Studies On Diterpenes Constituents And Anti Tumor Activity Of Rabdosia Excisa

More than 150 kinds of Rabdosia excisa were found in the world wide, butthere are 70 kinds of Rabdosia excisa and 21 kinks of Isodon excisus Var.coreanus were found in China. Rabdosia excisa(maxim) Hara is a plant of Labiata,and is mainly distributed in Northeast region of China, North Korea and far eastregion of Russia. The whole plant has activity in cure gastritis, arthralgia, traumaand toxicosis etc. According to the literature, the anti tumor activity of the constituents inRabdosia excisa were found. The constituents are ent-kaurene diterpenes, whichwere seprateded and identified in Rabdosia excisa(maxim) Hara also. The studyon the main chemical constituents of Rabdosia excisa(maxim) Hara began in1981, eleven compounds were seprateded and identified from the plant growing inNortheast region of China and North Korea. The eleven compounds areKamebakaurin, Kamebanin, Kamebacetal B, Excisunin A, Excisunin B, ExcisuninC, Excisunin D, Excisunin E, Excisunin F ,Excisunin G and ISOdonin B. Thesduty of pharmacology tests in vitro showed that Kamebakaurin, Kamebanin,Excisunin A and Excisunin B can effectively inhibit KB cells (humannasopharynx cancer ) and Hela cells (human venter cancer). Also, thepharmacology tests in vivo showed that the four compounds have obvious effectin anti S-180 (sarcoma 180), EAC (ascites cancer)and PS (P388 lymphleucocythemia).For the purpose of the further study on the chemical constituents andantitumor activity of diterpenes, the extraction and separation of Rabdosiaexcisa(maxim) Hara from Changbai mountain were performed, and the chemicalstructures of the constituents were identified by Nuclear magnetic resonace etc.The air-dried aerial parts of the Rabdosia excisa Hara (3Kg) were extracted withhot water (H2O). The extracting solution was passed through an AB-8 macroprousadsorption resin column and washed with ethanol-water solution (95%)successively. The EtOH eluate was evaporated in vacuum to give a residue ,which was partitioned between H2O and ethyl acetate (AcOEt) , the extract ofAcOEt partition was obtained, and then the H2O layer was treated with n-butanol(n-BuOH). The n-BuOH phase and AcOEt phase was evaporated in vacuum togive a residue, which was separated by HP-20 resin column chromatographyeluted with H2O-methanol gradient system. Four fraction were obtainedrespectively for each phase after the solvent was evaporated. Sixteen compoundswere gained after the fractions were separated by silica gel chromatography,ODS-HPLC and PTLC etc. By 1HNMR, 13CNMR, 1H-1H COSY, HMQC, HMBC,NOESY, MS, IR spectra analysis, the chemical structures of all the compoundswere identified as compound 1-excisanin H(3.5 mg),compound 2 -excisanin I(5.0 mg),compound 3-excisanin (J3.5 mg),compound 4-excisanin K(6.0mg),compound 5-excisanin L(9.2 mg),compound 6-excisanin A(262 mg),compound7-excisanin B(45mg),compound 8-excisanin D(8.0mg),compound 9-kamebakaurin (860.0 mg),compound 10-kamebanin(82.0 mg),compound 11-Robdokunmin C ( 74.0 mg),compound 12-RabdoserrinB(17.0 mg),compound-13reniformin A(1.0 mg),compound 14-reniformin C(8.0 mg),compound15-kamebacetal A(7.0 mg),compound 16-excisanin G(8.5 mg)。Compound 1~5and 11-15 are obtained from Rabdosia excisa Hara for the first time, compound1~5 are new compounds .The plane chemical structures of compound 1～5 areidentified by 1HNMR, 13CNMR, 1H-1H COSY, HMQC, HMBC, MS, IR etc.To determine four diterpenoids-Kamebakaurin(Ⅰ), excisanin A(Ⅱ),Rabdokunmin C(Ⅲ) and Kamebanin(Ⅳ) in the Rabdosia excisa (Maxim.) Hara,the RP-HPLC was used. The separation was performed on ZORBAX ExtendC18 column (250 mm × 4.6 mm,5 μm), using 23% acetonitrile and 77% watermixture as mobile phase with the rate of 1.2 mL?min-1 at 25 ℃, the wavelengthfor measurement was 230 nm. The results showed that the linearity was in therange of 0.1～0.4 μg(n=6) for all of them, and all the correlation coefficients were0.9999. The average recoveries for compound I～IV were 102.3%(RSD =1.0%) ,100.7 % (RSD =1.6%), 101.7 % (RSD =1.8%) and 97.3 % (RSD =2.2%)respectively(n=6) . This method is accurate, reliable and reproducible todetermine four diterpenoids-Kamebakaurin(Ⅰ), excisanin A(Ⅱ),RabdokunminC(Ⅲ) and Kamebanin(Ⅳ) in the Rabdosia excisa (Maxim.) Hara.The content of the four diterpenes-Kamebakaurin (Ⅰ), excisanin A(Ⅱ),Rabdokunmin C(Ⅲ) and Kamebanin(Ⅳ) in the Rabdosia excise with differentgrowth periods were determined by the above method ,the Results showed thattheir contents were variety in different growth periods in aerial parts of the plant.Kamebakaurin(Ⅰ) and excisanin A(Ⅱ) could reach their highest content in thefirst ten days of July;It suggested that Rabdokunmin C(Ⅲ) have a little variationfrom the begin of June to the begin of August, then the content would decrease;Kamebanin(Ⅳ) could reach their highest content in the middle ten days of June ,the content would decrease from then .So the total diterpenoids can reach theirhighest content in the first ten days of July.The anti tumor effect of the total diterpenes from Rabdosia excisa Hara invivo tests showed that 100mg /kg and 200mg/Kg can inhibit mice liver cancerH22,Lewis lung cancer and melanoma B16 in a dose-dependent manner. The moretotal diterpenes from Rabdosia excisa Hara were taken orally, the more enhancivethe inhibitory effect. At 100mg/Kg and 200mg/Kg, the average rates of inhibitorymice liver cancer H22 were 36.4% and 44.4%, the average rates of inhibitory miceLewis lung cancer were 41.40% and 48.43%, the average rates of inhibitory micemelanoma B16 were 39.02% and 48.95% respectively. However, the inhibitoryrate of low dose group(25mg/ml) was low and there is significance differencebetween it and Cyclophosphamide contractive group.Moreover, cytotoxicity effect on P388 cells of all the 16 diterpenes weretested by MTT method in vitro, the results showed that they all have stronginhibitory effect on P388 cell.