| Croton crassifolius(Euphorbiaceae)is a monecious undershrub widely distributed throughout South and Southeast China,Thailand,Vietnam,and Laos.The roots of C.crassifolius are used in folk and traditional Chinese medicine for treatment of snake bites,stomach ache,sternalgia,joint pain as well as pharyngitis,jaundice,rheumatoid arthritis.In Thailand,it has been used as an anticancer herbal medicine by the indigenous people for a long time.C.crassifolius were the 1990 edition of Guangxi medicine standard local recorded species.Its clinical application of the compound preparations are stomach Kang tablets,compound rheumatism Ning tablets,compound rheumatism Ning Injection and Shaolin Zhitong ointment etc.By the means of various chromatographic methods such as macroporous adsorption resin,silica gel,polyamide,ODS and reversed-phase preparative HPLC,forty-four compounds were isolated from the aqueous extract of the air dried flower buds of L.japonica.On the basis of analysis of the physical and chemical properties of compounds and combination with the MS,IR,OR,1D-NMR,2D-NMR and ECD methods,the chemical structures of all compounds were elucidated as:Crassifolius A(1),Crassifolius B(2),Crassifolius C(3),Crassifolius D(4),Crassifolius E(5),6S-OH-cyperenoic acid(6),Crassifoliuspyran A(7),Crassifoliuspyran B(8),Crassifterpenoid A(9),Teucvin(10),Isoteucvin(11),1,4-methano-3-benzoxepin-2(1H)-one(12),Penduliflaworosin(13),Chettaphanin I(14),Crassifolin H(15),Crassifoliusin A(16),methyl 9-(furan-3-yl)-2,7,13-trimethyl-4-oxo-10-oxatricyclo[5.3.3.01,6]trideca-5,8-diene-2-carboxyla te(17),Crassifolin F(18),Crassifolin D(19),Crassifolin C(20),Crassifolin J(21),Crassifolin L(22),Neoclerodan-5,10-en-19,6b;20,12-diolide(23),4-Methoxy-6-nonyl-2-pyrone(24),Crotonpyrones B(25),Crotonpyrones A(26),Cyperenoic acid(27),Cyperenol(28),Methyl cyperenoate(29),Spathulenol(30),Isodactyloxene A(31),1β,11-dihydroxy-5-eudesmene(32),(+)-4-Hydroxy-4,7-dimethyl-a-tetralone(33),6,7,8-Trimethoxycoumarin(34),Methyl ferulate(35),(2E)-3-(4-Hydroxy-3-methoxyphenyl)-2-propenoic acid(36),Des-O-methyllasiodiplodin(37),(2E,6E,12E)-3,7,13-trimethyl-10-(1-methylethenyl)-2,6,12-Cyclotetradecatrien-1-ol(38),(-)-Cembrene A(39),7-keto-sitosterol(40),7-ketositosterol(41),Aleuritolic acid(42),Acetyl aleuritolic acid(43),Lupeol(44).Among them,compound 1 exhibited good cytotoxicity with IC50 value of 17.91 μM against human liver tumor cell Hep3B.Following further studies of the anti-tumor mechanism of compound 1-induced cell growth inhibition,we found that compound 1 caused apoptotic cell death in Hep3B cells by detecting morphologic changes and Western blotting analysis.Then,MTT assay was applied to evaluate the cytotoxicity of the isolated compounds(7,8,24-26)against two liver cancer cell lines(HepG2 and MHCC97H).The result suggested that 1 displayed the highest cytotoxicity with an IC50 value of 9.8 μM against HepG2 cells.Moreover,there was no obvious cytotoxicity of 7,8,24-26 on normal liver cell line LO2.The morphological observation and Western blot analysis demonstrated that 7 could induce apoptosis via p53-mediated Ras/Raf/ERK suppression in HepG2 cells.In our research,methyl cyperenoate(MC)were transformed by Cunninghamella elegans AS 3.2028 for the first time.thirteen new metabolites(ZH 6-13)were isolated and identified.On the basis of analysis of the physical and chemical properties of compounds and combination with the MS,IR,OR,1D-NMR,2D-NMR and ECD methods,the chemical structures of all compounds were elucidated as:Methyl cyperenoate-9-dien-8-one(ZH-6),95-hydroxy-methyl cyperenoate(ZH-7),8R-hydroxy methyl-cyperenoate(ZH-8),8S=hydroxy-methyl cyperenoate(ZH-9),13R-hydroxy-methyl cyperenoate(ZH-10),10S-hydroxy-methyl cyperenoate(ZH-11),7S-hydroxy-methyl cyperenoate(ZH-12),methyl cyperenoate-8-one(ZH-13).Biotransformation metabolites ZH 1-5 were tested in vitro for antithrombotic activity by measuring the platelet count,together with the partial thromboplastin time(APTT),the thrombin time(TT)and the prothrombin time(PT).The biotransformation derivatives ZH 1-3 exhibited potent antithrombotic activity in vitro.Furthermore,the active compounds 1-3 were tested for their in vivo antithrombotic activity using a transgenic zebrafish model and compound 1 exhibited good antithrombotic activity in this model.In addition,cytotoxicity and anti-angiogenic activities of biotransformation products ZH 6-13 were evaluated by MTT assay and ELISA in HepG2 and MCF-7 cells.These results indicated that hydroxylated modification products 7-9 significantly inhibited VEGF release,which suggested the potential use of hydroxylated modification products for cancer therapy.Croton crassifolius is mainly distributed in South China and other areas in Southeast Asia.Its main bioactive constituents-clerodane diterpenoids and their analogues are widely distributed in plant.However,the quality control research of them in C.crassifolius and any other plants was rarely.Thus,the setting up of a reliable and comprehensive quality assessment method for these diterpenoids is.necessary.In this study,a rapid homogenate extraction and UHPLC-MS/MS method was firstly developed and validated for the determination of 9 clerodane diterpenoids[teucvin(10),1,4-methano-3-benzoxepin-2(1H)-one(12),chettaphanin-I(14),Methyl 9-(furan-3-yl)-2,7,13-trimethyl-4-oxo-10-oxatricyclo[5.3.3.01,6]trideca-5,8-diene-2-carboxyla to(17),crassifolin F(18),crassifolin D(19),crassifolin C(20),crassifolin L(22),neoclerodan-5,10-en-19,6b;20,12-diolide(23)]and their analogues in C.crassifolius.All calibration curves showed good linearity(r>0.9943)within the test ranges and the intra-and interday precisions and repeatability were all within required limits.This method only took 5 min to extract 9 diterpenoids in C.crassifolius and 12 min to quantify these components.The results indicated that the quantitative analysis based on UHPLC-MS/MS was a feasible method for quality control of clerodane diterpenoids and their analogues in C.crassifolius with potential perspective for quality control of complex matrices. |
