The Analysis Of Anti-cancer Active Components Of Pleurotus Eryngii And Its Killing Mechanism On Leukemia Cells

In recent years,due to changes in lifestyle and diet structure and due to the increasingly serious environmental pollution,malignant tumors have become the frequently occurring diseases that threaten people's health.According to WHO statistics,an annual number of 12 million people suffer from malignant tumors worldwide and the incidence rate will be increased by 50%in 2020.During the past half century,the curative methods of malignant tumor include surgery,radiotherapy and chemotherapy.However,while killing tumor cells,chemical drugs will harm normal cells,lower the restraining ability and immunity of bone marrow,and cause drug resistance,drug tolerance and varying degrees of mutagenic and genetic toxicity.In addition,some tumors are responseless to chemotherapy,which makes treatment more difficult.For clinical treatment of cancers,therefore,it is urgent to find methods and drugs with high curative effects and low side effects.After many encounters of hazards from chemically synthesized drugs,attention has been gradually diverted to the nature.It is a new prospective way and a hotspot to find anticancer and ancillary drugs with low side effects and unique curative effects from Chinese medicinal herbs.Chinese medicinal herbs have a wide medicinal source,low cost,long application history,few adverse effects,and low genetic toxicity.In the treatment of cancers,Chinese medicinal herbs can also fundamentally restore organic functions,improve immune system,and prolong survival period.Therefore,Chinese medicinal herbs are a hotspot in research and development of drugs against tumors.Chinese medicinal herbs provide rich bioactive materials and are a naturally formed "combination of chemicals".According to statistics,China has screened more than 3000 species of Chinese medicinal herbs belonging to 28 genera(families)for anticancer medicine,and among them,200 species contain active anticancer ingredients.However,bryophyta cannot be easily identified due to their microsize and cannot be easily collected because they are clustered and thick,so they are out of people's vision due to lack of direct economic values.In recent years,abundant compounds with unique structures and remarkable bioactivity were isolated from bryophyta.Many of them have potential application values and can be used as excellent leading compounds for development of new drugs.Polytrichum commune L.ex Hedw is one of the genus Polytrichum(Polytrichaceae)that has been used in Traditional Chinese Medicine(TCM)for its medicinal activity as treatment of diseases ranging from fever,hemostatic and traumatic injury to pneumonia,uterine prolapse and lymphocytic leukemia.The effects of Polytrichum plants on different cell lines were studied extensively.The alcohol and acid extracts of Polytrichum juniperum Hedw.(Polytrichaceae)could necrotize implanted Sarcoma 37 in CAF1 mice.In addition,ohioensin A which contains novel polycyclic skeleton was isolated from Polytrichum ohioense Ren.&Card.(Polytrichaceae),and showed cytotoxicity against 9PS murine leukemia and MCF-7 human breast tumor cells.Moreover.the ethanol extract of Polytrichum pallidisetum(Polytrichaceae)in the isolation of three novel benzonaphthoxanthenones and two novel cinnamoyl bibenzyls can significantly inhibit the growth of RPMI-7951 melanoma and U-251 glioblastoma multiforme.The unusual flavonone and benzonaphthoxanthenone(isolated from P.commune)showed IC50 values of 0.10(A549),1.7(HepG2),0.13(LOVO),0.11(MDA-MB-435),and 0.0022(6T-CEM).All above may provide new insights into the anti-cancer activities of P.commune,especially for leukaemia.In this paper,the medicinal bryophyta P.commune from the south of QinLing Mountains was studied.First,test tube method,paper chromatography,and high performance liquid chromatograph(HPLC)were used to analyze the chemical compositions of ethanol extracts from P.commune Hedw and the polar composition groups.Then different types of tumor cells were selected to screen the active anticancer ingredients in P.commune Hedw by using MTT.Finally flow cytometer,fluorescence microscope,scanning electron microscope,laser confocal microscope,Western blotting,and single cell gel electrophoresis were used to investigate the killing effects of ethanol extracts and the ethyl acetate composition group on L1210 cells,as well as the molecular mechanism.The results show that:1.Ethanol extract from PCLH(EPCLH)contain alkaloids,flavonoids,saponins,anthraquinones,and coumarins;the chloroform composition group is rich in alkaloids,the ethyl acetate composition group contains mainly flavonoids,and the n-butyl alcohol composition group contains abundant saponins.Typical chromatogram of HPLC indicated more than 17 distinct peaks in EPCLH;comparison of peak spectra between the polar composition groups showed that all groups were fully isolated from each other and completely conserved the chemical ingredients.2.EPCLH significantly restrained the proliferative activity of the in-vitro cultured murine leukemia L1210 cells,but showed low toxicity on MDA-MB-231 and MCF-7 human breast cancer cells.EPCLH could affect the growth of human nasopharyngeal carcinoma cells Eca-109 and human colorectal cancer cells SW-480,but showed no significant restraint on the proliferation of human colorectal cancer cells SW-620.Under the same conditions,EPCLH nearly had no impact on the growth of Human Embryonic Kidney 293 cells.3.EPCLH Ethyl-acetate Fraction(EEF)showed the most prominent killing effect on leukaemia cells,and the IC50 on L1210,K562 and U937 were 34.56,44.40 and 67.21 pg/mL,respectively.When HEK-293 cells were treated by EEF(15?55 pg/mL),its viability was not less than 90%.4.In comparison of cytotoxicity between ethanol extracts of sporophyte and gametocyte(collected from P.commune),the results showed that under the same doses,the sporophyte ethanol extracts showed significantly higher restraining effects on the proliferation of L1210 cells,K562 cells and U937 cells.5.After treatment by EPCLH,the L1210 cells showed apparent morphological changes in nucleus.Flow cytometry showed that the percentage of cells with PS eversion was in positive correlation with the dose of drugs,and the MMP was significantly reduced with the increasing dose of drug.Together with previous results,EPCLH could induce cell apoptosis in L1210 cells,and mitochondrial may play an important role in this action.6.EEF could induce mitochondria-dependent cell apoptosis in L1210 cells.Meanwhile,abundant ROS was detected in L1210 cells after EEF treatment,and the ROS scavenger NAC significantly relieved EEF-induced cell viability decline,MMP loss,and DNA fragmentation.In addition,EEF could also led to cell cycle arrest at S phase,injure cell membranes,and affect the intracellular calcium ion levels.