| Lonicera macrathoides Hands Mazz. which is rich in Hunan, the planting area reaches more than 300,000 ares. The trade name of bud medicine is "Xiangyinhua". Its output takes 40 percent market share all the time since the late 1990s. They are sold all parts of the country.It has been planting over thirty years, and also has gone through artificial selection and natural selection. It has been formed various germplasm resources about farmhouse breed and farmhouse strain and so on. But their qualities are not balanced, and names of article are so disordered that differentiate them difficultly. That is the reason why there are many difficulties in cultivation and management. At the same time, when people select improved variety, the main objective is focused on economic character like output and agronomic character like plant types, but never study the internal quality like the contents of active ingredients.Objective:L. macrathoides Hands Mazz. as a new resource, and its basic research is weak. This research was intended to use certified Flos Lonicera in Shandong and Henan as contrast, developed the contents of effective chemical composition of L. macrathoides Hands Mazz., such as Chlorogenic acid, Caffeic acid and volatile oil, as well as bacteriostatic effect, antibacterial effect, antipyretic effect, and other pharmacological effects and safety of comparative studies, attempted to establish the quality standard system of Xiang Yinhua; investigated the influence of combination of L. macrathoides Hands Mazz. and Scutellaria baicalensis Georgid on the main effective components, researched and established the HPLC chromatographic fingerprints of combination of L. macrathoides Hands Mazz., Flos Lonicera and S. baicalensis Georgid; developed the comparison research between heredity multiplicity of germplasm resources and the contents of active components, systemically arranged and scientifically evaluated L. macrathoides Hands Mazz. germplasm resources, and provided the scientific basis for the improved variety selective breeding and the GAP plant.Methods:The HPLC method was used for determining effective components of Chlorogenic acid and Caffeic acid. Volatile oil components were extracted by water distillation and was analyzed by GC-MS. Agar diffusion method and test tube dilution method were respectively used for researching vitro bacteriostatic effect and vitro bactericidal effect. Internal antibacterial effect was studied with NIH mouse and was determined its median lethal doses LD50. Antipyretic effect was studied with rat model induced by thermal. The HPLC method was used for determining of Chlorogenic acid and Baicalin in single medicine L. macrathoides Hands Mazz., Flos Lonicera, S. baicalensis Georgid with its cooking liquor and used SPSS 13.0 software to analyze. The similarities of fingerprints were analyzed by<Similarity Evaluation System for Chromatographic Fingerprint of TCM 2004>. Genetic diversity of germplasm resources of L. macrathoides Hands Mazz. was studied by inter-simple sequence repeat (ISSR) molecular markers, and POPOGENE32 software for cluster was used for analyzing amplified fragment datas. The HPLC method was used for comparing the contents of Chlorogenic acid, Caffeic acid, Luteolin, Rutin and Hyperin and other components from different germplasm resources of L. macrathoides Hands Mazz., and were established fingerprints of L. macrathoides Hands Mazz. "Xianglei Flos Lonicera ".Results:1、The percentages of the contents of Chlorogenic acid in L. macrathoide Hands Mazz. from the plant bases in Longhui, Xupu and Xinning of Hunan and Henan, Shandong as well as the medical market in Shandong were respectively 3.9±0.4537,2.6±0.1408,4.5±0.0182, 2.4±0.1913,2.1±0.1684,2.5±0.0506; the contents of Chlorogenic acid in L. macrathoide Hands Mazz. was higher than that in Flos Lonicera. The contents of Cafeic acid in L. macrathoide Hands Mazz. were almost the same as or a litter higher than that in Flos Lonicera. The volatile oil in L. macrathoide Hands Mazz.and Flos Lonicera both contained Linaiool, Geraniol, a-Terpineol and other seven same components. And the percentages of the contents of Linaiool were the highest one among them.2、L. macrathoides Hands Mazz. or Flos Lonicera could inhibit Staphylococcus aureus,β-streptococcus, Escherichia coli and other three kinds of bacteria, and had the strongest effect for Staphylococcus aureus andβ-steptococcus, and even killed Staphylococcus aureus. The lowest bactericidal concentration of medicine were significantly higher than the minimum inhibitory concentration of medicine. The antibacterial . effect of the decocta were stronger than one of the maceration. L. macrathoides Hands Mazz. which was from Longhui had the strongest antibacterial effect. L. macrathoides Hands Mazz. and Flos Lonicera could protect rat from infecting by staphylococcus, and L. macrathoides Hands Mazz. which was from Longhui had the best effect. L. macrathoides Hands Mazz. and Flos Lonicera had the same defervescence effect. Toxicities of medicine were small, toxicity of L. macrathoides Hands Mazz. was smaller.3、There were no influence on the content of main active ingredients like Chlorogenic acid and Baicalin, when L. macrathoide Hands Mazz. or Flos Lonicera was used with S. baicalensis Georgid.4、Take Chlorogenic acid as a reference peak, there were 12 fingerprint-peak in fingerprints of L. macrathoide Hands Mazz. and S. baicalensis Georgid water extract, Flos Lonicera and S. baicalensis Georgid water extract. The fingerprints of L. macrathoide Hands Mazz. and S. baicalensis Georgid water extract had 14 fingerprint-peak altogether, while the one of Flos Lonicera and S. baicalensis Georgid water extract had 13 fingerprint-peak in all. The similarities of fingerprints which were same medicinal material but different batch were more than 90 percent. The similarities of fingerprints between L. macrathoide Hands Mazz. with S. baicalensis Georgid water extract and Flos Lonicera with S. baicalensis Georgid water extract were basically over 90 percent. The fingerprints of L. macrathoide Hands Mazz. water extract compared with one of Flos Lonicera were more than four peaks, but the peak area were smaller. And two peaks disappeared after L. macrathoide Hands Mazz. have boiled with S. baicalensis Georgid.5、DNA-extracting method and the ISSR reaction system for L. macrathoide Hands Mazz. were optimized, and a stable as well as reliable way of identification of variety was set up. Then 15 valid primers were screened out, fingerprint warehouses of L. macrathoide Hands Mazz. were built and a tree diagram of L. macrathoide Hands Mazz. was constructed. These 18 stuffs were divided into 2 main groups, L. japonica Thunb. was one group, L. macrathoide Hands Mazz. was the other group. In L. macrathoide Hands Mazz. group, they were further divided into 5 subgroups.2 stuffs originated from Taihu were clustered together, conventional varieties in Yanbei and Longtan were respectively clustered one subgroup, Xianglei 6 and Xiangleiwang 2 clustered one subgroup, the rest stuffs were the fifth subgroup.6、There were extraordinary disparities in contents among different L. macrathoide Hands Mazz. germplasm, such as the contents of Chlorogenic acid were between 0.012% and 6.66%, a difference of 55.5 times among samples. Except 1,2,4,7,13 of samples, others were over 2005 version of the<People’s Republic of China Pharmacopoeia> required standards. The contents of Luteolin were between 0.009% and 0.079%, a difference of 55.5 times among samples, and the contents of all samples were below the 2005 edition of the<People’s Republic of China Pharmacopoeia> required standards. The contents of Cafeic acid were between 0.003% and 0.399%, a difference of 133 times among samples. The contents of Rutin were between 0.014% and 0.457%, a difference of more than 32 times among samples. The contents of Hypericum Coombe were between 0.007% and 0.096%, a difference of 13.7 times among samples. Conventional varieties had good internal qualities. The contents of active ingredients were generally higher in Taihu 1, Taihu 2, Xiangleiwang 1, Xiangleiwang 4, Baiyun 1 and Huawang 1. Chlorogenic acid far beyonded 2005 edition of the<People’s Republic of China Pharmacopoeia> required standard, suitable for promotion. The fingerprints of Xiangleiwang series of Hunan L. macrathoide Hands Mazz. were constructed, and had high similarities.Conclusion:1、The inherent quality and pharmacological action of are superior to Flos Lonicera. So L. macrathoide Hands Mazz. can completely replace Flos Lonicera to use.2、L. macrathoide Hands Mazz. germplasm resource are abundant. There are huge disparities in the contents of active ingredients and inner quality among different L. macrathoide Hands Mazz. germplasm.3、L. macrathoide Hands Mazz. is superior to Flos Lonicera, when they are respectively used with S. baicalensis Georgid. |
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