Studies On The Substance Basis Of Phytoestrogen From The Peel Of Punica Granatum L. And Analysis Different Pomegranats Cultivars With RAPD In Xinjiang

The peel of Punica granatum L. has been used in clinical practice with the activities of astringing intestine, hemostasis and deworming as a traditional Uygur natural medicine. It was reported that chemical constituents such as tannin, flavones, and alkaloid had been extracted from Punica granatum L. Besides tannin, the characteristic constituents in Punica, the potential phytoestrogen-like effects of flavones in pomegranates have becoming popular on study.As the replacement of estrogen, the safety and remarkable effects of phytoestrogen has aroused people's attention. Screening active constituents with the phytoestrogen-like effects from traditional Chinese medicine is possible and practical.According to the confusion of cultivars of Punica granatum L. in Xinjiang pomegranates, exact identification and rational classification of the cultivars of pomegranates are the basis of further utilization the pomegranates resource in Xinjiang.Objective:On the basis of screening the phytoestroge-like effects, to study the chemical constituents of the peel of Punica granatum L. and to determine the content of tannin the characteristic constituents in Punica. Applying the DNA molecular marker technique combined the chemistry analysis to clarify the molecular and chemistry characteristics of Punica granatum L. in Xinjiang.Methods1.Proliferation of MCF-7 assay and yeast two-hybrid assay were applied to screen phytoestrogen-like effects from eighteen natural medicine.2.Water-extracting, solvent-extracting and chromatography methods were applied to extract and separate chemical constituents from the peel of Punica granatum L. HPLC method was used to determine its content of tannin.3.Different random primers were used for the amplification in twenty-three samples of pomegranates from different localities. More polymorphic and better repetitive primers were selected in amplification reactions to construct the RAPD fingerprints of pomegranates from different cultivars according to cluster analysis.Results:1.The results of MCF-7 proliferation assay showed that the water-extraction from ten natural medicine (including Angelica acutiloba kitagawa var.sugiyamae Hikono. Ligusticum chuanxiong Hort. Paeonia lactiflora Pallas. Atractylodes macrocephala Koidz. Paeonia suffruticosa Andr. Cicer arietinum L. Punica granatum L . Ocimun basilicum L. Alisma orientale Juzepczuk. Cinnamomum cassia Blume.) stimulated proliferation of ER (+) MCF-7 (p<0.05) and indicated they had phytoestrogen-like effects. The results of yeast two-hybrid assay showed that the water-extraction from seven natural medicine (including Angelica acutiloba kitagawa var.sugiyamae Hikono. Ligusticum chuanxiong Hort. Paeonia lactiflora Pallas. Cicer arietinum L. Punica granatum L. Cinnamomum cassia Blume. Alisma orientale Juzepczuk.) enhanced the activity ofβ-galactosidase in yeast system and indicated they had phytoestrogen-like effects.2.13 chemical compounds were obtained from the peel of Punica granatum L .and their structures were elucidated by physical and chemical identification and spectral methods, including UV, IR, 1H-NMR and 13C-NMR. Ten of them were determined as gallic acid, methyl gallate, ellagic acid, (+) catechin, isoquerecitrin, D-mannitol, ursolic acid, Oleanolic acid,β-Sitosterol, Daucosterol. The result of content determination of tannin in pomegranates peel from five localities of Xinjiang by HPLC was 3.26mg/g, 4.53mg/g, 3.86mglg, 2.57mg/g and 2.94mg/g respectively, which indicated there were obvious difference between the samples from different localities.3.Genomic DNA of twenty-three samples from different cultivars of Punica granatum L. extracted by CTABⅠmethod amplified clear polymorphic bands and the suitable RAPD reaction system for pomegranate genomic DNA was settled as following, 25μl -reaction system with 2.5μl 10×buffer;10mMdNTP 0.5μl;16.5ng/μl primer 5μl;25mmol/l MgCl2 0.5μl;2.5u/μl Taq DNA polymerase 0.8μl;template DNA (approx.25ng/μl) 1μl;The program was as follows, peradventure in 94℃for 4 min;denature in 94℃for 1 min, anneal in 37℃for 1 min, extension in 72℃for 2 min, cycling number in 40, total extension in 72℃for 7min, and terminate reaction at 4℃.Fourteen primers with stable amplification were selected from fifty random primers and amplified polymorphic bands in 23 samples, then 7 of 14 primers ZY1, ZY4, ZY12, ZY23, ZY30, ZY41 and ZY42 were selected to use in the further analysis. Eighty-eight bands were obtained by amplification of the polymorphic primer and sixty-one bands (69%) among them were found to be polymorphic.RAPD reaction and cluster analysis were performed in 23 cultivars. Analyzed by statistics software according to similar coefficient, 23 cultivars could be divided into five clusters as following:clusterⅠ, including Korla sweet pomegranates and Kashi big seed sweet pomegranates;clusterⅡ, including Atushi sour pomegranates;clusterⅢ, including Kashi sour pomegranates and Yarkent sour pomegranates;clusterⅣ, including interspecific crossing cultivars such as Turpan sweet pomegranates and Kuche sour-sweet mixture pomegranates;clusterⅤ, including Piyama pomegranates and Chira pomegranates No.1. The above results showed that the genetic polymorphism of pomegranate cultivars had obvious difference, even the cultivars with same cross parenthood could be categorized by RAPD analysis.Conclusions:1.Proliferation of MCF-7 assay and yeast two-hybrid assay were applied for the first time to screen phytoestrogen-like effects from eighteen natural medicine and the water-extractions from 7 of 18 (including Punica granatum L.) showed phytoestrogen- like effects in both assays. It revealed that flavonoids constituents might be the effective substance in Punica granatum L., which provided theoretical basis for the utilization of phytoestrogenic plants resource.2.13 chemical compounds were obtained from the peel of Punica granatum L. in Xinjiang and the structures of 10 compounds were determined as gallic acid, methyl gallate, ellagic acid, (+) catechin, isoquerecitrin, D-mannitol, ursolic acid, Oleanolic acid,β-Sitosterol, Daucosterol. Theses researches filled the gaps in the blank of the study on chemical constituents of Punica granatum in Xinjiang.3.Content of gallic acid in the peel of Punica granatum L. from 5 localities were determined by HPLC method for the first time and the results showed the content of gallic acid were different among 5 samples from 5 localities, which provided the experimental basis for quality control of Punica granatum .4.By means of the platform of the molecular biology, RAPD fingerprint technique was applied for the cultivars study on Xinjiang's Punica granatum from different localities first the time. The result showed that the technique could be applied for analyzing the genetic distance of intraspecies and infra-species and provided evidence for solving the doubtful points of taxonomic identification on DNA molecular level.