Study on hydrolysis kinetics of azadirachtin and tructure analysis of hydrolysate
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摘要:目的
系统研究印楝素在水溶液中的水解。
方法硅胶柱层析法和半制备液相色谱法分离纯化w为44.56%的印楝素原药中的印楝素A，采用核磁共振仪和高效液相色谱定性、定量测定分离得到的印楝素A，建立一种检测水样中印楝素残留的高效液相色谱方法。
结果核磁共振仪和高效液相色谱测得印楝素A的质量分数分别为90.37%和91.82%。当印楝素添加水平为0.1、1.0和5.0 mg·kg-1时，水样中印楝素的平均回收率为92.53%~94.12%，变异系数为0.35%~0.84%，最小检测质量浓度为0.012 mg·L-1。印楝素在pH 4.0~6.0的缓冲溶液中稳定，当pH大于8.0时，印楝素降解加快，降解半衰期从pH 8.0的14.856 h降到pH 10.0的0.033 h。在pH 6.0的缓冲溶液中，25、35、45 ℃条件下印楝素的降解半衰期分别为24.68、13.69和2.36 d，而在pH 7.0的缓冲溶液中印楝素的降解半衰期分别为9.35、6.51和0.94 d。在pH 2.0的缓冲溶液中分离纯化水解产物得到印楝素A内酯衍生物。
结论印楝素在碱性环境下极不稳定，而在弱酸性环境中比较稳定。温度对印楝素的降解影响很大，随着温度的升高印楝素降解加快。
Abstract:ObjectiveTo study hydrolysis of azadirachtin in water systematically.
MethodAzadirachtin A from the 44.56% azadirachtin TC was isolated and purified by silica column chromatography and semi-preparative high performance liquid chromatography (HPLC). The chemical structure and content of isolated azadirachtin A were identified by nulear magnetic resonance (NMR) and HPLC. A method for determining azaditachtin residue in water by HPLC was established.
ResultThe mass fractions of azadirachtin A were 90.37% and 91.82% detected by NMR and HPLC respectively. When azadirachtin was added with the concentrations of 0.1, 1.0 and 5.0 mg·kg-1, the average recovery rates of azadirachtin from water samples ranged from 92.53% to 94.12%, the variation coefficients ranged from 0.35% to 0.84%, and the minimum detection limit was 0.012 mg·L-1. Azadirachtin was stable in buffer solutions with pH varying from 4.0 to 6.0. When pH was above 8.0, hydrolysis of azadirachtin was accelerated, and the degradation half-life was 14.856 h at pH 8.0 and declined to 0.033 h at pH 10.0. The degration half-lives of azadirachtin in buffer solutions at pH 6.0 were 24.68, 13.69 and 2.36 d under 25, 35 and 45 ℃ temperature respectively, while were 9.35, 6.51 and 0.94 d at pH 7.0. A lactone derivative of azadirachtin was obtained by isolating and purifing hydrolysate in buffer solution at pH 2.0.
ConclusionAzadirachtin is extremely unstable in alkaline environment while relatively stable in weak acid environment. Temperature has a great effect on the degradation of azadirachtin and the degradation accelerates as temperature increases.
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Keywords:
- azadirachtin /
- residue /
- hydrolysis kinetics /
- hydrolysate /
- silica column chromatography /
- HPLC /
- nuclear magnetic resonance
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图 3 印楝素水解产物印楝素A内酯衍生物的化学结构试
Figure 3. Structure of azadirachtin A lactone derivative of azadirachtin hydrolysate
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图 4 印楝素内脂衍生物的1H NMR和13C NMR图谱
Figure 4. 1H NMR and 13C NMR spectra of azadirachtin derivatives
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图 6 印楝素在pH 1.0、8.0、9.0、10.0缓冲溶液中的降解曲线
Figure 6. Degradation curves of azadirachtin in buffer solutions at pH 1.0, 8.0, 9.0 and 10.0
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图 7 印楝素在模拟天然水体不同温度下的降解曲线
Figure 7. Degradation curves of azadirachtin in simulated natural water at different temperatures
表 1 印楝素的核磁共振碳谱数据13C NMR
Table 1 13C NMR data of azadirachtin ×10-6
碳原子
编号化学位移 文献值[20] 实测值 C-1 70.7 70.6 C-2 29.8 29.7 C-3 67.1 67.0 C-4 45.6 45.6 C-5 37.1 36.9 C-6 74.5 74.3 C-7 76.5 76.5 C-8 50.4 50.3 C-9 44.9 44.7 C-10 52.6 52.5 C-11 104.2 104.2 C-12 169.5 169.8 C-13 70.1 70.1 C-14 68.6 68.8 C-15 73.9 74.0 C-16 25.1 25.0 C-17 48.7 48.7 C-18 20.8 21.0 C-19 69.1 69.0 C-20 83.6 83.4 C-21 107.5 107.6 C-22 108.8 108.7 C-23 147.0 146.8 C-28 73.0 72.9 C-29 166.2 166.3 C-30 18.4 18.5 3-C=O 173.3 173.5 CH3 21.4 21.4 29-OCH3 52.7 52.8 12-OCH3 53.1 53.2 O-Tig； C-1′ 171.8 171.9 C-2′ 128.8 128.6 C-3′ 137.5 137.9 C-4′ 14.2 14.3 C-5′ 11.9 11.9 
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表 2 印楝素的动力学分析
Table 2 Kinetic analysis of azadirachtin
pH 动力学方程 相关系数(r) 半衰期/d 1.0 Ct=44.31e -0.300 0t 0.999 8 0.10 2.0 Ct=42.57e-0.233 0t 0.997 6 2.98 3.0 Ct=44.61e-0.079 8t 0.997 4 8.68 4.0 Ct=44.03e-0.030 3t 0.994 8 22.87 5.0 Ct=44.85e-0.019 1t 0.991 2 36.33 6.0 Ct=44.36e-0.028 1t 0.997 6 24.68 7.0 Ct=43.04e-0.074 1t 0.997 7 9.35 8.0 Ct=43.73e-0.026 7t 0.975 4 0.62 9.0 Ct=45.16e-0.289 0t 0.998 8 0.10 10.0 Ct=44.99e-0.877 0t 0.999 9 0.03
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表 3 印楝素在模拟天然水体不同温度下的降解动力学方程
Table 3 Degradation kinetic equations of azadirachtin in simulated natural water at different temperatures
pH θ/℃ 动力学方程 相关系数(r) 半衰期/d 6.0 25 Ct=44.36e-0.028 1t 0.997 6 24.68 35 Ct=45.08e-0.050 6t 0.994 3 13.69 45 Ct=47.63e-0.294 0t 0.992 4 2.36 7.0 25 Ct=43.04e-0.074 1t 0.997 7 9.35 35 Ct=45.57e-0.106 0t 0.994 5 6.51 45 Ct=45.98e-0.737 0t 0.999 9 0.94
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表 4 印楝素在天然水体中的降解动力学方程
Table 4 Degradation kinetic equations of azadirachtin in natural water
水体 动力学方程 相关系数(r) 半衰期/d 珠江水 Ct=46.76e-0.056 9t 0.997 4 12.19 稻田水 Ct=45.98e-0.046 5t 0.989 7 14.91 地表水 Ct=45.72e-0.043 6t 0.996 6 15.90 湖水 Ct=44.79e-0.108 0t 0.994 2 6.43 水库水 Ct=44.96e-0.018 0t 0.995 0 37.06
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表 5 印楝素降解半衰期与水体理化性质的相关性分析
Table 5 Analysis of the correlation between the degradation half-life of azadirachtin and physical/chemical properties of water
理化性质 回归方程1) 相关系数(r) pH y＝183.570-23.537 0x 0.995 9 总磷含量 y＝20.995-11.123 0x 0.285 5 总氮含量 y＝27.190-2.367 8x 0.498 0 化学需氧量 y＝18.449-0.009 3x 0.044 7 硝酸盐氮含量 y＝20.352-2.726 1x 0.376 8 氨氮含量 y＝16.873+0.890 6x 0.319 1 1)y为印楝素半衰期，x为对应的理化性质。
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