Studies On The Chemical Constituents And Antitumor Activities Of The Fruiting Bodies Of Different Years Old Phellinus Vaninii

Yang Huang is a kind of fungus medicine.It has a variety of medicinal active ingredients and has medicinal and economic value.Its scientific name is Phellinus vaninii（abbreviated as P.v）.In this paper,the pharmacognostical identification of P.v was carried out firstly,and then the main active components,nutritional components and inorganic elements of P.v fruit bodies cultivated in different years（1-year-old,2-year-old and 3-year-old）were determined.At the same time,the antitumor activity of P.v fruit bodies cultivated in different years was studied,and the antitumor activity and mechanism of different extracts of P.v fruit bodies cultivated in3-year-old were studied Finally,the chemical constituents of petroleum ether were isolated and identified.The resources distribution,chemical composition,pharmacological activity and anti-tumor mechanism of P.v were reviewed by consulting literatures.There are mature artificial cultivation techniques and large cultivation area of P.v,but the research on the chemical constituents of P.v fruiting body is relatively less,which is still not comprehensive.From the perspective of modern research progress,the research on the main chemical constituents of P.v fruiting body,such as polysaccharides,flavonoids,sterols,is relatively more,and the research on other active constituents is relatively less.P.v has similar anti-tumor activity with other Sanghuangporus fungi.Through literature search,the pharmacological activity of P.v is only limited to anti-tumor,and its anti-tumor mechanism is rare.Through morphological,microscopic and molecular identification,the authenticity of P.v was identified,so as to ensure the accuracy of the experimental materials.Microscopically,the mycelial and spore structures of the fruiting bodies of P.v were observed,which were consistent with the reported characteristics.Meanwhile,the phylogenetic tree was constructed by the extraction,amplification,gel electrophoresis and gene sequencing of P.v DNA,and further verified that the experimental material was my P.v.There are some differences in chemical composition and nutritional composition between cultivated and wild P.v fruiting bodies of different years.Through analyzing the differences of main components content between cultivated and wild P.v fruiting bodies of different years,the acute toxicological experiment was carried out with the water extracts of different fruiting bodies.The results showed that the polysaccharide content was the highest in 3-year-old and Korean wild fruit bodies（0.83%and 0.86%）;the flavonoid content was the highest in 1-year-old and 3-year-old fruit bodies（0.46%and 0.49%）;the triterpene content was the highest in3-year-old fruit bodies（0.67%）;The content of sterols is the highest in the 1-year-old fruit body（0.27%）;the crude protein content is the highest in the 2-year-old（11.57%）;The content of total essential amino acids was the highest in the wild fruit body of Changbai Mountain,and the content of heavy metals did not exceed the standard.Acute toxicology experiments have shown that that when the maximum dosage of the fruit body of P.v was 360g·kg^-1,the diet,drinking water and behavioral activities of mice were normal,and there was no abnormal or pathological changes in all organs,and there was no obvious acute toxicity to mice.Different years of P.v fruit body powder has anti-tumor effect on H₂₂ tumor-bearing mice.By establishing a subcutaneous solid tumor model in mice,the powder of P.v fruit body of different growth years was given by intragastric administration at a dose of 800 mg/kg to observe the mice’s performance Growth status,organ index and tumor inhibition,then dissected the tumor tissue for hematoxylin-eosin（H&E）staining pathological section observation,and took blood from the orbit to detect the expression of related factors in the serum.The results showed that the fine powder of P.v fruit body has a certain inhibitory effect on tumor mice.Among them,the three-year-old P.v fruit body has the most obvious anti-tumor effect（38.12%）.Through the analysis of organ index of organs（lung,liver,kidney,spleen and thymus）,there was no significant difference between lung and liver（P>0.05）,and the kidney of different age P.v fruitbody groups was significantly atrophied compared with the model group Compared with the positive group,the spleen was significantly atrophied（P<0.01）,and the thymus was significantly increased（P<0.05）.The tumor tissue cells were observed by H&E staining.The3-year-old mice tumor tissue cells showed obvious cellular spaces or vacuoles,and the cell arrangement was loose.Compared with the model group,the serum levels of IL-2,IFN-γ,TNF-αand VEGF in each group were significantly decreased,and VEGF in the 3-year-old group was significantly decreased.The three-year-old P.v fruit body has the best tumor inhibition rate.Therefore,the mouse model was established with H₂₂ liver cancer cells and randomly divided into model group,CTX group,low,medium and high dose of P.v fruit body water extract group,ethanol extract group,petroleum ether In the extract group and the crude polysaccharide group,the administration was started 2 days after the modeling,and the tumor was inhibited for 10 days.Calculate the organ index and tumor inhibition rate of spleen,thymus and tumor tissue,and detect the content of TNF-α,INF-γ,and VEGF in serum;use hematoxylin-eosin（H&E）and immunohistochemical staining to observe the pathology of tumor tissue At the same time,Western blot（WB）was used to detect the expression level of related proteins in tumor tissues.The results showed that all extracts of P.v fruiting bodies have anti-tumor effects.Among them,petroleum ether extracts in the high,medium,and low groups have the best anti-tumor effects.The anti-tumor rates are73.21%,69.64%,and 61.90%,respectively.The contents of TNF-α,IFN-γ,and VEGF were increased.Observed by staining of tissue sections,the administration group can significantly improve the necrosis and ablation of tumor tissue,and the immunohistochemical results of the water extract group have negative regulation phenomenon,showing high expression of Bax and Bcl-2 protein,but its anti-tumor effect is poor.Western blot results showed that each protein showed a dose expression trend.Compared with the model group,the apoptosis genes Caspase-3,Caspase-9 and pathway genes NF-κb,JAK all showed high expression,and the mechanism of action may be through the mitochondrial pathway.After the drug is metabolized in the body,it causes the relevant apoptosis receptors（Bax,Bcl-2,TNF,etc.）in the cell to activate the Caspase-3 and Caspase-9 apoptotic proteins,causing nuclear chromatin or DNA to condense or degrade,and destroy tumor cells The normal proliferation of tumor cells induces tumor cell apoptosis and inhibits the growth of tumor tissues.Through GC-MS analysis,41 peaks were separated.Through CAS search,10 hydrocarbons,7 esters,18 alcohols and phenols,3 aldehydes and ketones were detected.Through the analysis of petroleum ether extract,9 common peaks were found,including 1 olefin,2 fatty acids and 2sterols There are four compounds and one ketone compound.At the same time,five monomers were obtained by systematic separation of petroleum ether extract.