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1 ( ) Journal of Hunan Agricultural University (Natural Sciences), Oct. 215, 41(5): DOI: /j.cnki.jhau 龙葵果生物碱提取纯化工艺研究 ( 154) * 摘要 6% 7% 8% 85% 9% 95% 1% 6% 7% 9% 95% 1% 8% 8% ( ) ( h) ( ) g/ml (1 2.5) g/ml 4 h (.824±.1) mg/g AB 8.3 mg/ml ph BV ph 3 7% 2. BV/h 9.44 关键词中图分类号 S 文献标志码 :A 文章编号 :17 132(215) Study on the extraction and purification conditions of alkaloids from the fruit of black nightshade (Solanum nigrum L.) Gao Xiaotang Wang Ping * (College of Forestry, Northeast Forestry University, Harbin 154, China) Abstract: Water, ethanol of 6%, 7%, 8%, 85%, 9%, 95% and 1% and methanol of 6%, 7%, 9%, 95% and 1% were used as solvent to test extracting rate of alkaloids from the fruit of black nightshade( Solanum nigrum L.). The result showed the ethanol of 8% had the highest extracting rate of alkaloids. Then, 8% ethanol was used as extraction solvent, different extraction temperature (2, 4, 6, 8, 1 ), extraction time (2, 3, 4, 5, 6, 7, 8 h) and ratio of material to solvent (1 5, 1 1, 1 2, 1 4, 1 6, 1 8, 1 1) g/ml were designed to study the optimum extraction conditions of alkaloids. The results showed that: the best extraction solvent was 8% ethanol; optimum extraction conditions were 57.5 of extraction temperature, (1 2.5) g/ml of material to solvent, 4 h of extraction time. On these conditions, the alkaloids extraction rate was (.824±.1) mg/g. By static adsorption and desorption testing of resins, the best resin was AB-8, the best purification conditions were.3 mg/ml of solution concentration, ph 9 of solution, 1 1 of diameter to length, 3. BV, ph 3 and 7% ethanol with the elution flow rate of 2. BV / h, the alkaloid purity could increase 9.44 times. Keywords: Solanum nigrum L. fruits; alkaloids; extraction and purification process; response surface methodology (Solanum nigrum L.) [1] [2 3] 收稿日期 : 修回日期 : 基金项目 ( ) 作者简介 (1991 ) tangxiaotusweet@163.com * @qq.com

2 522 ( ) 材料与方法 1.1 材料 ( ) ( ) ( ) ( ) HR24 ( ) RE 53A ( ) SHB IIIS ( ) 722 ( ) PHS 26 ph ( ) HHS ( ) TDL 4B W ( ) (1 5) cm ( ) AB 8 D11 X 5( ) HP2MGL NKA Ⅱ IR12Na( ) XAD 7( ( ) ) 1.2 方法龙葵果生物碱的提取 1) 19~9 nm 416 nm 416 nm.4 mg/ml μl 25 ml ph ml Markus Ganzera [4 5] 5 ml 2 h 416 nm [6] 2) [7 11] 1. g (1 2) g/ml 6% 7% 8% 85% 9% 95% 1% 6% 7% 9% 95% 1% 4 4 h 4 r/min 5 min 416 nm 3) ( h) ( ) ( ) g/ml 响应面优化龙葵果生物碱提取工艺 3 [12] [13] 龙葵果生物碱的纯化 1) 95% 24 h [14] 5% HCl 12 h 5% NaOH 12 h 2) 2. g 1 ml 2 ml 2 11 r/min 24 h 3 2 ml 6% 2 11 r/min 24 h [15] ( C C V Q e = e ) w C D= d Vd 1% C ( C C ) V e (μg/ml) C e

3 (μg/ml) C d (μg/ml) V (ml) V d (ml) w (g) 3) 2. g 1 ml 2 ml 2 11 r/min 1 h 3 2 ml 6% 2 11 r/min 1 h 4) 2. BV/h 4 ml / 1/1 [16] 5) ( mg/ml) (5% 6% 7% 8% 9%) ph( ) ph( ) ( BV/h) ( ) [17] =(m 1 /m ) 1% m (g) m 1 (g) 6) 2. BV ph 3. 7% 2. BV/h 4 ml/ 数据处理与分析 Excel Minitab Design Expert 8. 2 结果与分析 2.1 不同溶剂提取物中的生物碱得率 416 nm y=46.256x+.1 9 R 2 = mg/ml 6% 7% 8% 85% 9% 95% 1% (.27±.4) (.33±.3) (.64±.1) (.59±.1) (.48±.2) (.35±.1) (.24±.4) mg/g 7% 9% 95% 1% (.18±.3) (.3±.2) (.66±.2) (.65±.4) (.37±.1) mg/g (.22±.1) mg/g 8% 9% 8% 2.2 生物碱提取单因素试验结果 8% h (.4±.1) (.53±.1) (.64±.1) (.63±.2) (.65±.4) (.65±.2) (.65±.2) mg/g 4 h (.54±.1) (.65±.1) (.64±.1) (.52±.2) (.14±.1) mg/g 4 (1 5) (1 1) (1 2) (1 4) (1 6) (1 8) (1 1) g/ml (.31±.1) (.6±.4) (.64±.1) (.46±.3) (.4±.2) (.27±.1) (.26±.1) mg/g (1 2) g/ml 2.3 响应面优化龙葵果生物碱提取工艺全因子试验结果 Y= A.2 8B.119 8C+ (1).9 6AB.51 5AC+.12 3BC A B C (1) P <.1 R 2 =99.79%

4 524 ( ) (C) (A) (AC) 最陡爬坡试验结果 (1) 5 (1 2) g/ml 中心组合试验结果 Y= A C+.14AC.25A 2.9C 2 (2) P <. 1 P >.5 R 2 =97.21% [18] 1 Design Expert (1 2.54) g/ml.833 mg/g 图 1 提取温度和料液比交互作用下龙葵果生物碱得率的影响 Fig.1 Effects of temperature and liquid-solid ratio on alkaloids yield 模型的验证结果 57.5 (1 2.5) g/ml 4 h (.824±.1) mg/g 1.1% 2.4 最佳树脂筛选结果 1 AB 8 表 1 树脂静态吸附及解吸结果 Table 1 Absorption and desorption capabilities of the different macroporous resins to S. nigrum anthoyanins /nm /(m 2 g 1 ) /(μg g 1 ) /% NKA Ⅱ (128.9±.82)e (67.67±.7)d XAD (86.59±.46)f (92.95±.21)b HP2MGL (232.71±.55)b (48.36±.9)g X (56.1±.35)g (94.97±.11)a AB (247.41±.15)a (71.3±.34)c D (28.5±.7)c (6.4±.11)f IR12Na (16.8±.69)d (65.43±.16)e. 2.5 龙葵果生物碱吸附平衡和解吸平衡时间的测定 15 ( 2) ( 3) 2 3 AB 8 4 h 4 h /(μg g 1 ) /h 图 2 AB 8 树脂静态吸附曲线 Fig. 2 Static adsorption curve of AB 8

5 /% /h 图 3 AB 8 树脂静态解吸曲线 Fig 泄漏曲线的确定 Static desorption curve of AB 8 4 1/1.6 BV /(μg ml 1 ) /BV 图 4 泄漏曲线 Fig. 4 Leaked curve 2.7 不同条件对龙葵果生物碱纯化工艺的影响 mg/ml 25.45% 33.56% 63.17% 43.18% 33.41%.3 mg/ml.3 mg/ml ( ) 5% 6% 7% 8% 9% 18.83% 77.45% 9.98% 53.4% 33.41% 7% 7% ph % 51.53% 64.23% 73.85% 41.45% ph 9 ph 9 ph ph % 79.78% 76.18% 75.5% 34.62% ph 3 ph 3 ph BV/h 56.56% 54.23% 65.54% 52.95% 2. BV/h 2. BV/h % 76.3% 71.97% 洗脱剂体积的确定 ( 5) 5 2. BV 44.72% 3. BV 84.33% 4.6 BV 3. BV /(μg ml 1 ) /ml 3 结论与讨论图 5 洗脱曲线 Fig. 5 Eluent volume [12] ( 9% 8%) 4 h 4 h

6 526 ( ) (1 2) g/ml ph ph 7% (ph3) (ph9) H + 8% h (1 2.5) g/ml (.824±.1) mg/g AB 8 AB 8 4 h 4 h.6 BV.3 mg/ml ph BV ph 3 7% 2. BV/h 9.44 参考文献 : [1] [J] 24 2(6) [2] [J] 26 27(8) [3] [J] 27(5) [4] [J] 27(1) [5] Markus Ganzera Anja Krüger Michael Wink. Determination of quinolizidine alkaloids in different Lupinus species by NACE using UV and MS detection[j] Journal of Pharmaceutical and Biomedical Analysis 21 53(5) [6] [D] 25 [7] [J] 23(2) [8] Zhang Dong-Bo Chen Jian-Jun Zhang Li et al. Bioactive alkaloids from Palhinhaea cernua[j]. Phytochemistry Letters 214(1) [9] Sakornrat Thorroad Prateep Worawittayanont Nisachon Khunnawutmanotham et al Three new Lycopodium alkaloids from Huperzia carinata and Huperzia squarrosa[j] Tetrahedron 214 7(43) [1] Hanane Khalki Sylvia Navailles Camille L Piron et al A tobacco extract containing alkaloids induces distinct effects compared to pure nicotine on dopamine release in the rat[j] Neuroscience Letters [11] Cheng Gui-Guang Zhao Yun-Li Zhang Yu et al. Indole alkaloids from cultivated Vinca major[j]. Tetrahedron 214 7(45) [12] [J] (7) [13] [J] (5) [14] [J] 213(3) [15] Yang Jin Zhang Li Yan Zhu Gui Hua et al Separation and enrichment of major quinolizidine type alkaloids from Sophora alopecuroides using macroporous resins[j]. Journal of Chromatography B / [16] AB 8 [J] 27(12) [17] [D] 28 [18] [J] (9) 责任编辑 : 尹小红英文编辑 : 梁和

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g 16 6 2018 11 Chinese Journal of Bioprocess Engineering Vol. 16 No. 6 Nov. 2018 doi 10. 3969 /j. issn. 1672-3678. 2018. 06. 004 1 2 1 1 1. 330200 2. 330006 4 Box-Behnken 1 25 g /ml 121 92 min 2 6. 284%