Optimization of Extraction Technology of Total Triterpenoids from Inonotus sanghuang and Their Hypolipidemic and Antioxidant Activities
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摘要: 选用超声波法提取桑树桑黄子实体中的三萜类化合物，在单因素试验的基础上采用响应面法优选最佳方案，并对其体外降血脂和抗氧化活性进行了测定。结果表明，超声波法提取桑树桑黄总三萜的最优条件为：提取时间49 min、乙醇浓度为80%、提取温度为61 ℃，料液比为1:22（g/mL），总三萜得率可达12.32% ± 0.17%。质量浓度为80 mg/mL的总三萜体外结合甘氨胆酸钠、牛磺胆酸钠、胆酸钠的能力为同剂量考来烯胺的50.93%、52.14%和43.06%，说明其具有较好的体外降血脂活性。总三萜具有较好的还原能力，并能有效清除DPPH和ABTS自由基，其IC50值分别为0.304和0.520 mg/mL，表明其具有较好的体外抗氧化活性。Abstract: The ultrasonic-assisted extraction process of total triterpenoids from Inonotus sanghuang was employed in this paper. Based on the single factor experiments, the response surface methodology (RSM) was used to determine the optimal extraction condition, and its hypolipidemic and antioxidant activities were primarily evaluated. The results of optimum extraction conditions were as follow: Extraction time 49 min, ethanol concentration 80%, ultrasonic temperature 61 ℃, ratio of solid to liquid 1:22 (g/mL). Under these conditions, the yield of total triterpenoids was 12.32% ± 0.17%. The binding ability of 80 mg/mL triterpenoids to sodium glycylcholate, sodium taurocholate and sodium cholate in vitro was 50.93%, 52.14% and 43.06% of that of colenamine in the same dose, which indicated that the total triterpenoids of Inonotus sanghuang had the activity of lowering blood lipids in vitro. The iron ions reduction results showed that the total triterpenoids had certain reducing power. The IC50 values of scavenging capacities of DPPH and ABTS free radical was 0.304 and 0.520 mg/mL, respectively, which showed it possessed good antioxidants in vitro.
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Key words:
- Inonotus sanghuang /
- total triterpenoids /
- extraction /
- hypolipidemic /
- antioxidant activity
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图 1 料液比对总三萜得率的影响
Figure 1. Effect of solvent-to-solid ratio on extraction of total triterpenoids
图 2 提取时间对总三萜得率的影响
Figure 2. Effect of reaction time on extraction of total triterpenoids
图 3 提取温度对总三萜得率的影响
Figure 3. Effect of reaction temperature on extraction of total triterpenoids
图 4 乙醇浓度对总三萜得率的影响
Figure 4. Effect of reaction ethanol concentration on extraction of total triterpenoids
图 5 各因素交互作用的响应面图和等高线图
Figure 5. Response surface and contour plots of the interaction of various factors
图 6 桑树桑黄总三萜对胆酸盐的结合量
注：图中不同的字母表示不同质量浓度的总三萜对同一种胆酸盐的结合量差异显著（P<0.05）。
Figure 6. The binding amount of total triterpenoids to cholic acid salt in Inonotue sanghuang
图 7 桑树桑黄总三萜的DPPH自由基清除能力
Figure 7. DPPH free radical scavenging ability of total triterpenoids from Inonotus sanghuang
图 8 桑树桑黄总三萜的ABTS自由基清除能力
Figure 8. ABTS free radical scavenging ability of total triterpenoids from Inonotus sanghuang
图 9 VC和桑树桑黄总三萜的还原能力
Figure 9. The reduction capacity of VC and total triterpenoids from Inonotus sanghuang
表 1 响应面试验因素水平表
Table 1. Factors and levels table of the response surface experiment
水平 因素 A提取时间
（min）B乙醇浓度
（%）C提取温度
（℃）D料液比
(g/mL)−1 35 65 45 1:15 0 45 75 55 1:20 1 55 85 65 1:25 
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表 2 响应面设计与试验结果
Table 2. Design and results of response surface experiment
试验号 A B C D Y总三萜得率（%） 1 35 65 55 1:20 9.89 ± 0.26 2 55 65 55 1:20 10.18 ± 0.17 3 35 85 55 1:20 9.93 ± 0.18 4 55 85 55 1:20 11.16 ± 0.21 5 45 75 45 1:15 9.77 ± 0.15 6 45 75 65 1:15 10.24 ± 0.23 7 45 75 45 1:25 9.92 ± 0.14 8 45 75 65 1:25 11.71 ± 0.27 9 35 75 55 1:15 9.65 ± 0.25 10 55 75 55 1:15 10.14 ± 0.17 11 35 75 55 1:25 9.96 ± 0.15 12 55 75 55 1:25 11.59 ± 0.22 13 45 65 45 1:20 10.03 ± 0.19 14 45 85 45 1:20 9.98 ± 0.13 15 45 65 65 1:20 10.48 ± 0.25 16 45 85 65 1:20 11.97 ± 0.14 17 35 75 45 1:20 9.79 ± 0.20 18 55 75 45 1:20 10.32 ± 0.13 19 35 75 65 1:20 10.07 ± 0.24 20 55 75 65 1:20 11.34 ± 0.19 21 45 65 55 1:15 10.15 ± 0.11 22 45 85 55 1:15 10.86 ± 0.13 23 45 65 55 1:25 10.94 ± 0.09 24 45 85 55 1:25 11.13 ± 0.18 25 45 75 55 1:20 11.92 ± 0.11 26 45 75 55 1:20 11.86 ± 0.13 27 45 75 55 1:20 12.18 ± 0.15 28 45 75 55 1:20 11.94 ± 0.07 29 45 75 55 1:20 11.81 ± 0.11
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表 3 方差分析
Table 3. Analysis of variances
差异来源 平方和 自由度 均方 F值 P值 显著性 模型 19.32 14 1.38 30.86 <0.0001 ** A 2.47 1 2.47 55.13 <0.0001 ** B 0.94 1 0.94 21.03 0.0004 ** C 3.00 1 3 67.06 <0.0001 ** D 1.64 1 1.64 36.72 <0.0001 ** AB 0.22 1 0.22 4.94 0.0433 * AC 0.14 1 0.14 3.06 0.1021 AD 0.32 1 0.32 7.26 0.0174 * BC 0.59 1 0.59 13.25 0.0027 ** BD 0.068 1 0.068 1.51 0.2392 CD 0.44 1 0.44 9.74 0.0075 ** A2 5.67 1 5.67 126.81 <0.0001 ** B2 2.34 1 2.34 52.23 <0.0001 ** C2 3.51 1 3.51 78.37 <0.0001 ** D2 3.00 1 3 67.08 <0.0001 ** 残差 0.63 14 0.045 失拟项 0.55 10 0.055 2.68 0.1771 纯误差 0.081 4 0.02 总和 19.95 28 注：“*”表示差异显著，P<0.05，“**”表示差异极显著，P<0.01。
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表 4 桑树桑黄总三萜对胆酸盐的结合能力
Table 4. The binding capacity of total triterpenoids from Inonotus sanghuang to bile salts in vitro
指标名称 甘氨胆酸钠结合量（μmol/100 mg） 相对考来烯胺的结合率（%） 牛磺胆酸钠结合量（μmol/100 mg） 相对考来烯胺的结合率（%） 胆酸钠结合量（μmol/100 mg） 相对考来烯胺的结合率（%） 桑树桑黄总三萜 1.385 ± 0.071 50.93 ± 3.34 1.422 ± 0.067 52.14 ± 4.07 1.675 ± 0.034 43.06 ± 1.71 考来烯胺 2.726 ± 0.043 100.00 ± 1.57 2.734 ± 0.085 100.00 ± 3.11 3.893 ± 0.076 100.00 ± 2.02
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