Preparation and in vitro antifungal activity of Notopterygii Rhizoma et Radix extract microemulsion
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摘要: 采用乙醇溶液热萃取方法提取羌活根茎活性成分，通过稳定性等物理性能测试进行评价并确定微乳剂最优配方。微乳剂由质量分数 (下同)为15%的羌活提取物 (含羌活醇0.34%)、9%的环己酮、16%的HMK-2100、10%的乙醇、49.9%的水和0.1%的AF1506组成。理化性质测试结果表明，所制备的羌活提取物微乳剂各项指标均符合国家标准。为了比较研究其抑菌活性，同时还制备了5%羌活提取物 (含羌活醇0.11%)微乳剂和30%羌活提取物 (含羌活醇0.68%)乳油。采用菌丝生长速率法研究了羌活提取物不同剂型产品对小麦纹枯病菌Rhizoctonia cerealis、辣椒疫霉Phytophthora capsici、水稻稻瘟病菌Pyricularia grisea和水稻纹枯病菌Rhizoctonia solani的抑制作用。结果显示，15%羌活提取物微乳剂的抑菌效果显著，在用水稀释500倍 (羌活提取物质量浓度为300 mg/L，羌活醇质量浓度为6.75 mg/L)后施用，其对水稻稻瘟病菌和水稻纹枯病菌菌丝生长的抑制率均在90%以上。应用性能测试结果表明，羌活提取物微乳剂在绿萝叶片表面具有优异的铺展和润湿能力。Abstract: Hot ethanol-water solution was used to extract the active ingredients from the root of Notopterygium incisum Ting ex H. T. Chang (Notopterygii Rhizoma et Radix), and the microemulsion of the extracted active ingredient was prepared. The physical properties such as stability were tested to evaluate and determine the optimal formulation of the microemulsion. The microemulsion is composed of 15% Notopterygii Rhizoma et Radix extract (notopterol content 0.34%), 9% cyclohexanone, 16% HMK-2100, 10% ethanol, 49.9% water, and 0.1% AF1506. The results showed that the performances of the prepared Notopterygii Rhizoma et Radix extract microemulsion were in line with microemulsion standards. Meanwhile, 5% Notopterygii Rhizoma et Radix extract microemulsion (notopterol 0.11%) and 30% Notopterygii Rhizoma et Radix extract emulsion (notopterol 0.68%) were prepared. The mycelial growth rate method was used to evaluate the effects of different formulations on the four plant pathogens of Rhizoctonia cerealis, Phytophthora capsici, Pyricularia grisea and Rhizoctonia solani. The results showed that 15% Notopterygii Rhizoma et Radix extract microemulsion had significant fungi growth inhibition activities. After 500 times of dilution (Notopterygii Rhizoma et Radix extract 300 mg/L and notopterol 6.75 mg/L), the growth inhibition rates to P. grisea and R. solani were over 90%. Application performance tests showed that Notopterygii Rhizoma et Radix extract microemulsion had excellent spreading and wetting abilities on the leaf of green dill surface.
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图 1 羌活提取物中羌活醇高效液相色谱分析
Figure 1. HPLC chromatogram of notopterol extracted from Notopterygii Rhizoma et Radix
图 2 15%羌活提取物微乳剂100倍稀释液在绿萝同一叶片不同位置处的接触角
Figure 2. Contact angle of Notopterygii Rhizoma et Radix extract 15% ME 100-time dilution at different positions on the same leaf of green dill
图 3 15%羌活提取物微乳剂100倍稀释液的动态表面张力
Figure 3. The dynamic surface tension of 100 times diluted solution of Notopterygii Rhizoma et Radix extract 15% ME
表 1 有效成分羌活醇高效液相色谱分析流动相组成
Table 1. Mobile phase ratios in HPLC analysis of active ingredient notopterol
保留时间
Retention time/min流动相 A
Mobile phase A/%流动相 B
Mobile phase B/%0 15 85 14 35 65 20 35 65 35 60 40 40 75 25 42 15 85 50 15 85 
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表 2 羌活提取物原药含量对微乳剂稳定性影响
Table 2. The effects of Notopterygii Rhizoma et Radix extract content on the stability of microemulsion
质量分数
Mass fraction/%热贮稳定性Thermal storage
stability低温稳定性Low temperature
stability有效成分降解率Active ingredient
degradation rate/%提取物
Extract羌活醇Notopterol
环己酮 (溶剂) Cyclohexanone
(Solvent )HMK-2100 (乳化剂)HMK-2100 (Emulsifier) 5 0.11 15 20 透明 Transparent 合格 Qualified 6.50 10 0.23 15 20 透明 Transparent 合格 Qualified 6.10 15 0.34 15 20 透 明Transparent 合格 Qualified 4.34 20 0.45 15 20 浑浊 Turbid 合格 Qualified — 25 0.56 15 20 浑浊 Turbid 合格 Qualified — 30 0.68 15 20 浑浊 Turbid 合格 Qualified — 注：消泡剂AF1506质量分数0.1%，乙醇10%，水补足至100%。Note: The content of defoamer AF1506 is 0.1%, the ethanol 10%, and the water is added up to 100%.
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表 3 溶剂环己酮含量对微乳剂稳定性影响
Table 3. The effects of solvent cyclohexanone content on the stability of microemulsion
质量分数
Mass fraction/%热贮稳定性Thermal storage
stability低温稳定性Low temperature
stability有效成分降解率Active ingredient
degradation rate/%提取物
Extract羌活醇Notopterol
环己酮 (溶剂) Cyclohexanone
(Solvent )HMK-2100 (乳化剂)HMK-2100 (Emulsifier) 15 0.34 7 20 浑浊 Turbid 合格 Qualified — 15 0.34 9 20 透明 Transparent 合格 Qualified 3.76 15 0.34 11 20 透明 Transparent 合格 Qualified 8.22 15 0.34 13 20 透明 Transparent 合格 Qualified 1.69 15 0.34 15 20 透明 Transparent 合格 Qualified 4.34 15 0.34 17 20 透明 Transparent 合格 Qualified 6.49 注：消泡剂AF1506质量分数0.1%，乙醇10%，水补足至100%。Note: The content of defoamer AF1506 is 0.1%, the ethanol 10%, and the water is added up to 100%.
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表 4 乳化剂HMK-2100含量对微乳剂稳定性影响
Table 4. The effects of emulsifier HMK-2100 content on the stability of microemulsion
质量分数
Mass fraction/%热贮稳定性Thermal storage
stability低温稳定性Low temperature
stability有效成分降解率Active ingredient
degradation rate/%提取物
Extract羌活醇Notopterol
环己酮 (溶剂) Cyclohexanone
(Solvent )HMK-2100 (乳化剂)HMK-2100 (Emulsifier) 15 0.34 9 12 浑浊 Turbid 浑浊 Turbid — 15 0.34 9 14 浑浊 Turbid 浑浊 Turbid — 15 0.34 9 16 透明 Transparent 合格 Qualified 4.58 15 0.34 9 18 透明 Transparent 合格 Qualified 5.25 15 0.34 9 20 透明 Transparent 合格 Qualified 3.76 15 0.34 9 22 透明 Transparent 合格 Qualified 14.83 注：消泡剂AF1506质量分数0.1%，乙醇10%，水补足至100%。Note: The content of defoamer AF1506 is 0.1%, the ethanol 10%, and the water is added up to 100%.
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表 5 不同剂型配方的理化参数
Table 5. Physical and chemical parameters of different formulations
理化参数
Physical and chemical parameter15% 羌活提取物微乳剂
Notopterygii Rhizoma et Radix extract 15% ME30% 羌活提取物乳油
Notopterygii Rhizoma et Radix extract 30% EC外观 Exterior 深褐色均相透明液体
Dark brown homogeneous transparent liquid深褐色均相透明液体
Dark brown homogeneous transparent liquidpH 4~7 4~7 透明温度范围
Transparent temperature range−5~50 ℃ −5~50 ℃ 乳液稳定性 Emulsion stability 合格 Qualified 合格 Qualified 低温稳定性 (0 ℃ ± 2 ℃，7 d)
Low temperature stability合格 Qualified 合格 Qualified 热贮稳定性 (54 ℃ ± 2 ℃，14 d)
Thermal storage stability合格 Qualified 合格 Qualified 有效成分降解率
Active ingredient degradation rate/%< 5% < 5% 常贮稳定性 Room temperature storage stability 合格 Qualified 合格 Qualified
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表 6 不同羌活提取物剂型对植物病原真菌的抑制效果
Table 6. The inhibitory effects of different formulations of Notopterygii Rhizoma et Radix extract on plant pathogenic fungi
样品
Sample质量浓度Concentration/
(mg/L)羌活醇质量浓度 Concentration of
notopterol/(mg/L)抑制率Inhibition rate/% 小麦纹枯病菌
Rhizoctonia cerealis辣椒疫霉
Phytophthora capsici水稻稻瘟病菌
Pyricularia grisea水稻纹枯病菌
Rhizoctonia solani5% 羌活提取物微乳剂Notopterygii Rhizoma et
Radix extract 5% ME1000 22.50 74.4 87.1 100 92.6 500 11.25 66.4 71.4 97.2 88.9 250 5.63 45.3 57.1 94.6 85.2 15% 羌活提取物微乳剂Notopterygii Rhizoma et
Radix extract 15% ME1500 33.75 91.5 88.9 100 97.2 500 11.25 62.2 77.8 100 94.4 300 6.75 59.6 70.4 100 90.6 30% 羌活提取物乳油Notopterygii Rhizoma et
Radix extract 30% EC1000 22.50 82.9 81.5 100 91.7 500 11.25 76.8 79.3 100 88.5 300 6.75 57.4 77.8 100 86.3 噻呋酰胺 thiofuramide 50 — — — — 97.8 多菌灵 carbendazim 50 — 87.5 11.5 100 100
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