Reversing Multidrug Resistance Effect And Chemical Constituents Of Poria Cocos

Poria cocos is widely used in traditional Chinese medicine for its diuretic effect, sedative effect, tonic effect,anti-inflammatory activity,anticancer activity and immune enhancement. Its main components are polysaccharides and triterpenoids. In our previous research, the ethanolic extracts of 100 common traditional Chinese drugs, which were widely used in many prescriptions in treatment of cancer in China, were screened for reversing MDR of KBV200 cell line in vitro with MTT method. The results showed 9 extracts including the ethanolic extract of Poria cocos have MDR reversal activity.In this study, Poria cocos was chosen for isolating the active compounds and anti-MDR activities research:1) The powder of dried Poria cocos was extracted by 95% alcohol for two times. After concentration, the ethanolic extract of Poria cocos was suspended in water and extracted by petroleum, chloroform, ethyl acetate and n-butanol in turn. Silica gel column chromatography,Sephadex LH-20,TLC and preparative HPLC were used to isolate compounds from chloroform and ethyl acetate extractions. Physico-chemical properties and spectral methods were used to identify the structures of the components.Eight triterpenoids were isolated from the ethanolic extract of Poria cocos and identified by1H-NMR,13C-NMR,MS as 16 a -hydroxy -3,4-seco-lanosta-4(28),7,9(11),24(31)- tetraene-3,21-dioic acid -3-methy ester(compound 1),16a-hydroxy-3,4-seco-lanosta-4(28),7,9(11),24(31)- tetraene- 3,21-dioic acid(compound 2),Lanosta-7,9(11)-dien-21-oic acid ,3,16-dihydroxy -24-methylene-(3?,16a)-(9Cl)(compound 3),16a-hydroxy-3,4-seco-lanosta-4(28), 7,9(11),24-tetraene-3,21-dioic acid-3-methy ester(compound 4) ,lanosta-7,9(11),24- trien-21-oic acid,3-hydroxy,(3?)-(9Cl)(compound5),3?- acetoxy- 16a-hydroxy- lanosta-8,24(31)-diene-21-oic acid(compound 8),16a-hydroxy-3,4-seco-lanosta- 4(28),7,9(11),24- tetraene-3,21-dioic acid(compound 7) and lanosta-7,9(11)-dien- 21-oic acid ,16-hydroxy-24-methylene-3-oxo-(16a)-(9Cl)(compound 6).2) The toxicity of the eight trieterpenoids against KBV200 cells were tested by MTT assay at the concentration of 3.125μg/ml,12.5μg/ml,25μg/ml,50μg/ml and 200μg/ml respectively. The results showed that the compounds had little toxicity against KBV200 cells at the concentration lower than 12.5μg/ml, but if the concentration higher than 50μg/ml,the compounds had toxicity. 12.5μg/ml and 25μg/ml were chosen for the next researches.3) MTT assay was used to determine the effect on reversing multidrug resistance of the compounds isolated from Poria cocos. Different concentration of VCR was combined with the compounds at the concentration of 12.5μg/ml or 25μg/ml respectively.Survival ratio and reversal factor were calculated.The results showed that all the eight compounds had the effect on reversing multidrug resistance at different levels.Dehydrotumulosic acid had the best effect on reversing multidrug resistance and was chosen for the further research.4) Our results showed that vincristine combined with dehydrotumulosic acid caused an increase in apoptotic cell death of KBV200. Dehydrotumulosic acid did not change the expression level of P-gp,but it increased intracellular retention value of Rh123, and at the concentration of 25μg/ml, it could significantly increase intracellular accumulation of Rh123.