孙秀玉 1, 王英姿 1**, 乔延江 1, 冯艾灵 1, 段飞鹏 1, 周海燕 2 1. 100102 2. 101300 目的 : 优化金银花提取液的醇沉工艺, 规范清开灵注射液中金银花提取液的生产, 减少批次间 差异方法 : 采用正交试验, 以绿原酸和木犀草苷的含量为指标, 并采用多指标综合评分法处理数据, 优化 醇沉工艺中加醇时的药液温度 搅拌速度和加醇速度 3 个因素结果 : 金银花提取液的最佳醇沉工艺为加 醇时药液温度为 20, 搅拌速度 240 rpm, 加醇速度 1 倍生药量 /min 结论 : 优化的醇沉工艺稳定可行 : 清开灵注射液金银花正交试验醇沉工艺绿原酸木犀草苷 doi: 10.11842/wst.2014.01.035 中图分类号 :TQ460.6 文献标识码 :A 8 3 Lonicera japonica Thunb. 1 LC -20AT [1] SPD-20A VWD LC Solution [2,3] KQ5200 SHZ-D 110753-200413 11720-201106 Lonicera japonica Thunb. Fisher 收稿日期 :2013-03-21 * ** 修回日期 :2013-06-03 科学技术部国家 重大新药创制 项目 (2010ZX09502-002): 清开灵注射液安全性关键技术研究, 负责人 : 乔延江 ; 北京中医药大学创新团队 (2011-CXTD-13): 复方中药制药研究, 负责人 : 杜守颖 通讯作者 : 王英姿, 硕士生导师, 主要研究方向 : 中药制剂新技术与新剂型研究 187
2014 Vol.16 No.1 2 2.1 色谱条件 C 18 4.61 mm 250 mm 5 μm -0.4% 11 89 85% 327 nm 1.0 ml min -1 30 1 000 μl 25 ml 10 μl 1 000 70% 0.45 μm 1-0.5% 0~10 min 10%~20% 10~20 min 20%~22% 20~30 min 22%~28% 10 μl 20 000 2.1 5.25 10.5 2 2.2 对照品溶液的制备 50% Y X 105 μg ml -1 Y = 2 621.5X-23 973 r = 0.999 8 70% 100 0.021~6.080 μg μg ml -1 2.3 供试品溶液的制备 1 000mL 2 0.5 h 60 ml 75% ph 8.0 2.4 线性关系的考察 0.2 0.5 1.0 350 nm 1.0 ml min -1 30 2.0 4.0 7.0 ml 10 21 42 73.5 μg ml -1 10 μl 1.0 ml 25 10 5 2 ml A 绿原酸 B 绿原酸 1 A B HPLC 188
A 木犀草苷 B 木犀草苷 2 A B HPLC 1.5 ml 10 5 2 70% 4.0 10.0 15.0 20.0 30.0 50.0 75 μg ml -1 10 μl Y X RSD 1.93% 2.72% Y= 2 776.4X+1 958.3 r= 0.999 9 12 h 0.04~0.75 μg 2.8 加样回收率试验 2.5 精密度试验 10 μl 6 3 2 866 465 RSD 0.66% 3 537 930.17 RSD 0.23% 2.6 重复性试验 2.9 正交试验 6 2.9.1 正交试验设计 10 μl 1.621 2% RSD 1.53% 2.72% 2.7 稳定性试验 0 1 2 4 8 12 h 2.3 102.46% RSD 1.72% 97.21% RSD 0.90% 1 2 0.031 3% RSD A B C 3 3 189
2014 Vol.16 No.1 L 3 4 9 2.9.3 正交试验结果 3 3 2.9.2 实验方法 9 2 0.5 h X 1 X 2 60 ml 0.5 M = 0.5X 1 /1.660 4 + 0.5X 2 /0.045 3 100 75% ph 4 5 8.0 3 85% C>B>A C 1 >C 2 >C 3 B 3 >B 2 > 2.3 B 1 A 3 >A 2 >A 1 C [4] C 1 样品含量 /μg 加入量 /μg 测得量 /μg 回收率 /% 平均回收率 /% RSD/% 9.00 8.4 17.407 0 100.08 9.00 8.4 17.470 8 100.84 9.00 8.4 17.445 6 100.54 9.00 18.9 28.245 1 101.83 9.00 18.9 28.417 4 102.74 102.46 1.72 9.00 18.9 28.452 5 102.92 9.00 27.3 37.319 1 103.73 9.00 27.3 37.516 5 104.46 9.00 27.3 37.667 9 105.01 2 样品含量 /μg 加入量 /μg 测得量 /μg 回收率 /% 平均回收率 /% RSD/% 301 160 457.14 97.59 301 160 461.48 100.30 301 160 461.25 100.16 301 350 638.56 96.44 301 350 641.87 97.39 97.21 2.01 301 350 640.85 97.10 301 500 773.84 94.57 301 500 779.67 95.73 301 500 778.96 95.59 190
3 20 240 rpm 1 水平 /min加醇药液温度搅拌速度加醇速度 A / B/rpm C( 倍量 /min) 2.9.3 验证实验 1 2 3 20 30 40 80 160 240 1 4 7 3 A 1 B 3 C 1 3 98.73 ± 2.62 RSD 2.64% 6 P<0.05 A B 3 A 1 B 3 C 1 4 试验序号 药液温度 (A) 搅拌速度 (B) 加醇速度 (C) 绿原酸百分含量 /% 木犀草苷百分含量 /% 综合评分 (M) 1 1 1 1 1.391 7 0.040 3 86.40 2 1 2 2 1.428 3 0.038 5 85.53 3 1 3 3 1.366 5 0.037 6 82.64 4 2 1 2 1.405 7 0.039 4 85.85 5 2 2 3 1.326 0 0.037 9 81.80 6 2 3 1 1.660 4 0.045 3 100.00 7 3 1 3 1.343 6 0.035 9 80.17 8 3 2 1 1.629 7 0.044 1 97.78 9 3 3 2 1.522 5 0.041 1 91.28 K1 84.85 84.14 94.72 K2 89.22 88.37 87.55 K3 89.74 91.30 81.54 R 4.89 7.17 13.19 5 方差来源 离差平方和 自由度 均方 F P A 43.166 2 21.583 4.564 0.180 B 77.878 2 38.939 8.235 0.108 C 261.633 2 130.817 27.664 0.035 误差 9.457 2 4.729 注 :F 0.1 (2,2)=9.0,F 0.05 (2,2)=19.0 191
2014 Vol.16 No.1 6 编号绿原酸百分含量 /% 木犀草苷百分含量 /% 综合评分 x±s RSD/% 1 1.552 6 0.043 8 93.70 2 1.668 9 0.046 5 101.58 98.73±2.61 2.64 3 1.657 7 0.044 8 99.43 A 1 B 3 C 1 2010 20 240 rpm 1 / min 3 1. ( ). :,2010 205~206. 2..,1997,22 (4) 247~249. 3,..,2006,37 (3) 331~334. 4,..,2009,34(14) 1792~1794. Optimization of Alcohol Precipitation Techniques for Honeysuckle Extract of Qingkailing Injection by Orthogonal Test Sun Xiuyu 1, Wang Yingzi 1, Qiao Yanjiang 1, Feng Ailing 1, Duan Feipeng 1, Zhou Haiyan 2 (1. School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China; 2. Yabao Beizhongda (Beijing) Pharmaceutical Co., Ltd., Beijing 101300, China) Abstract: This study was aimed to optimize the alcohol precipitation techniques for honeysuckle extract in order to standardize the production of honeysuckle extract of Qingkailing Injection and reduce the differences among batches. The orthogonal test was applied in this study. The content of chlorogenic acid and galuteolin were taken as comprehensive indicators. Multi-index comprehensive scoring method was used in the data analysis. Three influencing factors, which were the fluid temperature, the stirring speed and the speed of adding alcohol, were optimized in the alcohol precipitation techniques. The results showed that the optimal alcohol precipitation techniques were when the fluid temperature was 20, the stirring speed was 240 rpm and the speed of adding alcohol was one time of the material per minute. It was concluded that the optimized alcohol precipitation process was stable and feasible. Keywords: Qingkailing Injection, honeysuckle, orthogonal test, alcohol precipitation, chlorogenic acid, galuteolin ( 责任编辑 : 李沙沙张志华, 责任译审 : 王晶 ) 192