| Tetrastigma hemsleyanum Diels et Gilg, belonging to the family Vitaceae, is a perennial herbaceous species native to China. It is mainly distributed in southern China. Because of the richness in anti-inflammatory, analgesic and antipyretic properties, its roots are used in the treatment of high fever, infantile febrile convulsion, pneumonia, asthma, hepatitis, nephritis, rheumatism, injuries and snake bite. The main active chemical constituents are flavonoids and polyphenols. As a commonly used antitumor herb medicine in the countryside, many researches have been carried out on the pharmacological activities and the chemical constituents. As a widely distributed plant, many researches showed that samples from different regions were different in pharmacognosy structure and pharmacological activities. Unfortunately, we know little about its secondary metabolites, particularly the comprehensive understanding of genetic variation and environmental factors regarding on the accumulation of secondary metabolites in T. hemsleyanum. In this study, the chemical diversity of T. hemsleyanum was evaluated in associations with genetic variation and environmental factors, to determine which contribute more on its secondary metabolic variation. Based on the research, we can elucidate the causes for varying qualities of T. hemsleyanum. Then we can put forward scientific suggestions about introducing, domesticating, breeding and quality assessment of T. hemsleyanum. The main results are as follows:(1) Four compounds (i.e. piceatannol, resveratrol, isoquercitrin, nicotiflorin) were identified from T. hemsleyanum by using spectroscopy method and mass spectrum technology. Through standard substance and recovery assay, the polydatin was determined in T. hemsleyanum. This is the first study reporting the isolation of piceatannol from T. hemsleyanum.(2) By applying HPLC-DAD technique and multivariate statistic analysis, the chemical diversity evaluation system of T. hemsleyanum was constructed based on chemical fingerprints and quantitative analysis of five indicative components. Based on the result, the T. hemsleyanum from 13 wild region of China were divided into three chemotypes.(3) Based on two sets of molecular analysis (cpDNA and SSR), the high level of genetic diversity was detected at the species level (cp DNA, h= 0.227; SSR, He= 0.428). Analysis of Molecular Variance (AMOVA) of two molecular markers indicated a significantly genetic differentiation among populations (cp DNA, Fst= 0.749, P< 0.001; SSR, Rst= 0.001, P< 0.001); Two lineages was divided based on two sets of molecular markers (cpDNA:Guangxi population was a lineage, the rest populations were the next lineage; SSR:populations of Ningbo and Kaihua in Zhejiang Province were a lineage, the rest populations were the next). The differentiation of chemical component of these two lineages didn’t agree with the genetic differentiation. The Mantel test of the relationship between the pairwise population genetic distance(FST for cpDNA and RST for SSR) and pairwise chemical distance (r=0.091, P=0.310 for cpDNA; r=0.122, P=0.204 for SSR) showed that population genetic differentiation did not correlate with the concentration of chemical components. This further confirmed the chemical component variations were less affected by genetics. Considerable chemical differentiations were observed among populations (P<0.007). Samples from Ningbo of Zhejiang Province, Lichuan of Hubei Province and Jinfo Mountain of Chongqing city possessed higher content of stilbene; while samples of Nandan of Guangxi Province and Jishou of Hunan Province contained high levels of flavonoids.(4) Effect of thirteen climate factors and six soil parameters on five indicative components were analyzed by generalized linear mixed models (GLMM). Variance partition (R software) was used to elucidate parameters regarding their contributions to chemical components variation. The result showed that chemical components was influenced more by climate factor (35%) compared to soil factors (28%). The relative humidity, annual precipitation and annual sunshine time were the main climate parameters on chemical components, while the pH value, available potassium, phosphorus and organic matter were the main soil parameters on chemical components.(5) The Box-Behnken design was used to optimize the extraction conditions for total phenolic content and ferric reducing antioxidant power (FRAP) from T. hemsleyanum. The optimum extraction conditions were as follows:70% ethanol concentration, 50℃ extraction temperature,47 min extraction time. Under these conditions, the total phenolic content and FRAP value were 5.51 mg gallic acid/g DW and 92.10 μmol FeSO4/g DW, respectively, which were in good agreement with the predicted value.(6)The total phenolic content, antioxidant capacities and antitumor activity of T. hemsleyanum roots were significantly influenced by the drying methods. Correlation analysis revealed that the antioxidant capacities (FRAP, Hydroxyl radical scavenging ctivity and DPPH scavenging activity) of the roots dried by different drying methods were positively correlated with their total phenolic content. The ethyl acetate parts of roots dried by different drying methods showed different cytotoxic activity on two cancer cell lines determined by MTT assay. For K562, the extract of ethyl acetate from roots if dried at 30℃, freeze-dried and shade-dried significantly inhibited tumor cell growth. And for A549, the obvious tumor cell inhibition occurred when extracts coming from roots dried at 30℃ and shade-dried. HPLC analysis showed that the higher content of piceatannol and resveratrol were detected in roots if dried at 30℃. Therefore, drying these herbs at 30℃ was recommended as the priority drying method.Conclusion:there are significantly differences on chemical compositions among different populations of T. hemsleyanum. The germplasms of Ningbo, Zhejiang Province and Jinfo Mountain, Chongqing city can be used as the preferred breeding materials based on high content of stilbene and high activity of in vitro antitumor. |
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