| Hepatocellular carcinoma(HCC),a predominant form of primary liver cancer,is the fifth most high morbidity cancer and the third-leading cause of cancer deaths in the world,herein remains one of the most frequently diagnosed malignancies worldwide.Unfortunately,there is the highest incidence of HCC and the highest number of deaths in China.Although significant progress has been made for HCC treatment,the poor prognosis and limited clinical options,however,still make it one of the deadliest cancers and thus require the alternative and effective drugs.Hence,it is very important and urgent to develop the effective drugs for the therapy of HCC.In recent several decades,natural products play an extremely significant role for the drug discovery and development to treat various diseases like cancer,due to their less toxicity and fewer side effects.It has been reported that natural products and their derived constituents account for over half of all anticancer drugs.Threfore,it is very important and necessary to continue to screen anticancer candidates from natural compounds.Juglans mandshurica Maxim is a member of Juglans genus from the Juglandaceae family.Its stem barks,fruits,leaves and roots have been used as a traditional Chinese medicine(TCM)to treat a variety of diseases such as bone tuberculosis,hordeolum,gastritis,diarrhea,diarrhea,dysentery and so on.In addition,the roots of J.mandshurica have been used as a folk medicine for treatment of cancer.In recent years,it has been widely concerned because of its prominent anti-tumor activity,however,most of the researches on pharmacological activities of J.mandshurica have focused largely on quinones.Additionally,research on its medical composition is not thorough and there are less reports on its anti-hepatocarcinoma activity.Picrasma quassiodes(D.Don)Benn.,a member of Picrasma genus from the Simaroubaceae family,is a widely used TCM for the treatment of hypertension,gastroenteritis,infection,diarrhea,sore throat and snakebite in China.In fact,it has served as an ingredient in many Chinese herbal preparations in China,such as Kumu injection and so on,due to its significant anti-inflammatory effect.To discover anti-hepatoma active compounds,the in vitro cytotoxicity for several extracts of Chinese medicines were evaluated in Hep3B and HepG2 cells by MTT assay,and we found that the 75%ethanol extract of the dried bark of J.mandshurica and the 95%ethanol extract prepared from the stems of P.quassioides exhibited more potent cytotoxicity towards hepatocellular carcinoma cells.On this basis,phytochemical investigation of J.mandshuric and P.quassioides were carried out under the guidance of bioactivity tracking and the underlying mechanism of anti-hepatoma active compounds were investigated in Hep3B and HepG2 cells.By means of various chromatographic methods,48 compounds were isolated from the ethanol extract of the dried bark of J.mandshurica.Among them,48 compounds were identified by extensive spectroscopic and spectrometric methods,including 10 alkaloids,11 coumarins,14 lignans,4 phenylpropanoids,2 flavones and 2 terpenoids and 5 other compounds as below:(+)-juglanaloid B(Al),(-)-juglanaloid B(A2),1’S,2’S-orixalone D(A3),4-methoxy-N-methyl-2-quinolone(A4),dectamine(A5),flindersine(A6),N-methylflindersine(A7),7,8-dimethoxy-5-methyl-2,3-methylenedioxybenzo[c]-phenanthridin-6(5H)-one(A8),2,3-methylenedioxy-10,11-dimethoxy-tetrahydroprotoberberine(A9),cryptopine(A10),juglansoside A(All),juglansoside B(A12),juglansin A(A13),1’R,2’R-5-methoxydecursidinol(A14),1’S,2’S-5-methoxydecursidinol(A15),5-methoxyseselin(A16),alloxanthoxyletin(A17),6-methoxy-7-geranyloxycoumarin(A18),isoschinilenol(A19),apigravin(A20),(+)-S-rutaretin methyl ether(A21),juglansoside C(A22),(+)-lyoniresinol(A23),(+)-lyoniresinol-9-O-β-D-glucopyranoside(A24),(-)-lyoniresinol-9-O-β-D-glucopyranoside(A25),7’,8’-threo,7,8-threo-1-[4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxymethyl)ethoxy]-3,5-dimethoxyphenyl]-1,2,3-propanetriol(A26),7’,8’-erythro-7,8-erythro-1-[4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxymethyl)ethoxy]-3,5-dimethoxyphenyl]-1,2,3-propanetriol(A27),(-)-medioresinol(A28),tortoside A(A29),(-)-syringarenol(A30),(+)-sesamin(A31),(-)-sesamin(A32),alangilignoside C(A33),(-)-lariciresinol(A34),(+)-5’-methoxylariciresinol(A35),syringin(A36),4-allyl-2-methoxyphenyl 6-O-β-D-apiosyl-(1→6)-β-D-glucoside(A37),eugenylglucoside(A38),4-O-geranyl-sinapyl alcohol(A39),hesperetin-7-O-β-D-glucopyranoside(A40),(R)-5-hydroxy-3’,4’,7-trimethoxy-lafanone(A41),4(15)-eudesmene-1β,6α-diol(A42),atractylenolide Ⅲ(A43),3,4,5-trimethoxyphenyl-1-O-β-D-glucopyranoside(A44),benzylβ-D-glucopyranoside(A45),8-(5R-oxo-2,5-dihydrofuran-2-yl)octanoic acid methyl ester(A46),8-(5S-oxo-2,5-dihydrofuran-2-yl)octanoic acid methyl ester(A47),methyl ester of azelaic acid(A48).Of all these isolates,9 compounds(Al-3,A11-A15 and A22)were new and 26 compounds were isolated from genus Juglans for the first time.The in vitro cytotoxic activities of all compounds were evaluated in two hepatocellular carcinoma cell lines(Hep3B and HepG2 cells)using MTT assay.Among the main components isolated from J.mandshurica,alkaloids and coumarins exhibited more potential against two hepatocellular carcinoma cells.While most of lignans had no cytotoxicity towards both Hep3B and HepG2 cells,suggesting that they may not be the anti-hepatoma active constituents of J.mandshurica.Further pharmacological research for alkaloids and coumarins form J.mandshurica demonstrated that an alkaloid A7 markedly caused apoptosis and autophagy,but without cell cycle arrest in HepG2 cells.Interestingly,only autophagic cell death was induced in A7-treated Hep3B cells.Coumarins A11-A12 markedly induced apoptosis in HepG2 cells,and A13,A17,A20 effectively caused apoptosis in Hep3B cells,suggesting that the anti-hepatoma activities of A11-A13,A17,A20 were associated with induction of apoptosis.Additionally,the preliminary SAR study was also conducted based on MTT results.By means of various chromatographic methods,11 compounds were isolated from the ethanol extract of the dried stems of P.quassiodes.All compounds were identified by extensive spectroscopic and spectrometric methods,including 10 lignans and 1 alkaloid as below:(+)-(7S,8R)-balanophonin(B1),(-)-(7R,8S)-balanophonin(B2),(+)-(7S,8R)-dehydrodiconiferyl alcohol(B3),(-)-(7R,8S)-dehydrodiconiferyl alcohol(B4),(+)-(7S,8R)-5-methoxyl-balanophonin(B5),(-)-(7R,8S)-5-methoxyl-balanophonin(B6),(+)-(7S,8R)-guaiacylglycerol-β-coniferyl aldehyde ether(B7),(-)-(7R,8S)-guaiacylglycerol-βconiferyl aldehyde ether(B8),(-)-(7R,8R)-guaiacylglycerol-β-coniferyl aldehyde ether(B9),(+)-(7S,8S)-guaiacylglycerol-β-coniferyl aldehyde ether(B10),6,12-dimethoxy-3-yl-βcarboline(B11).Of all these isolates,compound B10 was new and 9 compounds were isolated from genus Picrasma for the first time.The in vitro cytotoxic activities of all compounds were evaluated in two hepatocellular carcinoma cell lines(Hep3B and HepG2 cells)using MTT assay and the results suggested that all isolates from P.quassiodes displayed good cytotoxicity.In addition,some enantiomeric neolignans had enantioselective cytotoxicity.Further pharmacological research demonstrated that compound B11 from P.quassiodes exhibited the antitumor activities towards HCC cells through apoptosis induction.In addition,B11 triggered protective autophagy in HepG2 and Hep3B cells and blocking autophagy enhanced cell growth inhibitory effects and apoptosis induction of B11 in both HCC cells.Besides,in vivo,B11 significantly suppressed the HCC tumor growth in a nude mice xenograft model with fewer side effects.Taken together,this study provided important insights into the role of Bll as a potential antitumor agent for HCC,and the combination of DEC with autophagy inhibitors might improve its therapeutic effects on HCC.Moreover,The further studies on the underlying mechanisms responsible for the distinct cytotoxicity of enantiomeric lignans were investigated,and the results indicated that the different configurations of enantiomers B3 and B4 determined the enantioselective cytotoxicity on HepG2 cells through apoptosis induction and ROS generation.The absolute configurations of enantiomers B7 and B8 as well as B9 and B10 determined the enantioselective cytotoxicity on Hep3B cells through apoptosis induction and ROS generation,which were associated with the inactivation of MEK/ERK signaling pathway in B8-and B10-trsated cells. |
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