中药南蛇藤中雷公藤红素的提取及纯化工艺研究
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摘要
本论文对雷公藤红素的高效液相色谱(HPLC)定量方法的方法学进行了考察。比较了不同药材和药材不同部位中雷公藤红素总量,选择以湖南产南蛇藤根茎为提取原料,其雷公藤红素总含量为0.0286%。
对南蛇藤药材中雷公藤红素的回流提取工艺、超临界萃取工艺和超声波提取工艺进行对比,确定超声波提取工艺为最佳工艺,其优化条件为:10倍量的甲醇溶剂,超声功率400W,单次辐射时间8s(间歇时间5s),超声提取总时间为40min。超声波提取工艺验证,雷公藤红素的提取率可以达到90.8%,超声波循环提取工艺验证,提取率可以达到91.6%。
研究了样品中雷公藤红素的薄层层析和硅胶吸附柱层析。确定了薄层层析条件为:硅胶GF_(254),氯仿-甲醇(7:1)为展开剂,展开槽中上行展开,在紫外检测器365nm和254nm下观测。硅胶吸附柱的洗脱剂为石油醚-乙酸乙酯系统,洗脱梯度为:1:0(2V_0),5:1(2V_0),1:1(10 V_0),以建立的薄层层析条件进行检测,判定含有雷公藤红素的流分。高效液相检测可以得到纯度90.6%的雷公藤红素。
The quantitative analysis of tripterine by HPLC and its methodology were studied. Total contents of tripterine of different materials and parts were compared. Roots and stems of the Colastrus orbiculatus Thunb in Hunan were chosen and the total content of tripterine was 0.0286%.
Compared with refluxing extraction and supercritical extraction, ultrasonic extraction technology of tripterine from Colastrus orbiculatus Thunb was studied and the optimum extraction condition was: ten times of methanol to powder, ultrasonic power 400W, once radiation time 8s (interval time 5s), total time of ultrasonic extraction 40min. The yield of tripterine was 90.8% under the condition by ultrasonic extraction and 91.6% by ultrasonic circulation extraction.
Thin-layer chromatography (TLC) and silica gel adsorption chromatography of tripterine were studied. TLC condition was: silica gel GF_(254) as adsorbent, chloroform : methanol (7∶1) as developer. Tripterine was developed upwards in the separation chamber and detected under 365nm and 254nm. Eluant of silica gel adsorption column was petroleum ether-ethyl acetate. Elution gradient was 1∶0(2V_0), 5∶1(2V_0), 1∶1(10 V_0). The purity of tripterine obtained was 90.6%.
对南蛇藤药材中雷公藤红素的回流提取工艺、超临界萃取工艺和超声波提取工艺进行对比,确定超声波提取工艺为最佳工艺,其优化条件为:10倍量的甲醇溶剂,超声功率400W,单次辐射时间8s(间歇时间5s),超声提取总时间为40min。超声波提取工艺验证,雷公藤红素的提取率可以达到90.8%,超声波循环提取工艺验证,提取率可以达到91.6%。
研究了样品中雷公藤红素的薄层层析和硅胶吸附柱层析。确定了薄层层析条件为:硅胶GF_(254),氯仿-甲醇(7:1)为展开剂,展开槽中上行展开,在紫外检测器365nm和254nm下观测。硅胶吸附柱的洗脱剂为石油醚-乙酸乙酯系统,洗脱梯度为:1:0(2V_0),5:1(2V_0),1:1(10 V_0),以建立的薄层层析条件进行检测,判定含有雷公藤红素的流分。高效液相检测可以得到纯度90.6%的雷公藤红素。
The quantitative analysis of tripterine by HPLC and its methodology were studied. Total contents of tripterine of different materials and parts were compared. Roots and stems of the Colastrus orbiculatus Thunb in Hunan were chosen and the total content of tripterine was 0.0286%.
Compared with refluxing extraction and supercritical extraction, ultrasonic extraction technology of tripterine from Colastrus orbiculatus Thunb was studied and the optimum extraction condition was: ten times of methanol to powder, ultrasonic power 400W, once radiation time 8s (interval time 5s), total time of ultrasonic extraction 40min. The yield of tripterine was 90.8% under the condition by ultrasonic extraction and 91.6% by ultrasonic circulation extraction.
Thin-layer chromatography (TLC) and silica gel adsorption chromatography of tripterine were studied. TLC condition was: silica gel GF_(254) as adsorbent, chloroform : methanol (7∶1) as developer. Tripterine was developed upwards in the separation chamber and detected under 365nm and 254nm. Eluant of silica gel adsorption column was petroleum ether-ethyl acetate. Elution gradient was 1∶0(2V_0), 5∶1(2V_0), 1∶1(10 V_0). The purity of tripterine obtained was 90.6%.
引文
[1]阎克里,路平,方翠芬,等.中药南蛇藤的研究进展[J].西北药学杂志,2003.18(4):187-189
[2]张亮,张正竹,安登魁.雷公藤植物化学成分研究进展[J].中国药科大学学报 90.21,19(4):251
[3]“雷公藤”红素抗癌研究受关注[N]中国西部科技,2006.12:12
[4]武汉科学家首次发现雷公藤有望成为抗癌新药[N].中国制药息,2006.22(9):15
[5]中药雷公藤红素抗癌机理被证实[N].天然产物分离,2006.4(3):18-19
[6]张立平,靳风柱.雷公藤红素提取工艺的研究[J].中华医学全科杂志,2004.3(2):63-64
[7]Allison A.C.,Cacabelos R.,Lombardi R.M.,et al.Progress in neuro-psychopharmacology & biological psychiatry.Psychiatry,2001.25(7):1341-1357
[8]Jin HZ,Wang BY,Kim HS,et al.Anti-inflammatory constituents of Celastrus orbiculatus inhibit the NF-KappaB activation and NO production[J].J.Nat Prod,2002.65(1):89-91
[9]陈佩东,梁敬钰.南蛇藤属植物化学成分及活性研究进展[J].海峡药学,1999.11(4):3-6
[10]Anthony C.Allison,Ramon Cacabelos,Valter R.M.et al.Celastrol,a potent antioxidant and anti-inflammatory drug,as a possible treatment for Alzheimer's disease.Prog.Neuro-Psychop-harmacol.&Biol.psychiat,2001.25:1341-1357
[11]王明安,李增民,陈馥衡.南蛇藤根皮化学成分的研究[J].北京农业大学学报,1994.20(4):438
[12]袁玉英,顾芝萍,石其贤,等.南蛇藤素抑制豚鼠体外精子的受精能力[J].药学学报,1995.30(5):331-335
[13]马伟光,张滔,张超,等.有毒药物雷公藤的研究及展望[J].中华中医药杂志,2006.21(2):117-119
[14]张罗修,顾克显.雷公藤红素对某些免疫功能的影响[J].上海免疫学杂志,1986.6(5):277-279
[15]许晨,吴兆龙,张志刚,等。雷公藤红素防治狼疮性肾小球硬化的研究[J].中国中西医结合肾病杂志,2002.3(3):132
[16]张舰,许运明,王维明,等.南蛇藤提取物体内抗肿瘤作用的实验研究[J].中国中药杂志,2006.31(18):1514-1516
[17]黄煜伦,周幽心,周岱,等.雷公藤红素抑制体外血管生成的实验研究[J].中华肿瘤杂志,2003.25(5):429.
[18]周幽心,孙成法,许期年,等.雷公藤红素抑制血管内皮株增殖的体外研究[J].实用癌症杂志,2004.19(6):564.
[19]Folkman J.What is t he evidence that tumors are angiogenesis dependent?[J].Journal of the National Cancer Institute,1990.82(4):426.
[20]Fanelli M,Locopo N,Gattuso D,et al.Assessment of tumor vascularization:immuno histochemical and non-invase methods[J].Int J Biol Markers,1999.14(4):218.
[21]张丽娟,朱润庆,费雁,等.南蛇藤素对肿瘤血管的抑制作用[J].肿瘤防冶研究,2005.32(11):719-720
[22]Huanjie Yang,Di Chen,Qiuzhi Cindy Cui,Xiao Yuan,et al.Celastrol,a Triterpene Extracted from the Chinese "Thunder of God Vine," Is a Potent Proteasome Inhibitor and Suppresses Human Prostate Cancer Growth in Nude Mice.Cancer Res,2006.66:4758-4765.
[23]Wei He,Fu-Chth Huang,Ashvin Gavai,etal.Novel cytokine release inhibitors,part Ⅲ:Truncated analogs of celastrol[J].Bioorganic &medicinal Chemistry Letters,1998.8:3659-3664
[24]袁晓,卢大炎,王国亮,等.毛枝南蛇藤根皮中南蛇藤素得分离鉴定及含量测定.武汉植物学研究,1992.20(4)
[25]苗抗立,张晓康,董颖.雷公藤根皮三萜成分研究[J].天然产物研究与开发,1999.12(4):l一7
[26]夏焱,王文燕,张彦文,等.HPLC法测定雷公藤及其片剂中雷公藤红素[J].中草药,2005.36(8):1154-1156
[27]李红茹,李淑芬,段宏泉,等.超临界流体萃取雷公藤中有效成分的工艺优化fJl.天津大学学报,2007.40(3):269-274
[28]Shihua Wu,Cuirong Sun,Kuiwu Wang,et.al.Preparative isolation and purification of celastrol from Celastrus orbiculatus Thunb.By a new counter-current chromatography method with an upright coil planet centrifuge.Journal of Chromatography A,2004.1028:171-174
[29]夏志林,徐格青,郭舜民,等.雷公藤和昆明山海棠的薄层层析检查[J].中草药,1994.25(9):464-465
[30]中华人民共和国药典[S].北京:化学工业出版社,附录28
[2]张亮,张正竹,安登魁.雷公藤植物化学成分研究进展[J].中国药科大学学报 90.21,19(4):251
[3]“雷公藤”红素抗癌研究受关注[N]中国西部科技,2006.12:12
[4]武汉科学家首次发现雷公藤有望成为抗癌新药[N].中国制药息,2006.22(9):15
[5]中药雷公藤红素抗癌机理被证实[N].天然产物分离,2006.4(3):18-19
[6]张立平,靳风柱.雷公藤红素提取工艺的研究[J].中华医学全科杂志,2004.3(2):63-64
[7]Allison A.C.,Cacabelos R.,Lombardi R.M.,et al.Progress in neuro-psychopharmacology & biological psychiatry.Psychiatry,2001.25(7):1341-1357
[8]Jin HZ,Wang BY,Kim HS,et al.Anti-inflammatory constituents of Celastrus orbiculatus inhibit the NF-KappaB activation and NO production[J].J.Nat Prod,2002.65(1):89-91
[9]陈佩东,梁敬钰.南蛇藤属植物化学成分及活性研究进展[J].海峡药学,1999.11(4):3-6
[10]Anthony C.Allison,Ramon Cacabelos,Valter R.M.et al.Celastrol,a potent antioxidant and anti-inflammatory drug,as a possible treatment for Alzheimer's disease.Prog.Neuro-Psychop-harmacol.&Biol.psychiat,2001.25:1341-1357
[11]王明安,李增民,陈馥衡.南蛇藤根皮化学成分的研究[J].北京农业大学学报,1994.20(4):438
[12]袁玉英,顾芝萍,石其贤,等.南蛇藤素抑制豚鼠体外精子的受精能力[J].药学学报,1995.30(5):331-335
[13]马伟光,张滔,张超,等.有毒药物雷公藤的研究及展望[J].中华中医药杂志,2006.21(2):117-119
[14]张罗修,顾克显.雷公藤红素对某些免疫功能的影响[J].上海免疫学杂志,1986.6(5):277-279
[15]许晨,吴兆龙,张志刚,等。雷公藤红素防治狼疮性肾小球硬化的研究[J].中国中西医结合肾病杂志,2002.3(3):132
[16]张舰,许运明,王维明,等.南蛇藤提取物体内抗肿瘤作用的实验研究[J].中国中药杂志,2006.31(18):1514-1516
[17]黄煜伦,周幽心,周岱,等.雷公藤红素抑制体外血管生成的实验研究[J].中华肿瘤杂志,2003.25(5):429.
[18]周幽心,孙成法,许期年,等.雷公藤红素抑制血管内皮株增殖的体外研究[J].实用癌症杂志,2004.19(6):564.
[19]Folkman J.What is t he evidence that tumors are angiogenesis dependent?[J].Journal of the National Cancer Institute,1990.82(4):426.
[20]Fanelli M,Locopo N,Gattuso D,et al.Assessment of tumor vascularization:immuno histochemical and non-invase methods[J].Int J Biol Markers,1999.14(4):218.
[21]张丽娟,朱润庆,费雁,等.南蛇藤素对肿瘤血管的抑制作用[J].肿瘤防冶研究,2005.32(11):719-720
[22]Huanjie Yang,Di Chen,Qiuzhi Cindy Cui,Xiao Yuan,et al.Celastrol,a Triterpene Extracted from the Chinese "Thunder of God Vine," Is a Potent Proteasome Inhibitor and Suppresses Human Prostate Cancer Growth in Nude Mice.Cancer Res,2006.66:4758-4765.
[23]Wei He,Fu-Chth Huang,Ashvin Gavai,etal.Novel cytokine release inhibitors,part Ⅲ:Truncated analogs of celastrol[J].Bioorganic &medicinal Chemistry Letters,1998.8:3659-3664
[24]袁晓,卢大炎,王国亮,等.毛枝南蛇藤根皮中南蛇藤素得分离鉴定及含量测定.武汉植物学研究,1992.20(4)
[25]苗抗立,张晓康,董颖.雷公藤根皮三萜成分研究[J].天然产物研究与开发,1999.12(4):l一7
[26]夏焱,王文燕,张彦文,等.HPLC法测定雷公藤及其片剂中雷公藤红素[J].中草药,2005.36(8):1154-1156
[27]李红茹,李淑芬,段宏泉,等.超临界流体萃取雷公藤中有效成分的工艺优化fJl.天津大学学报,2007.40(3):269-274
[28]Shihua Wu,Cuirong Sun,Kuiwu Wang,et.al.Preparative isolation and purification of celastrol from Celastrus orbiculatus Thunb.By a new counter-current chromatography method with an upright coil planet centrifuge.Journal of Chromatography A,2004.1028:171-174
[29]夏志林,徐格青,郭舜民,等.雷公藤和昆明山海棠的薄层层析检查[J].中草药,1994.25(9):464-465
[30]中华人民共和国药典[S].北京:化学工业出版社,附录28