Research on Water Extraction Process of Flos Lonicerae-Fructus Forsythuae Based on Network Pharmacology and Design Space
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摘要:
目的 优选金银花-连翘药对水提取工艺。 方法 借助网络药理学对金银花-连翘潜在的活性成分进行初步筛选, 再结合《中国药典》2020年版一部金银花、连翘项下规定的质量标志物及参考文献确定指标性成分, 同时以金银花-连翘药对的出膏率及指标性成分的提取率作为水提取工艺的关键质量属性(Critical quality attributes, CQAs), 筛选关键工艺参数(Critical process parameters, CPPs), 通过Box-Behnken设计建立数学模型, 考察CQAs和CPPs之间的交互作用, 构建金银花-连翘水提取工艺的设计空间。 结果 筛选出酚酸类、连翘酯苷A和连翘苷作为金银花-连翘药对的指标成分, 饮片规格、提取时间、提取加水量为关键工艺参数。基于设计空间确定的最佳水提取工艺参数为: 连翘饮片规格0.8~1.2 cm, 一煎加12倍量水, 提取30~50 min, 二煎加10倍量水, 提取25~30 min。 结论 运用设计空间验证提取工艺所得的实测值与预测值基本一致, 表明建立的方法稳定可靠, 为金银花-连翘药对的工艺开发及质量控制提供思路。 Abstract:OBJECTIVE To optimize the water extraction process of Flos Lonicerae-Fructus Forsythuae and determine the range of water extraction process parameters. METHODS The active components were screened by network pharmacology, and the indicator ingredients were determined in combination with the quality markers under the relevant terms of Chinese Pharmacopoeia 2020 edition and the literature. Take extraction yield and the extraction rate of the indicative component as the critical quality attributes of the water extraction process to screen critical process parameters. The mathematical model was established by Box-Behnken experimental design to investigate the interaction between CQAs and CPPs and build the design space of the water extraction process of Flos Lonicerae-Fructus Forsythuae. RESULTS The extraction percentages of phenolic acids, forsythoside A and forsythin were screened as the index components; specifications of medicinal slices, extraction time and water addition were the key process parameters. Based on the establishment and optimization of the design space, the optimum water extraction process was obtained as follows: the medicinal slice of Lian-Qiao was broken into 0.8-1.2 cm, adding 12 times the amount of water in the first and extract for 30-50 min, 10 times the amount of water in the second and extract for 25-30 min. CONCLUSION The verification results show that the measured value obtained by using the design space method to optimize the water extraction process is close to the predicted value, indicating that the method is stable and reliable, which can provide ideas for its further process development and quality control for the couple medicines of Flos Lonicerae-Fructus Forsythuae. -
图 1 金银花-连翘药对成分靶点与疾病靶点韦恩图
Figure 1. Venn diagram of Flos Lonicerae-Fructus Forsythuae components genes and disease genes
图 2 金银花-连翘药对成分作用靶点GO功能分析
Figure 2. Analysis of GO function of ingredient action targets of Flos Lonicerae-Fructus Forsythuae
图 3 金银花-连翘药对成分作用靶点KEGG通路富集分析的20条通路气泡图
Figure 3. 20-Channel bubble diagram of KEGG pathway enrichment analysis of Flos Lonicerae-Fructus Forsythuae
图 4 金银花-连翘药对药物-成分-靶点-通路相互作用网络图
注: 红色圆点代表药物; 绿色八边形代表成分; 紫色菱形代表靶点; 黄色三角形代表通路。
Figure 4. The interaction network diagram of Flos Lonicerae-Fructus Forsythuae drug-component-target-pathway
图 5 供试品和对照品HPLC图
注: A.金银花-连翘药对供试品溶液特征图谱; B~D.对照品特征图谱; 1.新绿原酸; 2.绿原酸; 3.隐绿原酸; 4.咖啡酸; 5.连翘酯苷A; 6.4, 5-二-O-咖啡酰奎宁酸; 7.连翘苷
Figure 5. HPLC of test substance and reference substances
图 6 金银花-连翘药对水提取工艺筛选鱼骨图
Figure 6. Fishbone diagram for water extraction process parameters of Flos Lonicerae-Fructus Forsythuae
图 7 出膏率的等高线及响应面图
Figure 7. Contour analysis and response surface figure of content of extract rate
图 8 酚酸类成分提取率的等高线及响应面图
Figure 8. Contour analysis and response surface figure of the extraction rate of phenolic acids
图 9 连翘酯苷A提取率的等高线及响应面图
Figure 9. Contour analysis and response surface figure of the extraction rate of forsythoside A
图 10 连翘苷提取率的等高线及响应面图
Figure 10. Contour analysis and response surface figure of the extraction rate of forsythin
图 11 金银花-连翘药对水提取工艺设计空间
Figure 11. Design space of water extract process of Flos Lonicerae-Fructus Forsythuae
表 1 金银花-连翘药对主要活性成分基本信息
Table 1. The key active components of Flos Lonicerae-Fructus Forsythuae
组分 MOL ID 化合物 Pubchem CID OB/% DL/% 金银花 MOL000449 豆甾醇 5280794 43.83 0.76 MOL001494 Mandenol 5282184 42.00 0.19 MOL001495 亚麻酸乙酯 6371716 46.10 0.20 MOL002773 β-叶红素 5280489 37.18 0.58 MOL002914 Eriodyctiol(flavanone) 373261 41.35 0.24 MOL003006 (-)-(3R, 8S, 9R, 9aS, 10aS)-9-ethenyl-8-(beta-D-glucopyranosyloxy)-2, 3, 9, 9a, 10, 10a-hexahydro-5-oxo-5H, 8H-pyrano[4, 3-d]oxazolo[3, 2-a]pyridine-3-carboxylic acid_qt / 87.47 0.23 MOL003014 Secologanic dibutylacetal_qt / 53.65 0.29 MOL003036 ZINC03978781 11870462 43.83 0.76 MOL003044 Chryseriol 5280666 35.85 0.27 MOL003095 5-羟基-7-甲氧基-2-(3, 4, 5-三甲氧苯基)色酮 10970376 51.96 0.41 MOL003111 Centauroside_qt / 55.79 0.50 MOL003117 Ioniceracetalides B_qt / 61.19 0.19 MOL003128 Dinethylsecologanoside / 48.46 0.48 连翘 MOL000173 Wogonin 5281703 30.68 0.23 MOL000211 Mairin 64971 55.38 0.78 MOL000522 牛蒡子苷 100528 69.67 0.88 MOL000791 荷包牡丹碱 10237 81.25 0.57 MOL003283 (2R, 3R, 4S)-4-(4-Hydroxy-3-methoxy-phenyl)-7-methoxy-2, 3-dimethylol-tetralin-6-ol 160521 66.51 0.39 MOL003290 (3R, 4R)-3, 4-Bis[(3, 4-dimethoxyphenyl)methyl]oxolan-2-one 384877 52.30 0.48 MOL003295 (+)-松脂酚单甲基醚 / 53.08 0.57 MOL003305 连翘苷 24721571 36.40 0.86 MOL003306 ACon1_001697 21722915 85.12 0.57 MOL003308 (+)-松脂酚单甲基醚-4-D-β-葡萄糖苷 / 61.20 0.57 MOL003315 3Beta-acetyl-20, 25-epoxydammarane-24alpha-ol / 33.07 0.79 MOL003322 连翘酯醇 / 95.04 0.57 MOL003330 (-)-连翘脂素 3083590 42.06 0.74 MOL003347 Hyperforin 441298 44.03 0.61 MOL003370 Onjixanthone Ⅰ 5320290 34.45 0.84 共有及其它活性成分 MOL000006 木犀草素 5280445 36.16 0.25 MOL000098 槲皮素 5280343 46.43 0.28 MOL000358 β-谷甾醇 222284 36.91 0.75 MOL000414 咖啡酸 1549111 54.97 0.05 MOL000415 芦丁 5280805 3.20 0.68 MOL000422 山柰酚 5280863 41.88 0.24 MOL003065 隐绿原酸 9798666 10.48 0.33 MOL003066 新绿原酸 5280633 10.65 0.33 MOL003068 4, 5-二-O-咖啡酰奎宁酸 6474309 1.73 0.69 MOL003331 连翘酯苷A 5281773 3.05 0.61 MOL003871 绿原酸 1794427 13.61 0.31 
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表 2 各成分线性关系考察
Table 2. Linear relationship investigation of compontents
组分 回归方程 相关系数(r) 线性范围/(μg·mL-1) 新绿原酸 Y=10 524X-2 950.7 0.999 9 3.071~76.776 绿原酸 Y=13 228X-25 814 0.999 9 33.078~826.941 隐绿原酸 Y=11 080X-5 034.8 0.999 9 4.949~123.728 咖啡酸 Y=40 485X-3 206.6 0.999 9 1.998~49.950 连翘酯苷A Y=9 441.8X+32 643 0.999 9 42.343~1 058.585 4, 5-二-O-咖啡酰奎宁酸 Y=25 225X-4 685.5 0.999 9 6.242~156.059 连翘苷 Y=8 074.6X+12 605 0.999 9 9.137~228.424
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表 3 单因素提取规格考察
Table 3. Single factor extraction specification investigation
提取规格/cm 提取率/% 出膏率/% 酚酸类成分 连翘酯苷A 连翘苷 整个 2.14 1.52 0.10 26.61 1.5 1.93 2.71 0.16 29.93 1.0 2.09 3.50 0.20 31.70 0.8 1.96 3.98 0.24 32.46 0.5 1.95 3.30 0.15 32.46
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表 4 单因素提取时间考察
Table 4. Single factor extraction time investigation
提取时间/min 提取率/% 出膏率/% 酚酸类成分 连翘酯苷A 连翘苷 20+15 2.27 3.37 0.23 33.13 30+30 2.33 3.34 0.23 34.02 60+30 2.47 3.38 0.26 35.29 60+60 2.51 3.38 0.27 35.86 注: 30+30表示一煎煎煮30 min, 二煎煎煮30 min。
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表 5 Box-Behnken实验工艺参数及水平设计与结果
Table 5. Process parameters and levels design and results of Box-Behnken experiments
试验号 因素 响应值 X1/cm X2/min X3 Y1/% Y2/% Y3/% Y4/% 1 1.0 60+30 12+10 35.63 2.45 3.34 0.28 2 1.5 30+30 10+8 32.31 2.32 3.03 0.21 3 1.0 60+30 10+8 33.62 2.13 3.03 0.23 4 1.0 30+30 11+9 33.81 2.34 3.42 0.25 5 0.8 20+15 11+9 33.07 2.23 3.54 0.24 6 1.5 60+30 11+9 34.59 2.41 3.11 0.26 7 1.0 30+30 11+9 34.20 2.30 3.36 0.24 8 1.0 20+15 12+10 33.92 2.32 3.50 0.24 9 0.8 30+30 10+8 33.51 2.29 3.57 0.24 10 1.0 30+30 11+9 34.48 2.26 3.29 0.24 11 1.0 20+15 10+8 32.21 2.24 3.17 0.22 12 1.0 30+30 11+9 34.18 2.31 3.33 0.24 13 1.5 30+30 12+10 33.50 2.35 3.17 0.24 14 1.0 30+30 11+9 34.17 2.29 3.30 0.24 15 0.8 60+30 11+9 34.81 2.29 3.32 0.28 16 0.8 30+30 12+10 34.57 2.39 3.62 0.26 17 1.5 20+15 11+9 32.32 2.26 2.90 0.20 注: 60+30表示一煎煎煮60 min, 二煎煎煮30 min; 12+10表示一煎加12倍量水, 二煎加10倍量水。
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表 6 回归模型方差分析结果
Table 6. ANOVA results of regression model
来源 Y1 Y3 Y2 Y4 F P F P F P F P 模型 12.88 0.001 4 5.25 0.019 9 9.78 0.003 3 11.27 0.002 1 X1 11.38 0.011 9 2.37 0.167 3 60.12 0.000 1 23.27 0.001 9 X2 55.12 0.000 1 22.39 0.002 1 1.71 0.232 7 42.37 0.000 3 X3 38.64 0.000 4 13.19 0.008 4 12.23 0.010 0 27.69 0.001 2 X1X2 0.61 0.460 7 1.96 0.204 0 6.57 0.037 4 1.54 0.254 8 X1X3 0.04 0.853 6 1.19 0.312 0 0.29 0.608 3 0.39 0.554 8 X2X3 0.19 0.672 0 0.39 0.553 3 0.01 0.908 5 3.46 0.105 1 X12 6.49 0.038 3 0.00 1.000 0 0.18 0.681 5 0.06 0.806 4 X22 0.09 0.775 7 0.02 0.877 6 6.59 0.037 1 1.04 0.342 5 X32 2.74 0.141 7 5.74 0.047 8 0.37 0.560 2 0.79 0.402 6 失拟项 3.41 0.133 2 1.50 0.342 9 4.64 0.086 2 6.25 0.054 4
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表 7 设计空间验证试验点选取
Table 7. Selection of experimental verification points for design space
序号 X1/cm X2/min X3 试验点选取说明 YZ1 1.0 45+30 12+10 Y1、Y2、Y3、Y4均在95%置信区间内 YZ2 1.2 30+15 12+10 Y1、Y2、Y3、Y4部分在95%置信区间内, 部分在区间外 YZ3 1.5 20+15 12+10 Y1、Y2、Y3、Y4均不在95%置信区间内 注: 45+30表示一煎煎煮45 min, 二煎煎煮30 min; 12+10表示一煎加12倍量水, 二煎加10倍量水。
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表 8 设计空间验证结果(%)
Table 8. Results of verification test for design space(%)
序号 Y1 Y2 Y3 Y4 预测值 实测值 预测值 实测值 预测值 实测值 预测值 实测值 YZ1 35.12 35.24 2.33 2.37 3.44 3.58 0.27 0.26 YZ2 33.98 34.32 2.26 2.21 3.42 3.63 0.24 0.26 YZ3 33.56 33.13 2.22 2.27 3.08 3.18 0.21 0.23
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