| The extraction of Tephroseris kirilowii (Turcz.) Holub. by 60% ethanol, which mainly contained alkaloids and flavonoids, possessed inhibitory effects on leukemia LUIO cells. To further determine the pharmacological activity and toxicity of T. kirilowii and select its effective compositions, its alkaloids were analysed, the LDso value of its total alkaloids was determined, and the anti-tumor effects of an alkaloid from the plant were researched in the study.1. The extraction of total alkaloids and testing of A/-oxide from T. kirilowii The gum residues were extracted by cold and hot methanol from mills of T. kirilowii, acidified with 1%HC1 and filtrated to give acid liquid. The acid liquid was extracted with CH2Cl2 to remove impurity. Then, its pH was adjusted to 10-11 with 25% ammonia water to give basic liquid. The basic liquid was extracted with CH2Cl2. The total alkaloids were attained after CH2Cl2 being distilled. The basic liquid after being extracted by CH2Cl2 was extracted with ethyl actate and n-butanol in succession. The TV-oxide of alkaloids was tested by H2SO4Zn reduction and Ehrlich reagent detection to determine whether it existed in the plant. Results showed that the extraction rate of total alkaloids by cold and hot methanol was 0.067% and 0.126% respectively. The alkaloids from T. kirilowii existed mainly as free type. There was less or no TV-oxide in it.2. The analyses of total alkaloids from T. kirilowii by TLC and GC-MS The alkaloidal composition from T. kirilowii was analysed initially by TLC and GC-MS. TLC and GC-MS indicated that T. kirilowii contained at least 7 pyrrolizidine alkaloids (PAs). Of these alkaloids, senecionine(I), seneciphylline(II) and integerrimine(III) were the PAs with the structure of macrocyclic diesters.3. LD50 Determination of Alkaloids from I Kirilowii The LD50 of total alkaloids was determined using female KM mice by intraperitoneal injection according to improved Karber's method. The results showed that the LDso of total alkaloids was 74.52 +6.08mg/kg (mice, ip) with 95% confidence limit of 68.69~80.85mg/kg.4. Isolation, purification and determination of the alkaloids from T. kirilowii The alkaloids from T. kirilowii were isolated and purified by column chromatography, preparative TLC and recrystallization, and the isolated compounds were determined structurally by MS, IR and NMR. The results showed that two alkaloids (IV and VI) were isolated from T. kirilowii. Compound IV was determined as O7-angeloyl-heliotridine and its hydrolysate (V) was isolated and elucidated as heliotridine .5. The analyses of non-alkaloidal components in total alkaloids from T. kirilowii The non-alkaloidal components in total alkaloids from T. kirilowii were isolated and purified by preparative TLC and recrystallization, and the isolated compounds were determined by MS and NMR initially. The results showed that three phenylpropanoids (A4, B1 and C1) in total alkaloids from T. kirilowii were isolated.6. Study on anti-tumor effects using compound IV The inhibitory effects of compound IV on LI210 cells was analyzed by drawing up their diagram of growing curves. Growth inhibition of L1210, HepGa and HCC cells was measured by microculture tetrazolium (MTT) assay. The cell cycle of L1210 cells was tested by flow cytometry(FCM). The results showed that the slopes of growing curves and the maximums of growing density of L1210 cells exposed to compound IV decreased. Exposing L1210 cells to compound IV 80mg-L-1, the inhibitory rate was 18.18%. Exposing HepG2 and HCC cells to compound IV 320mg-L-1 , the inhibitory rates were 12.28% and 10.24% respectively. FCM indicated that L1210 cells in G2-M phase increased significantly (P<0.01) being exposed to compound IV 80mg-L-1 for 24h.Such conclusions could be attained through the study as following:* TLC and GC-MS indicated that T. kirilowii contained at least 7 pyrrolizi- dine alkaloids (PAs). Of these alkaloids, senecionine, seneciphylline and integerrimine with the structure of macrocyclic diesters were hepatotoxi...
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