【作者】 王跃虎；

【导师】 王建华；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2003， 硕士

【摘要】 狗舌草60％乙醇提取物具有抑制L₁₂₁₀细胞的作用，其中主要含有生物碱和黄酮类化合物。为了更好地评价狗舌草的药理活性和毒性并筛选出狗舌草的有效成分，本研究对狗舌草生物碱成分进行了分析，对其总碱的LD₅₀进行了测定，并用分离得到的一个单体生物碱进行了抗肿瘤试验。 1．狗舌草总生物碱的提取与氮氧化物的检测 利用冷浸法和热回流法从狗舌草粉中提取出浸膏，浸膏用1％HCl酸化，过滤，得酸水液。酸水液用CH₂Cl₂萃取除杂质后，用25％氨水调pH至10～11，得碱水液。碱水液用CH₂Cl₂萃取，回收CH₂Cl₂得总生物碱。CH₂Cl₂萃取后的碱水液分别用乙酸乙酯和正丁醇萃取。通过H₂SO₄-Zn还原反应和Ehrlich显色反应，检查狗舌草中是否含有生物碱氮氧化物。结果：通过冷浸法，狗舌草总碱提取率为0.067％；通过热回流法，总碱提取率为0.126％。狗舌草生物碱主要以游离型存在，氮氧化物很少或没有。 2．狗舌草总生物碱的TLC和GC-MS分析 利用TLC和GC-MS对狗舌草生物碱成分进行初步分析。分析表明，狗舌草至少含有7个双稠吡咯啶生物碱（PAs），其中千里光宁（Ⅰ）、千里光非灵（Ⅱ）和全缘千里光碱（Ⅲ）都是具有大环双酯结构的PAs。 3．狗舌草总生物碱LD₅₀测定 通过改良寇氏法、腹腔注射给药测定总生物碱对KM雌性小鼠的LD₅₀。结果：狗舌草总生物碱LD₅₀值为74.52±6.08mg/kg（KM小鼠，ip），95％可信限为68.69～80.85mg/kg。 4．狗舌草生物碱的分离、纯化与鉴定 通过柱层析、制备性TLC及重结晶法，对狗舌草生物碱进行分离、纯化，通过MS、IR和NMR对分离到的单体化合物进行结构鉴定。结果：从狗舌草总碱中分离到2个生物碱（Ⅳ和Ⅵ），化合物Ⅳ被鉴定为当归酰天芥菜定，其水解物之一被分离、鉴定为天芥菜定（Ⅴ）。 5．狗舌草总碱中非生物碱成分的分离与鉴定 通过制备性TLC及重结晶法，对狗舌草总碱中非生物碱成分进行分离、纯化，通过MS和NMR对分离到的单体化合物进行初步鉴定。结果：从狗舌草总碱中分离到3个苯丙酸类单体化合物（A4、B1和C1）。 6．化合物Ⅳ的抗肿瘤作用研究 通过细胞生长曲线的绘制，分析化合物Ⅳ对L₁₂₁₀细胞的抑制作用；通过MTT比色法，测定化合物Ⅳ对L₁₂₁₀、HepG₂和HCC细胞的抑制率；通过流式细胞术（FCM）检测化合物Ⅳ对L₁₂₁₀细胞周期的影响。结果：在化合物Ⅳ作用下，L₁₂₁₀细胞生长曲线斜率和最大生长密度降低。化合物Ⅳ80mg·L^-1对L₁₂₁₀细胞的抑制率为18.18％；化合物Ⅳ 320mg·L^-1对HepG₂和HCC细胞抑制率分别为12.28％和10.24％。FCM检测表明，化合物Ⅳ 80mg·L^-1作用24h后，L₁₂₁₀细ii西北农林科技大学2003届硕士学位论文:狗舌草生物碱及抗肿瘤活性成分研究胞的GZ一M期细胞明显增加（P<0.01）。 通过本研究，得到如下结论: *TLC及GC一MS显示，狗舌草中至少含有7个双稠毗咯睫生物碱。其中具有大环双酷结构的千里光宁、千里光非灵和全缘千里光碱为肝毒性双稠毗咯睫生物碱。 *首次从狗舌草中分离得到两个生物碱单体，其中之一鉴定为当归酞天芥菜定，另一个结构待定。当归酞天芥菜定是狗舌草中含量最高的生物碱。 *当归酞天芥菜定对白血病L121。细胞有一定的抑制作用，对肝癌HePGZ和HcC细胞作用较差。对L1210细胞的作用发生在细胞周期的GZ一M期。 *狗舌草总生物碱毒性较大，将狗舌草全草药用会有很大的风险。 *首次从狗舌草中分离得到三个苯丙酸类单体化合物，结构待定。更多还原

【Abstract】 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更多还原