The Study Of The Ent-kaurane Diterpenoids From Isodon Serra And Its Anti-hepatic Fibrosis Activity

ObjectiveIsodon serra(Lamiaceae)is a perennial herb belonging to the Lamiaceae family,also known as Yinchencao and Xiongdancao.It gets its name from the fact that it is usually grown in wet places near mountain valleys and that fresh leaves are rubbed with a yellowish-brown liquid.I.serra is distributed in central,southern and southwestern China,Guangxi,Guangdong and Fujian provinces are its main production areas.Biological activity studies performed on the species have shown that it has heat clearing and detoxifying,promoting blood circulation by removing blood stasis,antibacterial and anti-inflammatory treatment of various hepatitis,antitumor etc.As a common folk medicine,it also has a good effect on the prevention and treatment of acute cholecystitis and acute jaundice hepatitis and other liver and gall diseases.In addition,I.serra is widely used in clinical practice in Guangdong Province,and has been developed into a variety of health care products for preventing hepatitis,such as Xihuangcao infusion,Xihuangcao tea bag,etc.Nowadays,numerous chemical investigations on the genus Isodon have been carried out.The characteristic chemical constituents has been proved to be diterpenoids,which exhibit a wide range of biological activities,such as anti-tumor,anti-inflammatory and anti-hepatitis.However,the research on the anti-hepatic fibrosis activity of I.serra is still blank,especially,the anti-fibrosis activity of diterpenoids,the main active components,has not been reported.Therefore,in order to clarify the active material basis of anti-liver fibrosis in I.serra,obtain novel and active diterpenoids and enrich the structural types of diterpenoids,the chemical constituents of ent-kaurane diterpenoids and their anti-hepatic fibrosis activities from Ⅰ.serra were studied systematically in this paper.Methods1.The aerial parts of I.serra were powdered and extracted with 95%ethanol at room temperature,the combined solvent was concentrated to give a crude extract,which was partitioned with ethyl acetate and H2O.The ethyl acetate-soluble portion was subjected to column chromatography over silica gel,MCI,C-18,Sephadex LH-20 and preparative HPLC.The structures of the isolated diterpenoids were elucidated by extensive analysis of UV,IR,1H NMR,13C NMR,1H-1H COSY,HSQC,HMBC,NOESY and HRESI-MS.2.In order to evaluate the anti-hepatic fibrosis effect of compounds isolated from L serra,rat hepatic stellate cells(HSC-T6)were cultured in vitro,and TGF-β1(10 ng/mL)together with monomer compounds of different concentrations(10,2,0.4,0 μM)were given at the same time,meanwhile,the different concentrations monomer compounds without TGF-β1 were set as positive controls.MTT method was used to calculate the cells survival rate and observe the effect of each monomer compound on the inhibition of TGF-β1-induced HSC-T6 cells.Results1.31 compounds were isolated and identified as follows.14 7,20-epoxy ent-kauranes type diterpenoids:Effusanin E(1),Oridonin(2),Effusanin B(3),Lasiokaurin(4),Shikokianin A(5＊),Ponicidin(6),Hebeirubescensin K(7),Enmenol(8),Phyllostacin S(9＊),Rabdoternin B(10),Maoecrystal F(11),Rabdosianin A(12),Parvifolin G(13)and Shikokianin B(14＊);14 enmein type diterpenoids:Isodocarpin(15),Enmein(16),Epinodosin(17),Nodosin(18),Genervosin B(19),Serrin B(20),Serrin A(21),Rabdosin G(22),Carpalasionin(23),Epinodosinol(24);Sculponeatine E(25),Phyllostacin P(26＊),Phyllostacin Q(27＊)and Phyllostacin R(28＊);Two spiro-lactone type diterpenoids:Rabdonervosin B(29)and Rabdonervosin C(30);One enmein diterpene dimer:Bisjaponins C(31＊).Among the above compounds,compounds 5＊,9＊,14＊,26＊,27＊and 28＊are the six new ent-kaurane diterpenoids,and 31＊is one new enmein diterpene dimer.2.The anti-hepatic fibrosis activity of 31 ent-kaurane diterpenes was tesed in vitro.The results demonstrated that compounds 16,17,18,20,22,23,26,27 and 28 showed inhibitory effects on TGF-/β1-induced HSC-T6 cells proliferation.Among them,rabdosin G(22)exhibited excellent inhibitory ability on TGF-β1-induced HSC-T6 cells proliferation in a dose-dependent manner.The structure-activity relationship indicated that the α-methylene cyclopentanone of the D ring in the ent-kaurane diterpenoids is essential for the inhibition of TGF-β1-induced HSC-T6 cells proliferation.If the conjugated system of the double bond and the carbonyl unit on the D ring is broken,the activity of the compound will be greatly weakened or even disappeared.Additionally,the connected groups at the diterpenoids also affect the inhibition activity,in which the introduction of oxygen-containing groups(-OH/-OEt)at positions C-3,11 and 20 can enhance the activity of the compounds.ConclusionThe chemical constituents of I.serra were systematically studied in this paper,and a total of 31 ent-kaurane diterpenoids were identified,including six new ent-kaurane diterpenoids and one new symmetrical ent-kaurane diterpenoid dimer.Therefore the present study has greatly enriched the chemical constituents of I.serra and the structural types of the ent-kaurane diterpenoids in natural plants.Furthermore,the anti-hepatic fibrosis activity of the ent-kaurane diterpenoids was reported for the first time,which confirmed that diterpenoids were the basis of the anti-fibrosis pharmacodynamic substances in Ⅰ.serra.In the bioactivity experiment,the compounds 16,17,18,20,22,23,26,27 and 28 showed inhibitory effects on TGF-β1-induced HSC-T6 cells proliferation.Among them,rabdosin G(22)exhibited excellent inhibitory ability on TGF-β1-induced HSC-T6 cells proliferation in a dose-dependent manner.Therefore,the characteristic chemical constituents of ent-kaurane diterpenoids and their anti-liver fibrosis activities of the traditional liver protection Chinese herbal medicine I.serra were studied systematically in this paper,resulting in the discovery of lead compound of anti-liver fibrosis and providing the possibility for the development of new anti-liver fibrosis drugs.