Study on the Material Base of Tripterygium Glycosides Tablet against Human Acute Monocytic Leukemia
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摘要:
目的 发现雷公藤多苷片抗不同亚型血液肿瘤的敏感细胞株及其对敏感细胞株增殖抑制作用的物质基础。 方法 利用CCK-8法检测雷公藤多苷片及其提取物体外对肿瘤细胞增殖的影响; 构建敏感瘤株的体内异种移植瘤动物模型考察雷公藤多苷片对肿瘤细胞的抑制作用; 采用色谱分离技术对雷公藤多苷片提取物进行系统分离, 综合质谱和核磁共振数据鉴定化合物结构, 并考察单体化合物抗人急性单核细胞白血病(Acute monocytic leukemia, AMOL)活性; 进一步通过制备液相定向敲除雷公藤多苷片及其提取物中的雷公藤甲素(Triptolide, TP), 考察敲除TP前后雷公藤多苷片及其提取物的抗AMOL活性。 结果 细胞实验结果表明雷公藤多苷片有较强的抗AMOL活性; 体内异种移植瘤实验结果表明雷公藤多苷片可明显抑制AMOL细胞皮下异种移植瘤的生长; 进一步对雷公藤多苷片提取物进行系统分离及其细胞水平上的活性评价, 发现二萜类化合物TP抗AMOL活性最强; 定向敲除TP后的雷公藤多苷片及其提取物对AMOL细胞MV-4-11的IC50值分别升高8及13倍。 结论 雷公藤多苷片对AMOL细胞具有显著的抗白血病效应, 其中TP是其发挥药效的物质基础。 -
关键词:
- 雷公藤多苷片 /
- 化学成分 /
- 人急性单核细胞白血病 /
- 雷公藤甲素 /
- 抗肿瘤
Abstract:OBJECTIVE To discovery sensitive cell lines of tripterygium glycosides tablet against different subtypes of hematological tumors, and to find out the material base for the effect on proliferative inhibition against sensitive cell lines. METHODS The CCK-8 assay was used to assess the effect of tripterygium glycosides tablet and its extract on cell proliferation in vitro; Then, the in vivo subcutaneous xenograft models by injecting sensitive cells were constructed to test the inhibitory activity of tripterygium glycosides tablet; Furthermore, the extract of tripterygium glycosides was systemically chemical investigated by chromatographic separation techniques, mass spectrometry, and nuclear magnetic resonance, and anti-human acute monocytic leukemia (AMOL) activity was tested for each compound; In the end, preparative HPLC was used for the specific removal of triptolide (TP) from the extract of tripterygium glycosides. The anti-AMOL activity was measured and analyzed after removing TP from the formulations of T. wilfordii. RESULTS The results of cell experiments indicated that tripterygium glycosides exhibited potent anti-AMOL activity; Next, in vivo tumor xenograft models showed tripterygium glycosides significantly inhibited the growth of subcutaneous tumor xenografts of AMOL cells; Furthermore, chemical constituents of tripterygium glycosides extract were isolated systemically, the activity were evaluated at the cellular level. It was found that TP showed the strongest anti-AMOL activity. Finally, it was found that the IC50 values of the tripterygium glycosides tablet and its extract against AMOL cells increased 8 and 13 fold, respectively, after removing TP. CONCLUSION Tripterygium glycosides tablet shows significant inhibitory activity against acute monocytic leukemia cells, and its diterpenoid, triptolide, is the most active ingredient. -
表 1 TP定向敲除色谱条件
Table 1. TP directional knock-out chromatographic conditions
时间/min 流动相A/% 流动相B/% 0 65 35 40 30 70 41 0 100 45 0 100 46 65 35 50 65 35 表 2 雷公藤多苷片提取物作用于不同肿瘤细胞系48 h的IC50值(x±s,n=3)
Table 2. IC50 values of tripterygium glycosides extract against different tumor cell lines for 48 h (x±s, n=3)
细胞系名称 细胞系类型 雷公藤提取物/(μg·mL-1) SHI-1 AMOL细胞 1.69±0.01 MV-4-11 AMOL细胞 2.10±0.02 Kasumi-1 人急性原粒细胞白血病细胞 4.17±0.07 SU-DHL-10 人B细胞淋巴瘤细胞 5.18±0.03 K562 人慢性髓原白血病细胞 5.57±0.03 表 3 不同厂家雷公藤多苷片作用MV-4-11细胞48 h的IC50值(x±s,n=3)
Table 3. IC50 values of tripterygium glycosides tablet from different manufacturers against MV-4-11 cells for 48 h (x±s, n=3)
雷公藤多苷片 IC50/(μg·mL-1) 厂家1 3.33±0.01 厂家2 3.57±0.10 厂家3 5.64±0.02 厂家4 5.70±0.03 厂家5 5.55±0.37 表 4 雷公藤多苷片提取物的不同流分对MV-4-11细胞的增殖抑制率(x±s,%, n=3)
Table 4. The proliferation inhibition of fraction of tripterygium glycosides extract against MV-4-11 cells for 48 h(x±s, %, n=3)
流分 25 μg·mL-1 5 μg·mL-1 1 μg·mL-1 X3 99.92±0.10 92.70±0.08 1.52±3.00 X4 94.19±0.13 94.59±0.57 11.74±1.61 X5 96.05±1.06 91.80±0.42 1.45±1.35 X6 93.62±0.54 92.39±1.05 90.23±1.43 X7 95.18±0.09 92.49±0.62 89.37±1.38 X8 91.43±1.05 90.29±0.73 29.71±0.96 表 5 化合物1~15对MV-4-11细胞的增殖抑制率(x±s,%, n=3)
Table 5. The proliferation inhibition of compounds 1-15 against MV-4-11 cells for 48 h (x±s, %, n=3)
编号 化合物 25 μg·mL-1 5 μg·mL-1 1 μg·mL-1 1 雷酚内酯(Triptophnolide) 93.41±0.17 24.61±10.19 5.54±4.13 2 异雷酚新内酯(Isoneotriptophenolide) 66.54±1.14 -8.94±1.05 -23.48±3.64 3 雷酚萜醇(Triptonoterpenol) 91.37±1.30 27.67±15.60 12.01±9.58 4 Triptobenzene J 92.15±3.11 -4.44±6.44 -10.47±4.47 5 雷公藤甲素(Triptolide) 91.84±0.63 91.46±0.30 88.59±1.02 6 雷公藤晋碱(Wilforgine) -5.69±8.27 -33.66±12.28 -42.56±16.57 7 雷公藤定碱(Wilfordine) 16.21±10.07 9.19±11.47 3.28±13.39 8 雷公藤次碱(Wilforine) 55.09±4.53 17.49±1.24 15.39±0.68 9 雷公藤春碱(Wilfortrine) 12.77±0.97 9.31±5.95 -22.35±5.76 10 雷公藤新碱(Euonine) 7.00±1.00 -3.41±8.39 -6.82±2.16 11 Peritassine A 92.91±0.63 14.66±1.82 0.13±4.12 12 雷公藤红素(Celastrol) 94.17±0.29 77.40±0.41 73.72±0.29 13 雷公藤酚A(Wilforol A) 92.54±0.23 34.85±0.08 -22.92±1.38 14 雷公藤三萜酸A(Triptotriterpenic acid A) 89.65±0.35 2.39±0.27 -30.44±0.06 15 23-Nor-6-oxodemethylpristimerol 94.06±0.28 18.08±0.96 -52.65±0.20 16 雷公藤多苷片提取物(Tripterygium glycosides extract) 91.62±0.30 90.24±0.59 23.13±2.83 表 6 TP作用于不同肿瘤细胞系48 h的IC50值(x±s,n=3)
Table 6. IC50 values of TP against different tumor cell lines for 48 h (x±s, n=3)
细胞系名称 细胞系类型 TP/(ng·mL-1) SHI-1 AMOL细胞 1.37±0.11 MV-4-11 AMOL细胞 3.35±0.03 Kasumi-1 人急性原粒细胞白血病细胞 3.51±0.26 SU-DHL-10 人B细胞淋巴瘤细胞 11.41±1.22 K562 人慢性髓原白血病细胞 13.35±1.05 表 7 雷公藤多苷片及其提取物定向敲除TP前后作用MV-4-11细胞48 h的IC50值(x±s,μg·mL-1, n=3)
Table 7. IC50 values of tripterygium glycosides tablet and its extract against MV-4-11 cells for 48 h before or after targeted knock-out of TP (x±s, μg·mL-1, n=3)
雷公藤制剂 去除TP前 去除TP后 雷公藤多苷片(美通) 3.57±0.10 28.91±0.02 雷公藤多苷片提取物 2.10±0.02 28.21±0.22 -
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