High-efficiency separation and analysis of monosaccharides in Pueraria polysaccharides by pressurized capillary electrochromatography

doi:10.3724/SP.J.1123.2020.01005

Abstract

Abstract:

Pueraria polysaccharides have been proven to possess biological activities such as bacteriostasis, anti-oxidative, anti-tumor, and immunity boosting activities. The variation in the structure, composition, and amount of monosaccharides in these polysaccharides may lead to different spatial structures and biological activities. Therefore, extraction of Pueraria polysaccharides and determination of the monosaccharide composition are of great significance for activity analysis and quality control. Direct detection of saccharides is difficult because they are strongly polar and do not show absorption in the ultraviolet region. At present, the commonly used methods for saccharide detection are liquid chromatography-ultraviolet detection, gas chromatography-ultraviolet detection, and mass spectrometry. Pressurized capillary electrochromatography is a high-efficiency microseparation technology. In this study, two kinds of Pueraria polysaccharides were extracted by an ultrasonic-assisted method, and response surface methodology was performed to explore the conditions for ultrasonic-assisted extraction of polysaccharides from Pueraria. The interaction effects of four factors, the liquid-solid ratio, ultrasonic extraction time, ultrasonic extraction temperature, and ultrasonic power, on the extraction rate of the polysaccharides were analyzed. By combining the optimal conditions predicted by the software and the actual equipment conditions, the optimal extraction conditions for Pueraria polysaccharides were chosen as follows:ultrasonic extraction temperature, 90℃; liquid-solid ratio of Pueraria thomsonii Benth , 20 mL/g; liquid-solid ratio of Pueraria lobata Ohwi, 40 mL/g; ultrasonic extraction time, 30 min; ultrasonic power, 180 W. Through data fitting, the multiple quadratic regression equation of the four factors on the extraction rate of Pueraria polysaccharides was established. A novel method based on pressurized capillary electrochromatography for the separation and analysis of eight neutral monosaccharides has been established. The monosaccharides were derivatized by the 1-phenyl-3-methyl-5-pyrazolone pre-column derivatization method. The separation conditions for these monosaccharides were explored, and the buffer concentration, buffer pH, applied voltage, type of chromatographic column, and mobile phase ratio were optimized. Finally, the established pressurized capillary electrochromatography-ultraviolet detection method was applied to the detection and identification of two kinds of actual Pueraria polysaccharide samples. The results of response surface analysis showed that among the four experimental factors, ultrasonic extraction temperature had the greatest influence on the extraction rate of polysaccharides from the two kinds of Pueraria, followed by the liquid-solid ratio; the influence of the ultrasonic extraction time and ultrasonic power was relatively weak. The experimental conditions were determined as follows:the separation of eight neutral monosaccharide derivatives could be realized within 24 min on a Halo-2.7 μm core-shell C18 capillary column with acetonitrile-50 mmol/L ammonium acetate aqueous solution (18:82, v/v, pH 4.1) as the mobile phase, by detection at 250 nm under an applied voltage of-20 kV. The separation and detection speeds and the column efficiency achieved with this method were much better than those obtained with the traditional liquid chromatography method. The results show that the proposed method has a good linear relationship and good repeatability. The separation and identification results for the actual samples showed that the polysaccharides of Pueraria thomsonii Benth were mainly composed of glucose, mannose, rhamnose, and fucose in the molar ratio 1.00:0.16:0.14:0.07. The polysaccharides of Pueraria lobata Ohwi were mainly composed of glucose and mannose in the molar ratio 1.00:0.70. This study provides a novel method for the rapid and efficient separation and detection of neutral monosaccharides, and serves as a reference for analyzing the monosaccharide composition of Pueraria polysaccharides.

Key words: ultrasonic assistant extraction, pressurized capillary electrochromatography (pCEC), monosaccharide composition, Pueraria polysaccharides, response surface analysis

Xinyao XU, Soumia CHEDDAH, Yan WANG, Chao YAN. High-efficiency separation and analysis of monosaccharides in Pueraria polysaccharides by pressurized capillary electrochromatography[J]. Chinese Journal of Chromatography, 2020, 38(11): 1323-1331.

Figures/Tables 7

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Monosaccharide	Linear equation	r ²	RSD/% (retention time)	RSD/% (peak area)	LOD/ (mg/L, S/N =3)	LOQ/ (mg/L, S/N =10)
y : peak area; x : mass concentration, g/L.
Man	y =139.27x -5.00	0.9935	1.2	2.5	0.126	0.416
Rib	y =217.53x -11.54	0.9905	1.3	2.1	0.200	0.660
Rha	y =120.98x -7.41	0.9915	1.4	3.6	0.250	0.826
Glc	y =91.145x -3.61	0.9944	1.8	4.0	0.200	0.660
Gal	y =152.65x -6.22	0.9963	1.7	2.9	0.126	0.416
Xyl	y =185.43x -7.35	0.9918	1.2	3.3	0.150	0.496
Ara	y =219.69x -11.88	0.9915	1.2	1.7	0.200	0.660
Fuc	y =116.41x -6.11	0.9950	1.2	1.9	0.250	0.826

Monosaccharide	Linear equation	r ²	RSD/% (retention time)	RSD/% (peak area)	LOD/ (mg/L, S/N =3)	LOQ/ (mg/L, S/N =10)
y : peak area; x : mass concentration, g/L.
Man	y =139.27x -5.00	0.9935	1.2	2.5	0.126	0.416
Rib	y =217.53x -11.54	0.9905	1.3	2.1	0.200	0.660
Rha	y =120.98x -7.41	0.9915	1.4	3.6	0.250	0.826
Glc	y =91.145x -3.61	0.9944	1.8	4.0	0.200	0.660
Gal	y =152.65x -6.22	0.9963	1.7	2.9	0.126	0.416
Xyl	y =185.43x -7.35	0.9918	1.2	3.3	0.150	0.496
Ara	y =219.69x -11.88	0.9915	1.2	1.7	0.200	0.660
Fuc	y =116.41x -6.11	0.9950	1.2	1.9	0.250	0.826

Sample	Source	Sum of squares	df	Mean square	F -value	p -value (Prob＞F)	Significance
* significant at p -value＜0. 05; * * extremely significant at p -value＜0. 01; not significant: p -value＞0. 10.
Pueraria thomsonii	Model	53.97	14	3.85	30.95	＜0.0001	significant
Benth	A-liquid-solid ratio	1.58	1	1.58	12.72	0.0051	**
	B-time	0.00	1	0.00	0.00	0.9497
	C-temperature	27.71	1	27.71	222.51	＜0.0001	**
	D-power	0.65	1	0.65	5.22	0.0454	*
	AB	0.00	1	0.00	0.03	0.8702
	AC	0.00	1	0.00	0.01	0.9230
	AD	0.04	1	0.04	0.34	0.5743
	BC	3.75	1	3.75	30.09	0.0003	**
	BD	2.68	1	2.68	21.55	0.0009	**
	CD	0.17	1	0.17	1.35	0.2723
	A²	0.61	1	0.61	4.94	0.0505
	B²	3.50	1	3.50	28.09	0.0003	**
	C²	0.18	1	0.18	1.44	0.2582
	D²	6.54	1	6.54	52.50	＜0.0001	**
	Residual	1.25	10	0.12	-	-
	Lack of fit	1.12	6	0.19	5.77	0.0559	not significant
	Pure error	0.13	4	0.03	-	-
	Cor total	55.21	24	-	-	-
Pueraria lobata Ohwi	Model	16.14	14	1.15	10.59	0.0003	significant
	A-liquid-solid ratio	0.58	1	0.58	5.36	0.0431	^*
	B-time	0.03	1	0.03	0.24	0.6322
	C-temperature	8.52	1	8.52	78.29	＜0.0001	**
	D-power	0.27	1	0.27	2.44	0.1491
	AB	0.00	1	0.00	0.02	0.896
	AC	0.04	1	0.04	0.39	0.5482
	AD	0.01	1	0.01	0.07	0.8034
	BC	0.26	1	0.26	2.35	0.1561
	BD	0.48	1	0.48	4.43	0.0615
	CD	0.01	1	0.01	0.08	0.7792
	A²	0.00	1	0.00	0.00	0.9875
	B²	1.04	1	1.04	9.52	0.0115	*
	C²	0.19	1	0.19	1.76	0.2137
	D²	1.70	1	1.70	15.63	0.0027	**
	Residual	1.09	10	0.11	-	-
	Lack of fit	0.95	6	0.16	4.57	0.0815	not significant
	Pure error	0.14	4	0.04	-	-
	Cor total	17.23	24	-	-	-

Sample	Source	Sum of squares	df	Mean square	F -value	p -value (Prob＞F)	Significance
* significant at p -value＜0. 05; * * extremely significant at p -value＜0. 01; not significant: p -value＞0. 10.
Pueraria thomsonii	Model	53.97	14	3.85	30.95	＜0.0001	significant
Benth	A-liquid-solid ratio	1.58	1	1.58	12.72	0.0051	**
	B-time	0.00	1	0.00	0.00	0.9497
	C-temperature	27.71	1	27.71	222.51	＜0.0001	**
	D-power	0.65	1	0.65	5.22	0.0454	*
	AB	0.00	1	0.00	0.03	0.8702
	AC	0.00	1	0.00	0.01	0.9230
	AD	0.04	1	0.04	0.34	0.5743
	BC	3.75	1	3.75	30.09	0.0003	**
	BD	2.68	1	2.68	21.55	0.0009	**
	CD	0.17	1	0.17	1.35	0.2723
	A²	0.61	1	0.61	4.94	0.0505
	B²	3.50	1	3.50	28.09	0.0003	**
	C²	0.18	1	0.18	1.44	0.2582
	D²	6.54	1	6.54	52.50	＜0.0001	**
	Residual	1.25	10	0.12	-	-
	Lack of fit	1.12	6	0.19	5.77	0.0559	not significant
	Pure error	0.13	4	0.03	-	-
	Cor total	55.21	24	-	-	-
Pueraria lobata Ohwi	Model	16.14	14	1.15	10.59	0.0003	significant
	A-liquid-solid ratio	0.58	1	0.58	5.36	0.0431	^*
	B-time	0.03	1	0.03	0.24	0.6322
	C-temperature	8.52	1	8.52	78.29	＜0.0001	**
	D-power	0.27	1	0.27	2.44	0.1491
	AB	0.00	1	0.00	0.02	0.896
	AC	0.04	1	0.04	0.39	0.5482
	AD	0.01	1	0.01	0.07	0.8034
	BC	0.26	1	0.26	2.35	0.1561
	BD	0.48	1	0.48	4.43	0.0615
	CD	0.01	1	0.01	0.08	0.7792
	A²	0.00	1	0.00	0.00	0.9875
	B²	1.04	1	1.04	9.52	0.0115	*
	C²	0.19	1	0.19	1.76	0.2137
	D²	1.70	1	1.70	15.63	0.0027	**
	Residual	1.09	10	0.11	-	-
	Lack of fit	0.95	6	0.16	4.57	0.0815	not significant
	Pure error	0.14	4	0.04	-	-
	Cor total	17.23	24	-	-	-

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