高速逆流色谱分离制备无花果叶和长松萝中主要活性成分的研究
|
摘要
本文以无花果叶、长松萝为原料,用石油醚(60-90℃)进行粗提取,减压蒸发掉石油醚,得到无花果叶粗提物浸膏和长松萝粗提物。利用高速逆流色谱法进行分离纯化无花果叶、长松萝粗提物。通过多次试验,确定分离纯化无花果叶粗提物的高速逆流色谱溶剂体系为正己烷:乙酸乙酯:甲醇:水为(1:1:1:1 v/v),长松萝粗提物高速逆流色谱溶剂体系为正己烷:乙腈:乙酸乙酯:水(8:7:5:0.8 v/v)。通过高速逆流色谱的一次分离,一次进样无花果叶粗提物浸膏200mg可得到补骨脂素4.4 mg佛手内酯2.1 mg;一次进样长松萝粗提物25mg可获得松萝酸纯品11.2mg。高效液相色谱确定所得补骨脂素纯度为99.1%,佛手内酯纯度为98.2%,松萝酸纯度为99%以上。由质谱、核磁共振确定所得补骨脂素、佛手内酯、松萝酸的化学结构。将无花果叶80%醇提物和长松萝石油醚粗提物进行小鼠的急性亚急性及促生长实验。结果表明,无花果叶提取物属于无毒受试物,5.0g/kg.d的剂量对雄性小鼠有轻度促生长作用,对雌性小鼠无作用。无花果叶提取物5.0g/kg.d剂量可以使雌性和雄性小鼠肝脏重量显著增加,1.0g/kg.d剂量可明显提高雄性小鼠胸腺重量,对肾脏和脾脏无明显影响。长松萝粗提物属于低毒性受试物,长松萝提取物10mg/kg.d和50mg/kg.d剂量组小鼠生长状况均良好,能显著增加雄性幼鼠体重,同时对雌性幼鼠的生长也有促进作用。200mg/kg对雌性和雄性幼鼠生长有一定抑制作用。三个剂量均使雌性和雄性小鼠肝脏重量显著增加,高剂量组的效果更显著。该提取物对胸腺、肾脏和脾脏重量无明显影响。
In this paper, light petroleum (b.p.: 60℃-90℃) was used to extract Ficus carica L leaves and Usnea.longissima, and then evaporated to get the crude extract. High-speed counter-current Chromatography (HSCCC) was applied for the isolation and purification of psoralen and bergapten from the plant Ficus carica L leaves, for isolation and purification of usnic acid from Usnea.longissima. The two-phase solvent system for separating psoralen and bergapten from the plant Ficus carica L leaves by HSCCC was n-Hexane-ethyl acetate-methanol-water (1:1:1:1, v/v). The two-phase solvent system for separating usnic acid from Usnea.longissima by HSCCC was n-Hexane- acetonitrile - ethyl acetate -water (8:7:5:0.8 v/v). 4.4 mg of psoralen and 2.1 mg of bergapten were obtained from 200 mg of Ficus carica L leaves crude extract in one step separation. 11.2 mg of usnic acid was obtained from 25mg of Usnea.longissima crude extract. The purities of psoralen bergapten and usnic acid determined by HPLC which indicate that psoralen at 99.1%, bergapten at 98.2% purity, and usnic acid above 99%. The structures of psoralen bergapten and usnic acid were identified by 1H-NMR, 13C-NMR and MS. The crude extracts of Ficus carica L leaves by 80% ethanol and Usnea.longissima by light petroleum (b.p.: 60℃-90℃) were used to feed little mice, the mice’s growth performance was studied. The results indicated that the crude extract of Ficus carica L leaves was innocuous. 5.0 g/kg.d dosage can gently promote the growth of male mice, but have no effect on female mice. 5.0 g/kg.d dosage can obviously increase the weight of mice’s liver. 1.0 g/kg.d dosage can obviously increase the weight of male mice thymus, but no effect on kidney and spleen. The crude extract of Usnea.longissima was low noxious, 10 mg/kg.d and 50 mg/kg.d dosages can obviously increase the weight of little male mice, and also can promote the growth of little female mice. 200 mg/kg.d dosage can restrain the growth of little male and female mice. 10 mg/kg.d、50 mg/kg.d and 200 mg/kg dosages can observably increase the weight of rat liver especially high dosage. The crude extract of Usnea.longissima have no effect on thymus kidney and spleen.
In this paper, light petroleum (b.p.: 60℃-90℃) was used to extract Ficus carica L leaves and Usnea.longissima, and then evaporated to get the crude extract. High-speed counter-current Chromatography (HSCCC) was applied for the isolation and purification of psoralen and bergapten from the plant Ficus carica L leaves, for isolation and purification of usnic acid from Usnea.longissima. The two-phase solvent system for separating psoralen and bergapten from the plant Ficus carica L leaves by HSCCC was n-Hexane-ethyl acetate-methanol-water (1:1:1:1, v/v). The two-phase solvent system for separating usnic acid from Usnea.longissima by HSCCC was n-Hexane- acetonitrile - ethyl acetate -water (8:7:5:0.8 v/v). 4.4 mg of psoralen and 2.1 mg of bergapten were obtained from 200 mg of Ficus carica L leaves crude extract in one step separation. 11.2 mg of usnic acid was obtained from 25mg of Usnea.longissima crude extract. The purities of psoralen bergapten and usnic acid determined by HPLC which indicate that psoralen at 99.1%, bergapten at 98.2% purity, and usnic acid above 99%. The structures of psoralen bergapten and usnic acid were identified by 1H-NMR, 13C-NMR and MS. The crude extracts of Ficus carica L leaves by 80% ethanol and Usnea.longissima by light petroleum (b.p.: 60℃-90℃) were used to feed little mice, the mice’s growth performance was studied. The results indicated that the crude extract of Ficus carica L leaves was innocuous. 5.0 g/kg.d dosage can gently promote the growth of male mice, but have no effect on female mice. 5.0 g/kg.d dosage can obviously increase the weight of mice’s liver. 1.0 g/kg.d dosage can obviously increase the weight of male mice thymus, but no effect on kidney and spleen. The crude extract of Usnea.longissima was low noxious, 10 mg/kg.d and 50 mg/kg.d dosages can obviously increase the weight of little male mice, and also can promote the growth of little female mice. 200 mg/kg.d dosage can restrain the growth of little male and female mice. 10 mg/kg.d、50 mg/kg.d and 200 mg/kg dosages can observably increase the weight of rat liver especially high dosage. The crude extract of Usnea.longissima have no effect on thymus kidney and spleen.
引文
1. 北京中医学院. 中药化学[M]. 北京中医学院,1974.
2. 陈业高,于丽丽,黄荣.补骨脂化学成分的分离与鉴定. 云南化工, 2005,32(4).
3. 陈志霞,林励,孙冬梅. 中草药, 2005,27(12)1468-1469.
4. 戴德舜,王义明,罗国安.高速逆流色谱研究进展[J].分析化学,2001, 29(5):586～591.
5. 杜琪珍.低速逆流色谱仪研制和逆流色谱动态特性[博士学位论文].杭州:浙江大学.1999.
6. 国家药典委员会. 中华人民共和国药典[M] 北京:化学工业出版社,2000.
7. 国家药检委员会. 中药大词典[M] 上海:上海科学技术出版社1979.
8. 国家医药管理局中草药情报站.植物药有效成分手册,1986,867.
9. 国家中医药管理局《中华本草》编委会. 中华本草. 上海: 上海科学技术出版社, 1999.
10. 贺浪冲,焦欣庆,白硕可等. 反相高效液相色谱法测定血清中松萝酸浓度.分析化学 1996,24(7) 858.
11. 侯团章. 中草药提取物[M]. 北京: 中国医药科技出版社, 2004.
12. 胡元亮. 中药饲料添加剂的开发和利用. 第1版,北京化学工业出版社,2005;58-59.
13. 江苏新医学院 中药大词典,2555,1977.
14. 江苏新医学院, 中药大辞典 上海科技出版社,1986 年版: 341-343.
15. 靳菊情,董亚琳,贺浪冲. 松萝酸钠促皮肤创伤愈合的实验研究. 中药材2005,28(2) 109-111.
16. 靳菊情,丁东宁,欧阳雪宇,闫宝琦. 松萝酸的提取和抗癌活性研究. 西北药学杂志, 1996,11(5) 211-212.
17. 李荣钦,苏印泉,宋晓斌等. 松萝酸的防腐作用研究. 西北农林科技大学学报, 2006,34(5)123-125.
18. 马木提,地拉拉,阿不都拉. 无花果叶中补骨脂素的提取. 化学世界[J].2000,8:444-446.
19. 毛新伟 陈友地 杨伦等. 无花果叶中水溶性成分的提取及其急性毒性试验. 林产业化工通讯, 1999,33(3) 11-13.
20. 毛新伟, 陈友地,杨伦, 李秀玲.林产化工通讯[J].1999,33(3):11.
21. 毛新伟,陈友地,杨论,李秀龄. 无花果醇溶性成分的研究. 林产化工通讯[J].1999,33(1):3-6.
22. 孟正木,王佾先,纪江,钟维涛,等.无花果叶化学成分研究[J].中国药科大学学报,1996,27 (4):202-204.
23. 生药学杂志, 1975, 29(2): 147.
24. 四川科学技术出版社 药名手册 中国药房杂志社 第三军医大编 1997.7.
25. 苏印泉,王海宏,马养民,李荣钦,玉泉幸一郎,牟田信次.西藏长松萝浸提物抑菌作用研究.西北林学院学报[J].2006,21(5):154-155.
26. 王北婴,李仪奎. 中药新药研制开发技术与方法. 第1版,上海科学技术出版社,2001;788-791.
27. 王丽,阿地里江?阿不都拉,孙华,阿不都拉?阿巴斯.松萝酸研究进展.生物技术通讯[J].2005,16(4):472-473.
28. 乌宁奇,永格. 高效液相色谱法测定补肾酒中补骨脂素和异补骨脂素的含量. 时针国医国药,2005,16(12)1264-1265.
29. 向兰,刑东明,王伟,王如峰,杜力军. 马齿苋的化学成分研究进展. 亚太传统医药, 2006(7):65-68.
30. 向阳,张学琼,黄敏,张惠明. 高效液相色谱法测定驱白巴不期片中补骨脂素和异补骨脂素的含量. 中国现代应用药学杂志,2006,23(1) 60-63.
31. 杨帆,金描真,宋凤兰,刘丽娟,刘煌新. 补骨脂提取工艺的比较研究. 中国中药杂志, 2005,30(16)1290-1291.
32. 杨云,王浴铭,李国茹.开白康冲剂中补骨脂素的含量测定[J].河南中医药学刊,1998,5(2):
33. 姚三桃,杨滨,徐植灵等.气相色谱法测定补骨脂素中的有效成分[J].中国药学杂志,1996,31(7):394.
34. 叶文坚.薄层荧光扫描法测定补脾宜肠丸中补骨脂素的含量[J].中草药,1993,15(7):43.
35. 殷彩霞,李聪,彭莉,汤里里,杨明挚,张红,肖怡玲.云南丽江长茎松萝酚性成分研究.云南师范大学学报[J].1996,16(3):56-60.
36. 尹卫平, 等 新乡医学院学报, 1995, 12(4): 316～ 320.
37. 余得平. 不同含量乙醇提取补骨脂的比较. 时珍国医国药.中成药,2001,23(9):643.
38. 袁伯俊,王治乔. 新药临床前安全性评价与实践. 第 1 版,北京:军事医学出版社,1997;23.
39. 张天佑.逆流色谱技术[M].北京:北京科学技术出版社, 1991.362～366.
40. 赵爱云,吴神怡,杜桂彩. 无花果叶抑菌活性成分的实验研究. 青岛大学学报(自然科学版) [J].2005, 18(3): 37-40.
41. 赵萍,周海梅,吴云骥.无花果叶化学成分的研究)))无花果叶挥发油的研究[J].中草药,2004, 35(12):1341-1342.
42. 赵小钒,邵铁华,兰小霞.两种地衣植物的化合物分析.植物研究[J].2000,20(1):86-88.
43. 中草药成分化学[M] 北京:人民卫生出版社,1977.
44. 中草药通讯, 1975, (1): 18.
45. 中国药品生物制品鉴定所. 中国民族药志(第二卷). 北京:人民卫生出版社, 1990.
46. Abu-Mustafa E. A.. Phytochemistry,1964, 3 (6) : 701.
47. Evdokimoff.V.,Tucci,B.B.,Cavazzutti,G.,&Quercia,V.(1973)Boll.Chim.Farm.112,365-369.
48. Feige, G. B., Lumbsch, H.T., Huneek, S., &Elix, J.A.(1993) J.Chromatography A 646,417-427.
49. Fischer, r., &Midula,G.(1969) Arzneim. Forsch. 19, 2025-2026.
50. Ito Y. Efficient preparative countercurrent chromatography with a coil planet centrifuge[J].J. Chromatography A,1981,214:122.
51. Kim Ju Sun, Kim Jin Cheul, et al. Chemical Constituents of the Root of Dystaeniatakeshimana and Their Anti-Inflammatory Activity. Arch Pharm Res[J].2006,29(8):617-623
52. Rashid M.A., Majid M.A., Quader M.A.. Complete NMR assignments of (+)- usnic acid. Fitoterapia [J].1999,70
53. Planta. M ed. 1981, 43(2): 66.
54. Polonia,M.A.,Carneiro, A.F.,&Pinto, M.M.(1991)J.liq.Chromatogr.14,1447-1452.
55. Renmin Liu, Aifeng Li, Ailing Sun, Lingyi Kong Journal of Chromatography A,1057(2004) 225-228.
56. Ricardo M. Kuster, Robson R. Bernardo, Antonio J.R, et al. Furocoumarins from the Rhizomes of Dorstenia Brasiliensis, Phytochemistry[J].1994,36(1):221-223
57. Huneck S,Yoshimura I Identification of Lichen Substance Springer 1996..
58. Shingbal Dm. Indian J pharm Sci 1981,43:122.
59. Takeuchi Setsuo, et al.Agricultural and Biological Chemistry, 1978, 42 (7): 1449-1451.
60. YIN Weiping, CHEN Hongming, WANG Tianxin, CAI Mengshen. Chinese Traditional Herb Drugs 1997,28(1):3.
61. Yin Weiping, Chen Hongming, Wang Tianxin. Chinese Traditional Herb Drugs1998, 29(8):505.
62. Ito Yoichiro .Technical guidance for high-speed countercurrent chromatography[J]. Foods food ingredients journal of Japan.2003.208(9),709-724.
63. Ito.Yoichiro Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. Journal of Chromatography A[J]. 2005,1065:145-168.
64. Ito. Yoichiro Technical Guidance for High-speed Countercurrent Chromatography. Foods & Food Ingredients Journal of Japan[J].2003,208(9):709-724.
2. 陈业高,于丽丽,黄荣.补骨脂化学成分的分离与鉴定. 云南化工, 2005,32(4).
3. 陈志霞,林励,孙冬梅. 中草药, 2005,27(12)1468-1469.
4. 戴德舜,王义明,罗国安.高速逆流色谱研究进展[J].分析化学,2001, 29(5):586～591.
5. 杜琪珍.低速逆流色谱仪研制和逆流色谱动态特性[博士学位论文].杭州:浙江大学.1999.
6. 国家药典委员会. 中华人民共和国药典[M] 北京:化学工业出版社,2000.
7. 国家药检委员会. 中药大词典[M] 上海:上海科学技术出版社1979.
8. 国家医药管理局中草药情报站.植物药有效成分手册,1986,867.
9. 国家中医药管理局《中华本草》编委会. 中华本草. 上海: 上海科学技术出版社, 1999.
10. 贺浪冲,焦欣庆,白硕可等. 反相高效液相色谱法测定血清中松萝酸浓度.分析化学 1996,24(7) 858.
11. 侯团章. 中草药提取物[M]. 北京: 中国医药科技出版社, 2004.
12. 胡元亮. 中药饲料添加剂的开发和利用. 第1版,北京化学工业出版社,2005;58-59.
13. 江苏新医学院 中药大词典,2555,1977.
14. 江苏新医学院, 中药大辞典 上海科技出版社,1986 年版: 341-343.
15. 靳菊情,董亚琳,贺浪冲. 松萝酸钠促皮肤创伤愈合的实验研究. 中药材2005,28(2) 109-111.
16. 靳菊情,丁东宁,欧阳雪宇,闫宝琦. 松萝酸的提取和抗癌活性研究. 西北药学杂志, 1996,11(5) 211-212.
17. 李荣钦,苏印泉,宋晓斌等. 松萝酸的防腐作用研究. 西北农林科技大学学报, 2006,34(5)123-125.
18. 马木提,地拉拉,阿不都拉. 无花果叶中补骨脂素的提取. 化学世界[J].2000,8:444-446.
19. 毛新伟 陈友地 杨伦等. 无花果叶中水溶性成分的提取及其急性毒性试验. 林产业化工通讯, 1999,33(3) 11-13.
20. 毛新伟, 陈友地,杨伦, 李秀玲.林产化工通讯[J].1999,33(3):11.
21. 毛新伟,陈友地,杨论,李秀龄. 无花果醇溶性成分的研究. 林产化工通讯[J].1999,33(1):3-6.
22. 孟正木,王佾先,纪江,钟维涛,等.无花果叶化学成分研究[J].中国药科大学学报,1996,27 (4):202-204.
23. 生药学杂志, 1975, 29(2): 147.
24. 四川科学技术出版社 药名手册 中国药房杂志社 第三军医大编 1997.7.
25. 苏印泉,王海宏,马养民,李荣钦,玉泉幸一郎,牟田信次.西藏长松萝浸提物抑菌作用研究.西北林学院学报[J].2006,21(5):154-155.
26. 王北婴,李仪奎. 中药新药研制开发技术与方法. 第1版,上海科学技术出版社,2001;788-791.
27. 王丽,阿地里江?阿不都拉,孙华,阿不都拉?阿巴斯.松萝酸研究进展.生物技术通讯[J].2005,16(4):472-473.
28. 乌宁奇,永格. 高效液相色谱法测定补肾酒中补骨脂素和异补骨脂素的含量. 时针国医国药,2005,16(12)1264-1265.
29. 向兰,刑东明,王伟,王如峰,杜力军. 马齿苋的化学成分研究进展. 亚太传统医药, 2006(7):65-68.
30. 向阳,张学琼,黄敏,张惠明. 高效液相色谱法测定驱白巴不期片中补骨脂素和异补骨脂素的含量. 中国现代应用药学杂志,2006,23(1) 60-63.
31. 杨帆,金描真,宋凤兰,刘丽娟,刘煌新. 补骨脂提取工艺的比较研究. 中国中药杂志, 2005,30(16)1290-1291.
32. 杨云,王浴铭,李国茹.开白康冲剂中补骨脂素的含量测定[J].河南中医药学刊,1998,5(2):
33. 姚三桃,杨滨,徐植灵等.气相色谱法测定补骨脂素中的有效成分[J].中国药学杂志,1996,31(7):394.
34. 叶文坚.薄层荧光扫描法测定补脾宜肠丸中补骨脂素的含量[J].中草药,1993,15(7):43.
35. 殷彩霞,李聪,彭莉,汤里里,杨明挚,张红,肖怡玲.云南丽江长茎松萝酚性成分研究.云南师范大学学报[J].1996,16(3):56-60.
36. 尹卫平, 等 新乡医学院学报, 1995, 12(4): 316～ 320.
37. 余得平. 不同含量乙醇提取补骨脂的比较. 时珍国医国药.中成药,2001,23(9):643.
38. 袁伯俊,王治乔. 新药临床前安全性评价与实践. 第 1 版,北京:军事医学出版社,1997;23.
39. 张天佑.逆流色谱技术[M].北京:北京科学技术出版社, 1991.362～366.
40. 赵爱云,吴神怡,杜桂彩. 无花果叶抑菌活性成分的实验研究. 青岛大学学报(自然科学版) [J].2005, 18(3): 37-40.
41. 赵萍,周海梅,吴云骥.无花果叶化学成分的研究)))无花果叶挥发油的研究[J].中草药,2004, 35(12):1341-1342.
42. 赵小钒,邵铁华,兰小霞.两种地衣植物的化合物分析.植物研究[J].2000,20(1):86-88.
43. 中草药成分化学[M] 北京:人民卫生出版社,1977.
44. 中草药通讯, 1975, (1): 18.
45. 中国药品生物制品鉴定所. 中国民族药志(第二卷). 北京:人民卫生出版社, 1990.
46. Abu-Mustafa E. A.. Phytochemistry,1964, 3 (6) : 701.
47. Evdokimoff.V.,Tucci,B.B.,Cavazzutti,G.,&Quercia,V.(1973)Boll.Chim.Farm.112,365-369.
48. Feige, G. B., Lumbsch, H.T., Huneek, S., &Elix, J.A.(1993) J.Chromatography A 646,417-427.
49. Fischer, r., &Midula,G.(1969) Arzneim. Forsch. 19, 2025-2026.
50. Ito Y. Efficient preparative countercurrent chromatography with a coil planet centrifuge[J].J. Chromatography A,1981,214:122.
51. Kim Ju Sun, Kim Jin Cheul, et al. Chemical Constituents of the Root of Dystaeniatakeshimana and Their Anti-Inflammatory Activity. Arch Pharm Res[J].2006,29(8):617-623
52. Rashid M.A., Majid M.A., Quader M.A.. Complete NMR assignments of (+)- usnic acid. Fitoterapia [J].1999,70
53. Planta. M ed. 1981, 43(2): 66.
54. Polonia,M.A.,Carneiro, A.F.,&Pinto, M.M.(1991)J.liq.Chromatogr.14,1447-1452.
55. Renmin Liu, Aifeng Li, Ailing Sun, Lingyi Kong Journal of Chromatography A,1057(2004) 225-228.
56. Ricardo M. Kuster, Robson R. Bernardo, Antonio J.R, et al. Furocoumarins from the Rhizomes of Dorstenia Brasiliensis, Phytochemistry[J].1994,36(1):221-223
57. Huneck S,Yoshimura I Identification of Lichen Substance Springer 1996..
58. Shingbal Dm. Indian J pharm Sci 1981,43:122.
59. Takeuchi Setsuo, et al.Agricultural and Biological Chemistry, 1978, 42 (7): 1449-1451.
60. YIN Weiping, CHEN Hongming, WANG Tianxin, CAI Mengshen. Chinese Traditional Herb Drugs 1997,28(1):3.
61. Yin Weiping, Chen Hongming, Wang Tianxin. Chinese Traditional Herb Drugs1998, 29(8):505.
62. Ito Yoichiro .Technical guidance for high-speed countercurrent chromatography[J]. Foods food ingredients journal of Japan.2003.208(9),709-724.
63. Ito.Yoichiro Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. Journal of Chromatography A[J]. 2005,1065:145-168.
64. Ito. Yoichiro Technical Guidance for High-speed Countercurrent Chromatography. Foods & Food Ingredients Journal of Japan[J].2003,208(9):709-724.