【作者】 司红丽；

【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】 通过对狗舌草总黄酮的提取分离、定性、定量及毒性试验，对狗舌草总黄酮进行了系统的分析，并就其抗肿瘤活性进行评价。 1．狗舌草总黄酮的提取利用冷浸法和热回流法从狗舌草粉中提取出总黄酮。狗舌草热回流浸膏的得率明显高于冷浸法，但其总黄酮得率却低于冷浸法。本试验采用冷浸法提取狗舌草中总黄酮。 2．狗舌草总黄酮定性试验结合滤纸显色反应和薄层色谱检测结果，推断狗舌草总黄酮中主要含有黄酮类、黄酮醇类、二氢黄酮醇类和异黄酮类等，结构可能主要含有无5-OH黄酮类、5-OH，4′-OH黄酮类、5-OH，4′-OH，3-OR黄酮醇类、4′-羟基黄酮醇或7，4′-二羟基黄酮醇等。 3．狗舌草总黄酮定量试验 以芦丁为标样，蒸馏水为空白参比对照，在波长510nm处进行比色测定狗舌草中总黄酮为5.711％，为下一步的研究提供基本的理论指导。 4．狗舌草总黄酮LD₅₀测定通过改良寇氏法，用腹腔注射给药测定狗舌草总黄酮对KM雌性小鼠的LD₅₀值为1392.52±94.62mg·kg^-1，其毒性明显低于狗舌草60％乙醇提取物LD₅₀(791.22±170.17mg·kg^-1)及狗舌草属植物中可能含有的生物碱单体LD₅₀，属于低毒范围，因此可推断临床上狗舌草中毒主要由其中所含的双稠吡咯啶生物碱类物质引起，而与其中所含的黄酮类化合物相关性不大。其中所含的黄酮类化合物可能与狗舌草的抗癌活性有关。 5．狗舌草总黄酮对三种肿瘤细胞的作用 通过MTT比色法测定狗舌草总黄酮对L₁₂₁₀、Hep-G₂和HCC细胞的抑制率（IR）。狗舌草总黄酮对三种肿瘤细胞的IR均随浓度升高而增高，说明其生长抑制率和浓度正相关。不同浓度狗舌草总黄酮对Hep-G₂的IR最小，为不敏感；100.0μg·ml^-1狗舌草总黄酮对HCC的IR为中度敏感，其余不同浓度均为不敏感；10.0μg·ml^-1和100.0μg·ml^-1狗舌草总黄酮对淋巴性白血病细胞L₁₂₁₀的IR分别为中度敏感和高度敏感，其中半数致死浓度IC₅₀为7.756μg·ml^-1。说明狗舌草总黄酮对L₁₂₁₀有一定抑制作用，对肝癌细胞HCC及Hep-G₂作用不明显。 6．狗舌草总黄酮对L₁₂₁₀细胞的体外作用研究 由细胞生长曲线和mPDT测定结果表明，当狗舌草总黄酮浓度为10.0μg·ml^-1时，L₁₂₁₀细胞的生长缓慢，mPDT极显著增加（P＜0.01）。当狗舌草总黄酮浓度为100.0μg·ml^-1时，表现出较强的细胞毒性，使L₁₂₁₀细胞生长基本停滞。台盼蓝拒染率结果表明，不同浓度狗舌草总黄酮均可显著抑制L₁₂₁₀细胞的活性，且随着剂量的增加活细胞率减少。其中10.0和100.0μg·ml^-1的狗舌草总黄酮可显著抑制L₁₂₁₀细胞活性（P＜0.01）。说明狗舌草总黄酮能够抑制L₁₂₁₀细胞活性，且该作用具有剂量依赖性。狗舌草总黄酮还可使细胞形态有变圆、聚集倾向，随着剂量的增加，L₁₂₁₀细胞数量明显减少，聚集现象加剧，有大量的细胞碎ii西北农林科技大学2003届硕士学位论文:狗舌草总黄酮的提取及抗肿瘤作用研究片出现。流式细胞术检测，10.0陀·ml一，狗舌草总黄酮（TF）引起s期L:2:0细胞数目显著增加，引起S一GZ阻滞，出现S期的细胞不能正常向q期发展，从而阻断了细胞周期的正常发展。狗舌草总黄酮（TF）可能具有促进DNA断裂，激活了肿瘤细胞chk活性，加快已受损的肿瘤细胞的分裂速度，使损伤大量积累，导致细胞死亡的抗癌机理。更多还原

【Abstract】 The systematic analysis and anti-tumor activity were achieved through the extraction, determinant, quantitative and toxic test on the total flavonoids (TF) from Tephroseris kirilowii (Turcz.) Holub.1.The extraction of the total flavonoids (TF) from T. kirilowii The total flavonoids (TF) were extracted by cool and hot methanol methods from powder of T. kirilowii. The extraction rate of gum from the hot methanol method was higher than the cool leach method, but the content of the total flavonoids was lower. The cool leach method was applied in the experiment.2.The determinant test of the total flavonoids (TF) from T. kirilowii The total flavonoids (TF) from T.Kirihwii contain mostly flavones, flavonols, bihydroflavonols, isoflavones, etc. It concluded by the results of the color reaction of filter and the thin layer chromatography. The structures mainly contained de-5-OH flavones, 5-OH, 4’-OH flavones, 5-OH, 4’-OH, 3-OR flavonols, 4’-OH flavonols and 7, 4’-bihydro -xide flavonols.3. The quantitative test of the total flavonoids (TF) from T. kirilowiiThe result of colorimetric determination was 5.711% of the total flavonoids (TF) from T. Kirilowii by the spectrophotometry which mensurate on the wavelengh in 510nm with the rutin as mark and the distilled water as control. It could supply the basal theory for the next study.4. The LD50 determination of the total flavonoids (TF) from T. kirilowiiThe LD50 of the total flavonoids (TF) from T. Kirilowii was determined using female KM mice by intraperitoneal injection according to improved Karber’s method. The LD50 was 1392.52+94.62 mg·kg-1 which belong to lower toxicity. Its toxicity evidently lower than the LD50 of the extraction from T. Kirilowii using 60% ethanol solution and the alkaloid monomer. It concluded that the pyrrolizidine alkaloids lead to T.Kirilowii poisoning in clinic. The flavonoids from T. Kirilowii were likely to have the anti-tumor activity.5. The effect the total flavonoids (TF) from T. kirilowii on three tumor cellsThe inhibit rates were determined by MTT colorimetry method to study the total flavonoids (TF) from T. Kirilowii on three tumor cells. The inhibit rates (IR) increased with the concentration heightening that they were positive correlation. The results showed that the different concentration of total flavonoids was low sensitive to liver-cancer Hep-G2cells. At the concentration of 10.0ug·m-1 TF was medial sensitive to liver-cancer HCC cells. At the concentration of 10.0ug·ml-1 and 100.0ug·ml-1 were medial and high sensitive to leukemia L1210 cells respectively. And the half of inhibit concentration (IC50) of total flavonoids on leukemia L1210 cells was 7.756ug·ml-1. These results suggest that the total flavonoids (TF) from T. Kirilowii have higher anti-tumor activity to the L1210 cells than the HCC and Hep-G2 cells.6. The studies of the total flavonoids (TF) from T. kirilowii on the L1210 cells in vitroThe rate of growth was slow and mPDT increased greatly (P<0.01 ) at the concentration of 100ug·ml-1 of TF by studied on the growth curve and mPDT of the L1210 cells. At the concentration of 100.0ug·ml-1 of TF, it showed high cytotoxicity and made the growth stagnancy. The result of the resist-dye rate of trypan blue showed that the different concentration of total flavonoids (TF) could all restrain greatly the activity of L1210 cells. The rate of lively cell was decreased with the concentration increasing. At the concentration of 10.0ug·ml-1 and 100ug·ml-1 of TF possessed better inhibition to the activity of leukemia L1210 cells (P<0.01). It indicated that the total flavonoids (TF) inhibit the activity of L1210 cells and the inhibition rate had a positive interrelationship with the concentration. The conformation of L1210 cell had round and assemble tendency by the total flavonoids (TF). The amount decreased and assemble severed with the increasing concentration. It had lots of fragment in culture liquid. At the concentration of 10.0ug·ml-1 of TF, the amount of S phase L1210 cell increased great更多还原