Effect On Continuous Cropping Obstacle On Growth Of Polygonatum Odoratum And Its Polysaccharides On Antitumor Activity

Polygonatum odoratum is perennial herbs of the lily family. It is the dry rhizome of Polygonatum odoratum and the Hunan province of geoherbs, the country's largest production base in Hunan Shaodong polygonatum. In recent years, due to years of grow into a piece of Polygonatum odoratum, resulting in the serious diseases, reduce the yield of the crude drugs, the medicinal quality decline in.This phenomenon has caused great economic losses to the local Yaonong. At the same time, it has caused a serious threat to the sustainable development of the main producing areas of Polygonatum odoratum. But the existing research results, there is no research on the continuous cropping obstacle of Polygonatum odoratum. As a commonly used traditional Chinese medicine Polygonatum odoratum can nourishing lung yin, it is commonly used in the clinical treatment of lung cancer, esophageal cancer, stomach cancer and other tumors in the lung and stomach yin.Polygonatum odoratum polysaccharide is the main active ingredient in Polygonatum odoratum, studies have shown that Polygonatum Polysaccharide has obvious antitumor activity, but there is no report on the mechanism of anti-tumor activity.Therefore, the thesis is supported by the national science and Technology Department"Polygonatum, Fuling standardized planting and upgrading SOP". From Polygonatum allelopathy to begin to explore the reasons of continuous cropping obstacle of Polygonatum odoratum, in order to reveal the autotoxic mechanism of Polygonatum continuous cropping obstacles; it starts from the antitumor effect of Polygonatum odoratum, studied the antitumor activity of Polygonatum odoratum polysaccharide and its mechanism, It lays a foundation to overcome continuous cropping obstacles,promote the deep development of Polygonatum odoratum products and the Polygonatum odoratum industry sustainable development.In this paper, firstly, the seed germination and seedling growth of Polygonatum root exudates of allelopathy bioassay was carried out, then the GC-MS analytical technique of Polygonatum root exudates of isolation and identification based on, by exogenous addition method simulation of continuous cropping obstacles. By measuring the leaf photosynthetic parameters and protective enzyme activity and other physiological and biochemical indexes of allelopathic substances of Polygonatum odoratum seedlings in pot and plant growth in influence and Polygonatum odoratum rhizome yield and quality changes. Based, in human lung cancer A549 cells as target cells, using MTT method and FCM (flow cytometry) and immune group of method, ELISA and Western blot experiments, reveals the anti tumor activity of Polygonatum odoratum polysaccharide and its mechanism from the molecular level, cellular level and animal level three levels, to lay the foundation for the sustainable development of Polygonatum odoratum and the depth of the development.Objective:From Polygonatum odoratum of allelopathy began, and explores the reasons of continuous cropping obstacle of Polygonatum odoratum, reveal the mechanism of continuous cropping obstacle of Polygonatum odoratum possible autotoxicity induced; from Polygonatum odoratum and anti tumor effect of of Polygonatum odoratum polysaccharide anti-tumor activity and its mechanism, designed to overcome the continuous cropping obstacle of Polygonatum odoratum and promote products of Polygonatum odoratum depth development, polygonatum odoratum industry sustainable development lay the foundation.Method:1.Using the bioassay method to evaluate the allelopathic effects of root exudates of Polygonatum odoratum.2. Using GC-MS analysis technology of Polygonatum root exudates of isolation and identification,, the main component of exogenous method simulation of continuous cropping obstacles. By measuring the leaf photosynthetic parameters and protective enzyme activity and other physiological and biochemical indexes were observed in allelopathic effects of substances on the growth of Polygonatum odoratum seedlings in pot and the plant, the influence of Polygonatum odoratum rhizome yield and quality and content of polysaccharide with the coarse fraction changes.3. In human lung cancer A549 cells as target cells, using MTT assay, FCM (flow cytometry) and immune group of polymerase chain reaction (PCR) and experimental methods, from the molecular level, cellular level and animal level of different sources of Polygonatum odoratum polysaccharide anti-tumor activity and mechanism.4. Human lung cancer cell line A549 was inoculated into nude mice and randomly assigned to different treatments. The effect of tumor size and apoptosis was observed, the expression of Survivin and C-Myc, and the effect of beta-catenin pathway were observed.Results:1. Study on the effect of continuous cropping on the Allelopathy of Polygonatum odoratum Polygonatum odoratum rhizome water extraction liquid of the receptor (canola, corn, garlic) seed germination rate and radicle and plumule growth and there are obvious "promotion at low and high suppression" phenomenon, with the increase of the concentration, the root rot Fusarium solani has promotion effect. Polygonatum odoratum rhizosphere soil leaching extract could significantly inhibit the Polygonatum odoratum rhizome spore germination rate, seedling height and root fresh weight and with extraction solution concentration increased, enhanced inhibition. With the increase of concentration, polygonatum odoratum rhizome water immersion extract of garlic seed germination inhibition has the most significant effect (P<0.01), concentration of 0.025 and 0.05 on seed germination rate difference was not significant, had no statistical significance (P>0.05); to rape, maize root growth is obvious inhibitory effect. Extract the concentration of Polygonatum odoratum rhizome aqueous extract had strong inhibitory effect on the radicle length, on the root rot fungus eggplant Fusarium fungi hyphae growth plays a significant role in promoting. Provided liquid on the rhizome of hilum germination and seedling growth showed different inhibitory trend and with water extraction solution concentration increased, in the test of 0.g/ml concentration treatment, polygonatum odoratum sprouting rate, seedling height appear obvious growth inhibition of Polygonatum odoratum rhizosphere soil water leaching.2. Study on the correlation between physical and physiological activity of Polygonatum odoratum seedlings Polygonatum odoratum in rhizosphere soil of ethyl acetate extraction to 24 compounds were identified, including 16 acids,1 aldehydes, ketones, three lipids and two amide in solution. Arachidic acid, one of the fifteen alkyl acid, myristic acid are reported to be allelochemicals. Exogenous addition of different allelopathic substance to simulate continuous cropping stress, each treatment group between Polygonatum leaf chlorophyll a, chlorophyll b, total chlorophyll content difference significantly (P< 0.01). With increase of allelochemicals concentrations and between groups of chlorophyll a, chlorophyll b content was decreased; under the same concentration, mixed treatment groups of chlorophyll a content than a single sense of substances in the treatment group decreased significantly. Myristic acid in different concentrations of SOD activity in leaves of the fragrant solomonseal rhizome has the greatest impact; POD activity decreased with the increase of concentration in each treatment group, compared with CK, treatment of mixed group of POD activity of the weakest; pentadecanoic acid and 2-hydroxy ring pentadecanone on CAT activity difference was not significant (p> 0.05).3. Research on quality and the yield of Polygonatum cropping affecting Different allelochemicals treatment group of Polygonatum odoratum rhizome root fresh weight and root dry weight were significantly decreased (P< 0.01); 2-hydroxy ring pentadecanone treatment group with the concentration increased, root fresh weight and root dry weight change is not obvious (P> 0.05). Under the same high concentration conditions, compared with CK, the root fresh weight of each chemical treatment group decreased by-17.4%,-23.9%.-43.3% and-30.4% espectively, and the root dry weight decreased by -18.5%,-25.4%,-32.3% and 46.0%. Compared with CK, the effects of different chemical substances were not obvious (P>0.05) under low concentration. The content of polysaccharide in the 2-group was 8.3% and 7.9%, respectively, under moderate and high concentration.4. Study on the antitumor activity and mechanism of Polygonatum Polysaccharide in vitro For Polygonatum odoratum polysaccharide in vitro antitumor effects were studied by, MTT assay results show that Polygonatum odoratum polysaccharide on human lung cancer A549 cell growth inhibition, when the dose is 150 g/ml for 48h, the inhibition rate is as high as 76.30% hat of Polygonatum odoratum polysaccharide on tumor cell growth inhibition with. The effect of Polygonatum odoratum polysaccharide extract after 48 hours, compared with the negative control group, group of Polygonatum odoratum polysaccharide significantly induced apoptosis in A549 cells. The difference is significant (P< 0.01), lung adenocarcinoma A549 cells. Wnt3a, beta catenin protein and c-myc gene expression were lower (P< 0.05). Polygonatum odoratum polysaccharide extract suggest possible through down-regulation of intracellular Wnt3a, beta catenin, c-myc gene expression and blocking Wnt/beta catenin signaling process.5. The mechanism of Polygonatum Polysaccharide on tumor bearing mice Through the establishment of tumor bearing mice model and given saline treatment, tumor volume increases with the passage of time; and the physiological saline group compared, polygonatum odoratum polysaccharide intervention group, the tumor volume with increasing time is not obvious that Polygonatum odoratum polysaccharide extract can inhibit the growth of tumor cells. By pathological findings, polygonatum odoratum polysaccharide mice tumor tissue necrosis are heavier than normal saline group, there are large areas of necrosis, necrosis in the nucleus dissolved, cell fusion, unclear structure, indicating that Polygonatum odoratum polysaccharide can induce tumor cell necrosis. At the same time, tumor apoptosis related gene Survivin and C-Myc expression, suggesting that tumor cell apoptosis accelerated, and the decreased expression of cyclinDl, suggesting the proliferation of tumor cells decreased. In vivo and in vitro results consistent with the results of.Conclusion:1.Polygonatum odoratum rhizome water extraction liquid of the receptor (canola, corn, garlic) seed germination rate and radicle and plumule growth and there are obvious "promotion at low and high suppression" phenomenon, with the increase of the concentration, the root rot Fusarium solani has promotion effect. Polygonatum odoratum rhizosphere soil leaching extract could significantly inhibit the Polygonatum odoratum rhizome spore germination rate, seedling height and root fresh weight and with extraction solution concentration increased, enhanced inhibition.2. Polygonatum odoratum in rhizosphere soil of ethyl acetate extraction to 24 compounds were identified, including 16 acids,1 aldehydes, ketones, three lipids and two amide in solution. Arachidic acid, one of the fifteen alkyl acid, myristic acid with allelopathic activity. The root exudates of the allelochemicals accumulate, the stronger inhibition of chlorophyll and its enzyme system.3. Different sense of material processing of Polygonatum odoratum rhizome root fresh weight and root dry weight were significantly decreased, the polysaccharide content and coarse fractions vary, but the difference was not obvious, allelochemicals interference of Polygonatum odoratum growth regulation.4. Different sources of Polygonatum odoratum polysaccharide can promote the apoptosis of human lung cancer cell A549, inhibition of cell growth and proliferation; can reduce the tumor bearing mice tumor volume and quality, and promote cell apoptosis5. The adjusting and controlling gene survivin, c-myc and growth regulating signal regulation ofWnt/?-catenin-cyclinD1 expression, inhibition of proliferation and induction of apoptosis is the possible mechanism of Polygonatum odoratum polysaccharide antitumor.