植物学报 ›› 2018, Vol. 53 ›› Issue (3): 341-352.DOI: 10.11983/CBB17103
所属专题: 药用植物专辑 (2018年53卷3期)
李璐1, 安叶娟1, 乔春雷1, 杨晓华2,*(), 张华峰1,*(
), 薛梦莹1
收稿日期:
2017-05-23
接受日期:
2017-08-30
出版日期:
2018-05-01
发布日期:
2018-09-11
通讯作者:
杨晓华,张华峰
基金资助:
Li Lu1, An Yejuan1, Qiao Chunlei1, Yang Xiaohua2,*(), Zhang Huafeng1,*(
), Xue Mengying1
Received:
2017-05-23
Accepted:
2017-08-30
Online:
2018-05-01
Published:
2018-09-11
Contact:
Yang Xiaohua,Zhang Huafeng
摘要: 建立了药用植物心叶淫羊藿(Epimedium brevicornum)生物碱的超声波-微波协同提取工艺, 探讨了提取机理, 分析了生物碱的化学组成及其对人宫颈癌HeLa细胞增殖的抑制作用。采用正交试验法优化得到了淫羊藿生物碱的超声波-微波协同提取工艺: 浸泡时间为40分钟, 超声波-微波协同作用18分钟, 微波功率250 J∙s-1, 液固比为30 mL∙g-1, 乙醇溶液浓度为70%。超声波-微波协同提取法的提取率可达16.146 mg∙g-1, 显著高于超声波提取法、微波提取法和加热提取法。扫描电子显微镜观察结果表明, 超声波-微波协同作用可使淫羊藿叶片样品表面出现大量裂隙。激光粒度分析显示, 超声波-微波协同提取后, 中、低粒度范围的样品量明显增多, 而高粒度范围的样品量明显减少。薄层色谱和高效液相色谱分析结果表明, 淫羊藿生物碱含有木兰花碱, 不含小檗碱或含量很低。淫羊藿生物碱对HeLa细胞的增殖具有明显的抑制作用, 并且呈现出剂量依赖性。研究结果为药用植物淫羊藿资源的开发利用提供理论依据。
李璐, 安叶娟, 乔春雷, 杨晓华, 张华峰, 薛梦莹. 淫羊藿生物碱的超声波-微波协同提取及其 对HeLa细胞的抑制作用. 植物学报, 2018, 53(3): 341-352.
Li Lu, An Yejuan, Qiao Chunlei, Yang Xiaohua, Zhang Huafeng, Xue Mengying. Ultrasound-microwave-assisted Extraction of Alkaloids from Epimedium brevicornum and Their Inhibitory Effect on HeLa Cells. Chinese Bulletin of Botany, 2018, 53(3): 341-352.
图1 浸泡时间(A)、超声波-微波协同作用时间(B)、微波功率(C)、液固比(D)与乙醇溶液浓度(E)对淫羊藿生物碱提取率的影响不同小写字母表示差异显著(P<0.05)。
Figure 1 Effects of steeped time (A), ultrasound- microwave time (B), microwave power (C), liquid to solid ratio (D), and ethanol concentration (E) on extraction yield of Epimedium brevicornum alkaloidsDifferent lowercase letters indicate significant differences (P<0.05).
Test No. | Factor | Mean extraction yield (mg∙g-1) | |||||
---|---|---|---|---|---|---|---|
(A) Steeped time (min) | (B) Ultrasound- microwave time (min) | (C) Microwave power (J∙s-1) | Blank | (D) Liquid to solid ratio (mL∙g-1) | (E) Ethanol concentration (%) | ||
1 | A1 16 | B1 2 | C1 50 | 1 | D1 30 | E1 50 | 14.361 |
2 | A1 16 | B2 6 | C2 100 | 2 | D2 35 | E2 60 | 13.343 |
3 | A1 16 | B3 10 | C3 150 | 3 | D3 40 | E3 70 | 12.015 |
4 | A1 16 | B4 14 | C4 200 | 4 | D4 45 | E4 80 | 10.966 |
5 | A1 16 | B5 18 | C5 250 | 5 | D5 50 | E5 90 | 9.221 |
6 | A2 24 | B1 2 | C2 100 | 3 | D4 45 | E5 90 | 9.430 |
7 | A2 24 | B2 6 | C3 150 | 4 | D5 50 | E1 50 | 9.696 |
8 | A2 24 | B3 10 | C4 200 | 5 | D1 30 | E2 60 | 15.878 |
9 | A2 24 | B4 14 | C5 250 | 1 | D2 35 | E3 70 | 14.259 |
10 | A2 24 | B5 18 | C1 50 | 2 | D3 40 | E4 80 | 11.946 |
11 | A3 32 | B1 2 | C3 150 | 5 | D2 35 | E4 80 | 12.869 |
12 | A3 32 | B2 6 | C4 200 | 1 | D3 40 | E5 90 | 11.181 |
13 | A3 32 | B3 10 | C5 250 | 2 | D4 45 | E1 50 | 10.934 |
14 | A3 32 | B4 14 | C1 50 | 3 | D5 50 | E2 60 | 9.948 |
15 | A3 32 | B5 18 | C2 100 | 4 | D1 30 | E3 70 | 15.833 |
16 | A4 40 | B1 2 | C4 200 | 2 | D5 50 | E3 70 | 9.967 |
17 | A4 40 | B2 6 | C5 250 | 3 | D1 30 | E4 80 | 15.758 |
18 | A4 40 | B3 10 | C1 50 | 4 | D2 35 | E5 90 | 12.806 |
19 | A4 40 | B4 14 | C2 100 | 5 | D3 40 | E1 50 | 12.022 |
20 | A4 40 | B5 18 | C3 150 | 1 | D4 45 | E2 60 | 11.251 |
21 | A5 48 | B1 2 | C5 250 | 4 | D3 40 | E2 60 | 11.895 |
22 | A5 48 | B2 6 | C1 50 | 5 | D4 45 | E3 70 | 10.764 |
23 | A5 48 | B3 10 | C2 100 | 1 | D5 50 | E4 80 | 9.765 |
24 | A5 48 | B4 14 | C3 150 | 2 | D1 30 | E5 90 | 14.588 |
25 | A5 48 | B5 18 | C4 200 | 3 | D2 35 | E1 50 | 13.640 |
T1 | 59.906 | 58.522 | 59.824 | 60.817 | 76.417 | 60.652 | |
T2 | 61.209 | 60.741 | 60.393 | 60.779 | 66.917 | 62.315 | |
T3 | 60.766 | 61.398 | 60.418 | 60.791 | 59.059 | 62.839 | |
T4 | 61.803 | 61.784 | 61.632 | 61.196 | 53.345 | 61.303 | |
T5 | 60.652 | 61.891 | 62.086 | 60.753 | 48.598 | 57.226 | |
K1 | 11.981 | 11.704 | 11.965 | 12.163 | 15.284 | 12.131 | |
K2 | 12.242 | 12.148 | 12.079 | 12.156 | 13.383 | 12.463 | |
K3 | 12.153 | 12.280 | 12.084 | 12.158 | 11.812 | 12.568 | |
K4 | 12.361 | 12.357 | 12.326 | 12.239 | 10.669 | 12.261 | |
K5 | 12.131 | 12.378 | 12.414 | 12.151 | 9.720 | 11.445 | |
R | 0.379 | 0.674 | 0.449 | 0.089 | 5.564 | 1.123 |
表1 正交试验设计、结果与极差分析
Table 1 Design, result and extreme difference of orthogonal test
Test No. | Factor | Mean extraction yield (mg∙g-1) | |||||
---|---|---|---|---|---|---|---|
(A) Steeped time (min) | (B) Ultrasound- microwave time (min) | (C) Microwave power (J∙s-1) | Blank | (D) Liquid to solid ratio (mL∙g-1) | (E) Ethanol concentration (%) | ||
1 | A1 16 | B1 2 | C1 50 | 1 | D1 30 | E1 50 | 14.361 |
2 | A1 16 | B2 6 | C2 100 | 2 | D2 35 | E2 60 | 13.343 |
3 | A1 16 | B3 10 | C3 150 | 3 | D3 40 | E3 70 | 12.015 |
4 | A1 16 | B4 14 | C4 200 | 4 | D4 45 | E4 80 | 10.966 |
5 | A1 16 | B5 18 | C5 250 | 5 | D5 50 | E5 90 | 9.221 |
6 | A2 24 | B1 2 | C2 100 | 3 | D4 45 | E5 90 | 9.430 |
7 | A2 24 | B2 6 | C3 150 | 4 | D5 50 | E1 50 | 9.696 |
8 | A2 24 | B3 10 | C4 200 | 5 | D1 30 | E2 60 | 15.878 |
9 | A2 24 | B4 14 | C5 250 | 1 | D2 35 | E3 70 | 14.259 |
10 | A2 24 | B5 18 | C1 50 | 2 | D3 40 | E4 80 | 11.946 |
11 | A3 32 | B1 2 | C3 150 | 5 | D2 35 | E4 80 | 12.869 |
12 | A3 32 | B2 6 | C4 200 | 1 | D3 40 | E5 90 | 11.181 |
13 | A3 32 | B3 10 | C5 250 | 2 | D4 45 | E1 50 | 10.934 |
14 | A3 32 | B4 14 | C1 50 | 3 | D5 50 | E2 60 | 9.948 |
15 | A3 32 | B5 18 | C2 100 | 4 | D1 30 | E3 70 | 15.833 |
16 | A4 40 | B1 2 | C4 200 | 2 | D5 50 | E3 70 | 9.967 |
17 | A4 40 | B2 6 | C5 250 | 3 | D1 30 | E4 80 | 15.758 |
18 | A4 40 | B3 10 | C1 50 | 4 | D2 35 | E5 90 | 12.806 |
19 | A4 40 | B4 14 | C2 100 | 5 | D3 40 | E1 50 | 12.022 |
20 | A4 40 | B5 18 | C3 150 | 1 | D4 45 | E2 60 | 11.251 |
21 | A5 48 | B1 2 | C5 250 | 4 | D3 40 | E2 60 | 11.895 |
22 | A5 48 | B2 6 | C1 50 | 5 | D4 45 | E3 70 | 10.764 |
23 | A5 48 | B3 10 | C2 100 | 1 | D5 50 | E4 80 | 9.765 |
24 | A5 48 | B4 14 | C3 150 | 2 | D1 30 | E5 90 | 14.588 |
25 | A5 48 | B5 18 | C4 200 | 3 | D2 35 | E1 50 | 13.640 |
T1 | 59.906 | 58.522 | 59.824 | 60.817 | 76.417 | 60.652 | |
T2 | 61.209 | 60.741 | 60.393 | 60.779 | 66.917 | 62.315 | |
T3 | 60.766 | 61.398 | 60.418 | 60.791 | 59.059 | 62.839 | |
T4 | 61.803 | 61.784 | 61.632 | 61.196 | 53.345 | 61.303 | |
T5 | 60.652 | 61.891 | 62.086 | 60.753 | 48.598 | 57.226 | |
K1 | 11.981 | 11.704 | 11.965 | 12.163 | 15.284 | 12.131 | |
K2 | 12.242 | 12.148 | 12.079 | 12.156 | 13.383 | 12.463 | |
K3 | 12.153 | 12.280 | 12.084 | 12.158 | 11.812 | 12.568 | |
K4 | 12.361 | 12.357 | 12.326 | 12.239 | 10.669 | 12.261 | |
K5 | 12.131 | 12.378 | 12.414 | 12.151 | 9.720 | 11.445 | |
R | 0.379 | 0.674 | 0.449 | 0.089 | 5.564 | 1.123 |
Source | Sum of squares | dƒ | Mean square | F | P | Significance |
---|---|---|---|---|---|---|
(A) Steeped time (min) | 0.394 | 4 | 0.099 | 14.375 | 0.012 | * |
(B) Ultrasound-microwave time (min) | 1.537 | 4 | 0.384 | 56.051 | 0.001 | ** |
(C) Microwave power (J∙s-1) | 0.708 | 4 | 0.177 | 25.825 | 0.004 | ** |
Blank | 0.027 | 4 | 0.007 | |||
(D) Liquid to solid ratio (mL∙g-1) | 97.760 | 4 | 24.440 | 3564.343 | 0.0001 | ** |
(E) Ethanol concentration (%) | 3.896 | 4 | 0.974 | 142.039 | 0.0001 | ** |
Error | 0.027 | 4 | 0.007 | |||
Total | 104.323 | 28 |
表2 正交试验方差分析
Table 2 Variance analysis of orthogonal test
Source | Sum of squares | dƒ | Mean square | F | P | Significance |
---|---|---|---|---|---|---|
(A) Steeped time (min) | 0.394 | 4 | 0.099 | 14.375 | 0.012 | * |
(B) Ultrasound-microwave time (min) | 1.537 | 4 | 0.384 | 56.051 | 0.001 | ** |
(C) Microwave power (J∙s-1) | 0.708 | 4 | 0.177 | 25.825 | 0.004 | ** |
Blank | 0.027 | 4 | 0.007 | |||
(D) Liquid to solid ratio (mL∙g-1) | 97.760 | 4 | 24.440 | 3564.343 | 0.0001 | ** |
(E) Ethanol concentration (%) | 3.896 | 4 | 0.974 | 142.039 | 0.0001 | ** |
Error | 0.027 | 4 | 0.007 | |||
Total | 104.323 | 28 |
Method | Extraction yield (mg∙g-1) | RSD (%) | |||
---|---|---|---|---|---|
Replicate 1 | Replicate 2 | Replicate 3 | Mean | ||
Ultrasound-microwave assisted extraction | 15.915 | 16.262 | 16.262 | 16.146 a | 0.197 |
Ultrasound assisted extraction | 15.780 | 15.453 | 14.972 | 15.402 bc | 0.401 |
Microwave assisted extraction | 15.626 | 15.357 | 15.626 | 15.536 bc | 0.154 |
Heating extraction | 15.472 | 14.952 | 15.472 | 15.299 c | 0.296 |
表3 不同提取方法的提取效果比较
Table 3 Comparison of extraction yields between ultrasound-microwave assisted extraction and other extraction methods
Method | Extraction yield (mg∙g-1) | RSD (%) | |||
---|---|---|---|---|---|
Replicate 1 | Replicate 2 | Replicate 3 | Mean | ||
Ultrasound-microwave assisted extraction | 15.915 | 16.262 | 16.262 | 16.146 a | 0.197 |
Ultrasound assisted extraction | 15.780 | 15.453 | 14.972 | 15.402 bc | 0.401 |
Microwave assisted extraction | 15.626 | 15.357 | 15.626 | 15.536 bc | 0.154 |
Heating extraction | 15.472 | 14.952 | 15.472 | 15.299 c | 0.296 |
图2 不同提取方法处理后淫羊藿样品的扫描电子显微图像(A) 超声波-微波协同提取法; (B) 超声波提取法; (C) 微波提取法; (D) 加热提取法; (E) 未处理
Figure 2 Scanning electron micrographs of Epime- dium brevicornum leaf samples processed by different extraction methods(A) Ultrasound-microwave assisted extraction; (B) Ultrasound assisted extraction; (C) Microwave assisted extraction; (D) Heating extraction; (E) Untre- ated
图4 淫羊藿生物碱对人宫颈癌HeLa细胞增殖的抑制率(A) 24小时; (B) 48小时; (C) 72小时。不同小写字母表示差异显著(P<0.05)。
Figure 4 Inhibition rate of Epimedium brevicornum alkaloids on human cervical cancer HeLa cells(A) 24 h; (B) 48 h; (C) 72 h. Different lowercase letters indicate significant differences (P<0.05).
Concentration (μg∙mL-1) | Inhibition rate (%) | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Epimedium brevicornum alkaloids | Magnoflorine | Vinblastin sulfate | |||||||||
24 h | 48 h | 72 h | 24 h | 48 h | 72 h | 24 h | 48 h | 72 h | |||
0 | 0.000± 0.024 | 0.000± 0.140 | 0.000± 0.048 | 0.000± 0.015 | 0.000± 0.242 | 0.000± 0.035 | 0.000± 0.534 | 0.000± 0.101 | 0.000± 0.111 | ||
20 | 1.354± 0.048 | 1.239± 0.109 | 4.435± 0.077 | 6.150± 0.005 | 13.541± 0.504 | 15.408± 0.133 | 19.142± 0.075 | 23.967± 0.288 | 28.283± 0.316 | ||
50 | 7.472± 0.024 | 9.010± 0.298 | 11.156± 0.128 | 23.583± 0.071 | 31.737± 0.440 | 32.191± 0.105 | 30.362± 0.007 | 43.853± 0.273 | 42.140± 0.193 | ||
100 | 18.811± 0.025 | 27.799± 0.454 | 30.129± 0.162 | 42.173± 0.049 | 57.217± 0.426 | 59.882± 0.162 | 56.205± 0.102 | 67.590± 0.322 | 71.598± 0.177 | ||
200 | 28.827± 0.036 | 42.880± 0.283 | 67.410± 0.079 | 54.315± 0.094 | 72.024± 0.058 | 75.961± 0.061 | 77.961± 0.044 | 82.618± 0.061 | 91.609± 0.074 | ||
300 | 35.134± 0.084 | 67.263± 0.372 | 82.682± 0.208 | 67.496± 0.032 | 76.609± 0.181 | 88.300± 0.024 | 86.866± 0.045 | 90.539± 0.133 | 95.317± 0.100 | ||
400 | 47.724± 0.048 | 74.015± 0.058 | 94.238± 0.016 | 73.354± 0.125 | 78.255± 0.122 | 93.591± 0.011 | 89.346± 0.035 | 95.637± 0.110 | 97.515± 0.094 | ||
IC50 | >400 | 193.301 ±0.245 | 121.816 ±0.103 | 147.478 ±0.056 | 93.513± 0.282 | 77.672± 0.076 | 78.571± 0.120 | 57.159± 0.184 | 50.468± 0.152 | ||
IC25 | 130.490 ±0.041 | 71.873 ±0.245 | 56.048 ±0.103 | 48.857 ±0.056 | 31.541± 0.282 | 31.103± 0.076 | 29.803± 0.120 | 20.712± 0.184 | 18.863± 0.152 |
表4 淫羊藿生物碱对人宫颈癌HeLa细胞增殖的抑制作用(平均值±标准误)
Table 4 Inhibitory effect of Epimedium brevicornum alkaloids on human cervical cancer HeLa cells (means±SD)
Concentration (μg∙mL-1) | Inhibition rate (%) | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Epimedium brevicornum alkaloids | Magnoflorine | Vinblastin sulfate | |||||||||
24 h | 48 h | 72 h | 24 h | 48 h | 72 h | 24 h | 48 h | 72 h | |||
0 | 0.000± 0.024 | 0.000± 0.140 | 0.000± 0.048 | 0.000± 0.015 | 0.000± 0.242 | 0.000± 0.035 | 0.000± 0.534 | 0.000± 0.101 | 0.000± 0.111 | ||
20 | 1.354± 0.048 | 1.239± 0.109 | 4.435± 0.077 | 6.150± 0.005 | 13.541± 0.504 | 15.408± 0.133 | 19.142± 0.075 | 23.967± 0.288 | 28.283± 0.316 | ||
50 | 7.472± 0.024 | 9.010± 0.298 | 11.156± 0.128 | 23.583± 0.071 | 31.737± 0.440 | 32.191± 0.105 | 30.362± 0.007 | 43.853± 0.273 | 42.140± 0.193 | ||
100 | 18.811± 0.025 | 27.799± 0.454 | 30.129± 0.162 | 42.173± 0.049 | 57.217± 0.426 | 59.882± 0.162 | 56.205± 0.102 | 67.590± 0.322 | 71.598± 0.177 | ||
200 | 28.827± 0.036 | 42.880± 0.283 | 67.410± 0.079 | 54.315± 0.094 | 72.024± 0.058 | 75.961± 0.061 | 77.961± 0.044 | 82.618± 0.061 | 91.609± 0.074 | ||
300 | 35.134± 0.084 | 67.263± 0.372 | 82.682± 0.208 | 67.496± 0.032 | 76.609± 0.181 | 88.300± 0.024 | 86.866± 0.045 | 90.539± 0.133 | 95.317± 0.100 | ||
400 | 47.724± 0.048 | 74.015± 0.058 | 94.238± 0.016 | 73.354± 0.125 | 78.255± 0.122 | 93.591± 0.011 | 89.346± 0.035 | 95.637± 0.110 | 97.515± 0.094 | ||
IC50 | >400 | 193.301 ±0.245 | 121.816 ±0.103 | 147.478 ±0.056 | 93.513± 0.282 | 77.672± 0.076 | 78.571± 0.120 | 57.159± 0.184 | 50.468± 0.152 | ||
IC25 | 130.490 ±0.041 | 71.873 ±0.245 | 56.048 ±0.103 | 48.857 ±0.056 | 31.541± 0.282 | 31.103± 0.076 | 29.803± 0.120 | 20.712± 0.184 | 18.863± 0.152 |
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