi Nanchang)、No.30(Shanghai Chongmingdao)、No.31(Anhui Bozhou medicine market),5sample content is relatively low.6.2Accord with the previous content of mignonette element HPLC determination method, different origin samples of humulus scandens mignonette element content was determinated. Results showed that, NO.4(Guangdong Shaoguan)、 NO.4(Guangdong Shaoguan)、NO.8(Jiangsu Lianyungang)、NO.16(Sichuan Yibin)、 NO.23(Hebei Tangshan)、NO.28(Beijing Fengtai area),five samples of mignonette element content is higher; NO.2(Henan Nanyang)、NO.6(Shanxi Yuncheng)、 NO.10(Guizhou Guiyang)、NO.12(Guangxi Hechi)、NO.13(Chengdu Wenjiang area)、 NO.14(Chengdu Jinjiang area)、NO.15(Sichuan Xichang)、NO.18(Sichuan Pengzhou)、 NO.22(Chongqing Shapingba) and NO.27(Fujian Zhangzhou), in these10samples, mignonette content was not measured. This result is consistent with the results of TLC identification.6.3The HPLC determination of apigenin-7-O-β-D-glucosidase of Humulus scandens was determinated.Through the selection of detection wavelength and chromatographic conditions、the investigation of the preparation method of the test solution, as well as the precision, stability, repeatability, recovery test methodology validation, established apigenin-7-O-β-D-glucosidase HPLC determination method of Humulus scandens. Using the method determinated apigenin-7-O-β-D-glucosidase content of Humulus scandens from different regions, Results showed that, NO.1(Liaoning Fushun)、NO.3(Zhejiang Xianju)、NO.8(Jiangsu Lianyungang)、 NO.11(Hunan Changsha)、NO.16(Sichuan Yibin)、NO.21(Shandong Jinan)、 NO.23(Hebei Tangshan)、No.28(Beijing Fengtai area) and No.29(Chongqing Nanchuan), nine samples of apigenin-7-O-β-D-glucosidase element content is higher; N0.2(Henan Nanyang)、NO.10(Guizhou Guiyang)、NO.13(Chengdu Wenjiang area)、 NO.15(Sichuan Xichang)、NO.18(Sichuan Pengzhou) and NO.22(Chongqing Shapingba), in these6samples, mignonette content was not measured.This result is consistent with the results of TLC identification. |
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