| Traditional Chinese medicine Xuesanqi is the root tuber of Polygonum amplexicaule D. Don var. sinense Forb. It is the commonly used raw material for medicine among Enshi minorities in Hubei Province. It has many medical functions, such as improving blood circulation, maintaining normal menstrual flow, and curing various fractures, muscle injuries or pain from tissue swelling. Especially, Xuesanqi has been demonstrated to be an effective herb to treat rheumatism, atherosclerosis and to have antifungal activity. In addition, its distribution is very wide, there are a large number of wild and home-grown Xuesanqi in China. However, so far, there are sporadic studies about it, and lacks systematical research, which block its further applications. Therefore, the objective of the study is to isolate and identify its most effective compounds by various chromatographic techniques and to set up fingerprints of Xuesanqi from different areas in order to provide an academic basis for the scientific evaluation of the quality of Xuesanqi. On this basis, by means of modern pharmacological methods, it was tried to prove its different medical effects, including antifugal activity, mechanism of promoting the healing of bone fractures, and analgesic activity, etc, which would provide the scientific basis of developing new drug in the future.The main work and its innovations of this research are listed below:(1) Such chromatographic techniques, including TCL, normal-phase silica gel column, reverse-phase silica gel column, polydextran gel and semi-preparative high performance liquid colum were employed in this study. About 40 compounds were obtained by the above chromatographic techniques from EtOAc fraction of Xueshaqi. By means of modern spectroscopic methods (e.g. UV, IR, 1H-NMR, 13C-NMR, DEPT, EI-MS), 23 compounds were finally identified. Among them, 20 compounds were isolated for the first time from this plant. Moreover, 5 compounds were first obtained from Polygonaceae. A majority of these compounds belong to flavonoids and anthraquinones, which show great pharmacological activity.(2) Since the quality standards of Xuesanqi have not been established by now, on the basis of its chemical composition analysis, the fingerprints were set up by HPLC for the flavonoids of Xuesanqi from nine different regions. A total of 11 peaks were found, and their relative retention time and relative peak area ratio of the scope were determined. The results showed that each fingerprint of P. amplexicaule exhibited 90% of similarity despite of their different origin. Thus, the results met the quality requirements of fingerprint. The establishment of this method not only can help to identify Xuesanqi from different localities, but also can provide scientific evidence to formulate the quality standards of Xuesanqi.(3) The anti-fungal activity of secondary metabolites from Xuesanqi has been studied for the first time. Susceptibility disk assay was adopted to test the anti-fungal activity of ethyl acetate fraction and n-butanol fraction from Xuesanqi. In addition, its minimum inhibitory concentration was measured by agar dilution method and the effects of light, temperature, pH on its antifungal activity were examined by plate method. The results showed that the secondary metabolites of Xuesanqi had significant anti-fungal activity on pathogenic fungi and the deep drug-resistant strains. Its anti-fungal activity was rather stable and could not be easily interfered by the environmental factors, such as light, temperature, etc. Compared with ethyl acetate fraction, n-butanol fraction had better anti-fungal activity, even 0.013mg/ml n-butanol fraction showed strong anti-fungal effect.(4) To investigate Xuesanqi's pharmacological mechanism of promoting the healing of bone fractures in vitro, MC3T3-E1 cell line and osteoblasts from skull bones of mice were established to test the biological functions of the total extracts from Xuesanqi, petroleum ether fraction extracts, ethylacetate fraction extracts, n-butanol fraction extracts, water fraction and compound emodin-8-O-β-D-glucoside (EG) from ethylacetate fraction extracts. CCK-8 assay, PI staining and FACS were used to examine cell proliferation and cell cycle. The expression of alkaline phosphatase (ALP) and the content of prostaglandin E2 (PGE2) were detected by enzyme-linked immunosorbent assay (ELISA). The results of cell proliferation showed that 0.1-100μg/mL dose of total extracts, fraction extracts and compound from Xuesanqi could stimulate cell proliferation significantly (p<0.05). Total extracts and fraction extracts could increase the proportion S-phase of cells from 9.78 to 27.29%. Compound emodin-8-O-β--D-glucoside (EG) could increase the proportion S-phase of cells from 16.34% to 32.16%. Simultaneously, each extraction and compound EG had showed certain effects on the differentiation of osteoblasts, and exhibited a marked dosage dependent relationship. Especially, EG increased alkaline phosphatase (ALP) expression in MC3T3-E1 cells (0.1-100 ng/mL, p<0.01) and inhibited PGE2 production induced by TNF-αin osteoblasts (10-100 ng/mL). All the above results suggest that the mechanism of Xuesanqi to promote the healing of bone rheumatism and various fractures is that Xuesanqi directly stimulates the proliferation and differentiation of osteoblasts, and compound EG contributes most to these activities.(5) It was for the first time to investigate the analgesic activity of Xuesanqi in vivo. Hot- plate model and the acetic acid writhing test were adopted among the low, middle and high dosage groups from ethanol extracts, petroleum ether fraction extract, ethylacetate fraction extract, n-butanol fraction extract and water fraction, respectively. The results showed that 0.4 mg/mL, 0.8 mg/mL, 1.6 mg/mL dose of all extracts could significantly increase the pain threshold of licking hot-plate in mice (p<0.05) and markedly reduced the times of writhing induced by acetic acid (p<0.01). Moreover, the analgesic activity of water fraction and n-butanol fraction were much better than ethanol extracts, petroleum ether fraction extract, ethylacetate fraction extract. |
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