Pharmacognostical studies on Phyllanthus in Taiwan

• Main Authors: Lee Chao-Ying, 李昭瑩
• Other Authors: Chen Chung-Chuan
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/08837730059561390233

博士 === 中國醫藥大學 === 中國藥學研究所 === 93 === The genus Phyllanthus (Euphorbiaceae) has a great number of species, contains over 700 species, distributed in the tropical, subtropical and temperate areas. These species are widely distributed all over Taiwan at low altitude areas. According to the reports, the Phyllanthus plants consist of fourteen species in Taiwan, i.e. P. acidus, P. amarus, P. debilis, P. embergeri, P. emblica, P. hookeri, P. multiflorus, P. myrtifolius, P. oligospermus, P. tenellus, P. urinaria subsp. nudicarpus, P. urinaria subsp. urinaria, P. ussuriensis, P. virgatus. They are various but resemble in morphology that lead people confused and difficult to distinguish from each other. There are no obvious side effects and toxicity. For developing and using these resources, this study carried out to identify the histological characteristics of the stems and leaves in different species of Phyllanthus. The microscopic structure of stems indicated that P. emblica and P. myrtifolius had non-glandular hairs, all Phyllanthus species had bast fiber cells in the cortex layer except P. myrtifolius and P. oligospermus. P. debilis, P. emblica, P. tenellus, P. virgatus contained clustered crystals, and P. urinaria subsp. urinaria contained single crystals. The study on histological anatomy of leaves showed P. embergeri, P. hookeri, P. myrtifolius, P. urinaria subsp. nudicarpus, P. urinaria subsp. urinaria, P. ussuriensis and P. virgatus had non-glandular hairs. P. acidus had clustered crystals and single crystals, P. emblica, P. multiflorus and P. tenellus had single crystals, P. amarus, P. debilis, P. embergeri, P. hookeri, P. myrtifolius, P. oligospermus, P. urinaria subsp. nudicarpus and P. urinaria subsp. urinaria had clustered crystals. The differences among the morphology and histological anatomy structure of stems and leaves, it can provide an useful method to identify Phyllanthus plants. We set up a quantitative method to determine the quantity of gallic acid (GA). This method has a high degree of sensitivity, reproducibility, precision and recovery rate. In order to ensure the quality of the Phyllanthus, we can apply this method to evaluate the quality of these plants. We sampled several different Phyllanthus plants in Taiwan, and measured one of the major component - GA via high-performance liquid chromatography（HPLC）method. The result shows a great range of GA contained in these samples, although the quantity of GA in these samples varies in accordance with the place, season and organ of plant origin. The samples collected in southern Taiwan during summer contained more GA than samples from other place and other seasons, the GA content of Phyllanthus was the highest in the leaves than other parts. By analysing the peak height of 40 Phyllanthus species described above by HPLC method. We discovered that the peak height ratio of the chosen 12 peaks,it can distinguish Phyllanthus successfully. The entire internal transcribed spacer（ITS）region between 18s and 26S ribosomal DNA of the thirteen Phyllanthus species were amplified by polymerase chain reaction（PCR）. The primers for PCR,18D: 5'-CACACCGCCCGTCGCTCCTACCGA-3'and 28cc: 5'-ACTCGCCGTTACTAGGTGAA-3' were designed for amplification and sequencing. The length of PCR product was 830〜840 bp. Comparing the obtained sequence with the sequence of ITS from other higher plant species showed that the cloned sequence contained 87〜98 bp of 26S rDNA, ITS region, and 152〜154 bp of 18S rDNA. The length of ITS region was 580〜590 in Phyllanthus species,including 219〜224 bp of ITS1,163〜164 bp of 5.8S rDNA, and 197〜206 bp of ITS2. The G+C contents of ITS1, 5.8S rDNA and ITS2 regions were 45.7〜58.4, 53〜54.6 and 45.1〜54.9%, respectively. In this study,the sequence similarity of the rDNA region between Phyllanthus species were 79〜98%. It can provide a clear and definite evidence to identify Phyllanthus species. The effect of oral administration of Phyllanthus methanolic extracts (PME) or GA on the progression of acute liver damage induced by CCl4 in rats was examined by morphological and biochemical methods. With 0.5 g/kg dose, P. acidus, P. urinaria subsp. urinaria, GA, and with 1.0 g/kg dose, P. emblica, P. urinaria subsp. nudicarpus attenuated CCl4-induced increase in serum GOT. With 0.5 g/kg dose, P. acidus, P. urinaria subsp. nudicarpus, P. urinaria subsp. urinaria, GA, and with 1.0 g/kg dose, P. emblica, P. amarus, P. hookeri, P. tenellus attenuated CCl4-induced increase in serum GPT. Concurrently, P. acidus, P. multiflorus, P. embergeri, P. hookeri, P. tenellus and P. urinaria subsp. urinaria elevated the activity of liver reduced glutathione peroxidase (GSH-Px). Since the protective effects of P. acidus, P. emblica, P. myrtifolius, P. embergeri, P. urinaria subsp. nudicarpus, P. urinaria subsp. urinaria and GA correlate with a reduced liver infiltration and focal necrosis observed using histological method, these data demonstrate that P. acidus and P. urinaria subsp. urinaria possess potential hepatoprotective effect.