Bioactive Constituents And Anti-tumor Mechanism Of A Traditional Mongolian Medical Herb Clematis Aethusifolia Turcz

The dried aerial of Mongolian medical herb Clematis aethusifolia herbs is commonly used to“removal of lump and stagnation”in Mongolian medicine and were contained in"Ministerial Drug Standards for Mongolian Medicine",with functions against cold lump,indigestion,edema,wounds and hemorrhoids tumour,and modern clinical use for the treatment of various tumors,including cancer.Objective To study the antitumor activitives and consttituents of Mongolian medical herb Clematis aethusifolia Turcz,in order to find the relationship between the function“removal of lump and stagnation”and morden antitumor activitives,explain the material basis and mechanism of“removal of lump and stagnation”function,provide new ideas for the reasonable medication of traditional Mongolian medicine.Methods and results?1?Isolation and identification of chemical constituents:using Sephadex LH-20,macroporous resin,C₁₈ reversed-phase column chromatography and preparative HPLC methods to isolate alcohol extract of dried aerial of Clematis aethusifolia Turcz and indentify chemical constituents by 1D,2D-NMR,HRMS spectral methods and literature analysis methods.Fromthebutanol extraction layer and ethyl acetate extraction layer ofdried aerial of Clematis aethusifolia Turcz,15 compounds were isolated,which is ?3R?-6,7,8-trimethoxy-2,3,4,5-tetrahydrobenz-1-oxepin-3-ol?1?,3-O-p-coumaroyl-2-C-methyl-D-erythrono-1,4-lactone?2?,4-O-caffeoyl-2-C-methyl-D-erythrono acid?3?,4-O-Caffeoyl-2-C-methyl-D-erythrono-1,4-lactone?4?,?-?-4-?3,4-dihydroxyphenyl?-5-hydroxy-2?5H?-furanone?5?,caffeic acid?6?,1-O-caffeoyl-?-D-glucopyranose?7?,benzylalcohol a-L-arabinofuranosyl?1-6?-?-D-glucopyranoside?8?,aesculetine?9?,scopoline?10?,apigenin-7-O-?-D-glucopyranoside?11?,3?-O-methyl-apigenin--7-O-?-D-glucopyranoside?12?,vanillic acid?13?,protocatechuic acid?14?,butyl-?-D-fructofuranoside?15?.?2?Cytotoxicities:Using CCK-8 method to detect growth inhibition of 38 compounds from Clematis aethusifolia Turcz against common human tumor cell lines?human lung cancer cell line A549,human ovarian cancer cell line SK-OV-3 and human melanoma cell line,A-375neuroblastoma cell line SH-SY5Y,human colorectal adenocarcinoma cell line HCT-15?.1-O-caffeoyl-?-D-glucopyranose?7?,aesculetine?9?,apigenin-7-O-?-D-glucopyranoside?11?,3?-O-methyl-apigenin-7-O-?-D-glucopyranoside?12?,apigenin?16?have strong cytotoxic activities against parts of human tumor cell lines,the range of IC₅₀ is 15-60?M,other compounds have no obvious cytotoxic activities against five human tumor cell lines,IC₅₀>100?M.?3?Screening tumor target database:using Dell Power Edge R900 molecular simulation server to upload compounds from Clematis aethusifolia Turcz,using Auto Dock to dock molecular with 14 common clinical tumor targets to find related cancer targets with active inhibition.The target of molecular docking indicated that most isolated compounds from Clematis aethusifolia Turcz have strong inhibition against tumor cell signaling pathways protein kinase BRAF and PI3K.?4?Study on the antitumor mechanism:using q PCR method to detect tumor cells?m RNA expressions of related genes ERK₂,RAF,PDK₁ and AKT in PI3K-AKT-m TOR and RAS-RAF-MEK signaling pathway after aesculetine,apigenin-7-O-?-D-glucopyranoside and apigenin stimulated.The results showed that after aesculetine stimulated,A-375?m RNA expression of related genes ERK₂ expression decreased significantly,other genes?m RNA expression have no change;SH-SY5Y?m RNA expression of related genes PDK₁ expression decreased significantly,other genes?m RNA expression have no change.Afterapigenin-7-O-?-D-glucopyranoside stimulated,A-375?m RNA expression of related 4 genes ERK₂ expression no change;SH-SY5Y?m RNA expression of related genes PDK₁expression decreased significantly,other genes?m RNA expression have no change.After apigenin stimulated,A-375?m RNA expression of related genes ERK₂ expression decreased significantly,other genes?m RNA expression have no change; SH-SY5Y?m RNA expression of related genes ERK₂,PDK₁ and AKT expression decreased significantly,other genes?m RNA expression have no change.Using Western blot method to detect tumor cells'm RNA expressions of related proteins phospho-AKT,AKT, phospho-PDK1,PDK1,phospho-ERK?1/2?and ERK?1/2?in PI3K-AKT-m TOR and RAS-RAF-MEK signaling pathway after aesculetine,apigenin-7-O-?-D-glucopyranoside and apigenin stimulated.The results showed that after aesculetine stimulated,A-375 expression of all six related proteinshave no change;SH-SY5Y expression of related proteinsphospho-AKT,AKT and phospho-PDK1expression decreased significantly,other proteins expression have no change.Afterapigenin-7-O-?-D-glucopyranoside stimulated,A-375 expression of related proteins phospho-AKT,AKT,PDK1,phospho-ERK?1/2?and ERK?1/2?expression decreased significantly,other proteins expression have no change;SH-SY5Y expression of related protein AKT expression decreased significantly,other proteins expression have no change.Afterapigenin stimulated,all six related proteins expression decreased significantly;SH-SY5Y expression of related proteinsphospho-PDK1,PDK1 and phospho-ERK?1/2?expression decreased significantly,other proteins expression have no change.Conclusion Compounds 1–15 were isolated from Clematis aethusifolia Turcz for the first time,Compounds 1–3 were new compounds,compounds 4,5,8 and 15 were isolated from Ranunculaceae family for the first time,compounds 7,9,12 were isolated from Clematis genus for the first time.Compounds 9,11 and 16 have anti-tumor activities and inhibit tumor cell growth through PI3K-AKT-m TOR and/or RAS-RAF-MEK signaling pathway.