Ultrasound-microwave-assisted Extraction of Alkaloids from Epimedium brevicornum and Their Inhibitory Effect on HeLa Cells

doi:10.11983/CBB17103

Abstract

Abstract: A protocol of ultrasound-microwave-assisted extraction was developed to isolate alkaloids from the medicinal plant Epimedium brevicornum, and the extraction mechanism is discussed. The chemical composition of the alkaloid extract was analyzed. The inhibitory effect of E. brevicornum alkaloids on human cervical cancer HeLa cells was investigated. The optimal condition for ultrasound-microwave-assisted extraction of E. brevicornum alkaloids was achieved by using an orthogonal test: steeped time, 40 min; ultrasound-microwave time, 18 min; microwave power, 250 J∙s^-1; liquid-to-solid ratio, 30 mL∙g^-1; and ethanol concentration, 70%. Extraction yield of alkaloids by ultrasound- microwave-assisted extraction reached 16.146 mg∙g^-1, which was significantly higher than that by ultrasound-assisted extraction, microwave-assisted extraction and heating extraction. Scanning electron micrographs showed that the synergism between the ultrasound and microwave resulted in many interstices on the surface of leaf samples of E. brevicornum. Laser diffraction particle sizing analysis revealed that the amount of small and medium particles of leaf samples increased and that of large particles decreased after ultrasound-microwave-assisted extraction. Thin-layer chromatography and high-performance liquid chromatography revealed that the alkaloid extract contained magnoflorine, and berberine was not detectable. E. brevicornum alkaloids exhibited a significant dose-dependent inhibitory activity against human cervical cancer HeLa cells. The present research may be helpful for the exploitation and utilization of Epimedium resources.

Key words: Epimedium brevicornum, alkaloids, ultrasound-microwave-assisted extraction, mechanism, HeLa cells

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[J]. Chinese Bulletin of Botany, 2018, 53(3): 341-352.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB17103

https://www.chinbullbotany.com/EN/Y2018/V53/I3/341

Figures/Tables 8

References 33

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Test No.	Factor						Mean extraction yield (mg∙g^-1)
Test No.	(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 (%)	Mean extraction yield (mg∙g^-1)
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

Test No.	Factor						Mean extraction yield (mg∙g^-1)
Test No.	(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 (%)	Mean extraction yield (mg∙g^-1)
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

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 (%)
Method	Replicate 1	Replicate 2	Replicate 3	Mean	RSD (%)
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