| The Croton crassifolius Geisel.,belonging to the genus Croton(family Euphorbiaceae),is broadly distributed in south region of China and other areas in southeast Asia.It is pungent and bitter in flavour,warm in nature,and its roots have been used for the treatment of stomach pain,sore throat and cancer.Previous phytochemical investigations have revealed that C.crassifolius is abundant with terpenoids,including sesquiterpenes,diterpenoids and triterpenoids,in which clerodane-type diterpenoids are considered as the main and characteristic components.Pharmacological investigations have demonstated that C.crassifolius is a rich source of biological activities,including anti-inflammation,abirritation,and anti-gastric ulcer,and some of the terpenoids exhibited significant antitumor activities in vitro.In order to clarify the active constituents,search new or bioactive antitumor compounds,and complete its quality control study,we carried out a phytochemical investigation on the roots of it and evaluated the in vitro cytotoxicity of the terpenoids.Furthermore,we also established a method for the multicomponent quantitative determination of it.The MeOH extract of the roots of C,crassifolius were chromatographed on silica gel,sephadex LH-20,ODS,and semi-preparation HPLC to yield 39 compounds.Their structures were determined on the basis of IR,NMR,HR-ESI-MS,ORD,CD and X-ray crystallographic analysis.All compounds were elucidated as cracrosons D-H(1-5),1,4-methano-3-benzoxepin-2(1 H)-one(6),chettaphanin I(7),9-[2-(2(5H)-furanone-4-yl)ethyl]-4,8,9-trimethyl-1,2,3;4,5,6,7,8-octahydronaphthalen e-4-carboxylic acid(8),teucvidin(9),crassifolin H(10),isoteucvin(11),crassifolin L(12),teucvin(13),crassifolin K(14),crassifolin B(15),crassifolin I(16),spiro[furan-3-(2H),1'(2'H)-naphthalene]-5-carboxylic acid(17),keto-ester(18),crassifolin A(19),crassifolin E(20),ent-kaurane-3-oxo-16?,17-diol(21),aromadendrane-4?,1 Oa-diol(22),1?,11-dihydroxy-5-eudesmene(23),(1S,4S,S,,6R,7R,l0S)-1,4-dihydroxy maaliane(24),aromadendrane-4,?,10?-diol(25),cyperenol acid(26),cyperenol(27),taraxerol(28),acetyl aleuritolic acid(29),Lupeol(30),aleuritic acid(31),?-sitosterol(32),stigmasterol(33),ent-(16R)-16,17-dihydroxykauran-3-one(34),(?)-Oa-Hydroxy-1aH,7aH-guaia-4,11-dien-3-one(35),cryptomeridiol(36),(+)-alloaromadendrane-4?,10a-diol(37),mallotucin C(38),mallotucin D(39).Compound 1 was a new diterpenoid with a new skeleton,compounds 2-5 were new compounds,and compounds 18,21-22,24-25,34-37 were firstly isolated from the genus Croton.Compounds 1-31 were evaluated the in vitro cytotoxicity against Hep G2 cells via the MTT method with 5-FU as a positive control.Compound 30 exhibited weak cytotoxicity to Hep G2 cell.The IC50 values of 5-FU and 6 were 56.3 ± 6.4 and 78.4±6.1?M,respectively.An HPLC method was developed for the quantitative determination of chettaphanin I,teucvidin,crassifolin B,cyperenoic acid,crassifolin A,and cyperenol in C.crassifolius.This method was carried out on an Kromasil 100-5 C18 column(250 mm x 4.6 mm,5 ?m)using a mixture of MeCN-H2O(the H2O containing 0.02%trifluoroacetic acid)and under the following conditions:flow rate 1.0 mL/min,the detective wavelength was set at 210 nn,and the column temperature was set at 25 °C.The regression equations revealed good linear relationship(r? 0.999 7)between the peak areas of the compounds and their concentrations.The average recovery values of six compounds ranged from 98.48 to 102.50%,and the relative standard deviation(RSD)of precision,stability,repeatability,and average recoveries was within 2.00%.In conclusion,the described method revealed to be simple,repaid,reproducible accurate,and sensitive,which suggests that it could be used to the quality control for the C.crassifolius and its preparations. |
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