广西产红鱼眼叶有效成分及质量控制研究
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摘要
红鱼眼叶为大戟科叶下珠属植物无毛龙眼睛Phyllanthus reticulatus Poir. var. glaber Muell. Arg或龙眼睛Phyllanthus reticulatus Poir.的干燥叶,其味微涩,性平,归肝经,具有祛风、利湿、活血之功效,广西民间用于治疗糖尿病及其胃肠功能紊乱并发症、痢疾、肝炎等症,己被《广西壮族自治区壮药质量标准》(2008年版)收载。现代研究表明,红鱼眼叶具有一定的降血糖、抗肿瘤及抑菌等作用。但目前对红鱼眼叶化学成分、药效学研究的公开报道较少,其有效部位或有效成分尚不明确；现有的质量控制方法尚不完善。为了进一步合理开发利用红鱼眼叶药材,寻找其有效成分,揭示其降血糖、抗肿瘤及抑菌作用物质基础,并为药材质量标准的制订提供科学依据,本课题以药效学为指导,采用现代化学分离分析技术和手段对红鱼眼叶有效部位及有效化学成分、药材质量控制方法进行了系统研究。
化学成分预试验结果表明红鱼眼叶中可能含有蛋白质、糖类、鞣质、有机酸、黄酮类、酚类、蒽醌类、香豆素与内酯类、甾体类、三萜类、挥发油及油脂等成分。以不同极性溶剂对红鱼眼叶80%乙醇总提物进行部位分离后,采用柱色谱方法进行系统的化学成分分离与纯化,运用熔点测定、MS、1H-NMR、13C-NMR等方法进行结构鉴定。本课题从红鱼眼叶的石油醚提取物、乙酸乙酯提取物、正丁醇提取物中分离并鉴定了14个化合物,分别为：木栓酮(friedelin,1)、正三十烷醇(1-triacontanol,2)、羽扇豆醇(lupeol,3)、B-谷甾醇(β-sitosterol,4)、豆甾醇(stigmasterol,5)、21a-羟基木栓烷-3-酮(21a-hydroxyfriedelan-3-one,6)、3β-hydroxy-D:A-friedooleanan-27-oic acid(trichadenic acid B,7)、(?)果酸(ursolic acid,8)、B-胡萝卜苷(β-daucosterol,9)、没食子酸乙酯(ethyl gallate,10)、柯里拉京(corilagin,11)、鞣花酸(ellagic acid,12)、短叶苏木酚酸乙酯(ethyl brevifolincarboxylate,13)、没食子酸(gallic acid,14)。其中,化合物2、7-14均为首次从该植物中分得。
为阐明红鱼眼叶确切的药理作用,本课题以小鼠进行急性毒性试验,测得80%乙醇总提物的LDso为44.2g生药/kg,从而确定动物给药剂量；再对红鱼眼叶80%乙醇总提物、石油醚提取物、乙酸乙酯提取物、正丁醇提取物对链脲佐菌素所致糖尿病小鼠血糖的作用进行观察,结果表明,红鱼眼叶80%乙醇总提物、乙酸乙酯提取物均具有明显的降血糖作用。采用MTT法测定红鱼眼叶石油醚提取物、乙酸乙酯提取物、正丁醇提取物的含药血清分别对人肝癌细胞BEL-7404、人胃癌细胞SGC-7901增殖的抑制情况,结果表明,红鱼眼叶乙酸乙酯提取物、正丁醇提取物具有一定的体外抗肿瘤作用。采用琼脂平板打孔法和二倍稀释法测定红鱼眼叶80%乙醇总提物、石油醚提取物、乙酸乙酯提取物、正丁醇提取物的抑菌活性,结果表明,红鱼眼叶80%乙醇总提物、乙酸乙酯提取物、正丁醇提取物对金黄色葡萄球菌、金黄色葡萄球菌耐药株、宋内氏痢疾杆菌、绿脓杆菌、伤寒杆菌、大肠杆菌的生长均具有不同程度的抑制作用。以上结果提示乙酸乙酯提取物为红鱼眼叶降血糖有效部位；乙酸乙酯提取物、正丁醇提取物为红鱼眼叶抗肿瘤、抑菌有效部位。
为了寻找红鱼眼叶中抗肿瘤及抑菌有效成分,本课题对红鱼眼叶抗肿瘤及抑菌有效部位中分离得到的4个化合物：没食子酸、柯里拉京、没食子酸乙酯和短叶苏木酚酸乙酯进行抗肿瘤及抑菌活性的筛选。采用MTT法分别测定了4个化合物分别对人肝癌细胞BEL-7404和人胃癌细胞SGC-7901增殖的抑制情况,结果表明,没食子酸、柯里拉京、短叶苏木酚酸乙酯具有一定的体外抗肿瘤作用。采用二倍稀释法分别测定4个化合物对金黄色葡萄球菌、金黄色葡萄球菌耐药株、宋内氏痢疾杆菌、绿脓杆菌、伤寒杆菌、大肠杆菌生长的抑制作用,结果表明,没食子酸、柯里拉京、短叶苏木酚酸乙酯、没食子酸乙酯均有一定的抑菌作用。以上结果提示没食子酸、柯里拉京、短叶苏木酚酸乙酯为红鱼眼叶抗肿瘤、抑菌作用的主要有效成分。
本课题以广西不同产地的15批红鱼眼叶药材为研究对象,首次通过鉴别、检查、含量测定、指纹图谱等对红鱼眼叶药材的质量控制方法进行了系统研究。采用性状与显微鉴别法对红鱼眼的两种植物来源——无毛龙眼睛和龙眼睛的茎、叶进行对比鉴别研究。采用TLC法对红鱼眼叶中的没食子酸、柯里拉京进行鉴别研究,结果表明,该法简便、专属性较强。采用IR法鉴别红鱼眼叶药材,结果表明,广西不同产地红鱼眼叶药材的IR图谱相似,并确定了5个共有峰,可为药材的快速鉴别提供依据。采用GC-MS法分析红鱼眼叶的挥发油成分,结果表明,广西不同产地红鱼眼叶挥发油中主要含有植醇(9.17-48.90%)、植酮(2.09-20.90%)、法尼基丙酮(2.54-9.93%)、香叶基丙酮(0.31-6.77%)、异植醇(0.86-5.70%)、B-紫罗兰酮(0.67-4.92%)等成分。广西不同产地红鱼眼叶有关检查结果表明,水溶性浸出物为8.76-16.83%,醇溶性浸出物为6.26-10.88%,总灰分为4.93-9.99%,酸不溶性灰分为0.11-0.54%,水分为10.83-12.02%。采用HPLC法测定了红鱼眼叶中没食子酸、柯里拉京和鞣花酸的含量,结果表明,广西不同产地红鱼眼叶中没食子酸含量为2.138-4.201mg/g,柯里拉京含量为15.349-29.333mg/g,鞣花酸含量为2.903-8.009mg/g。采用HPLC法对红鱼眼叶药材进行了指纹图谱的研究,结果表明,广西不同产地红鱼眼叶药材化学组成相似,其相对比例较稳定,建立的指纹图谱共标定19个主要共有峰。
综上所述,本课题从石油醚提取物、乙酸乙酯提取物、正丁醇提取物中共分离鉴定了14个化合物,其中9个化合物首次从红鱼眼中分得。首次确定了红鱼眼叶乙酸乙酯提取物为降血糖有效部位；乙酸乙酯提取物、正丁醇提取物为红鱼眼叶抗肿瘤、抑菌有效部位。首次确定了没食子酸、柯里拉京、短叶苏木酚酸乙酯为红鱼眼叶抗肿瘤及抑菌作用的主要有效成分。通过生药、TLC、IR、GC-MS鉴别；浸出物、水分及灰分检查；含量测定；指纹图谱等,首次对红鱼眼叶药材质量控制方法进行系统研究。本课题为最终揭示红鱼眼叶降血糖、抗肿瘤、抑菌作用及其物质基础,建立药材质量的科学评价方法,实现药材可持续开发与利用提供了科学依据。
The leaves of Phyllanthus reticulatus Poir. var. glaber Muell. Arg. or Phyllanthus reticulatus Poir. are from Phyllanthus. The drug is mild, slightly bitter. It converges to liver channel. Its actions include dispelling wind, removing dampness and activating blood. It is used in folk medicinal practices of Guangxi for treatment of diabetes mellitus and gastrointestinal disorders of diabetics, shigella, hepatitis, etc. It has been embodied in 《Quality Standard for Zhuang Materia Medica of Guangxi Zhuang Autonomous Region》(2008edition). The mordern researches show that the leaves of Phyllanthus reticulatus possesse the hypoglycemic, antitumor and antibacterial activities. But researches on the plant about chemical constituents and pharmacology have been hardly reported. In order to obtain more comprehensive understanding of its effective components, reveal the substantial foundation of hypoglycemic, antitumor and antibacterial effects and provide scientific basis for establishing quality standard, the systematieally studies on the chemical components of effective fractions, and quality control methods have been carried out.
The pre-experiment of chemical components shows that the leaves of Phyllanthus reticulatus may contain protein, saccharides, tannins, organic acids, flavonoids, phenols, anthraquinones, coumarins and lactones, steroids, triterpenoids, volatile oil, etc. The80%ethanol extract obtained from the dried leaves have been divided into three fractions: petroleum ether extract, ethyl acetate extract and n-butanol extract. Compounds have been seperated and purified by chromatographic methods and identified by melting point determin-ation, MS,1H-NMR,13C-NMR, etc.14compounds have been isolated and identified from the leaves of Phyllanthus reticulatus:friedelin(1),1-triacontanol(2), lupeol(3), β-sitosterol(4), stigmasterol(5),21a-hydroxyfriedelan-3-one(6),3β-hydroxy-D:A-friedooleanan-27-oic acid (7), ursolic acid(8),β-daucosterol(9), ethyl gallate(10), corilagin(11), ellagic acid(12), ethyl brevifolincarboxylate(13), gallic acid(14). The compound2,7～14have been obtained from this plant for the first time.
The acute toxicity test of mice has been done to the80%ethanol extract of the leaves of Phyllanthus reticulatus, and LD50is44.2g drug/kg. The effects of the80%ethanol extract, petroleum ether extract, ethyl acetate extract and n-butanol extract on experimental diabetic mice induced by streptozotocin have been observed. The results indicate that80%ethanol extract and ethyl acetate extract have significant hypoglycemic effects. Antitumor experiments in vitro have been done by MTT assay to medicated serum from different extracts on human hepatoma cell BEL-7404, human gastric cancer cell SGC-7901. The results show that ethyl acetate extract and n-butanol extract have antitumor effects. Antibacterial activities of80%ethanol extract, petroleum ether extract, ethyl acetate extract and n-butanol extract have been tested in vitro by agar plate drilling test and double broth dilution method. The results display that80%ethanol extract, ethyl acetate extract and n-butanol extract have inhibition effects on growth of Staphylococcus aureus, Staphylococcus susceptive strain, Shigella sonnei, Pseudomonas aeruginosa, Salmonella enterica serovar Typhi, Escherichia coli. It can be infered that ethyl acetate extract is effective hypoglycemic fraction; ethyl acetate extract and n-butanol extract are antitumor and antibacterial fractions of the leaves of Phyllanthus reticulatus.
Furthermore, the antitumor and antibacterial effects in vitro of ethyl gallate, corilagin, ethyl brevifolincarboxylate, gallic acid have been investigated. The growth inhibition analyzed by MTT in the human hepatoma cell BEL-7404, human gastric cancer cell SGC-7901. The results show that corilagin, ethyl brevifolincarboxylate, gallic acid can inhibite the proliferation of tumor cells. Minimal inhibitory concentration was determined by double broth dilution method. The results indicate that corilagin, gallic acid and ethyl brevifolincarboxylate can inhibite the growth of Staphylococcus aureus, Staphylococcus susceptive strain, Shigella sonnei, Pseudomonas aeruginosa, Salmonella enterica serovar Typhi, Escherichia coli. It can be deduced that corilagin, gallic acid and ethyl brevifolincarboxylate are effective antitumor and antibacterial components of the leaves of Phyllanthus reticulatus.
For the first time,15batches leaves of Phyllanthus reticulatus have been analyzed to study quality control methods. The difference of stems and leaves bewteen Phyllanthus reticulatus Poir. var. glaber Muell. Arg. and Phyllanthus reticulatus Poir. has been studied by morphological and microscopical identification method. Corilagin and gallic acid have been identified by TLC which is simple and specific. The leaves of Phyllanthus reticulatus have been identified by IR. The results show the IR chromatograms of different samples are similar with5common peaks, and can provide basis for rapid identification. The volatile constituents from the leaves of Phyllanthus reticulatus have been analyzed by GC-MS. The results indicate the principal constituents are Phytol (9.17～48.90%),6,10,14-trimethyl-2-Pe-ntadecanon (2.09～20.90%),(5E,9E)-6,10,14-trimethyl-5,9,13-Pentadecatrien-2-one (2.54～9.93%),(E)-6,10-dimethylundeca-5,9-dien-2-one (0.31～6.77%), Isophytol (0.86～5.70%),4-(2,6,6-trimethyl--cyclohexen-l-yl)-3-Buten-2-one (0.67～4.92%), etc. The results of the examination show that the content of water extract, ethanol extract, total ash, acid insoluble ash and water is8.76～16.83%,6.26～10.88%,4.93～9.99%,0.11～0.54%,10.83～12.02%, respectively. The content of gallic acid, corilagin and ellagic acid in the leaves of Phyllanthus reticulatus is2.138～4.201mg/g,15.349～29.333mg/g,2.903～8.009mg/g respectively, which has been determined by HPLC. The research on HPLC fingerprint chromatogram of15batches leaves has been done. The mutual mode has been set up with19main common peaks. The chromatograms are similar, and the ratio of common peak's area of different samples is steady.
The research show firstly that ethyl acetate extract is effective hypoglycemic fraction; ethyl acetate extract and n-butanol extract are antitumor and antibacterial fractions of the leaves of Phyllanthus reticulates.14compounds have been identified and9compounds have been isolated from Phyllanthus reticulates for the first time. At the same time, the research also shows that corilagin, gallic acid and ethyl brevifolincarboxylate are effective antitumor and antibacterial components. For the first time, the study of quality control methods by pharmacognostic identification, TLC identification, IR identification, GC-MS identification; examination for extract, ash and water; HPLC content determination; HPLC fingerprint chromatogram has been done. The research provides the scientific basis to reveal the substantial foundation of hypoglycemic, antitumor and antibacterial effects, establish scientific evaluating method for drug quality, and achieve sustainable exploitation and utilization of Phyllanthus reticulatus.
化学成分预试验结果表明红鱼眼叶中可能含有蛋白质、糖类、鞣质、有机酸、黄酮类、酚类、蒽醌类、香豆素与内酯类、甾体类、三萜类、挥发油及油脂等成分。以不同极性溶剂对红鱼眼叶80%乙醇总提物进行部位分离后,采用柱色谱方法进行系统的化学成分分离与纯化,运用熔点测定、MS、1H-NMR、13C-NMR等方法进行结构鉴定。本课题从红鱼眼叶的石油醚提取物、乙酸乙酯提取物、正丁醇提取物中分离并鉴定了14个化合物,分别为：木栓酮(friedelin,1)、正三十烷醇(1-triacontanol,2)、羽扇豆醇(lupeol,3)、B-谷甾醇(β-sitosterol,4)、豆甾醇(stigmasterol,5)、21a-羟基木栓烷-3-酮(21a-hydroxyfriedelan-3-one,6)、3β-hydroxy-D:A-friedooleanan-27-oic acid(trichadenic acid B,7)、(?)果酸(ursolic acid,8)、B-胡萝卜苷(β-daucosterol,9)、没食子酸乙酯(ethyl gallate,10)、柯里拉京(corilagin,11)、鞣花酸(ellagic acid,12)、短叶苏木酚酸乙酯(ethyl brevifolincarboxylate,13)、没食子酸(gallic acid,14)。其中,化合物2、7-14均为首次从该植物中分得。
为阐明红鱼眼叶确切的药理作用,本课题以小鼠进行急性毒性试验,测得80%乙醇总提物的LDso为44.2g生药/kg,从而确定动物给药剂量；再对红鱼眼叶80%乙醇总提物、石油醚提取物、乙酸乙酯提取物、正丁醇提取物对链脲佐菌素所致糖尿病小鼠血糖的作用进行观察,结果表明,红鱼眼叶80%乙醇总提物、乙酸乙酯提取物均具有明显的降血糖作用。采用MTT法测定红鱼眼叶石油醚提取物、乙酸乙酯提取物、正丁醇提取物的含药血清分别对人肝癌细胞BEL-7404、人胃癌细胞SGC-7901增殖的抑制情况,结果表明,红鱼眼叶乙酸乙酯提取物、正丁醇提取物具有一定的体外抗肿瘤作用。采用琼脂平板打孔法和二倍稀释法测定红鱼眼叶80%乙醇总提物、石油醚提取物、乙酸乙酯提取物、正丁醇提取物的抑菌活性,结果表明,红鱼眼叶80%乙醇总提物、乙酸乙酯提取物、正丁醇提取物对金黄色葡萄球菌、金黄色葡萄球菌耐药株、宋内氏痢疾杆菌、绿脓杆菌、伤寒杆菌、大肠杆菌的生长均具有不同程度的抑制作用。以上结果提示乙酸乙酯提取物为红鱼眼叶降血糖有效部位；乙酸乙酯提取物、正丁醇提取物为红鱼眼叶抗肿瘤、抑菌有效部位。
为了寻找红鱼眼叶中抗肿瘤及抑菌有效成分,本课题对红鱼眼叶抗肿瘤及抑菌有效部位中分离得到的4个化合物：没食子酸、柯里拉京、没食子酸乙酯和短叶苏木酚酸乙酯进行抗肿瘤及抑菌活性的筛选。采用MTT法分别测定了4个化合物分别对人肝癌细胞BEL-7404和人胃癌细胞SGC-7901增殖的抑制情况,结果表明,没食子酸、柯里拉京、短叶苏木酚酸乙酯具有一定的体外抗肿瘤作用。采用二倍稀释法分别测定4个化合物对金黄色葡萄球菌、金黄色葡萄球菌耐药株、宋内氏痢疾杆菌、绿脓杆菌、伤寒杆菌、大肠杆菌生长的抑制作用,结果表明,没食子酸、柯里拉京、短叶苏木酚酸乙酯、没食子酸乙酯均有一定的抑菌作用。以上结果提示没食子酸、柯里拉京、短叶苏木酚酸乙酯为红鱼眼叶抗肿瘤、抑菌作用的主要有效成分。
本课题以广西不同产地的15批红鱼眼叶药材为研究对象,首次通过鉴别、检查、含量测定、指纹图谱等对红鱼眼叶药材的质量控制方法进行了系统研究。采用性状与显微鉴别法对红鱼眼的两种植物来源——无毛龙眼睛和龙眼睛的茎、叶进行对比鉴别研究。采用TLC法对红鱼眼叶中的没食子酸、柯里拉京进行鉴别研究,结果表明,该法简便、专属性较强。采用IR法鉴别红鱼眼叶药材,结果表明,广西不同产地红鱼眼叶药材的IR图谱相似,并确定了5个共有峰,可为药材的快速鉴别提供依据。采用GC-MS法分析红鱼眼叶的挥发油成分,结果表明,广西不同产地红鱼眼叶挥发油中主要含有植醇(9.17-48.90%)、植酮(2.09-20.90%)、法尼基丙酮(2.54-9.93%)、香叶基丙酮(0.31-6.77%)、异植醇(0.86-5.70%)、B-紫罗兰酮(0.67-4.92%)等成分。广西不同产地红鱼眼叶有关检查结果表明,水溶性浸出物为8.76-16.83%,醇溶性浸出物为6.26-10.88%,总灰分为4.93-9.99%,酸不溶性灰分为0.11-0.54%,水分为10.83-12.02%。采用HPLC法测定了红鱼眼叶中没食子酸、柯里拉京和鞣花酸的含量,结果表明,广西不同产地红鱼眼叶中没食子酸含量为2.138-4.201mg/g,柯里拉京含量为15.349-29.333mg/g,鞣花酸含量为2.903-8.009mg/g。采用HPLC法对红鱼眼叶药材进行了指纹图谱的研究,结果表明,广西不同产地红鱼眼叶药材化学组成相似,其相对比例较稳定,建立的指纹图谱共标定19个主要共有峰。
综上所述,本课题从石油醚提取物、乙酸乙酯提取物、正丁醇提取物中共分离鉴定了14个化合物,其中9个化合物首次从红鱼眼中分得。首次确定了红鱼眼叶乙酸乙酯提取物为降血糖有效部位；乙酸乙酯提取物、正丁醇提取物为红鱼眼叶抗肿瘤、抑菌有效部位。首次确定了没食子酸、柯里拉京、短叶苏木酚酸乙酯为红鱼眼叶抗肿瘤及抑菌作用的主要有效成分。通过生药、TLC、IR、GC-MS鉴别；浸出物、水分及灰分检查；含量测定；指纹图谱等,首次对红鱼眼叶药材质量控制方法进行系统研究。本课题为最终揭示红鱼眼叶降血糖、抗肿瘤、抑菌作用及其物质基础,建立药材质量的科学评价方法,实现药材可持续开发与利用提供了科学依据。
The leaves of Phyllanthus reticulatus Poir. var. glaber Muell. Arg. or Phyllanthus reticulatus Poir. are from Phyllanthus. The drug is mild, slightly bitter. It converges to liver channel. Its actions include dispelling wind, removing dampness and activating blood. It is used in folk medicinal practices of Guangxi for treatment of diabetes mellitus and gastrointestinal disorders of diabetics, shigella, hepatitis, etc. It has been embodied in 《Quality Standard for Zhuang Materia Medica of Guangxi Zhuang Autonomous Region》(2008edition). The mordern researches show that the leaves of Phyllanthus reticulatus possesse the hypoglycemic, antitumor and antibacterial activities. But researches on the plant about chemical constituents and pharmacology have been hardly reported. In order to obtain more comprehensive understanding of its effective components, reveal the substantial foundation of hypoglycemic, antitumor and antibacterial effects and provide scientific basis for establishing quality standard, the systematieally studies on the chemical components of effective fractions, and quality control methods have been carried out.
The pre-experiment of chemical components shows that the leaves of Phyllanthus reticulatus may contain protein, saccharides, tannins, organic acids, flavonoids, phenols, anthraquinones, coumarins and lactones, steroids, triterpenoids, volatile oil, etc. The80%ethanol extract obtained from the dried leaves have been divided into three fractions: petroleum ether extract, ethyl acetate extract and n-butanol extract. Compounds have been seperated and purified by chromatographic methods and identified by melting point determin-ation, MS,1H-NMR,13C-NMR, etc.14compounds have been isolated and identified from the leaves of Phyllanthus reticulatus:friedelin(1),1-triacontanol(2), lupeol(3), β-sitosterol(4), stigmasterol(5),21a-hydroxyfriedelan-3-one(6),3β-hydroxy-D:A-friedooleanan-27-oic acid (7), ursolic acid(8),β-daucosterol(9), ethyl gallate(10), corilagin(11), ellagic acid(12), ethyl brevifolincarboxylate(13), gallic acid(14). The compound2,7～14have been obtained from this plant for the first time.
The acute toxicity test of mice has been done to the80%ethanol extract of the leaves of Phyllanthus reticulatus, and LD50is44.2g drug/kg. The effects of the80%ethanol extract, petroleum ether extract, ethyl acetate extract and n-butanol extract on experimental diabetic mice induced by streptozotocin have been observed. The results indicate that80%ethanol extract and ethyl acetate extract have significant hypoglycemic effects. Antitumor experiments in vitro have been done by MTT assay to medicated serum from different extracts on human hepatoma cell BEL-7404, human gastric cancer cell SGC-7901. The results show that ethyl acetate extract and n-butanol extract have antitumor effects. Antibacterial activities of80%ethanol extract, petroleum ether extract, ethyl acetate extract and n-butanol extract have been tested in vitro by agar plate drilling test and double broth dilution method. The results display that80%ethanol extract, ethyl acetate extract and n-butanol extract have inhibition effects on growth of Staphylococcus aureus, Staphylococcus susceptive strain, Shigella sonnei, Pseudomonas aeruginosa, Salmonella enterica serovar Typhi, Escherichia coli. It can be infered that ethyl acetate extract is effective hypoglycemic fraction; ethyl acetate extract and n-butanol extract are antitumor and antibacterial fractions of the leaves of Phyllanthus reticulatus.
Furthermore, the antitumor and antibacterial effects in vitro of ethyl gallate, corilagin, ethyl brevifolincarboxylate, gallic acid have been investigated. The growth inhibition analyzed by MTT in the human hepatoma cell BEL-7404, human gastric cancer cell SGC-7901. The results show that corilagin, ethyl brevifolincarboxylate, gallic acid can inhibite the proliferation of tumor cells. Minimal inhibitory concentration was determined by double broth dilution method. The results indicate that corilagin, gallic acid and ethyl brevifolincarboxylate can inhibite the growth of Staphylococcus aureus, Staphylococcus susceptive strain, Shigella sonnei, Pseudomonas aeruginosa, Salmonella enterica serovar Typhi, Escherichia coli. It can be deduced that corilagin, gallic acid and ethyl brevifolincarboxylate are effective antitumor and antibacterial components of the leaves of Phyllanthus reticulatus.
For the first time,15batches leaves of Phyllanthus reticulatus have been analyzed to study quality control methods. The difference of stems and leaves bewteen Phyllanthus reticulatus Poir. var. glaber Muell. Arg. and Phyllanthus reticulatus Poir. has been studied by morphological and microscopical identification method. Corilagin and gallic acid have been identified by TLC which is simple and specific. The leaves of Phyllanthus reticulatus have been identified by IR. The results show the IR chromatograms of different samples are similar with5common peaks, and can provide basis for rapid identification. The volatile constituents from the leaves of Phyllanthus reticulatus have been analyzed by GC-MS. The results indicate the principal constituents are Phytol (9.17～48.90%),6,10,14-trimethyl-2-Pe-ntadecanon (2.09～20.90%),(5E,9E)-6,10,14-trimethyl-5,9,13-Pentadecatrien-2-one (2.54～9.93%),(E)-6,10-dimethylundeca-5,9-dien-2-one (0.31～6.77%), Isophytol (0.86～5.70%),4-(2,6,6-trimethyl--cyclohexen-l-yl)-3-Buten-2-one (0.67～4.92%), etc. The results of the examination show that the content of water extract, ethanol extract, total ash, acid insoluble ash and water is8.76～16.83%,6.26～10.88%,4.93～9.99%,0.11～0.54%,10.83～12.02%, respectively. The content of gallic acid, corilagin and ellagic acid in the leaves of Phyllanthus reticulatus is2.138～4.201mg/g,15.349～29.333mg/g,2.903～8.009mg/g respectively, which has been determined by HPLC. The research on HPLC fingerprint chromatogram of15batches leaves has been done. The mutual mode has been set up with19main common peaks. The chromatograms are similar, and the ratio of common peak's area of different samples is steady.
The research show firstly that ethyl acetate extract is effective hypoglycemic fraction; ethyl acetate extract and n-butanol extract are antitumor and antibacterial fractions of the leaves of Phyllanthus reticulates.14compounds have been identified and9compounds have been isolated from Phyllanthus reticulates for the first time. At the same time, the research also shows that corilagin, gallic acid and ethyl brevifolincarboxylate are effective antitumor and antibacterial components. For the first time, the study of quality control methods by pharmacognostic identification, TLC identification, IR identification, GC-MS identification; examination for extract, ash and water; HPLC content determination; HPLC fingerprint chromatogram has been done. The research provides the scientific basis to reveal the substantial foundation of hypoglycemic, antitumor and antibacterial effects, establish scientific evaluating method for drug quality, and achieve sustainable exploitation and utilization of Phyllanthus reticulatus.
引文
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