药学学报

Sample name	M_n/kDa	M_p/kDa	M_w/kDa	M_z/kDa	M_w/M_n
Green Forsythiae Fructus polysaccharides	7.844 ± 0.669 18	6.260 ± 0.943 59	13.036 ± 0.418 59	22.932 ± 0.736 77	1.662 ± 0.789 31
Ripe Forsythiae Fructus polysaccharides	10.833 ± 0.403 26	10.161 ± 0.998 14	11.990 ± 0.368 43	13.778 ± 0.789 25	1.107 ± 0.546 22

Sample name	M_n/kDa	M_p/kDa	M_w/kDa	M_z/kDa	M_w/M_n
Green Forsythiae Fructus polysaccharides	7.844 ± 0.669 18	6.260 ± 0.943 59	13.036 ± 0.418 59	22.932 ± 0.736 77	1.662 ± 0.789 31
Ripe Forsythiae Fructus polysaccharides	10.833 ± 0.403 26	10.161 ± 0.998 14	11.990 ± 0.368 43	13.778 ± 0.789 25	1.107 ± 0.546 22

Type of polysaccharide	Type of monosaccharide	Molar ratio	Content /μg·mL^-1
Green Forsythiae	Fuc	1.02%	3.635 0
Fructus	Rha	10.80%	35.310 1
polysaccharides	Ara	10.65%	38.081 0
	Gal	9.21%	36.129 6
	Glc	14.31%	56.135 9
	Xyl	1.84%	6.005 2
	Man	3.91%	15.352 3
	Gal-UA	47.15%	199.291 0
	Glc-UA	1.11%	4.670 9
Ripe Forsythiae	Fuc	0.97%	3.089 0
Fructus	Rha	7.90%	23.072 2
polysaccharides	Ara	10.95%	34.943 5
	Gal	5.59%	19.583 0
	Glc	14.37%	50.356 1
	Xyl	1.81%	5.274 6
	Man	2.64%	9.263 5
	Gal-UA	54.79%	206.762 2
	Glc-UA	1.01%	3.801 6

Type of polysaccharide	Type of monosaccharide	Molar ratio	Content /μg·mL^-1
Green Forsythiae	Fuc	1.02%	3.635 0
Fructus	Rha	10.80%	35.310 1
polysaccharides	Ara	10.65%	38.081 0
	Gal	9.21%	36.129 6
	Glc	14.31%	56.135 9
	Xyl	1.84%	6.005 2
	Man	3.91%	15.352 3
	Gal-UA	47.15%	199.291 0
	Glc-UA	1.11%	4.670 9
Ripe Forsythiae	Fuc	0.97%	3.089 0
Fructus	Rha	7.90%	23.072 2
polysaccharides	Ara	10.95%	34.943 5
	Gal	5.59%	19.583 0
	Glc	14.37%	50.356 1
	Xyl	1.81%	5.274 6
	Man	2.64%	9.263 5
	Gal-UA	54.79%	206.762 2
	Glc-UA	1.01%	3.801 6

Type of linkage	PMAAs	t_R/min	m/z	Molar ratio/%
t-Rha(p)	1, 5-di-O-Acetyl-6-deoxy-2, 3, 4-tri-O-methyl rhamnitol	4.706	59, 72, 89, 102, 115, 118, 131, 145, 162, 175	4.75
t-Ara(f)	1, 4-di-O-Acetyl-2, 3, 5-tri-O-methyl arabinitol	4.975	71, 87, 102, 118, 129, 145, 161	2.20
t-Xyl(p)	1, 5-di-O-Acetyl-2, 3, 4-tri-O-methyl xylitol	5.818	88, 101, 102, 118, 119, 161, 162	1.12
2-Rha(p)	1, 2, 5-tri-O-Acetyl-6-deoxy-3, 4-di-O-methyl rhamnitol	7.379	89, 100, 115, 130, 131, 175, 190	1.21
t-Glc(p)	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl glucitol	7.487	87, 102, 118, 129, 145, 161, 162, 205	4.90
3-Ara(f)	1, 3, 4-tri-O-Acetyl-2, 5-di-O-methyl arabinitol	8.032	87, 99, 113, 118, 129, 201, 233	1.08
t-Gal(p)-UA	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol	8.391	73, 89, 102, 118, 147, 162, 163, 207	10.10
t-Gal(p)	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol	8.392	87, 102, 118, 129, 145, 161, 162, 205	1.22
5-Ara(f)	1, 4, 5-tri-O-Acetyl-2, 3-di-O-methyl arabinitol	9.109	87, 102, 118, 129, 162, 189	2.59
3, 4-Rha(p)	1, 3, 4, 5-tetra-O-Acetyl-6-deoxy-2-O-methyl rhamnitol	9.909	87, 99, 113, 118, 129, 149, 173, 275	1.27
4-Man(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl mannitol	11.208	87, 102, 113, 118, 129, 162, 233	1.06
4-Gal(p)-UA	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol	12.002	87, 99, 102, 115, 118, 131, 162, 175, 235	41.06
4-Gal(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol	12.037	87, 102, 113, 118, 129, 162, 233	9.44
4-Glc(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl glucitol	12.303	87, 102, 113, 118, 129, 162, 233	8.54
6-Gal(p)	1, 5, 6-tri-O-Acetyl-2, 3, 4-tri-O-methyl galactitol	13.627	87, 99, 102, 118, 129, 162, 189, 233	1.14
3, 4-Gal(p)-UA	1, 3, 4, 5-tetra-O-Acetyl-2, 6-di-O-methyl galactitol	13.842	87, 118, 131, 143, 185, 205, 307	3.00
2, 4-Glc(p)	1, 2, 4, 5-tetra-O-Acetyl-3, 6-di-O-methyl glucitol	15.020	87, 88, 99, 113, 130, 190, 233	3.46
4, 6-Glc(p)	1, 4, 5, 6-tetra-O-Acetyl-2, 3-di-O-methyl glucitol	16.782	85, 102, 118, 127, 159, 162, 201, 261	1.89

Type of linkage	PMAAs	t_R/min	m/z	Molar ratio/%
t-Rha(p)	1, 5-di-O-Acetyl-6-deoxy-2, 3, 4-tri-O-methyl rhamnitol	4.706	59, 72, 89, 102, 115, 118, 131, 145, 162, 175	4.75
t-Ara(f)	1, 4-di-O-Acetyl-2, 3, 5-tri-O-methyl arabinitol	4.975	71, 87, 102, 118, 129, 145, 161	2.20
t-Xyl(p)	1, 5-di-O-Acetyl-2, 3, 4-tri-O-methyl xylitol	5.818	88, 101, 102, 118, 119, 161, 162	1.12
2-Rha(p)	1, 2, 5-tri-O-Acetyl-6-deoxy-3, 4-di-O-methyl rhamnitol	7.379	89, 100, 115, 130, 131, 175, 190	1.21
t-Glc(p)	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl glucitol	7.487	87, 102, 118, 129, 145, 161, 162, 205	4.90
3-Ara(f)	1, 3, 4-tri-O-Acetyl-2, 5-di-O-methyl arabinitol	8.032	87, 99, 113, 118, 129, 201, 233	1.08
t-Gal(p)-UA	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol	8.391	73, 89, 102, 118, 147, 162, 163, 207	10.10
t-Gal(p)	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol	8.392	87, 102, 118, 129, 145, 161, 162, 205	1.22
5-Ara(f)	1, 4, 5-tri-O-Acetyl-2, 3-di-O-methyl arabinitol	9.109	87, 102, 118, 129, 162, 189	2.59
3, 4-Rha(p)	1, 3, 4, 5-tetra-O-Acetyl-6-deoxy-2-O-methyl rhamnitol	9.909	87, 99, 113, 118, 129, 149, 173, 275	1.27
4-Man(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl mannitol	11.208	87, 102, 113, 118, 129, 162, 233	1.06
4-Gal(p)-UA	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol	12.002	87, 99, 102, 115, 118, 131, 162, 175, 235	41.06
4-Gal(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol	12.037	87, 102, 113, 118, 129, 162, 233	9.44
4-Glc(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl glucitol	12.303	87, 102, 113, 118, 129, 162, 233	8.54
6-Gal(p)	1, 5, 6-tri-O-Acetyl-2, 3, 4-tri-O-methyl galactitol	13.627	87, 99, 102, 118, 129, 162, 189, 233	1.14
3, 4-Gal(p)-UA	1, 3, 4, 5-tetra-O-Acetyl-2, 6-di-O-methyl galactitol	13.842	87, 118, 131, 143, 185, 205, 307	3.00
2, 4-Glc(p)	1, 2, 4, 5-tetra-O-Acetyl-3, 6-di-O-methyl glucitol	15.020	87, 88, 99, 113, 130, 190, 233	3.46
4, 6-Glc(p)	1, 4, 5, 6-tetra-O-Acetyl-2, 3-di-O-methyl glucitol	16.782	85, 102, 118, 127, 159, 162, 201, 261	1.89

Type of linkage	PMAAs	t_R/min	m/z	Molar ratio/%
t-Rha(p)	1, 5-di-O-Acetyl-6-deoxy-2, 3, 4-tri-O-methyl rhamnitol	4.698	59, 72, 89, 102, 115, 118, 131, 145, 162, 175	4.69
t-Ara(f)	1, 4-di-O-Acetyl-2, 3, 5-tri-O-methyl arabinitol	4.971	71, 87, 102, 118, 129, 145, 161	1.44
t-Ara(p)	1, 5-di-O-Acetyl-2, 3, 4-tri-O-methyl arabinitol	6.130	88, 101, 102, 118, 119, 161, 162	1.08
t-Glc(p)	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl glucitol	7.483	87, 102, 118, 129, 145, 161, 162, 205	6.20
t-Gal(p)-UA	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol	8.384	73, 89, 102, 118, 147, 162, 163, 207	12.09
t-Gal(p)	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol	8.387	87, 102, 118, 129, 145, 161, 162, 205	3.70
2, 3-Rha(p)	1, 2, 3, 5-tetra-O-Acetyl-6-deoxy-4-O-methyl rhamnitol	9.733	71, 89, 101, 131, 262	1.07
4-Man(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl mannitol	11.197	87, 102, 113, 118, 129, 162, 233	1.26
4-Gal(p)-UA	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol	12.005	87, 99, 102, 115, 118, 131, 162, 175, 235	48.75
4-Gal(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol	12.037	87, 102, 113, 118, 129, 162, 233	7.59
4-Glc(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl glucitol	12.310	87, 102, 113, 118, 129, 162, 233	7.46
3, 4-Gal(p)-UA	1, 3, 4, 5-tetra-O-Acetyl-2, 6-di-O-methyl galactitol	13.839	87, 118, 129, 143, 185, 203, 305	1.93
2, 4-Glc(p)	1, 2, 4, 5-tetra-O-Acetyl-3, 6-di-O-methyl glucitol	15.016	87, 88, 99, 113, 130, 190, 233	2.73

Type of linkage	PMAAs	t_R/min	m/z	Molar ratio/%
t-Rha(p)	1, 5-di-O-Acetyl-6-deoxy-2, 3, 4-tri-O-methyl rhamnitol	4.698	59, 72, 89, 102, 115, 118, 131, 145, 162, 175	4.69
t-Ara(f)	1, 4-di-O-Acetyl-2, 3, 5-tri-O-methyl arabinitol	4.971	71, 87, 102, 118, 129, 145, 161	1.44
t-Ara(p)	1, 5-di-O-Acetyl-2, 3, 4-tri-O-methyl arabinitol	6.130	88, 101, 102, 118, 119, 161, 162	1.08
t-Glc(p)	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl glucitol	7.483	87, 102, 118, 129, 145, 161, 162, 205	6.20
t-Gal(p)-UA	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol	8.384	73, 89, 102, 118, 147, 162, 163, 207	12.09
t-Gal(p)	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol	8.387	87, 102, 118, 129, 145, 161, 162, 205	3.70
2, 3-Rha(p)	1, 2, 3, 5-tetra-O-Acetyl-6-deoxy-4-O-methyl rhamnitol	9.733	71, 89, 101, 131, 262	1.07
4-Man(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl mannitol	11.197	87, 102, 113, 118, 129, 162, 233	1.26
4-Gal(p)-UA	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol	12.005	87, 99, 102, 115, 118, 131, 162, 175, 235	48.75
4-Gal(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol	12.037	87, 102, 113, 118, 129, 162, 233	7.59
4-Glc(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl glucitol	12.310	87, 102, 113, 118, 129, 162, 233	7.46
3, 4-Gal(p)-UA	1, 3, 4, 5-tetra-O-Acetyl-2, 6-di-O-methyl galactitol	13.839	87, 118, 129, 143, 185, 203, 305	1.93
2, 4-Glc(p)	1, 2, 4, 5-tetra-O-Acetyl-3, 6-di-O-methyl glucitol	15.016	87, 88, 99, 113, 130, 190, 233	2.73

No.	Metabolite	t_R/min	Formula	m/z	VIP	Model vs control	Fc	Treat vs model	Fc
1	Aspartate	1.474	C₄H₇NO₄	101.047 93	1.107 13	↑	1.570 184	↓	0.709 129
2	Choline	1.321	C₅H₁₄NO	102.115 93	1.359 75	↑	30.879 62	↓	0.325 997
3	Fumarate	1.749	C₄H₄O₄	116.011 03	2.129 89	↓	0.597 923	↑	1.417 095
4	L-Serine	1.418	C₃H₇NO₃	117.054 08	3.077 65	↓	0.432 520	↑	1.434 768
5	Succinate	3.916	C₄H₆O₄	118.026 72	2.632 80	↓	0.219 386	↑	2.587 650
6	L-Glutamate	1.845	C₅H₉NO₄	131.058 09	1.842 49	↓	0.561 708	↑	1.703 735
7	L-Asparagine	1.615	C₄H₈N₂O₃	132.052 30	4.309 88	↑	1.606 690	↓	0.657 017
8	Ornithine	1.402	C₅H₁₂N₂O₂	132.089 98	1.860 81	↑	4.409 108	↓	0.691 799
9	O-Acetylcholine	2.783	C₇H₁₆NO₂	144.126 36	4.465 79	↑	81.396 74	↓	0.562 737
10	L-Glutamine	2.358	C₅H₁₀N₂O₃	145.085 15	1.038 68	↓	0.639 575	↑	1.337 610
11	2-Oxoglutarate	2.062	C₅H₆O₅	146.021 70	1.899 38	↑	5.922 962	↓	0.451 885
12	1, 2-Diacyl-sn-glycerol	3.118	C₅H₆O₅R₂	172.084 91	1.659 93	↓	0.213 788	↑	2.958 079
13	Arginine	1.360	C₆H₁₄N₄O₂	174.111 80	5.694 11	↓	0.532 306	↑	1.422 239
14	Phosphatidylcholine	7.105	C₁₀H₁₈NO₈PR₂	254.126 58	2.746 34	↓	0.330 396	↑	2.227 042
15	N-Acetyl-L-aspartyl-L-glutamate	3.388	C₁₁H₁₆N₂O₈	268.106 06	1.383 01	↓	0.153 683	↑	4.795 126
16	LysW-glutamate	2.712	C₁₀H₁₅N₂O₇R	289.073 18	1.737 82	↓	0.640 030	↑	1.562 426
17	1-Acyl-sn-glycero-3-phosphocholine	2.189	C₉H₂₀NO₇PR	299.113 56	1.707 83	↑	2.405 316	↓	0.517 648
18	CDP-choline	1.499	C₁₄H₂₆N₄O₁₁P₂	488.107 50	1.968 60	↓	0.490 872	↑	1.566 374

No.	Metabolite	t_R/min	Formula	m/z	VIP	Model vs control	Fc	Treat vs model	Fc
1	Aspartate	1.474	C₄H₇NO₄	101.047 93	1.107 13	↑	1.570 184	↓	0.709 129
2	Choline	1.321	C₅H₁₄NO	102.115 93	1.359 75	↑	30.879 62	↓	0.325 997
3	Fumarate	1.749	C₄H₄O₄	116.011 03	2.129 89	↓	0.597 923	↑	1.417 095
4	L-Serine	1.418	C₃H₇NO₃	117.054 08	3.077 65	↓	0.432 520	↑	1.434 768
5	Succinate	3.916	C₄H₆O₄	118.026 72	2.632 80	↓	0.219 386	↑	2.587 650
6	L-Glutamate	1.845	C₅H₉NO₄	131.058 09	1.842 49	↓	0.561 708	↑	1.703 735
7	L-Asparagine	1.615	C₄H₈N₂O₃	132.052 30	4.309 88	↑	1.606 690	↓	0.657 017
8	Ornithine	1.402	C₅H₁₂N₂O₂	132.089 98	1.860 81	↑	4.409 108	↓	0.691 799
9	O-Acetylcholine	2.783	C₇H₁₆NO₂	144.126 36	4.465 79	↑	81.396 74	↓	0.562 737
10	L-Glutamine	2.358	C₅H₁₀N₂O₃	145.085 15	1.038 68	↓	0.639 575	↑	1.337 610
11	2-Oxoglutarate	2.062	C₅H₆O₅	146.021 70	1.899 38	↑	5.922 962	↓	0.451 885
12	1, 2-Diacyl-sn-glycerol	3.118	C₅H₆O₅R₂	172.084 91	1.659 93	↓	0.213 788	↑	2.958 079
13	Arginine	1.360	C₆H₁₄N₄O₂	174.111 80	5.694 11	↓	0.532 306	↑	1.422 239
14	Phosphatidylcholine	7.105	C₁₀H₁₈NO₈PR₂	254.126 58	2.746 34	↓	0.330 396	↑	2.227 042
15	N-Acetyl-L-aspartyl-L-glutamate	3.388	C₁₁H₁₆N₂O₈	268.106 06	1.383 01	↓	0.153 683	↑	4.795 126
16	LysW-glutamate	2.712	C₁₀H₁₅N₂O₇R	289.073 18	1.737 82	↓	0.640 030	↑	1.562 426
17	1-Acyl-sn-glycero-3-phosphocholine	2.189	C₉H₂₀NO₇PR	299.113 56	1.707 83	↑	2.405 316	↓	0.517 648
18	CDP-choline	1.499	C₁₄H₂₆N₄O₁₁P₂	488.107 50	1.968 60	↓	0.490 872	↑	1.566 374

青翘多糖和老翘多糖结构分析、抗炎活性及其机制研究

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赵小艳 ¹ , 郝志强 ²^,³^,⁴ , 武雅梅 ²^,³^,⁴ , 李科 ²^,³^,⁴^,^* , 李震宇 ²^,³^,⁴

药学学报 | 研究论文 2025,60(7): 2292-2302

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药学学报 |研究论文 2025 , 60 (7) : 2292 -2302

青翘多糖和老翘多糖结构分析、抗炎活性及其机制研究

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赵小艳¹, 郝志强²^,³^,⁴, 武雅梅²^,³^,⁴, 李科²^,³^,⁴^,^*, 李震宇²^,³^,⁴

作者信息

¹山西药科职业学院制药系, 山西太原 030031

²山西大学中医药现代研究中心, 山西太原 030006

³山西大学化学生物学与分子工程教育部重点实验室, 山西太原 030006

⁴地产中药功效物质研究与利用山西省重点实验室, 山西太原 030006

通讯作者:

李科, Tel/Fax: 86-351-7019297, E-mail: like@sxu.edu.cn

作者简介:

作者贡献:

赵小艳负责文中实验方案设计、实验实施、数据收集、结果分析、文章撰写等工作; 郝志强和武雅梅负责图形、文章修改等工作; 李科负责提供文章思路, 在文章写作方面进行指导和修改; 李震宇对整个实验内容给予监督, 并对文章进行修改完善。

Study on structure analysis, anti-inflammatory activity and mechanism of green Forsythiae Fructus polysaccharide and ripe Forsythiae Fructus polysaccharide

Xiao-yan ZHAO¹, Zhi-qiang HAO²^,³^,⁴, Ya-mei WU²^,³^,⁴, Ke LI²^,³^,⁴^,^*, Zhen-yu LI²^,³^,⁴

Affiliations

¹Shanxi Pharmaceutical Vocational College, Taiyuan 030031, China

²Modern Research Center of Traditional Chinese Medicine, Shanxi University, Taiyuan 030006, China

³Key Laboratory of Chemical Biology and Molecular Engineering, Ministry of Education, Shanxi University, Taiyuan 030006, China

⁴Shanxi Provincial Key Laboratory of Research and Utilization of Functional Substances in Traditional Chinese Medicine, Taiyuan 030006, China

出版时间: 2025-07-12 doi: 10.16438/j.0513-4870.2024-1209

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摘要

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连翘是大宗药材之一, 目前对于连翘的活性成分研究主要集中于连翘苷、连翘脂苷、连翘脂素等方面。而多糖越来越显现出其重要的药理功能, 这可能与连翘清热解毒、疏风散热的功效相关, 因此开展青翘多糖和老翘多糖的研究具有重要意义。本文通过凝胶色谱对青翘多糖和老翘多糖进行了分离纯化并对比分析了二者的结构, 通过脂多糖(lipopolysaccharide, LPS) 诱导的RAW264.7小鼠巨噬细胞过度炎症模型, 对青翘多糖和老翘多糖的抗炎活性进行了比较, 并通过细胞代谢组学初步探究了抗炎活性较强多糖的作用机制。实验结果发现, 青翘多糖的重均分子量大于老翘多糖, 青翘多糖和老翘多糖单糖组成中占比最多的单糖类型均为半乳糖醛酸(galacturonic acid, Gal-UA), 其次是葡萄糖(glucose, Glc)。甲基化结果表明两种多糖的主要连接方式均为4-Gal(p)-UA; 抗炎活性研究结果表明, 两种多糖均具有抗炎活性, 青翘多糖抗炎活性更强。代谢组学研究发现, 与对照组比较, 青翘多糖活性成分作用于RAW264.7细胞后, 细胞内共发现18种差异代谢物, 主要涉及精氨酸生物合成、丙氨酸代谢等途径, 这些途径与氧化应激、炎症反应密切相关。本文对青翘和老翘药效物质基础之一多糖进行了结构与活性比较, 为连翘资源开发奠定了基础。

关键词

青翘多糖 / 老翘多糖 / 结构表征 / 抗炎活性 / 代谢组学

Abstract

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Forsythia suspensa is one of the great medicinal herbs. At present, research on the active ingredients of Forsythia suspensa mainly focuses on Forsythin, Forsythiaside and Phillygenol, etc. Polysaccharides show increasingly significant pharmacological functions, which may be associated with the efficacy of Forsythia suspensa in clearing heat and removing toxins and dispersing wind and heat. Therefore, it is important to study the green Forsythiae Fructus polysaccharide and ripe Forsythiae Fructus polysaccharide. In this paper, we have separated and purified green Forsythiae Fructus polysaccharides and ripe Forsythia Fructus polysaccharides by gel chromatography and comparatively analysed the structures of the two groups. Comparison of the anti-inflammatory activity of green and ripe Forsythiae Fructus polysaccharides by lipopolysaccharide (LPS)-induced macrophage hyperinflammation model in RAW264.7 mice, the mechanism of action of polysaccharides with powerful anti-inflammatory activity has also been preliminarily investigated using cellular metabolomics. It was determined that the weight average molecular weight of green Forsythiae Fructus polysaccharide was significantly higher than that of ripe Forsythiae Fructus polysaccharide. The type of monosaccharide that accounted for the largest proportion of the monosaccharide composition of both green and ripe Forsythiae Fructus polysaccharides was Gal-UA, followed by Glc methylation results indicate that the major linkage of both polysaccharides is 4-Gal(p)-UA. The results of the anti-inflammatory activity study demonstrated that both polysaccharides exhibited anti-inflammatory activity, with green Forsythiae Fructus polysaccharide displaying a more powerful anti-inflammatory effect. A metabolomic study identified 18 distinct metabolites in RAW264.7 cells following the action of the active ingredient of green Forsythiae Fructus polysaccharide, compared with the control group. These metabolites were primarily involved in arginine biosynthesis, alanine metabolism, and other pathways that are closely associated with oxidative stress and the inflammatory response. This paper compares the structure and activity of polysaccharides, one of the bases for the medicinal effects of green Forsythiae Fructus and ripe Forsythiae Fructus, and provides a solid foundation for further studies on green Forsythiae Fructus and ripe Forsythiae Fructus.

Key words

green Forsythiae Fructus polysaccharide / ripe Forsythiae Fructus polysaccharide / structural characterization / anti-inflammatory activity / metabolomics

引用本文

赵小艳, 郝志强, 武雅梅, 李科, 李震宇. 青翘多糖和老翘多糖结构分析、抗炎活性及其机制研究. 药学学报, 2025 , 60 (7) : 2292 -2302 . DOI: 10.16438/j.0513-4870.2024-1209

Xiao-yan ZHAO, Zhi-qiang HAO, Ya-mei WU, Ke LI, Zhen-yu LI. Study on structure analysis, anti-inflammatory activity and mechanism of green Forsythiae Fructus polysaccharide and ripe Forsythiae Fructus polysaccharide[J]. Acta Pharmaceutica Sinica, 2025 , 60 (7) : 2292 -2302 . DOI: 10.16438/j.0513-4870.2024-1209

正文

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连翘是木犀科植物连翘[Forsythia suspensa (Thunb.) Vahl] 的干燥果实, 被喻为“疮家圣药”, 具有清热解毒、消肿散结、疏散风热的功效^[1]。对连翘本草考证发现, 从明代开始, 根据采收时间不同, 连翘分为青翘和老翘两种规格^[2]。秋季果实初熟尚带绿色时采收, 除去杂质, 蒸熟, 晒干, 习称“青翘”; 果实熟透时采收, 晒干, 除去杂质, 习称“老翘”。在民国和新中国成立初期, 老翘是连翘的主要规格, 经销全国乃至出口。然而随着改革开放和中成药需求和销量的增加, 抢青现象层出不穷, 现在市场上以青翘为主流规格。目前, 越来越多的研究者将连翘区分成青翘和老翘分别进行药理作用研究。通过对青翘和老翘药理作用进行文献调研发现, 青翘清热解毒的效果强于老翘。Sun等^[3]通过动物实验对青翘和老翘的抗炎、抗内毒素、发汗和保肝作用的差异进行研究, 实验结果表明, 青翘水煎液的抗急性炎症效果优于老翘, 青翘体外抗内毒素作用强于老翘, 青翘发汗作用明显, 而老翘保肝作用强于青翘。Zhang等^[4]通过对青翘和老翘的肺纤维化研究证明两者均能减少肺纤维化, 且老翘在减少胶原沉积方面优于青翘。

现代药理研究中, 连翘一般以连翘苷、连翘酯苷A和连翘酯素^{[5, 6]}为主要成分指标进行综合评价。在连翘提取物的制备方法(中国药典2020年版) 中, 多糖作为醇沉后的杂质被处理, 没有加以利用。随着现代药理研究的发展, 发现很多中药多糖具有明确的抗肿瘤、降血糖、抗突变、降血脂、抗病毒、保肝、抗氧化及抗衰老、抗炎等作用^[7]。如Chen等^[8]证明牛蒡子中性多糖可能通过沉默炎性细胞因子信号传导, 抑制Toll样受体4 (Toll-like receptor 4, TLR4)、髓样分化因子88 (myeloid differentiation factor 88, MyD88)、核因子κB (nuclear factor kappa-B, NF-κB) 信号通路的过度激活, 从而抑制炎症反应。Luo等^[9]研究虎杖多糖对RAW264.7细胞活性的影响以及在脂多糖诱导的炎症过程中的作用, 得出虎杖多糖可以减少脂多糖诱导的RAW264.7细胞中的肿瘤坏死因子α (tumor necrosis factor-α, TNF-α) 和白细胞介素-6 (interleukin-6, IL-6) 分泌, 抑制NF-κB信号通路。因此, 对青翘多糖和老翘多糖的抗炎活性进行对比, 并探究其作用机制是阐明连翘药效物质基础的关键问题之一。故本文以青翘和老翘中的多糖为主要研究对象, 对其进行结构鉴定, 确定其主要结构特征; 进行抗炎活性对比, 明确二者活性之间的差异; 进行细胞代谢组学分析, 探究其抗炎作用机制, 以期为进一步阐明连翘药效物质基础指明方向。

材料与方法

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仪器 ESJ30-5A电子天平(沈阳神宇龙腾天平有限公司); XH-T漩涡混合器(金坛市白塔新宝仪器厂); Reacti-thermo氮气吹扫仪、ICS 5000+离子色谱、UltiMate3000高效液相色谱仪、酶标仪Multiskan GO (美国Thermo Fisher Scientific公司); OPTILAB T-rex示差检测器、DAWN HELEOS-Ⅱ激光光散射检测器(美国Wyatt公司); Tissuelyser-48高通量组织破碎仪(上海净信实业发展有限公司); ZLS-1台式快速离心浓缩干燥器(湖南赫西仪器装备有限公司); LGJ-10冷冻干燥机(北京四环福瑞科仪科技发展有限公司); 6890A气相色谱仪、5977B质谱仪(美国Agilent公司); Milli-Q纯水仪(德国Merck公司); DGJ-10C冷冻干燥机(上海博登生物科技有限公司); RE 5298A旋转蒸发仪(上海亚荣生化仪器厂); explorer 100AKTA纯化系统、Sephacryl S-400HR 26mm×1000mm凝胶纯化柱(美国GE公司)。

材料与试剂 青翘、老翘、均采自山西省安泽县, 经山西大学中医药研究中心李科教授鉴定分别为木犀科连翘属植物连翘(Forsythia suspensa) 的干燥根。单糖标准品岩藻糖(Fuc, 批号LRAC0766)、鼠李糖(Rha, 批号LRAC0773)、阿拉伯糖(Ara, 批号LRAC0971)、半乳糖(Gal, 批号LRAC0966)、葡萄糖(Glc, 批号LRAC0967)、木糖(Xyl, 批号LRAC0970)、甘露糖(Man, 批号LRAC0968)、果糖(Fru, 批号LRAC0969)、核糖(Rib, 批号LRAC0972)、半乳糖醛酸(GalUA, 批号SLBR3842V)、葡萄糖醛酸(GlcUA, 批号SLBR3843V)、甘露糖醛酸(ManUA, 批号SLBR3840V)、古罗糖醛酸(GulUA, 批号SLBR3844V), 纯度均大于98%; 二甲基亚砜(DMSO, 批号S3442-250G)、氢氧化钠(色谱纯, 批号S5881-500G)、醋酸钠(分析纯, 批号S2889-250G)、1-环己基-2-吗啉乙基碳二亚胺甲基对甲苯磺酸盐(批号S3556-250G)、咪唑(批号S4669-500G)、硼氘化钠(批号S2866-100G)、乙酸酐(批号S2555-250G) 均购自Sigma公司。三氟乙酸(色谱纯, 批号CAEQ-4-018443-0250)、甲醇(分析纯, 批号CAEQ-4-003302-4000)、无水乙醇(批号CAEQ-4-003301-1000)、二氯甲烷(批号CAEQ-4-003325-2000)、氨水(批号CAEQ-4-002258-4000)、碘甲烷(批号CAEQ-4-014418-4000)、乙酸(批号CAEQ-4-015682-4000) 均购自上海安谱实验科技股份有限公司。硝酸钠(分析纯) 购自国药集团化学试剂有限公司。苯酚(批号240312)、硫酸(批号240411) 购自西陇科学股份有限公司。RAW264.7小鼠巨噬细胞从ATCC获得, MTT (批号M8182)、澳洲胎牛血清(批号11011-8611)、DMEM培养基(批号11995)、0.25%胰蛋白酶(批号T1300)、PBS磷酸盐缓冲液(批号P1020)、脂多糖(批号L8880) 均购自北京索莱宝科技有限公司。一氧化氮(NO, 批号S0025S) 检测试剂盒购自上海碧云天生物技术有限公司; 肿瘤坏死因子-α (TNF-α, 批号LV30537) 和白细胞介素-10 (IL-10, 批号LV30278) 的酶联免疫吸附测定试剂盒均购自武汉博士德生物工程有限公司。

青翘多糖和老翘多糖的提取 按照文献^[10]方法, 将烘干后样品用粉碎机粉碎, 并过60目筛。加入适量无水乙醇, 搅拌萃取脂溶性色素和部分杂质, 6 000 ×g离心10 min, 收集沉淀。向沉淀中加入适量纯水, 60 ℃水浴提取4 h, 6 000 ×g离心10 min, 收集上清提取液。沉淀残渣按照同样的步骤复提一遍。合并两次提取液, 真空旋蒸浓缩至原体积的1/10, 加入四倍体积无水乙醇4 ℃过夜醇沉。8 000 ×g离心10 min收集沉淀固体并冻干, 即得青翘和老翘粗多糖提取物。

青翘多糖和老翘多糖的纯化 根据参考文献^{[11, 12]}方法, 分别向青翘和老翘粗多糖提取物固体中加入纯水, 使粗多糖充分溶解, 加入木瓜蛋白酶, 过夜酶解。向水相中加入1/4体积三氯甲烷和正丁醇, 充分混匀, 收集上层水相。向液体中加入石油醚, 充分混匀, 收集下层水相。向水相中加入大孔树脂AB-8, 充分混匀, 过夜吸附, 收集液体, 3 000 Da透析袋透析48 h除去小分子组分, 多糖溶液冷冻干燥, 得到精制多糖。将其配成一定浓度的多糖母液, 10 000 ×g离心10 min, 取上清液过凝胶层析柱分离纯化, 流速1 mL·min^-1; 采用1.5倍柱体积纯水洗脱, 每10 mL收集一管, 收集所有洗脱液。每隔一管对管中洗脱液的总糖含量采用硫酸-苯酚法进行测定, 绘制凝胶纯化洗脱曲线。合并同一个洗脱峰对应的各收集管洗脱液, 旋蒸浓缩至原体积的1/5。冷冻干燥, 并采用硫酸-苯酚法鉴定凝胶纯化后的多糖含量。

青翘多糖和老翘多糖的分子量测定 如参考文献^{[13, 14]}, 将样品溶解在0.1 mol·L^-1 NaNO₃水溶液(含0.02% NaN₃, w/w) 中, 终浓度为1 mg·mL^-1, 并通过孔径为0.45 μm的过滤器过滤后上机检测。色谱条件: 采用凝胶排阻色谱柱OhpakSB-805HQ (300 mm × 8 mm) 和OhpakSB-803HQ (300 mm × 8 mm) 串联。柱温45 ℃, 进样量100 μL, 流动相A (0.02% NaN₃, 0.1 mol·L^-1 NaNO₃), 流速0.6 mL·min^-1, 洗脱梯度: 等度75 min。

青翘多糖和老翘多糖的单糖组成分析 根据参考文献^{[15, 16]}, 采用的是Thermo ICS5000+离子色谱系统(ICS5000+, 美国Thermo Fisher Scientific公司), 利用电化学检测器对单糖组分进行分析检测。取干净的色谱瓶, 称取适量多糖样品, 加入1 mL 2 mol·L^-1 TFA酸溶液, 121 ℃加热2 h。通氮气, 吹干。加入99.99%甲醇清洗, 再吹干, 重复甲醇清洗二三次。加入无菌水溶解, 转入色谱瓶中待测; 取适量的上清液, 旋转浓缩或氮气吹干。加入1 mL 2 mol·L^-1 TFA溶液, 121 ℃加热2 h。通氮气, 吹干。加入99.99%甲醇清洗, 再吹干, 重复甲醇清洗二三次。加入无菌水溶解, 转入色谱瓶中待测。标准品定量: 分别准确称取所需标准品后, 加入水配成10 mg·mL^-1标准溶液母液, 然后取适量标准品母液混合配制成最高指标浓度为60、50或40 μg·mL^-1的标准品混合溶液, 其中Fuc、Rha、Ara、Gal、Glc、Xyl配置成0.4、0.8、4、8、16、24、32、40 μg·mL^-1, Man配置成0.5、1、5、10、20、30、40、50 μg·mL^-1, Fru、Rib、Gal-UA、Gul-UA、Glc-UA、Man-UA配置成0.6、1.2、6、12、24、36、48、60 μg·mL^-1。测定条件: 采用Dionex^TM CarboPac^TM PA20 (150 mm × 3.0 mm, 10 μm) 液相色谱柱; 进样量为5 μL。流动相A为水, 流动相B为0.1 mol·L^-1的NaOH, 流动相C为0.1 mol·L^-1 NaOH和0.2 mol·L^-1 NaAc, 流速0.5 mL·min^-1; 柱温为30 ℃; 洗脱梯度: 0~26 min, 95% A、5% B; 26.1~42 min, 85% A、5% B、10% C; 42.1 min, 60% A、40% C; 52 min, 60% A、40% B, 52.1~60 min, 95% A、5% B。

连翘多糖的甲基化分析 称取样品5 mg, 加入1 mL纯水溶解, 加入1 mL 100 mg·mL^-1 1-环己基-2-吗啉乙基碳二亚胺甲基对甲苯磺酸盐, 反应2 h。加入1 mL 2 mol·L^-1的咪唑, 将样品平均分为两份, 分别加入1 mL 30 mg·mL^-1的NaBH₄和1 mL 30 mg·mL^-1的NaBD₄, 反应3 h。加入100 μL冰醋酸终止反应。透析48 h后冷冻干燥样品, 进行后续处理。样品中加入500 μL DMSO溶解。加入1 mg NaOH, 孵育30 min。加入50 μL碘甲烷溶液反应1 h。加入1 mL水和2 mL二氯甲烷, 涡旋混匀, 离心, 弃水相。重复水洗3次。吸取下层二氯甲烷相并用氮气吹干。加入100 μL 2 mol·L^-1 TFA, 121 ℃反应90 min。30 ℃蒸干。加入50 μL 2 mol·L^-1氨水, 50 μL 1 mol·L^-1 NaBD₄, 混匀, 室温下反应2.5 h。加入20 μL乙酸终止反应, 氮气吹干, 250 μL甲醇洗两次, 氮气吹干。加入乙酸酐250 μL, 涡旋混匀, 100 ℃反应2.5 h。加入1 mL水静置10 min。加入500 μL二氯甲烷, 涡旋混匀, 离心, 弃水相, 重复水洗3次。取下层二氯甲烷相, GC-MS上机检测。色谱条件: 色谱系统采用的是Agilent气相色谱系统(Agilent 6890A; Agilent Technologies, USA), 色谱柱: BPX70 (30 m × 0.25 mm × 0.25 µm, SGE, Australia)。进样量为1 μL, 分流比10∶1, 载气为高纯氦气; 柱温箱的初始温度为140 ℃保持2.0 min, 以3 ℃·min^-1程序升温至230 ℃, 保持3 min。质谱条件: 质谱系统采用美国Aiglent公司的四极杆质谱检测系统(Agilent 5977B; Agilent Technologies, USA), 采用电子轰击离子源(EI), 分析物在全扫描(SCAN) 模式下进行检测, 质量扫描范围m/z 50~350。

细胞培养 用含10% FBS胎牛血清的完全培养液将RAW264.7细胞配成单个细胞悬液, 以每孔10⁴个细胞的浓度接种到96孔板, 每孔体积100 μL。在5% CO₂、37 ℃条件下孵育24 h, 届时细胞单层铺满孔底。

MTT检测细胞毒性 实验分为: 空白组, 青翘多糖、老翘多糖给药组, 多糖给药组，浓度梯度分别为10、25、50、100、250、500 μg·mL^-1。每个浓度的药物设置3个复孔。给药完成后, 于培养箱培养24 h。次日取出96孔板, 向含细胞的96孔板中每孔加入10 μL 5 mg·mL^-1的MTT试剂, 再于培养箱中培养4 h。4 h后小心吸弃MTT试剂, 向每孔中加入100 μL DMSO试剂。室温下用摇床振荡10 min, 再于酶标仪490 nm下检测每孔吸光度。

IL-10、NO和TNF-α的水平测定 用含10% FBS胎牛血清的完全培养液将RAW264.7细胞配成单个细胞悬液, 以每孔10⁴个细胞的浓度接种到96孔板, 每孔体积100 μL。在5% CO₂、37 ℃条件下孵育24 h, 届时细胞单层铺满孔底。设置空白组, 炎症模型组, 以及青翘多糖、老翘多糖给药组(浓度梯度分别为10、25、50、100 μg·mL^-1), 每组均设置3个复孔, 药物预干预2 h后, 炎症模型组与多糖给药组加入适量LPS, 使得LPS终浓度为1 μg·mL^-1, 对照组加入等量的PBS后继续培养24 h; 24 h后取细胞培养基上清液, 4 ℃、1 000 ×g离心15 min, 取上清液, 根据ELISA试剂盒和NO快速检测盒的说明测定IL-10、NO和TNF-α的生成量。

代谢组学样品制备 实验分组: 空白组、模型组、青翘多糖给药组。将相同数量的RAW264.7细胞(每孔1×10⁵个) 于直径为10 cm的培养皿中培养, 每组设置6个重复。当细胞生长至90%时, 空白组加入10 mL DMEM培养基; 模型组加入适量LPS使其终质量浓度为1 μg·mL^-1; 给药组分别先加入100 μg·mL^-1青翘多糖预保护2 h, 然后加入适量LPS使其终质量浓度为1 μg·mL^-1。培养24 h后, PBS洗涤细胞3次, 用细胞刮刀刮下培养皿中的细胞, 4 ℃、1 200 r·min^-1离心10 min, 收集各组细胞沉淀, 于液氮中速冻, 并储存在-80 ℃下, 用于进一步细胞内代谢组学分析。

细胞内代谢物提取方法 将细胞4 ℃融化, 反复冻融5次后, 加入MeOH-CHCl₃-H₂O (8∶1∶1) 1 mL, 于冰上超声破碎15 min (超5 s, 停9 s), 4 ℃、1.3×10⁴ r·min^-1离心10 min, 收集上清。收集沉淀加入MeOH-CHCl₃-H₂O (8∶1∶1) 1 mL, 再次超声破碎15 min, 合并2次上清液冻干。于冻干后的细胞裂解物中加入50%乙腈60 μL复溶, 4 ℃、1.3×10⁴ r·min^-1离心10 min, 上清液置于液相小瓶中。另通过混合各自的等分试样作为QC样本。空白为提取溶剂。

色谱条件 流动相为10%甲酸水(A) 和乙腈(B)。梯度为: 0~2 min, 1% B; 2~8 min, 1%~35% B; 8~11 min, 35%~40% B; 11~14 min, 40%~60% B; 14~17 min, 60%~99% B; 17~19 min, 99%~1% B; 19~20 min, 1% B。体积流量0.2 mL·min^-1, 进样量为5 μL, 柱温40 ℃, Waters ACQUITYUPLC HSS T3色谱柱(100 mm × 2.1 mm, 1.7 μm)。

质谱条件 参照文献^[17]方法, 采用HESI离子化方式, 正极电压为3.5 kV, 负极2.5 kV。扫描模式为Full Scan/dd-MS2, 采集范围为m/z 100~1 500, 正负离子切换采集模式, 毛细管温度320 ℃, 加热器温度300 ℃, 鞘气体积流量35 arb, 辅助气体积流量10 arb。分辨率采用MS Full-Scan 35000 FWHM, MS/MS 17500 FWHM, 碰撞能量为12.5、25.0、37.5 eV。

数据处理 将采集到的原始LC-MS/MS数据文件导入Compound Discoverer 2.0软件(美国Thermo Fisher公司), 设置参数, 获取匹配和对齐的峰值数据, 将峰值数据导入SIMCA-P13.0软件进行主成分分析(PCA)、偏最小二乘判别分析(PLS-DA) 和正交偏最小二乘判别分析(OPLS-DA)。结合OPLS-DA的S-plot图中VIP > 1以及独立样本t检验筛选差异代谢物(以P < 0.05为标准筛选), 并对其进行鉴定。通过Metaboanalyst平台(https://dev.metaboanalyst.ca/) 进行通路分析^[18]。采用SPSS16.0软件对数据进行单因素方差分析, 各结果以

$ \overline{x} $

± s表示。

结果与分析

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1　青翘多糖和老翘多糖的纯化和结构解析

1.1　青翘多糖和老翘多糖的得率和总糖含量

通过水提醇沉法从青翘和老翘中得到青翘多糖和老翘多糖, 粗多糖经Sephacryl S-400HR柱色谱纯化, 各得到一个主要组分, 回收率均为16.0%。对青翘多糖和老翘多糖凝胶柱纯化前后的总糖含量进行测定, 结果表明青翘粗多糖的纯度为74.7%, 凝胶纯化后可以达到91.1%, 老翘粗多糖的纯度为71.2%, 凝胶纯化后达到88.9%。

1.2　连翘多糖的分子量测定

经凝胶排阻色谱-示差检测器, 多角度激光光散射仪检测, 根据Mark-Houwink方程计算出每个组分对应的分子量。由表1可得到青翘多糖的重均分子量和均分子量大于老翘多糖。从多糖样品的聚合物分散性指数(M_w/M_n) 可以看出青翘多糖和老翘多糖的PDI较小, 分子量分布较均匀。

1.3　连翘多糖的单糖组成测定

图1所示为标准样品离子色谱图和样品离子色谱图, 根据样品特征图谱的保留时间, 将单糖混合对照品图谱与样品特征图谱进行对比, 可以确定青翘多糖和老翘多糖由Fuc、Rha、Ara、Gal、Glc、Xyl、Man、Gal-UA和Glc-UA 9种单糖组成, 如表2所示。若岩藻糖的物质的量为1, 青翘多糖中各单糖组成比例约为1.00∶10.68∶10.44∶9.03∶14.03∶1.80∶3.83∶46.23∶1.09, 老翘多糖中各单糖组成比例约为1.00∶8.14∶11.29∶5.76∶14.81∶1.87∶2.72∶56.48∶1.04。综上可知, 青翘多糖和老翘多糖中单糖含量最高的均为Gal-UA。

1.4　连翘多糖的甲基化分析

甲基化分析是确定多糖中单糖单元连接方式的最常用方法。青翘多糖甲基化分析结果显示t-Rha(p), t-Ara(f), t-Xyl(p), 2-Rha(p), t-Glc(p), 3-Ara(f), t-Gal(p)-UA, t-Gal(p), 5-Ara(f), 3, 4-Rha(p), 4-Man(p), 4-Gal(p)-UA, 4-Gal(p), 4-Glc(p), 6-Gal(p), 3, 4-Gal(p)-UA, 2, 4-Glc(p), 4, 6-Glc(p) 的摩尔比为4.75∶2.20∶1.12∶1.21∶4.90∶1.08∶10.10∶1.22∶2.59∶1.27∶1.06∶41.06∶9.44∶8.54∶1.14∶3.00∶3.46∶1.89。其中4-Gal(p)-UA含量占比最高, 推测为主链, 同时存在t-Rha(p), t-Glc(p), t-Gal(p)-UA, t-Gal(p), 4-Glc(p)等分支结构。老翘多糖甲基化分析结构显示t-Rha(p), t-Ara(f), t-Ara(p), t-Glc(p), t-Gal(p)-UA, t-Gal(p), 2, 3-Rha(p), 4-Man(p), 4-Gal(p)-UA, 4-Gal(p), 4-Glc(p), 3, 4-Gal(p)-UA, 2, 4-Glc(p) 的摩尔比为4.69∶1.44∶1.08∶6.20∶12.09∶3.70∶1.07∶1.26∶48.75∶7.59∶7.46∶1.93∶2.73。其中4-Gal(p)-UA含量占比最高, 推测为主链。同时存在t-Rha(p), t-Glc(p), t-Gal(p)-UA, 4-Gal(p), 4-Glc(p) 等分支结构。具体内容如图2, 表3、4所示。

2　连翘多糖的抗炎活性实验

2.1　MTT法检测药物毒性

如图3所示, 两种多糖在0~100 μg·mL^-1之间培养24 h无明显细胞毒性(存活率在90%以上, P < 0.001), 当药物浓度为250~500 μg·mL^-1, 细胞存活率降低。因此, 在0~100 μg·mL^-1选择合适浓度进行后续炎症因子的测定。

2.2　NO、IL-10、和TNF-α水平的测定

如图4所示, 与空白组相比, LPS组的NO、IL-10、和TNF-α显著增加, 表明LPS诱导的小鼠单核巨噬细胞RAW264.7细胞炎症模型构建成功。在25~100 μg·mL^-1时, 青翘多糖和老翘多糖对NO、TNF-α均具有抑制作用, 且在100 μg·mL^-1时抑制效果最强, 与模型组比较差异有统计学意义(P < 0.05), 其中青翘多糖比老翘多糖抑制效果更加明显。青翘多糖和老翘多糖在0~100 μg·mL^-1时可以促进IL-10的产生, 且均在50 μg·mL^-1时促进效果达到最强, 与模型组比较差异有统计学意义(P < 0.05), 其中青翘多糖比老翘多糖促进效果更加明显。

3　代谢组学分析

根据上述实验, 青翘多糖的抗炎活性比老翘多糖的抗炎活性更强, 故本实验对青翘多糖进行细胞代谢组学分析, 以探究其抗炎作用机制。

3.1　细胞代谢组学分析方法可靠性检测

为检测分析方法的重复性和稳定性, 对细胞的QC样本进行了聚类分析, PCA结果表明, 所有QC样本都在标准偏差(SD) 的2倍范围之内(图5), 同时, QC样本RSD < 30%的变量占78.31%。结果表明, 该分析方法的稳定性和重复性很高, 适合大批量进样。

3.2　细胞代谢组学数据统计分析

从PLS-DA得分图中可以看出细胞代谢物在对照组、给药组与QC组、模型组之间能明显分开(图6A), 表明RAW264.7细胞在一定程度上能够被青翘多糖调控。模型评价指标显示左侧所有Q² (R²) 低于右边原始值, Q²所在蓝色回归线与左侧纵坐标相交于零以下, 表明模型验证有效。OPLS-DA有助于筛选出给药后的差异代谢物(图6C)。通过S-plot图(图6D) 结合VIP > 1以及独立样本t检验P < 0.05筛选出细胞中的差异代谢物。由表5可以看出, 与对照组相比, 给药后反丁烯二酸、L-丝氨酸、琥珀酸盐等11个代谢物水平显著升高。天冬氨酸盐、胆碱、L-天冬素等7个代谢物水平显著降低。使用MetaboAnalyst对得到的18个差异代谢物进行通路分析, 图中圆点颜色越深表示参与该通路代谢物越多, 圆点越大表示该通路作用越明显, 结果见图7。差异代谢物相关通路共有30条, 综合Holm P值、错误发现率(false discoveryrate, FDR) 值和Impact值发现, 以下3条代谢通路最为重要, 分别为精氨酸生物合成、丙氨酸、天冬氨酸和谷氨酸代谢、甘油磷脂代谢。

讨论

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连翘作为山西“十大道地药材”之一, 自古以来便作为传统中药材, 以产量高、品质好的特点行销海内外, 其中安泽、陵川连翘更以个大饱满、药用价值高的优势, 成功申报中国国家地理标志产品。本文对连翘不同采收时间得到的青翘和老翘的结构及生物活性进行了比较, 并深入探究了其发挥作用的机制, 为连翘资源的开发利用奠定了坚实基础。

重均分子量是中药多糖以及多糖制剂的一个重要参数, 研究其分布对于探究多糖药效作用具有重要意义。多项研究发现, 多种中药多糖的抗炎活性与其重均分子量呈正相关。Zhang等^[19]发现, 取代度相近时, 重均分子量较大的硫酸化灵芝β-葡聚糖对LPS诱导RAW264.7巨噬细胞释放NO的抑制率较高。Ying等^[20]从紫芝子实体水提取物中分离得到了30%、50%、70%乙醇沉淀的粗多糖样品, 其中30%的重均分子量更大, 体外刺激巨噬细胞释放NO和促进小鼠脾淋巴细胞增殖的活性较50%和70%的更好。Liu等^[21]对不同生长期的金耳多糖的结构及抗炎活性进行了研究, 研究发现金耳多糖抗炎活性与重均分子量的大小呈正相关。本文对青翘多糖与老翘多糖的分子量进行了测定, 发现青翘多糖的重均分子量大于老翘多糖, 且在体外抗炎活性的对比中发现青翘多糖与老翘多糖均有抗炎活性, 但青翘多糖的体外抗炎活性更强, 因此, 重均分子量较大可能是青翘多糖的体外抗炎活性强于老翘多糖的原因之一。

单糖组成对多糖的生物活性有显著影响, 大部分植物多糖由阿拉伯糖、半乳糖、葡萄糖、鼠李糖、甘露糖、木糖、岩藻糖以及半乳糖醛酸、葡萄糖醛酸等组成^[22]。有文献证明半乳糖醛酸含量多的多糖在抗炎方面具有更好的活性。Hou等^[23]从青梅果汁中提取得到含有半乳糖醛酸的可溶性果胶, 可显著抑制TNF-α、干扰素-γ (interferon-γ, IFN-γ)、IL-6在LPS诱导的RAW264.7细胞中的释放量, 增加IL-10的分泌, 起到抗炎作用。Popov等^[24]从甜椒中分离的果胶多糖辣椒素的主要成分是半乳糖醛酸, 小鼠口服后发现辣椒素半乳糖醛酸片段可减少脂多糖刺激的TNF-α的释放并增加IL-10的产生。Ovodova等^[25]从新鲜芹菜中分离出来一种含半乳糖醛酸达81%的果胶多糖, 可提高接受致死剂量LPS小鼠的存活率, 通过减少白细胞介素-1β、增加白细胞介素-10起到抗内毒素作用, 还可以减少LPS注射后迁移到腹膜腔的中性粒细胞的数量。本文对青翘多糖和老翘多糖进行了单糖组成测定, 结果发现青翘多糖和老翘多糖均由Fuc、Rha、Ara、Gal、Glc、Xyl、Man、Gal-UA和Glc-UA等9种单糖组成, 其中青翘多糖和老翘多糖中半乳糖醛酸均为含量最高的单糖组分, 因此推测青翘多糖和老翘多糖抗炎活性与半乳糖醛酸的存在有关。

中药多糖以其结构的多样性和复杂性而闻名, 多糖的单糖组成、糖苷键、分支程度和相对分子质量等都能对其功效产生影响。在这些因素中, 糖苷键(链上糖单元之间的连接类型) 作为多糖链局部构象的关键, 决定了多糖的空间形态和生物活性^[26]。Wu等^[27]通过对甘草多糖(Glycyrrhiza polysaccharides, GPS) 进行结构表征, 证明GPS-1是一种以→4)-α-D-Gal(p)-UA-(1→为主链的HG型果胶多糖, GPS-1与NDV疫苗联合使用可提升蛋鸡外周血和脾脏淋巴细胞中辅助性T细胞CD4⁺和杀伤性T细胞CD8⁺T细胞的比例, 促进IL-2、IL-4、IL-17和IFN-γ的分泌, 提高免疫球蛋白IgA及IgG的表达水平, 表现出良好的免疫增强作用。本文中青翘多糖甲基化分析结构显示主链主要由4-Gal(p)-UA组成, 占比最高(41.06%), 同时含有4-Glc(p) 和少量4-Man(p), 老翘多糖甲基化分析结构显示主链主要由4-Gal(p)-UA (48.75%) 组成, 同时存在t-Gal(p)-UA、4-Gal(p)、4-Glc(p) 等分支结构。4-Gal(p)-UA和4-Glc(p) 通常能够维持多糖的空间稳定性, 便于与免疫系统中的模式识别受体(如TLRs: Toll-like receptors或CLRs: C-type lectin receptors) 相互作用, 从而触发抗炎信号通路^[28], 使多糖发挥抗炎作用。

本文对比了青翘多糖和老翘多糖的体外抗炎活性, 发现青翘多糖的体外抗炎活性更强, 但其抗炎作用机制尚不明确, 因此本文选用细胞代谢组学从代谢的层面上揭示其作用机制, 发现青翘多糖主要通过调节精氨酸生物合成, 丙氨酸、天冬氨酸和谷氨酸代谢, 甘油磷脂代谢发挥抗炎活性。精氨酸能够直接参与尿素循环, 而尿素循环与三羧酸循环相关, 因此可以在一定程度上通过调节线粒体功能从而参与炎症反应^{[29, 30]}。本研究中巨噬细胞RAW264.7造模后细胞内精氨酸水平和空白组相比显著下调, 青翘多糖给药后回调, 提示青翘多糖通过调节精氨酸生物合成和精氨酸代谢途径发挥抗炎作用。丙氨酸-天冬氨酸-谷氨酸代谢途径共涉及谷氨酸、丙氨酸、柠檬酸盐、天冬氨酸、2-酮戊二酸5种生物标志物, 在机体代谢中发挥着重要作用^[31]。甘油磷脂是一个重要的生物学指标。作为细胞膜的主要脂质成分, 其含量的波动直接反映脂质代谢紊乱^[32], 研究发现这可能与氧化应激和炎症反应相关。上述代谢通路涉及能量代谢、氨基酸代谢及脂质代谢等方面, 可能是青翘多糖发挥抗炎药效的主要途径。后续需通过转录组学和蛋白质组学进行进一步验证, 从而全面解释其作用机制。

参考文献

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文献

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参考文献引证文献

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Li K, Lv TT, Cui RQ, et al. Investigation of mechanism of Qingqiao and Laoqiao on acute lung injury rats based on serum metabolomics [J]. Chin J Exp Tradit Med Form (中国实验方剂学杂志), 2024, 30: 89-97.

Li SF, Zhang LW, Zhan ZL. Herbal textual research on Forsythiae Fructus in famous classical formulas [J]. Chin J Exp Tradit Med Form (中国实验方剂学杂志), 2022, 28: 111-122.

Sun YN. Preliminary Studies on Serum Pharmacochemistry and Pharmacodynamics Systematic Comparison of the Green Forsythiae Fructus and Ripe Forsythiae Fructus (青翘与老翘药效学比较及血清药物化学初步研究) [D]. Taiyuan: Shanxi Provincial Institute of Traditional Chinese Medicine, 2016.

Zhang QQ, Zhang BB, Yang F, et al. Differential effects of Qingqiao and Laoqiao on bleomycin-induced pulmonary fibrosis in mice by principal component analysis [J]. China J Chin Mater Med (中国中药杂志), 2024, 49: 4128-4138.

Cao RR, Li HP, Huang YJ, et al. Determination of effective components in different Forsythia suspensa by HPLC-ESI/MS [J]. J Henan Univ Sci Technol Nat Sci (河南科技大学学报·自然科学版), 2015, 36: 101-104.

Mu Y, Liu B, Zhang X, et al. Analysis of constituents in different parts of Forsythia suspensa by UPLC-Q-TOF-MS and evaluation of their anti-inflammatory activity [J]. China J Chin Mater Med (中国中药杂志), 2024, 49: 968-980.

Lv JL, Huo YJ, Zhang Y. Latest advancements research on pharmacological activity of polysaccharides in Chinese medicine [J]. Chem Reagents (化学试剂), 2025, 47: 1-8.

Chen XL, Li WW, Chen Q, et al. Structure analysis and anti-inflammatory activity evaluation of neutral polysaccharides from Arctium lappa L. [J]. Sci Technol Food Ind (食品工业科技), 2023, 44: 45-54.

Luo LG, Wang D, Meng XQ, et al. Protein removal process and anti-inflammation effect of polysaccharides from Polygonum cuspidatum [J]. Cent South Pharm (中南药学), 2024, 22: 1999-2005.

Gui LL, Wang SK, Wang J, et al. Effects of forsythin extract in Forsythia leaves on intestinal microbiota and short-chain fatty acids in rats fed a high-fat diet [J]. Food Sci Hum Wellness, 2024, 13: 659-667.

Du BX, Fu YP, Wang X, et al. Isolation, purification, structural analysis and biological activities of water-soluble polysaccharide from Glehniae Radix [J]. Int J Biol Macromol, 2019, 128: 724-731.

Zhang H, Li CC, Lai PF, et al. Fractionation, chemical characterization and immunostimulatory activity of β-glucan and galactoglucan from Russula vinosa Lindblad [J]. Carbohydr Polym, 2021, 256: 117559.

Hu T, Huang QL, Wong K, et al. Structure, molecular conformation, and immunomodulatory activity of four polysaccharide fractions from Lignosus rhinocerotis sclerotia [J]. Int J Biol Macromol, 2017, 94: 423-430.

Zheng ZM, Huang QL, Ling CQ. Water-soluble yeast β‑glucan fractions with different molecular weights: extraction and separation by acidolysis assisted-size exclusion chromatography and their association with proliferative activity [J]. Int J Biol Macromol, 2019, 123: 269-279.

Wang L, Zhang B, Xiao J, et al. Physicochemical, functional, and biological properties of water-soluble polysaccharides from Rosa roxburghii Tratt fruit [J]. Food Chem, 2018, 249: 127-135.

Zhu MQ, Huang RM, Wen P, et al. Structural characterization and immunological activity of pectin polysaccharide from kiwano (Cucumis metuliferus) peels [J]. Carbohydr polym, 2021, 254: 117371.

Liang ML, Zhao F, Fang Y, et al. Hippocampus metabolomic research on antidepressant mechanism of petroleum ether fraction of Bupleuri Radix by LC-MS in rats [J]. Chin Tradit Herb Drugs (中草药), 2018, 49: 2291-2301.

Pang Z, Lu Y, Zhou G, et al. MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation [J]. Nucleic Acids Res, 2024, 52: 398-406.

Zhang Z, Liu YF, Tang QJ, et al. Sulfated derivatives of Ganoderma lucidum β-glucan inhibits NO production in lipopolysaccharide-induced RAW264.7 macrophage [J]. Acta Edulis Fungi (食用菌学报), 2018, 25: 105-112.

Ying YJ, Zhang JS, Tang QJ, et al. Properties and in vitro immunological activities of polysaccharides from Ganoderma sinense fruit bodies [J]. Acta Edulis Fungi (食用菌学报), 2016, 23: 34-39.

Liu LP, Liu YF, Sun HW, et al. Effects of cultivation time on structural characteristics and anti-inflammatory activity of polysaccharides from Naematelia aurantialba [J]. Acta Edulis Fungi (食用菌学报), 2024, 31: 42-50.

Liu C, Wu XH, Liu J, et al. Research progress on anti-inflammatory activity mechanism and structure-activity relationship of plant polysaccharides [J]. Sci Technol Food Ind (食品工业科技), 2022, 43: 415-425.

Hou JL, Zhu Z, Xie MH, et al. Characteristics of water-soluble pectin from greengage and its anti-inflammatory activity in LPS-induced RAW246.7 cells [J]. Sci Technol Food Ind (食品工业科技), 2022, 43: 401-409.

Popov SV, Ovodova RG, Golovchenko VV, et al. Chemical composition and anti-inflammatory activity of a pectic polysaccharide isolated from sweet pepper using a simulated gastric medium [J]. Food Chem, 2011, 124: 309-315.

Ovodova RG, Golovchenko VV, Popov SV, et al. Chemical composition and anti-inflammatory activity of pectic polysaccharide isolated from celery stalks [J]. Food Chem, 2009, 114: 610-615.

Zhao N, Han Z, Jian YL, et al. Research progress on structural characterization and quality evaluation of traditional Chinese medicine polysaccharides [J]. Chin Tradit Herb Drugs (中草药), 2024, 55: 7491-7506.

Wu Y. Study on the Chemical Structure and Immune-enhancing Effect of GPS-1 and Its Mechanism (甘草多糖GPS-1化学结构和免疫增强作用及其机制的研究) [D]. Nanjing: Nanjing Agricultural University, 2022.

Chen G, Jiang N, Zheng J, et al. Structural characterization and anti-inflammatory activity of polysaccharides from Astragalus membranaceus [J]. Int J Biol Macromol, 2023, 241: 124386.

Wu G, Bazer FW, Burghardt RC, et al. Proline and hydroxyproline metabolism: implications for animal and human nutrition [J]. Amino Acids, 2011, 40: 1053-1063.

Sun XY. Study on Toxic Mechanism of Atmospheric Submicron Particles Basedon Metabolomics (基于代谢组学的大气亚微米颗粒物毒性机制研究) [D]. Hangzhou: Zhejiang University of Technology, 2020.

Liu YY, Wang KD, Xia DN, et al. Study on the mechanism of sarsasapogenin on HepG2 cells based on cell metabolomics technology [J]. J Shenyang Pharm Univ (沈阳药科大学学报), 2023, 40: 164-172.

Wong BS, Camilleri M, Carlson P, et al. Increased bile acid biosynthesis is associated with irritable bowel syndrome with diarrhea [J]. Clin Gastroenterol Hepatol, 2012, 10: 1009-1015.

2025年第60卷第7期

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doi: 10.16438/j.0513-4870.2024-1209

接收时间：2024-12-06
首发时间：2026-09-20
出版时间：2025-07-12

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收稿日期：2024-12-06
修回日期：2025-05-07

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¹山西药科职业学院制药系, 山西太原 030031

²山西大学中医药现代研究中心, 山西太原 030006

³山西大学化学生物学与分子工程教育部重点实验室, 山西太原 030006

⁴地产中药功效物质研究与利用山西省重点实验室, 山西太原 030006

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李科, Tel/Fax: 86-351-7019297, E-mail: like@sxu.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Sample name	M_n/kDa	M_p/kDa	M_w/kDa	M_z/kDa	M_w/M_n
Green Forsythiae Fructus polysaccharides	7.844 ± 0.669 18	6.260 ± 0.943 59	13.036 ± 0.418 59	22.932 ± 0.736 77	1.662 ± 0.789 31
Ripe Forsythiae Fructus polysaccharides	10.833 ± 0.403 26	10.161 ± 0.998 14	11.990 ± 0.368 43	13.778 ± 0.789 25	1.107 ± 0.546 22

Sample name

M_n/kDa

M_p/kDa

M_w/kDa

M_z/kDa

M_w/M_n

Green Forsythiae Fructus polysaccharides

7.844 ± 0.669 18

6.260 ± 0.943 59

13.036 ± 0.418 59

22.932 ± 0.736 77

1.662 ± 0.789 31

Ripe Forsythiae Fructus polysaccharides

10.833 ± 0.403 26

10.161 ± 0.998 14

11.990 ± 0.368 43

13.778 ± 0.789 25

1.107 ± 0.546 22

Type of polysaccharide	Type of monosaccharide	Molar ratio	Content /μg·mL^-1
Green Forsythiae	Fuc	1.02%	3.635 0
Fructus	Rha	10.80%	35.310 1
polysaccharides	Ara	10.65%	38.081 0
	Gal	9.21%	36.129 6
	Glc	14.31%	56.135 9
	Xyl	1.84%	6.005 2
	Man	3.91%	15.352 3
	Gal-UA	47.15%	199.291 0
	Glc-UA	1.11%	4.670 9
Ripe Forsythiae	Fuc	0.97%	3.089 0
Fructus	Rha	7.90%	23.072 2
polysaccharides	Ara	10.95%	34.943 5
	Gal	5.59%	19.583 0
	Glc	14.37%	50.356 1
	Xyl	1.81%	5.274 6
	Man	2.64%	9.263 5
	Gal-UA	54.79%	206.762 2
	Glc-UA	1.01%	3.801 6

Type of polysaccharide

Type of monosaccharide

Molar ratio

Content /μg·mL^-1

Green Forsythiae

Fuc

1.02%

3.635 0

Fructus

Rha

10.80%

35.310 1

polysaccharides

Ara

10.65%

38.081 0

Gal

9.21%

36.129 6

Glc

14.31%

56.135 9

Xyl

1.84%

6.005 2

Man

3.91%

15.352 3

Gal-UA

47.15%

199.291 0

Glc-UA

1.11%

4.670 9

Ripe Forsythiae

Fuc

0.97%

3.089 0

Fructus

Rha

7.90%

23.072 2

polysaccharides

Ara

10.95%

34.943 5

Gal

5.59%

19.583 0

Glc

14.37%

50.356 1

Xyl

1.81%

5.274 6

Man

2.64%

9.263 5

Gal-UA

54.79%

206.762 2

Glc-UA

1.01%

3.801 6

Type of linkage	PMAAs	t_R/min	m/z	Molar ratio/%
t-Rha(p)	1, 5-di-O-Acetyl-6-deoxy-2, 3, 4-tri-O-methyl rhamnitol	4.706	59, 72, 89, 102, 115, 118, 131, 145, 162, 175	4.75
t-Ara(f)	1, 4-di-O-Acetyl-2, 3, 5-tri-O-methyl arabinitol	4.975	71, 87, 102, 118, 129, 145, 161	2.20
t-Xyl(p)	1, 5-di-O-Acetyl-2, 3, 4-tri-O-methyl xylitol	5.818	88, 101, 102, 118, 119, 161, 162	1.12
2-Rha(p)	1, 2, 5-tri-O-Acetyl-6-deoxy-3, 4-di-O-methyl rhamnitol	7.379	89, 100, 115, 130, 131, 175, 190	1.21
t-Glc(p)	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl glucitol	7.487	87, 102, 118, 129, 145, 161, 162, 205	4.90
3-Ara(f)	1, 3, 4-tri-O-Acetyl-2, 5-di-O-methyl arabinitol	8.032	87, 99, 113, 118, 129, 201, 233	1.08
t-Gal(p)-UA	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol	8.391	73, 89, 102, 118, 147, 162, 163, 207	10.10
t-Gal(p)	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol	8.392	87, 102, 118, 129, 145, 161, 162, 205	1.22
5-Ara(f)	1, 4, 5-tri-O-Acetyl-2, 3-di-O-methyl arabinitol	9.109	87, 102, 118, 129, 162, 189	2.59
3, 4-Rha(p)	1, 3, 4, 5-tetra-O-Acetyl-6-deoxy-2-O-methyl rhamnitol	9.909	87, 99, 113, 118, 129, 149, 173, 275	1.27
4-Man(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl mannitol	11.208	87, 102, 113, 118, 129, 162, 233	1.06
4-Gal(p)-UA	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol	12.002	87, 99, 102, 115, 118, 131, 162, 175, 235	41.06
4-Gal(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol	12.037	87, 102, 113, 118, 129, 162, 233	9.44
4-Glc(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl glucitol	12.303	87, 102, 113, 118, 129, 162, 233	8.54
6-Gal(p)	1, 5, 6-tri-O-Acetyl-2, 3, 4-tri-O-methyl galactitol	13.627	87, 99, 102, 118, 129, 162, 189, 233	1.14
3, 4-Gal(p)-UA	1, 3, 4, 5-tetra-O-Acetyl-2, 6-di-O-methyl galactitol	13.842	87, 118, 131, 143, 185, 205, 307	3.00
2, 4-Glc(p)	1, 2, 4, 5-tetra-O-Acetyl-3, 6-di-O-methyl glucitol	15.020	87, 88, 99, 113, 130, 190, 233	3.46
4, 6-Glc(p)	1, 4, 5, 6-tetra-O-Acetyl-2, 3-di-O-methyl glucitol	16.782	85, 102, 118, 127, 159, 162, 201, 261	1.89

Type of linkage

PMAAs

t_R/min

m/z

Molar ratio/%

t-Rha(p)

1, 5-di-O-Acetyl-6-deoxy-2, 3, 4-tri-O-methyl rhamnitol

4.706

59, 72, 89, 102, 115, 118, 131, 145, 162, 175

4.75

t-Ara(f)

1, 4-di-O-Acetyl-2, 3, 5-tri-O-methyl arabinitol

4.975

71, 87, 102, 118, 129, 145, 161

2.20

t-Xyl(p)

1, 5-di-O-Acetyl-2, 3, 4-tri-O-methyl xylitol

5.818

88, 101, 102, 118, 119, 161, 162

1.12

2-Rha(p)

1, 2, 5-tri-O-Acetyl-6-deoxy-3, 4-di-O-methyl rhamnitol

7.379

89, 100, 115, 130, 131, 175, 190

1.21

t-Glc(p)

1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl glucitol

7.487

87, 102, 118, 129, 145, 161, 162, 205

4.90

3-Ara(f)

1, 3, 4-tri-O-Acetyl-2, 5-di-O-methyl arabinitol

8.032

87, 99, 113, 118, 129, 201, 233

1.08

t-Gal(p)-UA

1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol

8.391

73, 89, 102, 118, 147, 162, 163, 207

10.10

t-Gal(p)

1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol

8.392

87, 102, 118, 129, 145, 161, 162, 205

1.22

5-Ara(f)

1, 4, 5-tri-O-Acetyl-2, 3-di-O-methyl arabinitol

9.109

87, 102, 118, 129, 162, 189

2.59

3, 4-Rha(p)

1, 3, 4, 5-tetra-O-Acetyl-6-deoxy-2-O-methyl rhamnitol

9.909

87, 99, 113, 118, 129, 149, 173, 275

1.27

4-Man(p)

1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl mannitol

11.208

87, 102, 113, 118, 129, 162, 233

1.06

4-Gal(p)-UA

1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol

12.002

87, 99, 102, 115, 118, 131, 162, 175, 235

41.06

4-Gal(p)

1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol

12.037

87, 102, 113, 118, 129, 162, 233

9.44

4-Glc(p)

1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl glucitol

12.303

87, 102, 113, 118, 129, 162, 233

8.54

6-Gal(p)

1, 5, 6-tri-O-Acetyl-2, 3, 4-tri-O-methyl galactitol

13.627

87, 99, 102, 118, 129, 162, 189, 233

1.14

3, 4-Gal(p)-UA

1, 3, 4, 5-tetra-O-Acetyl-2, 6-di-O-methyl galactitol

13.842

87, 118, 131, 143, 185, 205, 307

3.00

2, 4-Glc(p)

1, 2, 4, 5-tetra-O-Acetyl-3, 6-di-O-methyl glucitol

15.020

87, 88, 99, 113, 130, 190, 233

3.46

4, 6-Glc(p)

1, 4, 5, 6-tetra-O-Acetyl-2, 3-di-O-methyl glucitol

16.782

85, 102, 118, 127, 159, 162, 201, 261

1.89

Type of linkage	PMAAs	t_R/min	m/z	Molar ratio/%
t-Rha(p)	1, 5-di-O-Acetyl-6-deoxy-2, 3, 4-tri-O-methyl rhamnitol	4.698	59, 72, 89, 102, 115, 118, 131, 145, 162, 175	4.69
t-Ara(f)	1, 4-di-O-Acetyl-2, 3, 5-tri-O-methyl arabinitol	4.971	71, 87, 102, 118, 129, 145, 161	1.44
t-Ara(p)	1, 5-di-O-Acetyl-2, 3, 4-tri-O-methyl arabinitol	6.130	88, 101, 102, 118, 119, 161, 162	1.08
t-Glc(p)	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl glucitol	7.483	87, 102, 118, 129, 145, 161, 162, 205	6.20
t-Gal(p)-UA	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol	8.384	73, 89, 102, 118, 147, 162, 163, 207	12.09
t-Gal(p)	1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol	8.387	87, 102, 118, 129, 145, 161, 162, 205	3.70
2, 3-Rha(p)	1, 2, 3, 5-tetra-O-Acetyl-6-deoxy-4-O-methyl rhamnitol	9.733	71, 89, 101, 131, 262	1.07
4-Man(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl mannitol	11.197	87, 102, 113, 118, 129, 162, 233	1.26
4-Gal(p)-UA	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol	12.005	87, 99, 102, 115, 118, 131, 162, 175, 235	48.75
4-Gal(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol	12.037	87, 102, 113, 118, 129, 162, 233	7.59
4-Glc(p)	1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl glucitol	12.310	87, 102, 113, 118, 129, 162, 233	7.46
3, 4-Gal(p)-UA	1, 3, 4, 5-tetra-O-Acetyl-2, 6-di-O-methyl galactitol	13.839	87, 118, 129, 143, 185, 203, 305	1.93
2, 4-Glc(p)	1, 2, 4, 5-tetra-O-Acetyl-3, 6-di-O-methyl glucitol	15.016	87, 88, 99, 113, 130, 190, 233	2.73

Type of linkage

PMAAs

t_R/min

m/z

Molar ratio/%

t-Rha(p)

1, 5-di-O-Acetyl-6-deoxy-2, 3, 4-tri-O-methyl rhamnitol

4.698

59, 72, 89, 102, 115, 118, 131, 145, 162, 175

4.69

t-Ara(f)

1, 4-di-O-Acetyl-2, 3, 5-tri-O-methyl arabinitol

4.971

71, 87, 102, 118, 129, 145, 161

1.44

t-Ara(p)

1, 5-di-O-Acetyl-2, 3, 4-tri-O-methyl arabinitol

6.130

88, 101, 102, 118, 119, 161, 162

1.08

t-Glc(p)

1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl glucitol

7.483

87, 102, 118, 129, 145, 161, 162, 205

6.20

t-Gal(p)-UA

1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol

8.384

73, 89, 102, 118, 147, 162, 163, 207

12.09

t-Gal(p)

1, 5-di-O-Acetyl-2, 3, 4, 6-tetra-O-methyl galactitol

8.387

87, 102, 118, 129, 145, 161, 162, 205

3.70

2, 3-Rha(p)

1, 2, 3, 5-tetra-O-Acetyl-6-deoxy-4-O-methyl rhamnitol

9.733

71, 89, 101, 131, 262

1.07

4-Man(p)

1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl mannitol

11.197

87, 102, 113, 118, 129, 162, 233

1.26

4-Gal(p)-UA

1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol

12.005

87, 99, 102, 115, 118, 131, 162, 175, 235

48.75

4-Gal(p)

1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl galactitol

12.037

87, 102, 113, 118, 129, 162, 233

7.59

4-Glc(p)

1, 4, 5-tri-O-Acetyl-2, 3, 6-tri-O-methyl glucitol

12.310

87, 102, 113, 118, 129, 162, 233

7.46

3, 4-Gal(p)-UA

1, 3, 4, 5-tetra-O-Acetyl-2, 6-di-O-methyl galactitol

13.839

87, 118, 129, 143, 185, 203, 305

1.93

2, 4-Glc(p)

1, 2, 4, 5-tetra-O-Acetyl-3, 6-di-O-methyl glucitol

15.016

87, 88, 99, 113, 130, 190, 233

2.73

No.	Metabolite	t_R/min	Formula	m/z	VIP	Model vs control	Fc	Treat vs model	Fc
1	Aspartate	1.474	C₄H₇NO₄	101.047 93	1.107 13	↑	1.570 184	↓	0.709 129
2	Choline	1.321	C₅H₁₄NO	102.115 93	1.359 75	↑	30.879 62	↓	0.325 997
3	Fumarate	1.749	C₄H₄O₄	116.011 03	2.129 89	↓	0.597 923	↑	1.417 095
4	L-Serine	1.418	C₃H₇NO₃	117.054 08	3.077 65	↓	0.432 520	↑	1.434 768
5	Succinate	3.916	C₄H₆O₄	118.026 72	2.632 80	↓	0.219 386	↑	2.587 650
6	L-Glutamate	1.845	C₅H₉NO₄	131.058 09	1.842 49	↓	0.561 708	↑	1.703 735
7	L-Asparagine	1.615	C₄H₈N₂O₃	132.052 30	4.309 88	↑	1.606 690	↓	0.657 017
8	Ornithine	1.402	C₅H₁₂N₂O₂	132.089 98	1.860 81	↑	4.409 108	↓	0.691 799
9	O-Acetylcholine	2.783	C₇H₁₆NO₂	144.126 36	4.465 79	↑	81.396 74	↓	0.562 737
10	L-Glutamine	2.358	C₅H₁₀N₂O₃	145.085 15	1.038 68	↓	0.639 575	↑	1.337 610
11	2-Oxoglutarate	2.062	C₅H₆O₅	146.021 70	1.899 38	↑	5.922 962	↓	0.451 885
12	1, 2-Diacyl-sn-glycerol	3.118	C₅H₆O₅R₂	172.084 91	1.659 93	↓	0.213 788	↑	2.958 079
13	Arginine	1.360	C₆H₁₄N₄O₂	174.111 80	5.694 11	↓	0.532 306	↑	1.422 239
14	Phosphatidylcholine	7.105	C₁₀H₁₈NO₈PR₂	254.126 58	2.746 34	↓	0.330 396	↑	2.227 042
15	N-Acetyl-L-aspartyl-L-glutamate	3.388	C₁₁H₁₆N₂O₈	268.106 06	1.383 01	↓	0.153 683	↑	4.795 126
16	LysW-glutamate	2.712	C₁₀H₁₅N₂O₇R	289.073 18	1.737 82	↓	0.640 030	↑	1.562 426
17	1-Acyl-sn-glycero-3-phosphocholine	2.189	C₉H₂₀NO₇PR	299.113 56	1.707 83	↑	2.405 316	↓	0.517 648
18	CDP-choline	1.499	C₁₄H₂₆N₄O₁₁P₂	488.107 50	1.968 60	↓	0.490 872	↑	1.566 374

No.

Metabolite

t_R/min

Formula

m/z

VIP

Model vs control

Treat vs model

Aspartate

1.474

C₄H₇NO₄

101.047 93

1.107 13

↑

1.570 184

↓

0.709 129

Choline

1.321

C₅H₁₄NO

102.115 93

1.359 75

↑

30.879 62

↓

0.325 997

Fumarate

1.749

C₄H₄O₄

116.011 03

2.129 89

↓

0.597 923

↑

1.417 095

L-Serine

1.418

C₃H₇NO₃

117.054 08

3.077 65

↓

0.432 520

↑

1.434 768

Succinate

3.916

C₄H₆O₄

118.026 72

2.632 80

↓

0.219 386

↑

2.587 650

L-Glutamate

1.845

C₅H₉NO₄

131.058 09

1.842 49

↓

0.561 708

↑

1.703 735

L-Asparagine

1.615

C₄H₈N₂O₃

132.052 30

4.309 88

↑

1.606 690

↓

0.657 017

Ornithine

1.402

C₅H₁₂N₂O₂

132.089 98

1.860 81

↑

4.409 108

↓

0.691 799

O-Acetylcholine

2.783

C₇H₁₆NO₂

144.126 36

4.465 79

↑

81.396 74

↓

0.562 737

L-Glutamine

2.358

C₅H₁₀N₂O₃

145.085 15

1.038 68

↓

0.639 575

↑

1.337 610

2-Oxoglutarate

2.062

C₅H₆O₅

146.021 70

1.899 38

↑

5.922 962

↓

0.451 885

1, 2-Diacyl-sn-glycerol

3.118

C₅H₆O₅R₂

172.084 91

1.659 93

↓

0.213 788

↑

2.958 079

Arginine

1.360

C₆H₁₄N₄O₂

174.111 80

5.694 11

↓

0.532 306

↑

1.422 239

Phosphatidylcholine

7.105

C₁₀H₁₈NO₈PR₂

254.126 58

2.746 34

↓

0.330 396

↑

2.227 042

N-Acetyl-L-aspartyl-L-glutamate

3.388

C₁₁H₁₆N₂O₈

268.106 06

1.383 01

↓

0.153 683

↑

4.795 126

LysW-glutamate

2.712

C₁₀H₁₅N₂O₇R

289.073 18

1.737 82

↓

0.640 030

↑

1.562 426

1-Acyl-sn-glycero-3-phosphocholine

2.189

C₉H₂₀NO₇PR

299.113 56

1.707 83

↑

2.405 316

↓

0.517 648

CDP-choline

1.499

C₁₄H₂₆N₄O₁₁P₂

488.107 50

1.968 60

↓

0.490 872

↑

1.566 374