Studies On Pharmacognosy Of Ardisia Primulaefolia,Ardisia Mamillata And Antitumor Activity

Ardisia primulaefolia and Ardisia mamillata are plants of Myrsinaceae Ardisia, and are both mainly from in China. These two plants widely distributed in China and are in the state of natural growth; and there are still no relevant reports about the study of the two plants’growth status, environmental conditions, the quality of medicine, chemical composition and the active parts.The aim of this study is to provide guides line to botany, varietal identification, quality standard setting up and chemistry component research of Ardisia primulaefolia and Ardisia mamillata, and in vitro anti-tumor activity study on different extraction of Ardisiaprimulaefolia.Objective:The aim of this study is to provide guide line to varietal identification, quality control standard setting up and chemistry component research through botany, pharmacognosy, drug quality and chemistry component research of Ardisia primulaefolia and Ardisia mamillata, and give valuable ideas to discovery of new anti-tumor drug by study the vitro anti-tumor effect of different extracts of Ardisia primulaefolia fox the first time. Methods:1、Using experimental methods of pharmaceutical botany, pharmaceutical plant cultivation and pharmacognosy to study the biological characteristics of Ardisia primulaefolia and Ardisia mamillata,and observing the appearance and microstructure of the two plants. According to the quality standard of Chinese medicine in Chinese Drug2010, moisture, ash and alcoholic solvent extraction of Ardisia primulaefolia and Ardisia mamillata are measured. The chemistry component is identified using chemistry component experiment system and total saponin content is measured using ultraviolet and visible spectrophotometry.2、Anti-tumor effect of HeLa cell line and A549cell line is studied using MTT method. Morphological changes of apoptosis was observed under microscope;and apoptosis rate and cell cycle of HeLa cells and A549cells of different extraction fraction of Ardisia primulaefolia are detected by flow cytometry. In order to observe the effect of NBE to cervical cancer, the xenografts cervical cancer model in nude mice was successful established by adopting hypodermic inoculating with HeLa cells.Results:1、Ardisia primulaefolia and Ardisia mamillata of Guangxi province are mainly distributed in Darning Mountain Nature Reserve, Nanning City and Gulongshan Nature Reserve, Baise City, Longsheng huaping Nature Reserve, Guilin, and some original recorded distribution area are extinguished now. Studies of Medicinal Plant show that Ardisia primulaefolia and Ardisia mamillata are essentially similar in biological characteristics.2、Ardisia primulaefolia and Ardisia mamillata are very similar in morphology, but Ardisia mamillata have creeping woody rhizomes, and its leaves do not form a rosette, the hair at the base of the leaf surface is uplift, like a small tumor. Transverse microscopic study shows the main characteristics of two plants. The roots’structure of transverse section shows that Ardisia primulaefolia’ cortex much wider than Ardisia mamillata, and they both contain secretory cavities; their cambium layer is obvious, pith broad, and containing secretory cavities. The stem sructure of transverse section shows that Ardisia primulaefolia’ cortex contain fewer secretory cavities, but the two plants’cambium layers are both not obvious, pith broader, and with secretory cavities. Leaves sructure of transverse section of Ardisia primulaefolia and Ardisia mamillata are similar.3、The moisture content of Ardisia primulaefolia and Ardisia mamillata is between6.01%-8.68%. Among roots, stems and leaves parts of herbs, roots contain the highest moisture content, leaves contain the lowest moisture content; roots and stems of Ardisia mamillata both have higher moisture content. The total ash content of the two plants is between2.45%-10.67%, the roots, stems, leaves’total ash content of Ardisia mamillata are higher than Ardisia primulaefolia. Two plants’alcohol extract content and water extracts are8.53%-14.44%and6.12%-21.40%, respectively; the roots, stems, leaves’alcohol extract and water extracts of Ardisia mamillata are both higher than Ardisia primulaefolia.4、Ardisia primulaefolia and Ardisia mamillata both possess polysaccharides, glycosides, amino acids, protein and peptide, saponins, tannins, phenols, quinones, flavonoids and the two plant possess different components, such ascoumarin, Lactone, alkaloids, cardiac glycosides, fats and oils. The total saponin content of Ardisia primulaefolia and Ardisia mamillata were determined, and Ardisia primulaefolia got higher total saponin content than Ardisia mamillata.5、Crude extract of Ardisia primulaefolia was fractionated into three fractions, petroleum ether extract,aqueous extract and n-butanol extract. The results of HeLa cell line and A549cell line using MTT assay show that aqueous extract and n-butanol extract both have effective antitumor potential. Cell viability of A549cell lineinhibited by A.primulaefolia crude extract is in a dose dependent growth, Cell viability of HeLa cell lineinhibited by A.primulaefolia crude extract is in a dose and time dependent growth. The cell morphous and cell cycle canbe induced by two fractions of A.primulaefolia, drug effect to cells can be observed under microscope compared with control group. The cell morphous of HeLa cell line and A549cell line was altered significantly and the cell density was decreased obviously by contrast phase microscope, and the cells number was decreased obviously. The flow cytometer results showed that the two fractions of A.primulaefolia could not only induce apoptosis, but also stoppage the cell cycle. The results showed that HeLa cell line and A549cell line can be blocked in G0/G1phasa by two fractions of A.primulaefolia.NBE has effective anti-tumor effect on xenografts cervical cancer in nude mice. After treatment by the NBE, the weight of the positive control group was slightly lower than the control group and treated group. Comparing to control group, the high-dose group and positive control group inhibited tumor growth in tumor-bearing mice significantly, and the difference is obvious;the medium and low-dose group have weaker inhibition of tumor growth. In a certain range, the inhibition rate of xenograft cervical cancer in nude mice by the treatment of NBE is in a dose-dependent manner.Conclusion:1. Ardisia primulaefolia and Ardisia mamillata are plants of Myrsinaceae Ardisia and they are both mainly from China, widely distributed, but the distribution of a small number of adult plants sharp drop in recent years; protecting the environment and selecting excellent plants to cultivate can optimize both plant germplasm, and the biological characteristics of the two plantsis basically similar to each other.2. The morphologic features and microscopic characteristics of Ardisia primulaefolia and Ardisia mamillata are similar. The roots’structure of transverse section shows that two plants’pith are both broad, and obvious.The difference is that the cortex of roots’Structure of transverse section of Ardisia primulaefolia’is much wider than Ardisia mamillata. The stem structure of transverse section shows that Ardisia primulaefolia’ cortex and pith both containa few secretory cavities.3. Moisture content results show that among the roots, stems, leavesparts of Ardisia primulaefolia and Ardisia mamillata,,roots have the highest moisture content, and leaves have the lowest moisture content; The three parts’total ash content of Ardisia mamillataare all higher than Ardisia primulaefolia. The roots, stems, leaves’alcohol extract and water extracts of Ardisia mamillataare all higher than Ardisia primulaefolia. Because of the characteristics of plant, sunshine time and other factors, the experiment data have some difference, but the relative standard deviation is very samll.4. Ardisia primulaefolia and Ardisia mamillatapossess a variety of common chemical composition, and some different chemical composition, such as cardiac glycosides, alkaloids, and so on. The total saponin content of Ardisia primulaefolia is much higher than Ardisia mamillata.5. The MTT assay results show that all three extractionpart of A.primulaefolia have effective antitumor potentialin a dose-dependent response on HeLa cell line and A549cell line. And comparing with control group, effect to cells of all three extraction part of A.primulaefolia can be observed under the microscope. The cell morphous of HeLa cell line and A549cell line was changed significantly. The flow cytometer results show that all extraction part of A.primulaefolia can induce apoptosis of HeLa cell line and A549cell line and can be blocked in G0/G1phasa. After treatment by the NBE, all NBE dose group have improved the general characteristics of tumor-bearing mice, the inhibition of the xenograft cervical cancer model in nude mice by NBE is expressed as decreasing of tumor volume.