| Chenopodium ambrosioides L.is an annual or perennial herb of the genus Amaranthus,and is also known as the grass,red zealand and scented grass.It is a traditional medicinal plant in many countries.C.ambrosioides can be used as a whole herb,with various effects such as anti-oxidation,anti-bacterial,anti-tumor.It is often used to treat skin eczema,phlegm,rheumatism,mouth sores and snake bites.In the past,the study of the C.ambrosioides was based on the whole plant as the extraction material,and mainly focused on the biological activity function of its essential oil.There are few reports on the total flavonoids of C.ambrosioides.Flavonoids are a class of substances with a variety of biological functions.The composition and content of the flavonoids vary depending on the extraction method,extraction solvent,and extraction site.Therefore,in this study,different polar organic solvents such as petroleum ether,ethyl acetate and n-butanol and distilled water were used to separate the solvent.Microwave ultrasonic assisted extraction was used to extract total flavonoids from C.ambrosioides seeds.The content of quercetin and kaempferol was determined by HPLC.The content of anti-tumor activity was evaluated by MTT method and the anti-tumor mechanism was further explored.In order to provide reference for the development and utilization of sichuan C.ambrosioides.The research contents are as follows:（1）Preparation of total flavonoids from C.ambrosioides seeds:petroleum ether,ethyl acetate,n-butanol and distilled water were used as solvent separately or sequentially.Microwave ultrasonic combined synthesis/extraction extraction and D101 macroporous adsorption resin were used to obtain petroleum ether extract（S1）,ethyl acetate extract（S2）,n-butanol extract（S3）,distilled water extract（S4）,petroleum ether-ethyl acetate extract（S5）,petroleum ether-ethyl acetate-n-butanol extract（S6）and petroleum ether-ethyl acetate-n-butanol-distilled water extract（S7）.The results showed that the extraction rate of S6 was the highest,being 3.97%.Determination of total flavonoids showed that the highest content of S6 was 96.39mg/g.HPLC method showed that the content of quercetin in S2 and S6 was was higher,0.602 mg/g and 0.531 mg/g,respectively;the content of kaempferol in S2 and S6 was higher,0.462 mg/g and 0.479 mg/g,respectively.The results showed that the total flavonoids extracted by petroleum ether-ethyl acetate-n-butanol were the highest,and the content of quercetin and kaempferol was higher.（2）Evaluation of anti-tumor activity of total flavonoids from C.ambrosioides seeds:MTT assay was used to determine the effect of S1-S7 on the proliferation of human normal liver L02 cells,human liver cancer Hep G-2 cells,Hep3b cells,SMMC-7721 cells,human cervical cancer Hela cells and human colon cancer HT29cell.The results showed that the seven extracts had different degrees of inhibition on six kinds of cells in a dose-dependent manner（P<0.05）,but the inhibitory effect on normal cells L02 was small.The anti-tumor effect of S2,S3,S5 and S6 was better than the other three,but the dissolution effect of S2 and S5 was poor.Combined with the extraction rate of total flavonoids and the content of quercetin and kaempferol,we chose S6,petroleum ether-ethyl acetate-n-butanol extract of C.ambrosioides seeds（PEN）to subsequent studies.SMMC-7721 cells and Hela cells are most sensitive to PEN.In this study,SMMC-7721 cells were selected as target cells for PEN anti-tumor mechanism research.Under inverted microscope,PEN treated SMMC-7721 cells for24 h.With the increase of concentration,the adhesion and refraction of SMMC-7721cells decreased,and gradually collapsed to suspension death.Colony formation experiments showed that the colony forming ability of SMMC-7721 cells gradually decreased.（3）PEN induced SMMC-7721 cell cycle arrest.The cell cycle was detected by PI staining flow cytometry.The number of cells in G1and S phase of SMMC-7721cells in PEN treatment group was significantly decreased,and the number of cells in G2phase was increased in a dose-dependent manner,indicating that PEN could block SMMC-7721 cells in G2phase.（4）PEN induced autophagy in SMMC-7721 cells.Ultrastructural observation of cells by transmission electron microscopy revealed many autophagic vacuoles,and autophagosomes in SMMC-7721 cells,indicating that PEN induced autophagy in SMMC-7721 cells.Western blot analysis showed that the expression of Beclin 1,LC3and AMPK was up-regulated and the expression of m TOR protein was decreased in SMMC-7721 cells with the increase of PEN concentration,indicating that PEN induced AMPK-m TOR signaling pathway-mediated autophagy in SMMC-7721 cells.（5）PEN induced apoptosis in SMMC-7721 cells.After staining with phalloidin,the fluorescence intensity of SMMC-7721 cells gradually decreased with the increase of PEN treatment concentration,and the cell volume shrinkage became smaller,and the microfilaments were dispersed in the cells.The disorder of microfilaments appeared in the high concentration PEN（0.7 mg/m L）treatment group.AO/EB double staining showed that early apoptotic cells（VA）,late apoptotic cells（NVA）and necrotic cells（NVNA）were present in the PEN-treated group,and the number of NVA was the highest after 0.7 mg/m L PEN treatment.The apoptosis rate was further detected by Annexin V/PI double staining.It was found that the apoptosis and necrotic cells in SMMC-7721 cells increased with the increase of PEN concentration.The ultrastructure of cells observed by transmission electron microscopy showed that the morphology of SMMC-7721 cells changed the nuclear membrane shrinks,the chromatin in the nucleus accumulates,the mitochondria swell and the sputum breaks after PEN treatment.Mitochondrial membrane potential（MMP）measurements showed that the number of cells emitting green fluorescence gradually increased with the increase of PEN treatment concentration,indicating that MMP of SMMC-7721cells decreased.Reactive oxygen species（ROS）assay showed that the relative content of ROS in the PEN-treated group increased,and the relative content of ROS was significantly different from that of the control group after treatment with 0.3 mg/m L and 0.7 mg/m L PEN（P<0.05）.Western blot analysis showed that the expression levels of Caspase 3,Caspase 9 and Bad protein in SMMC-7721 cells were up-regulated with the increase of PEN concentration,while the expression of Bcl-2protein was decreased.This indicates that PEN induces mitochondrial initiation of endogenous apoptotic in SMMC-7721 cells.In summary,total flavonoids extracted from C.ambrosioides seeds had certain anti-tumor activity,and tumor cells were more sensitive than normal cells.Among them,SMMC-7721 cells and Hela cells are most sensitive to PEN.PEN inhibited the proliferation of SMMC-7721 cells;induced morphological changes of SMMC-7721cells,cytoskeletal reorganization;and induced AMPK-m TOR signaling pathway-mediated autophag and mitochondrial-induced endogenous apoptosis in SMMC-7721cells,suggesting that PEN can induce autophagic apoptosis in SMMC-7721 cells. |
