陈良华, 等 : 福建绞股蓝皂苷的优化提取及其抗肝癌活性 陈良华 1, 郑志忠 1, 张树峰 1,3, 谌迪 1, 明艳林 1, 陈清西 2, 童庆宣 *1 (1. /, 361002;2., 361005;3., 361021) : 绞股蓝素有 南方人参 美誉, 含有与人参皂苷相同骨架达玛烷型结构的三萜皂苷通过不同体积分数乙醇溶液优化提取绞股蓝皂苷, 测定其抗癌活性, 为后续活性化合物的分离奠定基 础利用 HPLC 分析绞股蓝的皂苷成分并确定提取绞股蓝皂苷的乙醇体积分数, 正交实验优化提 取方法, 进而 MTT 法测定提取的绞股蓝皂苷抗肝癌活性 HPLC 分析确定用体积分数 75 % 乙醇溶液提取绞股蓝总皂苷 (GYP), 体积分数 45% 乙醇提取极性绞股蓝皂苷 (GYP1), 体积分数 90 % 乙醇提取弱极性绞股蓝皂苷 (GYP 2 ); 正交实验结果表明在 70 水浴中以 30 质量的体积分数 45 % 乙醇加热提取 12 h 为最佳条件提取 GYP 1, 在 60 水浴中以 30 质量的体积分数 90 % 乙醇加 热提取 6 h 为最佳条件提取 GYP 2 ; 利用 MTT 法测定 GYP GYP 1 和 GYP 2 对肝癌细胞的抑制作用,GYP 和 GYP 2 对肝癌细胞 SMCC7721 具有抗癌活性, 半抑制率 IC 50 值分别为 (165.83±14.12) μg/ml 和 (113.97±9.26) μg/ml,gyp 1 没有抗癌活性, 表明绞股蓝抗癌药效成分主要是由弱极性绞股蓝皂苷发挥功效 : 绞股蓝皂苷 ; 优化提取 ; 抗肝癌活性 :R 284.2 :A :1673 1689(2012)011 1203 06 Optimum Extraction and Anti-Hepatoma Activity of Gypenosides CHEN Liang-hua 1, ZHENG Zhi-zhong 1, ZHANG Shu-feng 1,3, CHEN Di 1, MING Yan-lin 1, CHEN Qing-xi 2, TONG Qing-xuan *1 (1. Key Laboratory of Xiamen City for Plant Introduct / Quarantine and Plant Product,Xiamen Overseas Chinese Subtropical Plant Introduction Garden,Xiamen 361002,China; 2. School of Life Sciences,Xiamen University,Xiamen 361005,China; 3. College of Chemical Engineering,Huaqiao University,Xiamen 361021,China) Abstract: Gynostemma pentaphyllum is known as "Southern Ginseng". It contains triterpenoid saponins of the dammarane -type structure with the same skeleton of ginsenosides. In this manuscript, the saponin components of Gynostemma pentaphyllum were analyzed by HPLC,which choosed the concentration of ethanol extraction,and it was found that that adopted to the extract gypenosides(gyp) was from 75 % ethanol,polar gypenosides(gyp1) from 45% ethanol,and lower : 2011-11-03 : (3502Z20061143) : (1977 ),,,, E-mail:seafollow@126.com * : (1956 ),,,, E-mail:xmqingxuan@yahoo.com.cn 2012 31 11 1203
CHEN Liang-hua,et al: Optimum Extraction and Anti-Hepatoma Activity of Gypenosides Research Article polar gypenosides (GYP 2 ) from 90% ethanol. Furthermore,the optimum extraction parameters for GYP 1 and GYP 2 were studied by Orthogonal method and listed as follows: GYP 1 was extracted with thirty fold-weight 45 % enthanol in 70 water bath for 12 h,and GYP 2 was extracted with thirty fold-weight 90% ethanol in 60 water bath for 6 h. Anti-hepatoma activity was determined tests by MTT and the results showed that GYP and GYP 2 can inhibit hepatoma cell SMCC7721 with IC 50 value 165.83 ±14.12 μg/ml and 113.97 ±9.26 μg/ml,respectively. However,GYP 1 without any anticancer activity. The result showed that Gynostemma pentaphyllum exhibited the anti-hepatoma activity caused by lower polar saponins. Keywords: Gypenosides, optimum extraction, anti-hepatoma activity (Gynostemma pentaphyllum Thnmb Mak.),,, 14 2,2001 ( ), [1] 1 20 70, (Gypenoside), 1.1 169 201 : [2], ; SMCC7721 Bel7402: ; Rb 1 :,, 1.2 :Agilent 1200,Agilent ;, :MK3, Thermo ; :, GPY HyClone ;RPMI1640 DMEM : Rb l Rb l Rd F2, GIBCO ; : Sangon, ; : Merck ;3-(4,5- - [3] [4] [5] 2)-2,5- (MTT): Sigma [6], ; 1.3 [7-8], 1.3.1 绞股蓝皂苷的提取 [9-10],, HPLC,, 3~5 cm,, 4 50 g,, 20 1 L, 45 %,75 %, 95 % 3 ; 60 ; 12 h,,,,, [11] 1.3.2 不同极性绞股蓝皂苷的提取和正交优化, 4 10 g, 1204 Journal of Food Science and Biotechnology Vol.31 No.11 2012
陈良华, 等 : 福建绞股蓝皂苷的优化提取及其抗肝癌活性 15 %,30 %,45 %, HPLC ; 4 10 g 2.1 HPLC, 70%,80%,90%,100%, HPLC, ;,, 3, HPLC, L 9 (3 3 ), : 1.3.3 绞股蓝皂苷 HPLC 分析 :, Agilent Eclipse XDB-C18 (150 mm 4.6 mm);, HPLC :25 ; :203 nm; :1.0 ml/min; 20 μl, : (A)/ (B), ( 1a), 0~8 min:a,80%,b, 2 20% ;8 ~20 min:a,65%,b,35% ;20 ~35 min:a, 75% 30%,B,70% ;35 ~40 min:a,30%,b,70% ;40 ~50 (GYP), 20~ min:b,100% ;50 ~60 min:b,100% ;60 ~70 min:a, 30 min (GYP 1 ), 80%,B,20% 45~60 min 1.3.4 绞股蓝皂苷质量分数测定 [12] (GYP 2 ), 2 mg/ml GYP 1 GYP 2, Rb 1,, 15%,30%,45%,, 70%,80%,90%, : 200 μl, 800 μl 5 % -,60 45% GYP 1 15 min,, ( 1b), 90% GYP 2 3 ml, 550 nm, ( 1c) 1.3.5 MTT 法测定绞股蓝皂苷抗癌活性 MTT (0 6.25 12.5 25 50 100 200 μg/ml) (GYP) (GYP 1 ) (GYP 2 72 h 20~30 min 45~60 min 2 100%, HPLC :, SMCC77221 Bel7402 :, 0.25%, 5 10 4 /ml, 96, 100 μl 24 h, 100 μl, 4, 72 h,, MTT (0.5 mg/ml) 200 μl, 4 h, DMSO 200 μl 10 min, 570 nm (A 570 nm ) =(A -A )/A 100% 2 2012 31 11 1205
CHEN Liang-hua,et al: Optimum Extraction and Anti-Hepatoma Activity of Gypenosides Research Article 1 HPLC Fig.1 HPLC analysis of gypenoside GYP 2, 2.2, 45% A 3 B 3 C 2, 60, 30 GYP 1,, 90% 6 h, 9 3,, 3.21%,, 3 GYP 1, 1 R, 2 (GYP 2 ) GYP 1 A ( Tab.2 Design and result of orthogonal test )>B( )>C( ), GYP 1,, 1 10 40 3 2.16 A 3 B 3 C 3, 70, 30 2 10 50 6 2.19 45% 12 h, 3 10 60 9 2.22,3 3.12%, 3.26% 3.32%, 3.23% Tab.1 1 (GYP 1 ) Design and result of orthogonal test A B ( ) / C /h 1 10 50 4 1.70 2 10 60 8 1.82 3 10 70 12 2.07 4 20 50 8 2.87 5 20 60 12 2.96 6 20 70 4 3.04 /% 2.3 90% GYP 2,, 3,, 3 GYP 2, 2 R, GYP 2 A( )>B( )>C( ), A ( ) / B C /h 4 20 40 6 2.26 5 20 50 9 2.30 6 20 60 3 2.33 7 30 40 9 3.04 8 30 50 3 3.12 9 30 60 6 3.21 K 1 2.19 2.49 2.53 / K 2 2.30 2.54 2.56 / K 3 3.12 2.59 2.52 / R 0.93 0.10 0.03 / /% 7 30 50 12 3.08 8 30 60 4 3.10 9 30 70 8 3.18 K 1 1.86 2.55 2.62 / K 2 2.96 2.62 2.62 / K 3 3.12 2.77 2.70 / R 1.26 0.22 0.09 / 2.4 MTT (GYP) (GYP 1 ) (GYP 2 SMCC77221( 2a) Bel7402( 2b) 2 : 72 h,gyp GYP 2 SMCC7721, IC 50 (165.83±14.12) μg/ml (113.97±9.26) μg/ml, 1206 Journal of Food Science and Biotechnology Vol.31 No.11 2012
陈良华, 等 : 福建绞股蓝皂苷的优化提取及其抗肝癌活性 GYP 1 0 ~200 μg/ml GYP 2 0~200 μg/ml SMCC 50 %, Bel7402, 50 %, GYP, GYP 2, SMCC, Bel7402 ;GYP GYP 1 Fig.2 2 Inhibition effect of gypenoside on the growth of Hepatocellular carcinoma cells in vitro 3,,,,,,,,,, [13] 45%, 90% ;, 70 30, 45% 12 h, 60 30 90% 6 h : [ 1 ] Valentina R N,Tom H H,Van H T,et al. Chemistry and pharmacology of Gynostemma pentaphyllum [J]. Phytochemistry Review,2005,4:197-219. [ 2 ] Ji H K,Yong N H. Dammarane-type saponins from Gynostemma pentaphyllum[j]. Phytochemistry,2011,72:1453-1459. [ 3 ] Lu Kung-wen,Tsai M L,Chen Jung-chou,et al. Gypenosides inhibited invasion and migration of human tongue cancer SCC4 cells through down-regulation of NF-kappaB and matrix metalloproteinase-9[j]. Anticancer Research,2008,28(2):1093-1099. [ 4 ] Yeo J,Kang Y J,Jeon S M,et al. Potential hypoglycemic effect of an ethanol extract of Gynostemma pentaphyllum in C57BL/ 2012 31 11 1207
CHEN Liang-hua,et al: Optimum Extraction and Anti-Hepatoma Activity of Gypenosides Research Article KsJ-db/db mice[j]. Journal of Medicinal Food,2008,11:709-716. [ 5 ],,. [J].,2007,13:7-8. CHEN Ji -xiang,zhang Jian -guo,zhang Li,et al. Study on liver protective effect of total saponins of Gynostemma pentaphyllum[j]. China Pharmaceuticals,2007,13:7-8. (in Chinese) [ 6 ],,,. D- [J].,2006,19(5):497-499. LIU Guo-hui,YAO dan-dan,lu Jia-yi,et al. The effect mechanism of Gynostemma Pentaphyllum Makino to hippoocampus in aged rats induced by D-galactose[J]. Journal Medicine Theory & Practice,2006,19(5):497-499. (in Chinese) [ 7 ] Chen Jung-chou,Lu Kung-wen,Tsai M L,et al. Gypenosides induced G0/G1 arrest via CHk2 and apoptosis through endoplasmic reticulum stress and mitochondria-dependent pathways in human tongue cancer SCC-4 cells[j]. Oral Oncology,2009,45(3): 273-283. [ 8 ] Schild L,Chen Bing -huei,makarov P,et al. Selective induction of apoptosis in glioma tumour cells by a Gynostemma pentaphyllum extract[j]. Phytomedicine,2010,17(8):589-597. [ 9 ] Nguyen P H,Gauhar R,Hwang S L,et al. New dammarane -type glucosides as potential activators of AMP -activated protein kinase (AMPK) from Gynostemma pentaphyllum[j]. Bioorganic & Medicinal Chemistry,2011,19(21):6254-6260. [10] Tom H H,Valentina R N,Noeris K S,et al. A novel LXR -α activator identified from the natural product Gynostemma pentaphyllum[j]. Biochemical Pharmacology,2005,70(9):1298-1308. [11],. [J].,1992,21(1):51-53. CHEN Zhong-ren,ZHANG Yong-tian. The natural resource of Gynostemma pentaphyllum in Fujian province[j]. Subtropical Plant Science,1992,21(1):51-53.(in Chinese) [12],,. 6 [J].,2006,15(3):26-27. JIANG Wei-zhe,ZHOU Yan-wen,LI Jin-shen. Comparision of total saponin contents in gynostemma cultivated of Guangxi[J]. China Pharmaceuticals,2006,15(3):26-27.(in Chinese) [13],. [J].,2008,27(2):14-18. LIU Xue-xiang,CHEN Jian-wei. Research progress of chinese medicine effective component biotransformation[j]. Journal of Food Science and Biotechnology,2008,27(2):14-18.(in Chinese) 1208 Journal of Food Science and Biotechnology Vol.31 No.11 2012