Studies On Effective Components And Quality Control Methods Of Leaves Of Phyllanthus Reticulatus In Guangxi

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.