新疆石榴皮植物雌激素物质基础及石榴品种RAPD分析研究
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摘要
石榴Punica granatum L.,隶属于石榴科(Punicaceae)石榴属(Punica)。石榴的干燥果皮是其药用部位,具有涩肠、止血、驱虫的功能,用于久泻、久痢、便血、虫积腹痛。据报道,石榴中含有鞣质类、黄酮类及生物碱类成分,尤其是黄酮类成分以具有植物雌激素的活性而成为研究热点之一。
植物雌激素作为雌激素的替代品,以其安全性及显著的效果,受到越来越多的关注,从天然药物中筛选具有雌激素作用的药物,并进一步筛选其有效成分是实际可行的。针对新疆石榴品种分类相对混乱现象,对现有石榴品种准确鉴定和合理分类是进一步利用石榴资源的基础。
目的:
利用人乳腺癌细胞MCF-7细胞增殖实验与酵母双杂交实验对新疆石榴皮等18种天然药物的植物雌激素作用进行了初步筛选,并对其中具有雌激素活性的新疆石榴皮进行了化学成分的研究。采用分子标记技术和化学分析手段相结合的方法,阐明石榴的分子特征和化学特征,从而为鉴别新疆石榴品种提供实验依据。
方法:
研究内容分为三个部分:
1.采用人乳腺癌细胞MCF-7细胞增殖实验与酵母双杂交(Yeast two-hybrid)实验对新疆石榴皮、罗勒、红景天、阿里红、鹰嘴豆、枸杞、肉苁蓉、琐阳、鼠尾草、当归、川芎、芍药、桃仁、牡丹皮、桂枝、泽泻、白术、茯苓等18种天然药物进行植物雌激素活性的筛选。
2.采用水提取、系统溶剂萃取,分别进行Diaion HP-20、MCI Gel CHP-20、Sephadex LH-20、硅胶等柱层析及制备薄层层析,提取新疆石榴皮样品中的化学成分,并运用HPLC法测定鞣质类成分的含量。
3.选取不同随机引物对不同产地的23个石榴样本进行RAPD指纹图谱分析。优化RAPD条件后,从中选出多态性高、重复性好的引物对不同样本进行检测,并根据条带的差异进行聚类分析,构建不同石榴品种的RAPD指纹图谱。
结果:
1.MCF-7细胞增殖实验结果表明:当归、川芎、芍药、白术、牡丹皮、鹰嘴豆、新疆石榴皮、罗勒、泽泻、桂枝水提取物可促使ER(+)MCF-7细胞的增殖(P<0.05),提示它们具有植物雌激素样活性;酵母双杂交(Yeast two-hybrid)系统实验结果表明:当归、川芎、牡丹皮、鹰嘴豆、石榴皮、泽泻、桂枝、水提取物均能使酵母系统中的β-半乳糖苷酶的活性增强(P<0.05),提示它们具有植物雌激素样活性。
2.从新疆石榴皮中提取分离结果得到13个化合物。根据化合物的理化性质及UV、IR、1H-NMR、13C-NMR等波谱方法,鉴定了10个化合物的结构,分别为:没食子酸(gallic acid)、没食子甲酯(methyl gallate)、鞣花酸(ellagic acid)、(+)-儿茶素[(+)catechin]、异槲皮苷(isoquerecitrin)、D-甘露醇(D-mannitol)、熊果酸(ursolic acid)、齐墩果酸(Oleanolic acid)、β-谷甾醇(β-Sitosterol)、胡萝卜苷(Daucosterol);并运用HPLC方法测定新疆5个石榴主产地中没食子酸含量为疏附县:3.26mg/g、疏勒县:4.53mg/g、叶城:3.86mg/g、皮山县:2.57mg/g、策勒县:2.94mg/g。结果表明不同产地的化学成分的含量具有明显差异。
3.CATBⅠ法提取23份石榴基因组DNA能扩增出清晰的多态性带,确定了适合石榴RAPD反应的反应体系为:10×buffer:2.5μl;dNTP(10mM):0.5μl;引物(10μM):5μl;MgCl2(25mmol/l):0.5μl;Taq DNA聚合酶:0.8μl(2.5μ/μl);模板DNA(约25ng/μl):1μl;总体积25μl;剩余的用ddH2O来补充;确定了适合石榴RAPD反应的扩增程序为:94℃预变性4 min;94℃变性1min,37℃退火1min,72℃延伸2 min,40循环;72℃总延伸7min后在4℃终止反应。从50个引物中筛选出能稳定扩增的7个引物ZY1、ZY4、ZY12、ZY23、ZY30、ZY41、ZY42,用这些引物对23个石榴品种进行扩增,共扩增出88条带,61条具有多态性,多态性比例为69%。
对新疆石榴23个品种进行了RAPD扩增,根据23个品种的Nei相似系数进行聚类分析的结果:能将23个品种聚为5类:第1类包括库尔勒市甜石榴与喀什市大籽甜石榴等甜石榴品种;第2类是阿图什市酸石榴;第3类包括喀什市阿奇克阿娜尔、叶城酸石榴等酸石榴品种;第4类包括吐鲁番市甜石榴、库车酸甜石榴等种间杂交和来源不详品种;第5类包括皮亚曼石榴和策勒1号等新疆优质石榴品种。本研究结果表明:石榴品种间的遗传多态性有明显差异,即使父母本相同的品种,也能分开。
结论:
1.首次采用人乳腺癌细胞MCF-7细胞增殖实验和酵母双杂交技术,对18种天然药物进行植物雌激素活性的筛选。结果表明新疆石榴皮、罗勒、鹰嘴豆、当归、川芎、芍药、白术、牡丹皮、泽泻、桂枝等7种天然药物的水提物在两种检测方法中均表现出植物雌激素样活性,为今后雌激素类植物资源的开发提供了理论支持。
2.首次对新疆石榴皮进行了化学成分研究,从中分离得到13个化合物,鉴定了其中10个化合物的结构,依次为没食子酸、没食子甲酯、鞣花酸、儿茶素、异槲皮苷、齐墩果酸、熊果酸、β-谷甾醇、胡萝卜苷。所有的化合物均为从新疆甜石榴中首次获得。本研究填补了新疆石榴皮化学成分研究的空白。
3.首次利用HPLC定量方法对5个新疆主产区的石榴样品的没食子酸含量进行了测定,结果显示:5个产区的石榴样品的没食子酸含量不同,其中疏勒县最高为4.53 mg/g,皮山县最低为2.57 mg/g,这为石榴皮药材全面的质量控制提供了实验依据。
4.借助分子生物学平台,首次采用RAPD指纹图谱技术,对新疆不同产地的石榴进行品种研究,结果表明该技术能较好地用于分析种内或种下样品的遗传距离,为解决种下分类鉴定疑点问题提供DNA分子水平的证据。
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.
Methods
1.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.
植物雌激素作为雌激素的替代品,以其安全性及显著的效果,受到越来越多的关注,从天然药物中筛选具有雌激素作用的药物,并进一步筛选其有效成分是实际可行的。针对新疆石榴品种分类相对混乱现象,对现有石榴品种准确鉴定和合理分类是进一步利用石榴资源的基础。
目的:
利用人乳腺癌细胞MCF-7细胞增殖实验与酵母双杂交实验对新疆石榴皮等18种天然药物的植物雌激素作用进行了初步筛选,并对其中具有雌激素活性的新疆石榴皮进行了化学成分的研究。采用分子标记技术和化学分析手段相结合的方法,阐明石榴的分子特征和化学特征,从而为鉴别新疆石榴品种提供实验依据。
方法:
研究内容分为三个部分:
1.采用人乳腺癌细胞MCF-7细胞增殖实验与酵母双杂交(Yeast two-hybrid)实验对新疆石榴皮、罗勒、红景天、阿里红、鹰嘴豆、枸杞、肉苁蓉、琐阳、鼠尾草、当归、川芎、芍药、桃仁、牡丹皮、桂枝、泽泻、白术、茯苓等18种天然药物进行植物雌激素活性的筛选。
2.采用水提取、系统溶剂萃取,分别进行Diaion HP-20、MCI Gel CHP-20、Sephadex LH-20、硅胶等柱层析及制备薄层层析,提取新疆石榴皮样品中的化学成分,并运用HPLC法测定鞣质类成分的含量。
3.选取不同随机引物对不同产地的23个石榴样本进行RAPD指纹图谱分析。优化RAPD条件后,从中选出多态性高、重复性好的引物对不同样本进行检测,并根据条带的差异进行聚类分析,构建不同石榴品种的RAPD指纹图谱。
结果:
1.MCF-7细胞增殖实验结果表明:当归、川芎、芍药、白术、牡丹皮、鹰嘴豆、新疆石榴皮、罗勒、泽泻、桂枝水提取物可促使ER(+)MCF-7细胞的增殖(P<0.05),提示它们具有植物雌激素样活性;酵母双杂交(Yeast two-hybrid)系统实验结果表明:当归、川芎、牡丹皮、鹰嘴豆、石榴皮、泽泻、桂枝、水提取物均能使酵母系统中的β-半乳糖苷酶的活性增强(P<0.05),提示它们具有植物雌激素样活性。
2.从新疆石榴皮中提取分离结果得到13个化合物。根据化合物的理化性质及UV、IR、1H-NMR、13C-NMR等波谱方法,鉴定了10个化合物的结构,分别为:没食子酸(gallic acid)、没食子甲酯(methyl gallate)、鞣花酸(ellagic acid)、(+)-儿茶素[(+)catechin]、异槲皮苷(isoquerecitrin)、D-甘露醇(D-mannitol)、熊果酸(ursolic acid)、齐墩果酸(Oleanolic acid)、β-谷甾醇(β-Sitosterol)、胡萝卜苷(Daucosterol);并运用HPLC方法测定新疆5个石榴主产地中没食子酸含量为疏附县:3.26mg/g、疏勒县:4.53mg/g、叶城:3.86mg/g、皮山县:2.57mg/g、策勒县:2.94mg/g。结果表明不同产地的化学成分的含量具有明显差异。
3.CATBⅠ法提取23份石榴基因组DNA能扩增出清晰的多态性带,确定了适合石榴RAPD反应的反应体系为:10×buffer:2.5μl;dNTP(10mM):0.5μl;引物(10μM):5μl;MgCl2(25mmol/l):0.5μl;Taq DNA聚合酶:0.8μl(2.5μ/μl);模板DNA(约25ng/μl):1μl;总体积25μl;剩余的用ddH2O来补充;确定了适合石榴RAPD反应的扩增程序为:94℃预变性4 min;94℃变性1min,37℃退火1min,72℃延伸2 min,40循环;72℃总延伸7min后在4℃终止反应。从50个引物中筛选出能稳定扩增的7个引物ZY1、ZY4、ZY12、ZY23、ZY30、ZY41、ZY42,用这些引物对23个石榴品种进行扩增,共扩增出88条带,61条具有多态性,多态性比例为69%。
对新疆石榴23个品种进行了RAPD扩增,根据23个品种的Nei相似系数进行聚类分析的结果:能将23个品种聚为5类:第1类包括库尔勒市甜石榴与喀什市大籽甜石榴等甜石榴品种;第2类是阿图什市酸石榴;第3类包括喀什市阿奇克阿娜尔、叶城酸石榴等酸石榴品种;第4类包括吐鲁番市甜石榴、库车酸甜石榴等种间杂交和来源不详品种;第5类包括皮亚曼石榴和策勒1号等新疆优质石榴品种。本研究结果表明:石榴品种间的遗传多态性有明显差异,即使父母本相同的品种,也能分开。
结论:
1.首次采用人乳腺癌细胞MCF-7细胞增殖实验和酵母双杂交技术,对18种天然药物进行植物雌激素活性的筛选。结果表明新疆石榴皮、罗勒、鹰嘴豆、当归、川芎、芍药、白术、牡丹皮、泽泻、桂枝等7种天然药物的水提物在两种检测方法中均表现出植物雌激素样活性,为今后雌激素类植物资源的开发提供了理论支持。
2.首次对新疆石榴皮进行了化学成分研究,从中分离得到13个化合物,鉴定了其中10个化合物的结构,依次为没食子酸、没食子甲酯、鞣花酸、儿茶素、异槲皮苷、齐墩果酸、熊果酸、β-谷甾醇、胡萝卜苷。所有的化合物均为从新疆甜石榴中首次获得。本研究填补了新疆石榴皮化学成分研究的空白。
3.首次利用HPLC定量方法对5个新疆主产区的石榴样品的没食子酸含量进行了测定,结果显示:5个产区的石榴样品的没食子酸含量不同,其中疏勒县最高为4.53 mg/g,皮山县最低为2.57 mg/g,这为石榴皮药材全面的质量控制提供了实验依据。
4.借助分子生物学平台,首次采用RAPD指纹图谱技术,对新疆不同产地的石榴进行品种研究,结果表明该技术能较好地用于分析种内或种下样品的遗传距离,为解决种下分类鉴定疑点问题提供DNA分子水平的证据。
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.
Methods
1.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.
引文
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