Study On The Chemical Constituents And Biological Activities Of Ardisia Punctata Lindl.

In 2020,there were 19.29 million new cancer cases worldwide,of which 4.57 million were in China,accounting for 23.7 percent of the global total.The incidence and mortality of malignant tumors continue to rise in China,and the annual medical expenditure caused by malignant tumors exceeds 220 billion yuan.The burden of malignant tumors is increasing day by day in China,and the prevention and control situation is still very serious.It is an important way to search for antitumor components from natural products.At present,anti-tumor drugs in natural plants are found in four aspects:based on tumor cell proliferation and apoptosis targets,based on inflammatory regulation targets,based on inhibition of cancer invasion and metastasis,based on new anticancer targets,etc.Based on the investigation of a large number of medicinal plants and the preliminary research work of the research group,17 plants were selected as candidate plants,and their anti-tumor activities in vitro were preliminarily screened,and it was found that Parilla has the strongest inhibitory effect on tumor cell proliferation.After comprehensive analysis,parilla was finally selected as the research object.Ardisia punctata Lindl.is a plant of the genus Amaranthaceae,it mainly distributed in tropical and subtropical regions,and it mainly distributed south of the Yangtze River basin in China,including Guangdong,Guangxi,Zhejiang,Jiangxi,Fujian and Hunan provinces.It has the effect of promoting blood circulation,regulating menstruation,dispelling wind and dehumidifying,and is often used for the treatment of symptoms such as amenorrhea,dysmenorrhea,rheumatism and numbness.Its main components are isocoumarins,lignans,flavonoids,benzoquinones,organic acids,saponins,etc.The plants belonging to the same genus,such as Ardisia crenata Sims,Ardisia crispa（Thunb.）A.DC.,Ardisia mamillata Hance,Ardisia japonica（Thunb）Blume,and Ardisia gigantifolia Stapf,have been found to have certain anti-tumor activities.However,there are few reports on the related studies of Ardisia punctata Lindl.At present,and it lacks of systematic study of its chemical composition.In order to better develop and utilize the resources of Ardisia lindleyana and search for its active components,the chemical components of Ardisia lindleyana were systematically studied and its biological activities were evaluated.In this paper,and 60 kg Of Ardisia lindleyana was extracted by hot reflux extraction and cold extraction method with 80%ethanol was used as the solvent medium.The obtained extract were extracted with petroleum ether,ethyl acetate and n-butanol,respectively.The ethyl acetate extract and n-butanol extract were systematically separated and purified by silica gel chromatography,ODS column chromatography,Sephadex LH-20 gel column chromatography,AB-8 macroreticular resin column chromatography,MCI pore adsorption resin column chromatography,preparative HPLC,recrystallization and other multi-dimensional separation techniques,a total of 51 compounds were obtained.The structures of 51 compounds,including 5 new compounds and 46 known compounds,were identified by physical and chemical properties,¹H-NMR,¹³C-NMR,IR,MS,HSQC,HMBC,DEPT,¹H-¹H COSY,NOESY,single crystal diffraction and circular binary chromatography.They are ardisiapunine A（1）,ardisiapunine B（2）,ardisiapunine C（3）,ardisiapunine D（4）,ardisiapunine E（5）,bergenin（6）,4-O-gallanyl bergenin（7）,11-O-gallanyl bergenin（8）,11-O-(3’,4’-dimethylgaleoyl bergenin（9）,11-O-（4’-methoxy galeoyl）-bergenin（10）,11-O-syringoyl bergenin（11）,mycophenolic acid（12）,munjistin（13）,quercetin（14）,daucosterol（15）,liriodendrin（16）,（2S,3S,4R）-2-[（2R）-2-hydroxytetracosanoylamino]-1,3,4-octadecanetriol（17）,（2S,3S,4R,10E）-2-[（2R）-2-hydroxytetracosanoylamino]10-octadecene-1,3,4-triol（18）,（2S）-1-O-heptatriacontanoyl glycerol（19）,N-dotriacontanol（20）,（+）-isolariciresinol-9-O-β-glucopyranoside（21）,（+）-isolariciresinol-9’-O-β-glucopyranoside（22）,（+）-5’-methoxyisolariciresinol-9-O-β-D-glucopyranose（23）,（+）-5’-methoxyisolariciresinol-9’-O-β-D-glucopyranose（24）,（+）-Lyoniresinol-3α-O-β-D-glucopyranoside（25）,syringin（26）,phenethyl alcohol-β-D-（2’-O-β-D-glucopyranosyl）glucopyranoside（27）,D-fructofuranosyl-β（2→6）-α-D-glucopyranoside（28）,1-Ribityl-2,3-diketo-1,2,3,4-Tetrahydro6,glucopyranoside（27）,D-fructofuranosyl-β（2→6）-α-D-glucopyranoside（28）,1-ribityl-2,3-diketo-1,2,3,4-tetrahydro-6,7-dimethyl-quinoxaline（29）,lumichrome（30）,Uracil（31）,n-trans-feruloyl tyramine（32）,vanilic acid（33）,isovanilic acid（34）,5-hydroxy-2-methoxybenzoic acid（35）,3,4-dimethoxy-5-hydroxybenzoic acid（36）,syringic acid（37）,ethyl-O-α-L-rhamnoside（38）,ethyl-O-β-D-pyranoside（39）,ethyl-O-β-D-glucopyranoside（40）,ethyl-O-α-L-pyranarabinoside（41）,n-decane mercaptan（42）,nonyl valerate（43）,ardisiacrispin B（44）,ardisiacrispin A（45）,3-O-α-L-arabinopyranosyl cyclamiretin A（46）,ardisianoside G（47）,ardisimamilloside F（48）,ardisicrenoside A（49）,ardicrenin（50）,and ardisicrenoside B（51）,respectively.Using He La,Mc F-7,H460,Hep G2,B16F10,A549 and U87 tumor cells as tumor models in vitro,the inhibitory activities of the isolated compounds on tumor cell proliferation were preliminarily screened by MTT assay.The results showed that multiple compounds could inhibit the proliferation of tumor cells in vitro,among which the new compounds 1,3 and 5 had different degrees of inhibition on the proliferation of the above 7 tumor cells in vitro.The inhibitory effects of new compound 1 on tumor cell proliferation in vitro were as follows:B16F10>Hep G2>He La>A549>U87>H460>Mc F-7,with IC₅₀ values of 31.03,37.18,55.65,65.90,73.01,75.06 and 97.22μM,respectively.The inhibitory effects of new compound 3on tumor cell proliferation in vitro were as follows:Hep G2>B16F10>Mc F-7>He La>U87>A549>H460,with IC₅₀ values of 12.40,17.51,21.73,24.96,25.50,29.86 and 33.09μM,respectively.The inhibitory effects of compound 5 on tumor cell proliferation in vitro were as follows:MCF-7>H460>B16F10>He La>Hep G2>U87,with IC50 values of 8.47,10.83,12.46,13.23,34.85 and 54.54μM,respectively.The effects of compounds 5,44 and 46 on tumor cell morphology,proliferation activity,cell cycle,apoptosis and expression of apoptosis-related proteins were evaluated by MTT assay,flow cytometry,western blot and other biological techniques.The results of cell morphological observation showed that compounds 5,44 and 46changed the morphology of the cells,the nucleus showed pyknosis,the cell membrane and the nucleus ruptured,and the apoptotic bodies gradually increased,and all showed a dose-effect relationship.Cell cycle experiment showed that compound 5could increase G2/M phase cells from 14.08%to 33.86%（25μM）.Compound 44could increase G2/M phase cells from 14.08%to 37.22%（3.13μM）,and compound46 could increase G2/M phase cells from 14.08%to 29.29%（25μM）,all showed a dose-response relationship.The results of cell apoptosis assay showed that compound5 could increase the cell apoptosis rate from 13.62%to 45.37%（25μM）,compound44 could increase the cell apoptosis rate from 13.62%to 18.62%（0.78μM）,and compound 46 could increase the apoptosis rate from 13.62%to 48.73%（25μM）,all showed a dose-response relationship.Results of cell apoptosis related protein assay showed that He La cell apoptosis related protein expression levels were significantly changed after different concentrations of compounds 5,44 and 46,Bcl-2 and Pro-caspase 3 expression was down-regulated,Bax and cleaved caspase 3 expression was up-regulated,and all were dose-dependent.MTT assay,Griess assay,microscope and other methods were used to evaluate the in vitro anti-inflammatory activity of monomer compounds isolated from LPS-stimulated RAW 264.7 cells.Compounds 2,5,6,7,8,9,10,22,37 and 46 can effectively modulate the level of NO produced by LPS-stimulated RAW264.7 cells to exert anti-inflammatory effect in vitro.