两面针药材对肿瘤细胞NCI-H460和SK-OV-3“谱—效”关系的研究
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摘要
目的：以芸香科植物两面针(Zanthoxylum nitidum (Roxb.) DC.)为研究对象,以NCI-H460、SK-OV-3两种肿瘤细胞为药效模型,构建两面针的指纹图谱。在两面针的HPLC指纹图谱和其抗肿瘤活性的基础上,通过数据处理,找到两面针“化学谱-药效”之间的对应关系。
方法：
1.两面针药材抗肿瘤活性部位的筛选：两面针药材成分复杂,分别甲醇、乙醇、乙酸乙酯、氯仿、石油醚5种不同极性的溶剂对两面针药材进行提取,得到不同溶剂部位提取物；采用MTT法,以人大细胞肺癌细胞NCI-H460和人卵巢癌细胞SK-OV-3为模型,对不同极性提取物进行活性筛选,确定两面针抗肿瘤的活性部位。
2.两面针药材抗肿瘤活性的测定：采用MTT法对43个产地的两面针药材氯仿提取物进行NCI-H460和SK-OV-3两种肿瘤细胞的增殖抑制检测,用酶标仪测得OD值,再计算抑制率。
3.建立两面针药材的HPLC指纹图谱：根据化学指纹图谱研究的要求,运用国家药典委员会颁布的中药色谱指纹图谱相似度评价系统软件,建立并评价两面针药材抗肿瘤活性部位的HPLC指纹图谱。
4.构建两面针药材谱-效关系模型：采用后退法,将两面针药材HPLC指纹图谱中峰面积进行量化,与MTT法测得两面针药材抗肿瘤活性的结果结合,构建两面针药材抗肿瘤“化学谱—药效”关系。
结果：
1.两面针药材抗肿瘤活性部位的筛选：两面针抗肿瘤活性的有效成分主要存在于氯仿部位,该部位含有氯化两面针碱、鹅掌楸碱等多种生物碱成分。
2.两面针药材提取物具有明显的体外抗肿瘤作用,对肺癌细胞和卵巢癌细胞有明显的增殖抑制作用。
3.建立两面针药材的HPLC指纹图谱：经相似度、精密度、重复性及稳定性等方法检测,所建立的两面针药材HPLC指纹能全面的反应两面针药材氯仿部位主要成分的分布特征。
4.统计结果表明,HPLC指纹图谱种的24个指纹峰中,有15个峰对肿瘤细胞NCI-H460有作用,其谱-效关系为：Y=32.703-12.216X2-8.102X4+13.553X5+15.953X6-3.802X8+10.979X9-9.449X11+10.503X14-6.900X15-3.519X16+11.878X17+14.683X18-8.121X21-8.482X22+4.737X23(R2=0.923)；有15个峰对肿瘤细胞SK-OV-3有作用,其谱-效关系为：Y=74.908+18.784X4-10.686X5+10.615X6+2.889X8-5.170X1o-7.046X11-7.498X12+4.217X13-8.057X15-9.265X18-15.088X20-4.015X22+3.356X7+5.175X16-2.424X24(R2=0.938)。10批药材的验证表明所得的谱-效关系计算值与实际值十分接近。
结论：两面针的SK-OV-3细胞和NCI-H460细胞抗肿瘤谱-效方程具有统计学意义。通过方程可以估计两面针药材的肿瘤细胞增殖抑制率,从而达到评价两面针药材的抗肿瘤作用水平,从药效角度反映两面针药材质量的目的。
Objective:Zanthoxylum nitidum DC. and cancer cells NCI-H460, SK-OV-3 were studied and the fingerprint was established. On the basis of the Zanthoxylum nitidum DC. HPLC fingerprint and its anti-tumor activity, backward-elimimation procedure were applied to find the correlation between Zanthoxylum nitidum DC. fingerprint and pharmacodynamics.
Methods:
1.Screening of the part of Zanthoxylum nitidum DC. with antitumor activity:Five different polar dissolvants:methanol, ethanol, acetoacetate, chloroform and ligarine were used to abstract the Zanthoxylum nitidum DC.. MTT was used to detect the extracts of Zanthoxylum nitidum DC. on NCI-H460 and SK-OV-3 in vitro.
2. Study on the anti-tumor activity of Zanthoxylum nitidum DC.:The cytotoxicity of Zanthoxylum nitidum DC. from 43 metropolis on NCI-H460 and SK-OV-3 were detected by MTT assay in vitro.
3. The estabishment of Zanthoxylum nitidum DC..HPLC fingerprint: According to the requirements of the chemical fingerprint, estabish the HPLC fingerprint of Zanthoxylum nitidum DC.. The similarity evaluation of chromatograms was processed by chromatographic fingerprint evaluation system software which promulgated by Pharmacopoeia Commission.
4. Study on the correlation between characteristic of Zanthoxylum nitidum (Roxb.)DC. fingerprint and pharmacodynamics:at the premise of the acquired quantitateve fingerprint characteristic and pharmacodynamic data, backward-elimimation procedure was applied to find the correlation between Zanthoxylum nitidum DC. fingerprint and pharmacodynamics.
Results:
1. Screening of the part of Zanthoxylum nitidum DC. with anti-tumor activity:The active ingredient of Zanthoxylum nitidum DC. in anti-tumor activity mainly in the chloroform part. The chloroform part contained nitidine chlorine, liriodendrin and other alkaloids.
2. The extracts of Zanthoxylum nitidum DC. had significant inhibitory activious in vitro on NCI-H460 and SK-OV-3.
3. The estabishment of Zanthoxylum nitidum DC. HPLC fingerprint:The Zanthoxylum nitidum DC. HPLC fingerprint could comprehensive response to the distribution characteristics of the main component.
4. It could be concluded that there were 15 peaks have effective on NCI-H460 in the Zanthoxylum nitidum DC. HPLC fingerprint. The equation of the fingerprint and pharmacodynamics:Y=32.703-12.216X2-8.102X4+13.553X5 +15.953X6-3.802X8+10.979X9-9.449X11+10.503X14-6.900X15-3.519X16+11.878 X17+14.683X18-8.121X21-8.482X22+4.737X23 (R2=0.923); There was 15 peaks had effective on SK-OV-3 in the Zanthoxylum nitidum DC. HPLC fingerprint. The equation of the fingerprint and pharmacodynamics:Y=74.908+18.784X4--10.686X5+10.615X6+2.889X8-5.170X10-7.046X11-7.498X12+4.217X13-8.057X15-9.265X18-15.088X20-4.015X22+3.356X7+5.175X16-2.424X24 (R2=0:938). It showed that the calculated value were very close to the actual value compared to the datas of 10 origins Zanthoxylum nitidum DC.
Conclusions:The equation of the Zanthoxylum nitidum DC. fingerprint and pharmacodynamics is significant statistically. The cytotoxicity can be estimated through the equation of the Zanthoxylum nitidum DC. fingerprint and pharmacodynamics.
方法：
1.两面针药材抗肿瘤活性部位的筛选：两面针药材成分复杂,分别甲醇、乙醇、乙酸乙酯、氯仿、石油醚5种不同极性的溶剂对两面针药材进行提取,得到不同溶剂部位提取物；采用MTT法,以人大细胞肺癌细胞NCI-H460和人卵巢癌细胞SK-OV-3为模型,对不同极性提取物进行活性筛选,确定两面针抗肿瘤的活性部位。
2.两面针药材抗肿瘤活性的测定：采用MTT法对43个产地的两面针药材氯仿提取物进行NCI-H460和SK-OV-3两种肿瘤细胞的增殖抑制检测,用酶标仪测得OD值,再计算抑制率。
3.建立两面针药材的HPLC指纹图谱：根据化学指纹图谱研究的要求,运用国家药典委员会颁布的中药色谱指纹图谱相似度评价系统软件,建立并评价两面针药材抗肿瘤活性部位的HPLC指纹图谱。
4.构建两面针药材谱-效关系模型：采用后退法,将两面针药材HPLC指纹图谱中峰面积进行量化,与MTT法测得两面针药材抗肿瘤活性的结果结合,构建两面针药材抗肿瘤“化学谱—药效”关系。
结果：
1.两面针药材抗肿瘤活性部位的筛选：两面针抗肿瘤活性的有效成分主要存在于氯仿部位,该部位含有氯化两面针碱、鹅掌楸碱等多种生物碱成分。
2.两面针药材提取物具有明显的体外抗肿瘤作用,对肺癌细胞和卵巢癌细胞有明显的增殖抑制作用。
3.建立两面针药材的HPLC指纹图谱：经相似度、精密度、重复性及稳定性等方法检测,所建立的两面针药材HPLC指纹能全面的反应两面针药材氯仿部位主要成分的分布特征。
4.统计结果表明,HPLC指纹图谱种的24个指纹峰中,有15个峰对肿瘤细胞NCI-H460有作用,其谱-效关系为：Y=32.703-12.216X2-8.102X4+13.553X5+15.953X6-3.802X8+10.979X9-9.449X11+10.503X14-6.900X15-3.519X16+11.878X17+14.683X18-8.121X21-8.482X22+4.737X23(R2=0.923)；有15个峰对肿瘤细胞SK-OV-3有作用,其谱-效关系为：Y=74.908+18.784X4-10.686X5+10.615X6+2.889X8-5.170X1o-7.046X11-7.498X12+4.217X13-8.057X15-9.265X18-15.088X20-4.015X22+3.356X7+5.175X16-2.424X24(R2=0.938)。10批药材的验证表明所得的谱-效关系计算值与实际值十分接近。
结论：两面针的SK-OV-3细胞和NCI-H460细胞抗肿瘤谱-效方程具有统计学意义。通过方程可以估计两面针药材的肿瘤细胞增殖抑制率,从而达到评价两面针药材的抗肿瘤作用水平,从药效角度反映两面针药材质量的目的。
Objective:Zanthoxylum nitidum DC. and cancer cells NCI-H460, SK-OV-3 were studied and the fingerprint was established. On the basis of the Zanthoxylum nitidum DC. HPLC fingerprint and its anti-tumor activity, backward-elimimation procedure were applied to find the correlation between Zanthoxylum nitidum DC. fingerprint and pharmacodynamics.
Methods:
1.Screening of the part of Zanthoxylum nitidum DC. with antitumor activity:Five different polar dissolvants:methanol, ethanol, acetoacetate, chloroform and ligarine were used to abstract the Zanthoxylum nitidum DC.. MTT was used to detect the extracts of Zanthoxylum nitidum DC. on NCI-H460 and SK-OV-3 in vitro.
2. Study on the anti-tumor activity of Zanthoxylum nitidum DC.:The cytotoxicity of Zanthoxylum nitidum DC. from 43 metropolis on NCI-H460 and SK-OV-3 were detected by MTT assay in vitro.
3. The estabishment of Zanthoxylum nitidum DC..HPLC fingerprint: According to the requirements of the chemical fingerprint, estabish the HPLC fingerprint of Zanthoxylum nitidum DC.. The similarity evaluation of chromatograms was processed by chromatographic fingerprint evaluation system software which promulgated by Pharmacopoeia Commission.
4. Study on the correlation between characteristic of Zanthoxylum nitidum (Roxb.)DC. fingerprint and pharmacodynamics:at the premise of the acquired quantitateve fingerprint characteristic and pharmacodynamic data, backward-elimimation procedure was applied to find the correlation between Zanthoxylum nitidum DC. fingerprint and pharmacodynamics.
Results:
1. Screening of the part of Zanthoxylum nitidum DC. with anti-tumor activity:The active ingredient of Zanthoxylum nitidum DC. in anti-tumor activity mainly in the chloroform part. The chloroform part contained nitidine chlorine, liriodendrin and other alkaloids.
2. The extracts of Zanthoxylum nitidum DC. had significant inhibitory activious in vitro on NCI-H460 and SK-OV-3.
3. The estabishment of Zanthoxylum nitidum DC. HPLC fingerprint:The Zanthoxylum nitidum DC. HPLC fingerprint could comprehensive response to the distribution characteristics of the main component.
4. It could be concluded that there were 15 peaks have effective on NCI-H460 in the Zanthoxylum nitidum DC. HPLC fingerprint. The equation of the fingerprint and pharmacodynamics:Y=32.703-12.216X2-8.102X4+13.553X5 +15.953X6-3.802X8+10.979X9-9.449X11+10.503X14-6.900X15-3.519X16+11.878 X17+14.683X18-8.121X21-8.482X22+4.737X23 (R2=0.923); There was 15 peaks had effective on SK-OV-3 in the Zanthoxylum nitidum DC. HPLC fingerprint. The equation of the fingerprint and pharmacodynamics:Y=74.908+18.784X4--10.686X5+10.615X6+2.889X8-5.170X10-7.046X11-7.498X12+4.217X13-8.057X15-9.265X18-15.088X20-4.015X22+3.356X7+5.175X16-2.424X24 (R2=0:938). It showed that the calculated value were very close to the actual value compared to the datas of 10 origins Zanthoxylum nitidum DC.
Conclusions:The equation of the Zanthoxylum nitidum DC. fingerprint and pharmacodynamics is significant statistically. The cytotoxicity can be estimated through the equation of the Zanthoxylum nitidum DC. fingerprint and pharmacodynamics.
引文
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