药学学报

No.	RT/min	M	Metabolic pathway	Calculated [M-X]^-/[M+X]⁺ (m/z)	Observed[M-X]^-/[M+X]⁺ (m/z)	Error (10^-6)	CE/eV	Ion mode	MS², data, m/z, (ralative, intensity, %)
M1	8.373	DZYS	Iso	461.072 0	461.072 5	1.08	30	Neg	61.987 9 (36), 262.069 2 (33), 285.040 0 (100),
M2	7.751	DZYS	-GlcA	285.039 9	285.040 6	2.46	30	Neg	79.957 2 (100), 124.007 8 (34), 292.122 1 (26)
M3	8.062	DZYS	+SO₃	541.028 8	541.029 2	0.74	30	Neg	113.024 3 (22), 285.040 3 (100), 461.071 8 (26)
M4	12.728	DZYS	+Me	475.087 6	475.088 3	1.47	20	Neg	113.024 3 (82), 200.129 8 (78), 303.087 5 (100)
M5	8.761	DZYS	+H₂	463.087 7	463.088 4	1.51	30	Neg	113.024 9 (10), 166.999 0 (18), 287.057 6 (100)
M6	7.285	DZJS	-OH	429.082 2	429.080 5	-3.96	30	Neg	124.007 4 (100), 316.122 5 (35), 445.190 1 (10)
M7	8.463	DZJS	-GlcA	269.045 0	269.045 8	2.97	30	Neg	96.960 4 (100), 117.034 8 (9), 269.046 0 (3)
M8	8.451	DZJS	+SO₃	525.033 9	525.034 7	1.52	30	Neg	113.024 5 (32), 269.045 9 (100), 461.072 9 (13)
M9	10.629	DZJS	+H₂	447.092 8	447.093 4	1.34	30	Neg	96.960 3 (8), 255.080 7 (100), 459.206 2 (39)
M10	7.943	DZJS	+Me	459.092 7	459.094 6	4.14	20	Neg	113.024 7 (50), 283.062 0 (100), 388.995 5 (23)
M11	3.864	CA	+SO₃	258.991 2	258.992 1	3.48	30	Neg	79.957 3 (3), 135.045 3 (100), 179.035 1 (8)
M12	10.317	CA	+GSH	486.118 2	486.121 9	7.61	30	Neg	96.960 4 (3), 255.081 0 (6), 413.201 8 (100)
M13	4.874	CA	+GlcA	355.066 5	355.067 2	1.97	30	Neg	61.988 2 (21), 135.045 4 (100), 179.035 3 (62)
M14	7.683	CA	+Me	193.050 0	193.049 8	-1.04	20	Neg	59.013 6 (3), 109.066 1 (6), 160.040 6 (100)
M15	2.877	CA	-OH	163.039 5	163.040 2	4.29	20	Neg	65.039 2 (2), 106.042 1 (100), 119.050 0 (31)
M16	4.918	CA	+H₂	181.050 1	181.050 7	3.31	30	Neg	79.957 5 (18), 107.050 6 (100), 108.053 8 (5)
M17	7.131	3-CQA	+Me	367.102 9	367.104 2	3.54	30	Neg	59.013 7 (23), 96.960 5 (100), 171.103 3 (8)
M18	4.866	3-CQA	+GlcA	529.119 3	529.120 7	2.65	20	Neg	187.007 6 (100), 188.010 8 (4), 242.997 7 (23)
M19	4.252	3-CQA	+H₂	355.103 0	355.103 3	0.84	30	Neg	80.964 9 (8), 137.061 2 (100), 181.050 9 (3)
M20	9.462	1, 5-CQA	-OH	499.124 1	499.128 1	8.01	30	Neg	113.024 6 (9), 285.041 0 (100), 417.083 3 (22)
M21	11.095	1, 5-CQA	+Me	529.134 6	529.137 7	5.86	30	Neg	254.072 5 (42), 371.148 5 (100), 411.184 8 (11)
M22	10.248	1, 5-CQA	+H₂	517.134 7	517.135 1	0.77	30	Neg	137.060 8 (4), 181.050 8 (100), 365.087 9 (20)
M23	1.982	1, 5-CQA	De	191.055 6	191.055 6	0.00	30	Neg	79.957 1 (51), 109.029 9 (11), 125.024 1 (100)
M24	2.338	MD-2H-A	-Me	313.071 3	313.075 2	12.46	40	Neg	79.957 3 (100), 96.960 5 (41), 124.007 4 (24)
M25	2.695	MLP-A	-OH	323.092 0	323.090 7	-4.02	40	Neg	59.013 4 (27), 79.957 4 (100), 124.007 5 (48)
M26	2.371	MLP-2H-B	-OH	311.128 4	311.129 3	2.89	40	Neg	79.957 4 (100), 106.980 8 (9), 135.045 4 (16)
M27	2.818	MLP-2H-B	-Me-OH	297.112 7	297.113 7	3.37	40	Neg	59.013 5 (100), 71.013 5 (43), 143.107 6 (31)
M28	3.322	MLP-2H-B	+Me	341.138 9	341.137 6	-3.81	40	Neg	79.957 1 (100), 124.007 2 (24), 153.129 1 (8)
M29	1.783	MD-D	-Me	355.081 9	355.083 3	3.94	40	Neg	59.013 0 (14), 135.044 7 (100), 179.033 9 (9)
M30	10.849	RO-D	+Me	867.474 2	867.470 8	-3.92	30	Neg	61.987 5 (11), 112.985 1 (100), 783.487 2 (3)
M31	7.836	RO-C	-OH	879.474 3	879.470 5	-4.32	30	Neg	61.987 6 (10), 112.985 1 (100), 391.284 2 (4)
M32	10.548	RO-C	+H₂	897.484 8	897.481 2	-4.01	20	Neg	61.987 8 (5), 112.985 2 (100), 130.943 0 (21)
M33	5.245	Rg1	-OH	783.489 5	783.489 0	-0.64	40	Neg	165.055 5 (100), 287.164 2 (23), 405.262 7 (29)
M34	11.489	Rg1	+Me	813.500 0	813.498 4	-1.97	30	Neg	61.988 0 (1), 89.024 1 (6), 637.431 2 (100)
M35	10.242	Rd	-Me	931.526 7	931.526 8	0.11	30	Neg	61.987 7 (46), 885.521 2 (100), 915.530 3 (6)
M36	11.390	20S-Rg2	-Me-OH	753.478 9	753.477 6	-1.73	40	Neg	89.023 7 (100), 149.045 1 (58), 283.264 1 (21)
M37	11.062	20R-Rh2	+GlcA	797.468 8	797.467 2	-2.01	40	Neg	61.987 5 (100), 89.024 3 (94), 149.045 3 (45)
M38	2.055	DSZ-A	-OMe	387.217 1	387.220 4	8.52	30	Pos	61.039 7 (14), 83.085 5 (11), 316.311 6 (100)
M39	1.860	DSZ-A	+OH	433.222 6	433.224 7	4.85	30	Pos	88.111 8 (57), 100.111 7 (100), 226.107 0 (9)
M40	3.448	DSZ-B	+OH	417.191 3	417.189 6	-4.07	30	Pos	81.069 8 (59), 109.101 2 (100), 123.117 2 (48)
M41	3.036	SS-A	+OH	449.217 5	449.213 1	-9.79	30	Pos	85.028 4 (43), 146.027 0 (100), 204.123 6 (43)
M42	2.286	SS-A	+2O-2H	463.196 8	463.195 1	-3.67	30	Pos	70.064 9 (6), 114.065 9 (12), 285.074 9 (100)
M43	4.908	SS-A	-OMe	403.212 0	403.210 9	-2.73	30	Pos	145.101 1 (16), 302.114 7 (55), 413.266 2 (100)
M44	5.479	SS-A	-OH	417.227 7	417.226 8	-2.16	30	Pos	357.278 6 (2), 403.283 4 (5), 415.281 7 (100),
M45	5.205	SS-A	+GlcA	609.254 7	609.258 1	5.58	30	Pos	59.059 8 (11), 114.065 7 (100), 149.132 8 (15)
M46	3.727	SS-B	-Me	403.175 7	403.173 6	-5.21	30	Pos	57.069 3 (100), 114.065 5 (46), 234.148 7 (71)
M47	3.497	SS-B	+OH	433.186 2	433.184 9	-3.00	30	Pos	84.080 7 (26), 125.096 0 (52), 203.110 3 (100)
M48	5.254	SS-B	-OMe	387.180 7	387.179 3	-3.62	30	Pos	72.080 9 (100), 111.080 4 (35), 350.305 5 (28)
M49	5.081	SS-B	-OH	401.196 4	401.195 5	-2.24	30	Pos	242.093 3 (20), 302.114 8 (100), 355.262 1 (32)
M50	4.074	SS-B	-OH	499.233 2	499.230 2	-6.01	30	Pos	76.075 0 (41), 114.065 7 (100), 136.047 0 (20)
M51	4.202	GMS-D	-OH	515.228 1	515.225 3	-5.43	30	Pos	60.080 3 (23), 85.028 4 (100), 162.112 5 (85)

No.	RT/min	M	Metabolic pathway	Calculated [M-X]^-/[M+X]⁺ (m/z)	Observed[M-X]^-/[M+X]⁺ (m/z)	Error (10^-6)	CE/eV	Ion mode	MS², data, m/z, (ralative, intensity, %)
M1	8.373	DZYS	Iso	461.072 0	461.072 5	1.08	30	Neg	61.987 9 (36), 262.069 2 (33), 285.040 0 (100),
M2	7.751	DZYS	-GlcA	285.039 9	285.040 6	2.46	30	Neg	79.957 2 (100), 124.007 8 (34), 292.122 1 (26)
M3	8.062	DZYS	+SO₃	541.028 8	541.029 2	0.74	30	Neg	113.024 3 (22), 285.040 3 (100), 461.071 8 (26)
M4	12.728	DZYS	+Me	475.087 6	475.088 3	1.47	20	Neg	113.024 3 (82), 200.129 8 (78), 303.087 5 (100)
M5	8.761	DZYS	+H₂	463.087 7	463.088 4	1.51	30	Neg	113.024 9 (10), 166.999 0 (18), 287.057 6 (100)
M6	7.285	DZJS	-OH	429.082 2	429.080 5	-3.96	30	Neg	124.007 4 (100), 316.122 5 (35), 445.190 1 (10)
M7	8.463	DZJS	-GlcA	269.045 0	269.045 8	2.97	30	Neg	96.960 4 (100), 117.034 8 (9), 269.046 0 (3)
M8	8.451	DZJS	+SO₃	525.033 9	525.034 7	1.52	30	Neg	113.024 5 (32), 269.045 9 (100), 461.072 9 (13)
M9	10.629	DZJS	+H₂	447.092 8	447.093 4	1.34	30	Neg	96.960 3 (8), 255.080 7 (100), 459.206 2 (39)
M10	7.943	DZJS	+Me	459.092 7	459.094 6	4.14	20	Neg	113.024 7 (50), 283.062 0 (100), 388.995 5 (23)
M11	3.864	CA	+SO₃	258.991 2	258.992 1	3.48	30	Neg	79.957 3 (3), 135.045 3 (100), 179.035 1 (8)
M12	10.317	CA	+GSH	486.118 2	486.121 9	7.61	30	Neg	96.960 4 (3), 255.081 0 (6), 413.201 8 (100)
M13	4.874	CA	+GlcA	355.066 5	355.067 2	1.97	30	Neg	61.988 2 (21), 135.045 4 (100), 179.035 3 (62)
M14	7.683	CA	+Me	193.050 0	193.049 8	-1.04	20	Neg	59.013 6 (3), 109.066 1 (6), 160.040 6 (100)
M15	2.877	CA	-OH	163.039 5	163.040 2	4.29	20	Neg	65.039 2 (2), 106.042 1 (100), 119.050 0 (31)
M16	4.918	CA	+H₂	181.050 1	181.050 7	3.31	30	Neg	79.957 5 (18), 107.050 6 (100), 108.053 8 (5)
M17	7.131	3-CQA	+Me	367.102 9	367.104 2	3.54	30	Neg	59.013 7 (23), 96.960 5 (100), 171.103 3 (8)
M18	4.866	3-CQA	+GlcA	529.119 3	529.120 7	2.65	20	Neg	187.007 6 (100), 188.010 8 (4), 242.997 7 (23)
M19	4.252	3-CQA	+H₂	355.103 0	355.103 3	0.84	30	Neg	80.964 9 (8), 137.061 2 (100), 181.050 9 (3)
M20	9.462	1, 5-CQA	-OH	499.124 1	499.128 1	8.01	30	Neg	113.024 6 (9), 285.041 0 (100), 417.083 3 (22)
M21	11.095	1, 5-CQA	+Me	529.134 6	529.137 7	5.86	30	Neg	254.072 5 (42), 371.148 5 (100), 411.184 8 (11)
M22	10.248	1, 5-CQA	+H₂	517.134 7	517.135 1	0.77	30	Neg	137.060 8 (4), 181.050 8 (100), 365.087 9 (20)
M23	1.982	1, 5-CQA	De	191.055 6	191.055 6	0.00	30	Neg	79.957 1 (51), 109.029 9 (11), 125.024 1 (100)
M24	2.338	MD-2H-A	-Me	313.071 3	313.075 2	12.46	40	Neg	79.957 3 (100), 96.960 5 (41), 124.007 4 (24)
M25	2.695	MLP-A	-OH	323.092 0	323.090 7	-4.02	40	Neg	59.013 4 (27), 79.957 4 (100), 124.007 5 (48)
M26	2.371	MLP-2H-B	-OH	311.128 4	311.129 3	2.89	40	Neg	79.957 4 (100), 106.980 8 (9), 135.045 4 (16)
M27	2.818	MLP-2H-B	-Me-OH	297.112 7	297.113 7	3.37	40	Neg	59.013 5 (100), 71.013 5 (43), 143.107 6 (31)
M28	3.322	MLP-2H-B	+Me	341.138 9	341.137 6	-3.81	40	Neg	79.957 1 (100), 124.007 2 (24), 153.129 1 (8)
M29	1.783	MD-D	-Me	355.081 9	355.083 3	3.94	40	Neg	59.013 0 (14), 135.044 7 (100), 179.033 9 (9)
M30	10.849	RO-D	+Me	867.474 2	867.470 8	-3.92	30	Neg	61.987 5 (11), 112.985 1 (100), 783.487 2 (3)
M31	7.836	RO-C	-OH	879.474 3	879.470 5	-4.32	30	Neg	61.987 6 (10), 112.985 1 (100), 391.284 2 (4)
M32	10.548	RO-C	+H₂	897.484 8	897.481 2	-4.01	20	Neg	61.987 8 (5), 112.985 2 (100), 130.943 0 (21)
M33	5.245	Rg1	-OH	783.489 5	783.489 0	-0.64	40	Neg	165.055 5 (100), 287.164 2 (23), 405.262 7 (29)
M34	11.489	Rg1	+Me	813.500 0	813.498 4	-1.97	30	Neg	61.988 0 (1), 89.024 1 (6), 637.431 2 (100)
M35	10.242	Rd	-Me	931.526 7	931.526 8	0.11	30	Neg	61.987 7 (46), 885.521 2 (100), 915.530 3 (6)
M36	11.390	20S-Rg2	-Me-OH	753.478 9	753.477 6	-1.73	40	Neg	89.023 7 (100), 149.045 1 (58), 283.264 1 (21)
M37	11.062	20R-Rh2	+GlcA	797.468 8	797.467 2	-2.01	40	Neg	61.987 5 (100), 89.024 3 (94), 149.045 3 (45)
M38	2.055	DSZ-A	-OMe	387.217 1	387.220 4	8.52	30	Pos	61.039 7 (14), 83.085 5 (11), 316.311 6 (100)
M39	1.860	DSZ-A	+OH	433.222 6	433.224 7	4.85	30	Pos	88.111 8 (57), 100.111 7 (100), 226.107 0 (9)
M40	3.448	DSZ-B	+OH	417.191 3	417.189 6	-4.07	30	Pos	81.069 8 (59), 109.101 2 (100), 123.117 2 (48)
M41	3.036	SS-A	+OH	449.217 5	449.213 1	-9.79	30	Pos	85.028 4 (43), 146.027 0 (100), 204.123 6 (43)
M42	2.286	SS-A	+2O-2H	463.196 8	463.195 1	-3.67	30	Pos	70.064 9 (6), 114.065 9 (12), 285.074 9 (100)
M43	4.908	SS-A	-OMe	403.212 0	403.210 9	-2.73	30	Pos	145.101 1 (16), 302.114 7 (55), 413.266 2 (100)
M44	5.479	SS-A	-OH	417.227 7	417.226 8	-2.16	30	Pos	357.278 6 (2), 403.283 4 (5), 415.281 7 (100),
M45	5.205	SS-A	+GlcA	609.254 7	609.258 1	5.58	30	Pos	59.059 8 (11), 114.065 7 (100), 149.132 8 (15)
M46	3.727	SS-B	-Me	403.175 7	403.173 6	-5.21	30	Pos	57.069 3 (100), 114.065 5 (46), 234.148 7 (71)
M47	3.497	SS-B	+OH	433.186 2	433.184 9	-3.00	30	Pos	84.080 7 (26), 125.096 0 (52), 203.110 3 (100)
M48	5.254	SS-B	-OMe	387.180 7	387.179 3	-3.62	30	Pos	72.080 9 (100), 111.080 4 (35), 350.305 5 (28)
M49	5.081	SS-B	-OH	401.196 4	401.195 5	-2.24	30	Pos	242.093 3 (20), 302.114 8 (100), 355.262 1 (32)
M50	4.074	SS-B	-OH	499.233 2	499.230 2	-6.01	30	Pos	76.075 0 (41), 114.065 7 (100), 136.047 0 (20)
M51	4.202	GMS-D	-OH	515.228 1	515.225 3	-5.43	30	Pos	60.080 3 (23), 85.028 4 (100), 162.112 5 (85)

基于预测数据库发现中药灯盏生脉在大鼠体内的代谢产物的新分析策略研究

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李元元 ^# , 郑博文 ^# , 马聪玉 , 生宁 ^* , 张金兰 ^*

药学学报 | 研究论文 2023,58(8): 2468-2475

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药学学报 |研究论文 2023 , 58 (8) : 2468 -2475

基于预测数据库发现中药灯盏生脉在大鼠体内的代谢产物的新分析策略研究

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李元元^#, 郑博文^#, 马聪玉, 生宁^*, 张金兰^*

作者信息

中国医学科学院、北京协和医学院药物研究所, 天然药物活性物质与功能国家重点实验室, 北京 100050

通讯作者:

*生宁, E-mail: shengcc@imm.ac.cn;

张金兰, E-mail: zhjl@imm.ac.cn

Novel strategy in Dengzhan Shengmai capsule metabolites analysis based on the prediction database

Yuan-yuan LI, Bo-wen ZHENG, Cong-yu MA, Ning SHENG^*, Jin-lan ZHANG^*

Affiliations

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

出版时间: 2023-08-12 doi: 10.16438/j.0513-4870.2023-0528

文章导航

摘要

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灯盏生脉胶囊是一种复方中成药, 由灯盏细辛、人参、麦冬和五味子四味药材组成, 包含了黄酮类、木脂素类、皂苷类及有机酸类等药效成分。其临床应用广泛, 主要用于治疗缺血性心脑血管疾病、痴呆等疾病。本研究提出了一种基于预测数据库对多成分中药代谢产物鉴定的研究策略, 并以灯盏生脉胶囊为例展开研究。具体研究策略为: 基于灯盏生脉预测代谢产物数据库, 使用高分辨率UPLC-Q-TOF/MS分析方法分析给药前后大鼠胆汁、尿液和粪便等生物样本, 通过分析结果与数据库对比识别可能的代谢产物, 并基于原型成分质谱裂解规律、一级和二级高分辨离子信息对代谢产物进行鉴定。最终在大鼠胆汁、尿液和粪便样本中共鉴定51个灯盏生脉代谢产物, 主要类别为黄酮类、有机酸类和木脂素类代谢产物。本研究为中药及其复方体内代谢产物鉴定及代谢途径分析提供了新策略, 为全面识别和鉴定活性成分代谢产物, 以深入了解中药体内代谢机制提供了切实可行的手段。实验遵守中国医学科学院药物研究所的动物实验伦理委员会制定的规程(动物实验伦理审查编号: No. 00003645)。

关键词

灯盏生脉 / 超高效液相色谱-飞行时间质谱联用 / 代谢产物鉴定 / 中药代谢

Abstract

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Dengzhan Shengmai capsule, as a compound Chinese patent medicine, consists of four herbs: Herba Erigerontis, Ginseng, Ophiopogon, and Schisandrae Chinensis Fructus, and contains significant components of flavonoids, lignans, saponins, and organic acids. It is widely used clinically to treat cerebrovascular diseases such as chronic cerebral hypoperfusion and dementia with remarkable efficacy. This study proposes a research strategy for multi-component traditional Chinese medicine metabolites based on prediction databases and unfolds the analysis using Dengzhan Shengmai capsule as an example. Using the UPLC-Q-TOF/MS method, the analytical method was established and detected biological samples such as urine, feces, and bile of rats before and after administration based on the prediction of theoretical metabolites of Dengzhan Shengmai capsule. The possible secondary fragment ion information of metabolites was identified by comparing the detected results with prediction databases. The metabolites were identified based on the archetypal component mass spectrometric cleavage law and multistage mass spectrometric data. 51 metabolites, mainly flavonoid, organic acid, and lignan constituents, were finally identified from rat biosamples based on 306 theoretical metabolites of Dengzhan Shengmai capsule. This study provides a new strategy for the identification of metabolites in vivo and the analysis of metabolic pathways of TCM. The study complied with the procedures established by the Animal Experiment Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences and passed the animal experiment ethics examine (No. 00003645).

Key words

Dengzhan Shengmai / UPLC-Q-TOF-MS / identification of metabolites / metabolism of TCM

引用本文

李元元, 郑博文, 马聪玉, 生宁, 张金兰. 基于预测数据库发现中药灯盏生脉在大鼠体内的代谢产物的新分析策略研究. 药学学报, 2023 , 58 (8) : 2468 -2475 . DOI: 10.16438/j.0513-4870.2023-0528

Yuan-yuan LI, Bo-wen ZHENG, Cong-yu MA, Ning SHENG, Jin-lan ZHANG. Novel strategy in Dengzhan Shengmai capsule metabolites analysis based on the prediction database[J]. Acta Pharmaceutica Sinica, 2023 , 58 (8) : 2468 -2475 . DOI: 10.16438/j.0513-4870.2023-0528

正文

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中药在我国具有悠久的历史, 其成分的复杂性为作用机制及其药效物质的诠释带来较大挑战^[1]。传统中药多为口服用药, 进入体内的多成分决定了中药发挥药效作用的多靶点和多途径的特点^[2]。如何全面准确地对主要中药成分及其代谢物进行识别和鉴定是中药代谢研究的难点之一。随着分析技术的快速发展, 研究人员通过探索并开发适合中药多成分分析的新技术, 有力地推动了中药多成分吸收和代谢研究^[3-5], 但由于中药成分复杂, 体内吸收利用程度差异大, 体内代谢途径多样, 对于体内代谢产物的快速识别和鉴定仍存在困难, 是中药代谢研究中需要突破的关键问题之一。

灯盏生脉胶囊是复方中成药, 由灯盏细辛与生脉饮名方组合, 生脉饮是经典的方剂, 由人参、麦冬和五味子三味药材组成, 出自金元时期李东垣的《内外伤辨惑论》。该药具有益气养阴、活血健脑的功效, 临床上应用广泛, 在治疗慢性心/脑灌注不足、痴呆等疾病上疗效显著^[6-8]。灯盏细辛作为灯盏生脉胶囊中的重要药材, 主要含有黄酮类、咖啡酰奎宁酸、木脂素以及皂苷类化合物等^[9]。其中黄酮类及咖啡酰奎宁酸类化合物的神经保护作用具有广泛的报道, 包括黄酮类化合物灯盏乙素调节GABA能突触功能并抑制小胶质细胞介导的神经炎症^[10-12]; 二咖啡酰奎宁酸类化合物在体外研究中可以有效降低Aβ诱导的氧化应激及细胞损伤等^{[13, 14]}。

本研究以灯盏生脉胶囊为例, 开发了一种中药代谢产物分析研究的新策略。通过分析中药成分体内代谢规律建立灯盏生脉胶囊多成分代谢产物理论数据库, 并根据该数据库建立针对性分析方法对生物样本中的代谢产物进行快速发现, 采集可能的代谢产物的高分辨质谱数据, 通过高分辨质谱数据与理论数据库对比识别样品中的代谢产物并记录保留时间。进一步采集识别代谢产物的二级高分辨质谱碎片信息, 综合分析一级和二级高分辨质谱数据及原型成分的裂解规律, 鉴定大鼠灌胃灯盏生脉后的体内代谢产物, 这个分析策略实现了快速识别和鉴定中药多成分体内代谢物。

材料与方法

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仪器

安捷伦1290液相色谱串联6550 iFunnel Q-TOF四极杆飞行时间质谱仪(美国Agilent Technologies公司); XP205型METTLER TOLEDO十万分之一电子天平(瑞士Mettler Toledo公司); VORTEX-2 GENIE涡旋混合器(美国Scientific Industries公司); KQ2200DV型数控超声仪(昆山市超声仪器有限公司)。

试剂与药品

灯盏生脉喷干粉(批号: 20190023) 由云南生物谷药业股份有限公司提供; 灯盏生脉胶囊原型成分标准品: 咖啡酸(CA; 批号: HC321511)、隐绿原酸(4-CQA; 批号: HR8112S1)、新绿原酸(5-CQA; 批号: HR2042211)、绿原酸(3-CQA; 批号: HS143011)、异绿原酸C (4, 5-CQA; 批号: HA19115B2)、1, 3-二咖啡酰奎宁酸(1, 3-CQA; 批号: HS20545B2)、1, 5-二咖啡酰奎宁酸(1, 5-CQA; 批号: HS20725B1)、灯盏甲素(DZJS; 批号: HR2105W9)、灯盏乙素(DZYS; 批号: HR2141B1)、麦冬黄烷酮D (MD-D; 批号: HR2713W2)、甲基麦冬黄酮A (MLP-A; 批号: HM2711W1)、甲基麦冬二氢高异黄酮A (MPL-2H-A; 批号: HM2712W1)、甲基麦冬二氢高异黄酮B (MLP-2H-B; 批号: HM2712W2)、麦冬二氢高异黄酮A (MD-2H-A; 批号: HR2717W1)、人参皂苷R-Rg2 (R-Rg2; 批号: HR4189W2)、人参皂苷20S-Rg2 (20S-Rg2; 批号: HG027746198)、人参皂苷20S-Rg3 (20S-Rg3; 批号: HG027161198)、人参皂苷Rb1 (Rb1; 批号: HG027159198)、人参皂苷Re (Re; 批号: HG027166198)、人参皂苷Rb2 (Rb2; 批号: HG027160198)、人参皂苷Rc (Rc; 批号: HG027168198)、人参皂苷Rd (Rd; 批号: HR2072W7)、人参皂苷S-Rh1 (S-Rh1; 批号: HR17711S1)、人参皂苷R-Rh1 (R-Rh1; 批号: HG027748198)、人参皂苷20R-Rh2 (20R-Rh2; 批号: HG027746198)、人参皂苷Rg1 (Rg1; 批号: HG027445198)、麦冬皂苷C (RO-C; 批号: HS20613B1)、麦冬皂苷D (RO-D; 批号: HS20611B1)、麦冬皂苷D' (RO-D'; 批号: HR1112W9)、五味子甲素(DSZ-A; 批号: HS19906B1)、五味子醇甲(SS-A; 批号: HS19910B3)、五味子酯甲(SST-A; 批号: HS20127B1)、五味子乙素(DSZ-B; 批号: HR0933W2)、五味子醇乙素(SS-B; 批号: HS19911B1)、五味子酯乙素(SST-B; 批号: HS2069W2)、五味子丙素(DSZ-C; 批号: HS19906B3)、戈米辛G (GMS-G; 批号: HR1131W2)、戈米辛D (GMS-D; 批号: HS19117B1)、戈米辛E (GMS-E; 批号: HS201125B2)、戈米辛O (GMS-O; 批号: HR0397W1) 均购自宝鸡市辰光生物科技有限公司, HPLC纯度均大于98%; 质谱级甲醇(批号: 198728)、色谱级乙酸乙酯(批号: 203771)、色谱级异丙醇(批号: 207073) 均购自美国Fisher Scientific公司; 质谱级甲酸(批号: 191747) 购自美国Thermo公司; 色谱级乙酸铵(批号: MKCC8724) 购自美国Sigma-Aldrich公司; 纯净水(杭州娃哈哈公司)。

实验动物

SPF级雄性大鼠, 约8周龄, 体重220 ± 10 g, 购自北京维通利华实验动物技术有限公司, 许可证号SCXK (京) 2016-0006。大鼠在中国医学科学院药物研究所动物房清洁间中饲养, 每天12 h光照, 温度为23 ± 2 ℃, 湿度为55% ± 5%, 自由饮水进食。实验遵守中国医学科学院药物研究所的动物实验伦理委员会制定的规程(动物实验伦理审查编号: No. 00003645)。

色谱条件

使用Waters ACQUITY UPLC BEH C18色谱柱(2.1 mm × 100 mm, 1.7 μm); 预柱为Waters ACQUITY UPLC BEH C18 VanGuard Pre-column (2.1 mm × 5 mm, 1.7 μm); 柱温: 30 ℃; 梯度洗脱。

有机酸类及灯盏细辛黄酮类成分流动相为0.1%甲酸水(A)-0.1%甲酸乙腈(B), 洗脱梯度为: 0.0~5.0 min, 10%~25% B; 5.0~7.0 min, 25%~30% B; 7.0~8.0 min, 30% B; 8.0~9.0 min, 30%~40% B; 9.0~12.0 min, 40%~50% B; 12.0~15.0 min, 50%~100% B; post run: 1 min; 流速0.3 mL·min^-1; 进样量: 5 μL。

皂苷类和麦冬黄酮类成分流动相为0.1 mmol·L^-1乙酸铵水(A)-0.1 mmol·L^-1乙酸铵乙腈(B), 洗脱梯度为: 0.0~1.0 min, 20%~60% B; 1.0~2.0 min, 60%~65% B; 2.0~7.0 min, 65%~80% B; 7.0~12.0 min, 80%~90% B; 12.0~15.0 min, 90%~100% B; post run: 1 min; 流速0.3 mL·min^-1; 进样量: 5 μL。

木脂素类成分流动相为0.1 mmol·L^-1乙酸铵水(A)-0.1 mmol·L^-1乙酸铵乙腈(B), 洗脱梯度为0.0~3.0 min, 20%~80% B; 3.0~4.0 min, 80%~85% B; 4.0~8.0 min, 85%~95% B; 8.0~8.5 min, 95%~100% B; 8.5~10.0 min, 100% B; post run: 1 min; 流速: 0.3 mL·min^-1; 进样量: 5 μL。

质谱条件

使用Dual AJS ESI离子源; 正离子模式下分析木脂素类化合物, 负离子模式下分析有机酸、黄酮及皂苷类化合物; 鞘气温度: 350 ℃; 鞘气流速: 12 L·min^-1; 干燥气温度: 280 ℃; 干燥气流速: 12 L·min^-1; 雾化气压: 45 psi; 毛细管电压: 2 000 V; 喷嘴电压500 V; 一级质谱采集范围: m/z 100~1 600; 二级质谱采集范围: m/z 50~1 600; 分辨率: 40 000。

给药液制备

取灯盏生脉喷干粉2 g, 置10 mL量瓶中, 加生理盐水溶解定容, 涡旋3 min, 超声15 min后得到0.2 g·mL^-1灯盏生脉给药液, 临用前新鲜配制。

生物样品采集

雄性SD大鼠置于代谢笼中, 禁食12 h, 自由饮水, 收集大鼠尿液及粪便样品后灌胃给予灯盏生脉给药液(0.72 g·kg^-1) 分别收集大鼠给药后0~4、4~8、8~12、12~24 h时间段尿液和粪便样品, 尿液样品记录体积, 粪便样品烘干后存于-80 ℃冰箱待分析^[10]。

雄性SD大鼠禁食12 h后, 5 mL·kg^-1腹腔注射20% (w/v) 乌拉坦麻醉后固定于手术板。上腹部正中线开口约2 cm, 在分离胆总管, 并在近肝端插管固定。缝合腹肌及皮肤后塑料管引至腹腔外, 收集大鼠空白胆汁样品后灌胃给予灯盏生脉给药液(0.72 g·kg^-1), 分别收集大鼠给药后0~4、4~8、8~12、12~24 h时间段的胆汁样品, 记录体积, 样品放置在-80 ℃冰箱直至分析。

生物样品处理

精密移取1 mL大鼠胆汁/尿液样品或1 g大鼠粪便样品, 加入8.0 mL含1%甲酸的乙酸乙酯/异丙醇(3∶7, v/v) 溶液, 超声提取30 min, 涡旋提取15 min, 4 500 r·min^-1离心20 min。取上清液, 氮气吹干后, 残渣加入50%甲醇100 μL, 涡旋3 min复溶, 13 000 r·min^-1离心5 min, 取上清液转移至液相小瓶中, UHPLC-Q-TOF-MS仪器进样分析。

数据处理

应用安捷伦MassHunter Qualitative Software 10.0软件, 采用上述建立的UPLC-Q-TOF-MS定性分析方法, 分析经过前处理的生物样品(尿、粪、胆汁), 分别获得原型成分和代谢产物的保留时间、一级高分辨质谱数据和二级高分辨质谱数据等信息。通过与标准品比对、代谢物理论数据库比较、高分辨质量数并结合二级高分辨质谱数据的信息鉴定灯盏生脉的体内代谢产物。

结果

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1 理论代谢产物数据库建立

根据Ⅰ相代谢转化类型包括羟基化(+O)、甲基羧化(+2O-2H)、去甲基化(-CH₃)、去羟基化(-O)、去甲氧基化(-OCH₃)、去甲基去羟基化(-CH₂-O)、还原(+H₂) 和脱葡萄糖醛酸化(-C₆H₈O₆); Ⅱ相代谢转化类型包括葡萄糖醛酸结合(+C₆H₈O₆)、谷胱甘肽结合(+C₁₀H₁₇N₃O₆S)、硫酸结合(+SO₃) 和甲基化结合(+CH₃) 等对灯盏生脉中主要成分: 黄酮类、有机酸、木脂素以及皂苷类成分可能的代谢产物进行预测, 建立理论数据库, 数据库信息共包括309个理论代谢产物: 黄酮类代谢产物51个; 有机酸类代谢产物60个; 皂苷类代谢产物105个及木脂素类代谢产物93个。

黄酮类以灯盏乙素为例, 灯盏乙素可能存在7个体内代谢产物(图 1A), 包括灯盏乙素还原代谢产物、灯盏乙素脱葡萄糖醛酸化代谢产物(灯盏乙素苷元)、灯盏乙素同分异构体、灯盏乙素硫酸化代谢产物、灯盏乙素谷胱甘肽结合产物、灯盏乙素甲基化代谢产物和灯盏乙素去羟基化代谢产物等。

木脂素类化合物如五味子甲素则理论上包含五味子甲素羟基化代谢产物、五味子甲素去甲基化代谢产物、五味子甲素去甲氧基代谢产物、五味子甲素羟基化去甲基化代谢产物等(图 1B)。

有机酸类以4, 5-CQA为例(图 1C), 其可能存在9个体内代谢产物, 包括4, 5-CQA葡萄糖醛酸化代谢产物、4, 5-CQA硫酸酯化代谢产物、4, 5-CQA谷胱甘肽结合代谢产物、4, 5-CQA甲基化代谢产物、4, 5-CQA脱羟基代谢产物、4, 5-CQA还原代谢产物、4, 5-CQA分解为咖啡酸、4, 5-CQA分解为奎尼酸以及4, 5-CQA分解的奎尼酸脱水代谢产物等。

皂苷类以人参皂苷Rg1为例(图 1D), 包含人参皂苷Rg1葡萄糖醛酸化代谢产物、人参皂苷Rg1硫酸酯化代谢产物、人参皂苷Rg1谷胱甘肽结合代谢产物、人参皂苷Rg1甲基化代谢产物、人参皂苷Rg1脱甲基代谢产物、人参皂苷Rg1脱羟基代谢产物、人参皂苷Rg1还原代谢产物以及人参皂苷Rg1脱甲基脱羟基代谢产物等。

2 代谢产物分析方法建立

综合考虑灯盏生脉胶囊中不同类型化合物及其代谢物的理化性质不同, 分别在正、负离子模式下对代表性原型成分色谱及质谱条件进行了优化。结果显示, 流动相体系可显著改善不同类型成分的质谱响应, 通过对不同流动相条件下各类化合物总离子流图(TIC) 的对比, 发现有机酸类和灯盏细辛等黄酮类化合物在水-甲醇流动相基础上添加0.1%甲酸可显著增强质谱响应; 麦冬黄酮类、皂苷类和木脂素类化合物添加0.1 mmol·L^-1乙酸铵可显著增强质谱响应(图 2)。

为提高目标化合物的电离效率, 使用各类成分代表性化合物的对照品配制了混合标准溶液(500 ng·mL^-1), 进行质谱参数优化。各类成分代表性化合物在不同二级碰撞电压(CE) 下峰面积如图 3所示。优化后最终的质谱参数为采用ESI源正离子模式分析木脂素类化合物, CE电压为25 eV; 负离子模式分析有机酸类、黄酮类和皂苷类化合物, CE电压分别为15、25和25 eV。鞘气温度: 350 ℃; 鞘气流速: 12 L·min^-1; 干燥气温度: 280 ℃; 干燥气流速: 12 L·min^-1; 雾化气压: 45 psi; 毛细管电压: 2 000 V; 喷嘴电压: 500 V。

基于实验室前期研究, 采用了乙酸乙酯/异丙醇(3∶7, v/v) 溶剂体系提取生物样品(胆汁、尿液和粪便) 中代谢物, 考察了乙酸乙酯/异丙醇(3∶7, v/v) 提取溶剂中加入不同比例的甲酸或乙酸(图 4) 的提取效果, 比较了各类成分代表性化合物在不同比例有机酸提取条件下的峰面积, 最终选择含1%甲酸的乙酸乙酯/异丙醇溶液(3∶7, v/v) 提取各类大鼠生物样品中代谢产物。

3 给药后大鼠胆汁、尿液和粪便中代谢产物分析

基于上述确定的分析方法, 对大鼠给药后0~4、4~8、8~12、12~24 h时间段采集的生物样品进行分析, 采集高分辨一级质谱数据, 并将所得数据与代谢产物理论数据库进行对比, 扣除空白生物样品中的干扰性成分。识别大鼠灌胃灯盏生脉后胆汁、尿液及粪便中可能的代谢产物, 记录其保留时间, 进一步在质谱Auto MS/MS模式下采集相关二级质谱数据。通过与40种灯盏生脉原型标准品和二级质谱信息对照, 总结不同类型灯盏生脉代谢产物裂解规律。最终在大鼠生物样品中共鉴定到可能的代谢产物51个(表 1), 其中有机酸类和灯盏细辛黄酮类代谢产物23个(图 5), 皂苷类和麦冬黄酮类代谢产物14个(图 6) 及木脂素类代谢产物14个(图 7)。结果显示灯盏生脉在大鼠体内的代谢产物主要代谢途径包括: 羟基化(+O)、去甲基化(-CH₃)、去羟基化(-O)、还原(+H₂)、脱葡萄糖醛酸化(-C₆H₈O₆)、葡萄糖醛酸结合(+C₆H₈O₆)、硫酸酯化(+SO₃) 和甲基化结合(+CH₃) 等。同时, 根据已鉴定的代谢产物结果在大鼠血浆中发现了15个代谢产物。

讨论

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本研究探索了一种新的中药多成分代谢产物的分析策略, 首先预测各类型成分可能的体内代谢产物, 覆盖多种结构类型的成分, 建立中药多成分Ⅰ相和Ⅱ相代谢产物的理论数据库。建立生物样本代谢产物的液质联用分析方法, 并优化分析条件, 采用优化后的方法采集生物样本中代谢产物一级和二级高分辨质谱数据。将生物样本中采集的代谢产物一级质谱数据与理论数据库对比, 发现和识别生物样本中可能的代谢产物, 然后采集不同碰撞电压下生物样本中已识别的代谢产物二级碎片离子信息, 并基于原型成分质谱裂解规律和代谢产物的多级质谱数据对代谢产物进行鉴定。

以中成药灯盏生脉胶囊为例, 依据Ⅰ相和Ⅱ相代谢转化规律建立理论代谢产物数据库: 包括黄酮类、有机酸类、皂苷类和木脂素类成分理论代谢产物309个。分别针对有机酸类及灯盏细辛黄酮类成分、皂苷类及麦冬黄酮类以及木脂素类成分优化了前处理及色谱质谱条件, 并分析大鼠给药前后的生物样品。按照建立的代谢产物分析策略运行, 最终在大鼠生物样本中共鉴定到代谢产物共51个, 其中胆汁样品中鉴定到的代谢产物共14个; 尿液样品中鉴定到的代谢产物共28个; 粪便样品中鉴定到的代谢产物共30个。

中药多成分体内代谢的研究是诠释中药物质基础和药效机制的重要内容。通过对中药多成分在体内的吸收、分布、代谢和排泄过程的深入研究, 可以更全面清晰地分析中药在体内移行变化规律, 为阐释中药药效过程和机制、开发针对性治疗新药起到至关重要的作用。本研究开发的理论代谢产物数据库、各类成分针对性分析方法、生物样本代谢产物鉴定分析的代谢产物研究模式为今后中药多成分代谢产物研究提供了新的研究策略, 并可进一步迁移方法至三重四极杆质谱等进行代谢产物定量分析, 为深入研究多成分体内代谢模式及移行变化规律提供有力技术支撑。

作者贡献: 李元元负责主要的实验设计、部分实验操作和数据统计分析; 郑博文负责文献检索、数据统计分析和论文撰写; 马聪玉负责部分实验操作、文献检索; 生宁指导实验设计; 张金兰负责构思、设计本研究; 生宁、张金兰进行论文修改和基金获得。

利益冲突: 所有作者无任何利益冲突。

基金

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中国医学科学院医学与健康科技创新工程项目(2022-I2M-2-002)
国家自然科学青年基金资助项目(82204341)
科技部重点研发计划(2019YFC1708901)

参考文献

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2023年第58卷第8期

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文章信息

doi: 10.16438/j.0513-4870.2023-0528

接收时间：2023-04-29
首发时间：2025-11-21
出版时间：2023-08-12

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收稿日期：2023-04-29
修回日期：2023-07-13

基金

中国医学科学院医学与健康科技创新工程项目(2022-I2M-2-002)

国家自然科学青年基金资助项目(82204341)

科技部重点研发计划(2019YFC1708901)

作者信息

中国医学科学院、北京协和医学院药物研究所, 天然药物活性物质与功能国家重点实验室, 北京 100050

通讯作者:

*生宁, E-mail: shengcc@imm.ac.cn;

张金兰, E-mail: zhjl@imm.ac.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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TxT

No.	RT/min	M	Metabolic pathway	Calculated [M-X]^-/[M+X]⁺ (m/z)	Observed[M-X]^-/[M+X]⁺ (m/z)	Error (10^-6)	CE/eV	Ion mode	MS², data, m/z, (ralative, intensity, %)
M1	8.373	DZYS	Iso	461.072 0	461.072 5	1.08	30	Neg	61.987 9 (36), 262.069 2 (33), 285.040 0 (100),
M2	7.751	DZYS	-GlcA	285.039 9	285.040 6	2.46	30	Neg	79.957 2 (100), 124.007 8 (34), 292.122 1 (26)
M3	8.062	DZYS	+SO₃	541.028 8	541.029 2	0.74	30	Neg	113.024 3 (22), 285.040 3 (100), 461.071 8 (26)
M4	12.728	DZYS	+Me	475.087 6	475.088 3	1.47	20	Neg	113.024 3 (82), 200.129 8 (78), 303.087 5 (100)
M5	8.761	DZYS	+H₂	463.087 7	463.088 4	1.51	30	Neg	113.024 9 (10), 166.999 0 (18), 287.057 6 (100)
M6	7.285	DZJS	-OH	429.082 2	429.080 5	-3.96	30	Neg	124.007 4 (100), 316.122 5 (35), 445.190 1 (10)
M7	8.463	DZJS	-GlcA	269.045 0	269.045 8	2.97	30	Neg	96.960 4 (100), 117.034 8 (9), 269.046 0 (3)
M8	8.451	DZJS	+SO₃	525.033 9	525.034 7	1.52	30	Neg	113.024 5 (32), 269.045 9 (100), 461.072 9 (13)
M9	10.629	DZJS	+H₂	447.092 8	447.093 4	1.34	30	Neg	96.960 3 (8), 255.080 7 (100), 459.206 2 (39)
M10	7.943	DZJS	+Me	459.092 7	459.094 6	4.14	20	Neg	113.024 7 (50), 283.062 0 (100), 388.995 5 (23)
M11	3.864	CA	+SO₃	258.991 2	258.992 1	3.48	30	Neg	79.957 3 (3), 135.045 3 (100), 179.035 1 (8)
M12	10.317	CA	+GSH	486.118 2	486.121 9	7.61	30	Neg	96.960 4 (3), 255.081 0 (6), 413.201 8 (100)
M13	4.874	CA	+GlcA	355.066 5	355.067 2	1.97	30	Neg	61.988 2 (21), 135.045 4 (100), 179.035 3 (62)
M14	7.683	CA	+Me	193.050 0	193.049 8	-1.04	20	Neg	59.013 6 (3), 109.066 1 (6), 160.040 6 (100)
M15	2.877	CA	-OH	163.039 5	163.040 2	4.29	20	Neg	65.039 2 (2), 106.042 1 (100), 119.050 0 (31)
M16	4.918	CA	+H₂	181.050 1	181.050 7	3.31	30	Neg	79.957 5 (18), 107.050 6 (100), 108.053 8 (5)
M17	7.131	3-CQA	+Me	367.102 9	367.104 2	3.54	30	Neg	59.013 7 (23), 96.960 5 (100), 171.103 3 (8)
M18	4.866	3-CQA	+GlcA	529.119 3	529.120 7	2.65	20	Neg	187.007 6 (100), 188.010 8 (4), 242.997 7 (23)
M19	4.252	3-CQA	+H₂	355.103 0	355.103 3	0.84	30	Neg	80.964 9 (8), 137.061 2 (100), 181.050 9 (3)
M20	9.462	1, 5-CQA	-OH	499.124 1	499.128 1	8.01	30	Neg	113.024 6 (9), 285.041 0 (100), 417.083 3 (22)
M21	11.095	1, 5-CQA	+Me	529.134 6	529.137 7	5.86	30	Neg	254.072 5 (42), 371.148 5 (100), 411.184 8 (11)
M22	10.248	1, 5-CQA	+H₂	517.134 7	517.135 1	0.77	30	Neg	137.060 8 (4), 181.050 8 (100), 365.087 9 (20)
M23	1.982	1, 5-CQA	De	191.055 6	191.055 6	0.00	30	Neg	79.957 1 (51), 109.029 9 (11), 125.024 1 (100)
M24	2.338	MD-2H-A	-Me	313.071 3	313.075 2	12.46	40	Neg	79.957 3 (100), 96.960 5 (41), 124.007 4 (24)
M25	2.695	MLP-A	-OH	323.092 0	323.090 7	-4.02	40	Neg	59.013 4 (27), 79.957 4 (100), 124.007 5 (48)
M26	2.371	MLP-2H-B	-OH	311.128 4	311.129 3	2.89	40	Neg	79.957 4 (100), 106.980 8 (9), 135.045 4 (16)
M27	2.818	MLP-2H-B	-Me-OH	297.112 7	297.113 7	3.37	40	Neg	59.013 5 (100), 71.013 5 (43), 143.107 6 (31)
M28	3.322	MLP-2H-B	+Me	341.138 9	341.137 6	-3.81	40	Neg	79.957 1 (100), 124.007 2 (24), 153.129 1 (8)
M29	1.783	MD-D	-Me	355.081 9	355.083 3	3.94	40	Neg	59.013 0 (14), 135.044 7 (100), 179.033 9 (9)
M30	10.849	RO-D	+Me	867.474 2	867.470 8	-3.92	30	Neg	61.987 5 (11), 112.985 1 (100), 783.487 2 (3)
M31	7.836	RO-C	-OH	879.474 3	879.470 5	-4.32	30	Neg	61.987 6 (10), 112.985 1 (100), 391.284 2 (4)
M32	10.548	RO-C	+H₂	897.484 8	897.481 2	-4.01	20	Neg	61.987 8 (5), 112.985 2 (100), 130.943 0 (21)
M33	5.245	Rg1	-OH	783.489 5	783.489 0	-0.64	40	Neg	165.055 5 (100), 287.164 2 (23), 405.262 7 (29)
M34	11.489	Rg1	+Me	813.500 0	813.498 4	-1.97	30	Neg	61.988 0 (1), 89.024 1 (6), 637.431 2 (100)
M35	10.242	Rd	-Me	931.526 7	931.526 8	0.11	30	Neg	61.987 7 (46), 885.521 2 (100), 915.530 3 (6)
M36	11.390	20S-Rg2	-Me-OH	753.478 9	753.477 6	-1.73	40	Neg	89.023 7 (100), 149.045 1 (58), 283.264 1 (21)
M37	11.062	20R-Rh2	+GlcA	797.468 8	797.467 2	-2.01	40	Neg	61.987 5 (100), 89.024 3 (94), 149.045 3 (45)
M38	2.055	DSZ-A	-OMe	387.217 1	387.220 4	8.52	30	Pos	61.039 7 (14), 83.085 5 (11), 316.311 6 (100)
M39	1.860	DSZ-A	+OH	433.222 6	433.224 7	4.85	30	Pos	88.111 8 (57), 100.111 7 (100), 226.107 0 (9)
M40	3.448	DSZ-B	+OH	417.191 3	417.189 6	-4.07	30	Pos	81.069 8 (59), 109.101 2 (100), 123.117 2 (48)
M41	3.036	SS-A	+OH	449.217 5	449.213 1	-9.79	30	Pos	85.028 4 (43), 146.027 0 (100), 204.123 6 (43)
M42	2.286	SS-A	+2O-2H	463.196 8	463.195 1	-3.67	30	Pos	70.064 9 (6), 114.065 9 (12), 285.074 9 (100)
M43	4.908	SS-A	-OMe	403.212 0	403.210 9	-2.73	30	Pos	145.101 1 (16), 302.114 7 (55), 413.266 2 (100)
M44	5.479	SS-A	-OH	417.227 7	417.226 8	-2.16	30	Pos	357.278 6 (2), 403.283 4 (5), 415.281 7 (100),
M45	5.205	SS-A	+GlcA	609.254 7	609.258 1	5.58	30	Pos	59.059 8 (11), 114.065 7 (100), 149.132 8 (15)
M46	3.727	SS-B	-Me	403.175 7	403.173 6	-5.21	30	Pos	57.069 3 (100), 114.065 5 (46), 234.148 7 (71)
M47	3.497	SS-B	+OH	433.186 2	433.184 9	-3.00	30	Pos	84.080 7 (26), 125.096 0 (52), 203.110 3 (100)
M48	5.254	SS-B	-OMe	387.180 7	387.179 3	-3.62	30	Pos	72.080 9 (100), 111.080 4 (35), 350.305 5 (28)
M49	5.081	SS-B	-OH	401.196 4	401.195 5	-2.24	30	Pos	242.093 3 (20), 302.114 8 (100), 355.262 1 (32)
M50	4.074	SS-B	-OH	499.233 2	499.230 2	-6.01	30	Pos	76.075 0 (41), 114.065 7 (100), 136.047 0 (20)
M51	4.202	GMS-D	-OH	515.228 1	515.225 3	-5.43	30	Pos	60.080 3 (23), 85.028 4 (100), 162.112 5 (85)

No.

RT/min

Metabolic pathway

Calculated [M-X]^-/[M+X]⁺ (m/z)

Observed[M-X]^-/[M+X]⁺ (m/z)

Error (10^-6)

CE/eV

Ion mode

MS², data, m/z, (ralative, intensity, %)

8.373

DZYS

Iso

461.072 0

461.072 5

1.08

Neg

61.987 9 (36), 262.069 2 (33), 285.040 0 (100),

7.751

DZYS

-GlcA

285.039 9

285.040 6

2.46

Neg

79.957 2 (100), 124.007 8 (34), 292.122 1 (26)

8.062

DZYS

+SO₃

541.028 8

541.029 2

0.74

Neg

113.024 3 (22), 285.040 3 (100), 461.071 8 (26)

12.728

DZYS

+Me

475.087 6

475.088 3

1.47

Neg

113.024 3 (82), 200.129 8 (78), 303.087 5 (100)

8.761

DZYS

+H₂

463.087 7

463.088 4

1.51

Neg

113.024 9 (10), 166.999 0 (18), 287.057 6 (100)

7.285

DZJS

-OH

429.082 2

429.080 5

-3.96

Neg

124.007 4 (100), 316.122 5 (35), 445.190 1 (10)

8.463

DZJS

-GlcA

269.045 0

269.045 8

2.97

Neg

96.960 4 (100), 117.034 8 (9), 269.046 0 (3)

8.451

DZJS

+SO₃

525.033 9

525.034 7

1.52

Neg

113.024 5 (32), 269.045 9 (100), 461.072 9 (13)

10.629

DZJS

+H₂

447.092 8

447.093 4

1.34

Neg

96.960 3 (8), 255.080 7 (100), 459.206 2 (39)

M10

7.943

DZJS

+Me

459.092 7

459.094 6

4.14

Neg

113.024 7 (50), 283.062 0 (100), 388.995 5 (23)

M11

3.864

+SO₃

258.991 2

258.992 1

3.48

Neg

79.957 3 (3), 135.045 3 (100), 179.035 1 (8)

M12

10.317

+GSH

486.118 2

486.121 9

7.61

Neg

96.960 4 (3), 255.081 0 (6), 413.201 8 (100)

M13

4.874

+GlcA

355.066 5

355.067 2

1.97

Neg

61.988 2 (21), 135.045 4 (100), 179.035 3 (62)

M14

7.683

+Me

193.050 0

193.049 8

-1.04

Neg

59.013 6 (3), 109.066 1 (6), 160.040 6 (100)

M15

2.877

-OH

163.039 5

163.040 2

4.29

Neg

65.039 2 (2), 106.042 1 (100), 119.050 0 (31)

M16

4.918

+H₂

181.050 1

181.050 7

3.31

Neg

79.957 5 (18), 107.050 6 (100), 108.053 8 (5)

M17

7.131

3-CQA

+Me

367.102 9

367.104 2

3.54

Neg

59.013 7 (23), 96.960 5 (100), 171.103 3 (8)

M18

4.866

3-CQA

+GlcA

529.119 3

529.120 7

2.65

Neg

187.007 6 (100), 188.010 8 (4), 242.997 7 (23)

M19

4.252

3-CQA

+H₂

355.103 0

355.103 3

0.84

Neg

80.964 9 (8), 137.061 2 (100), 181.050 9 (3)

M20

9.462

1, 5-CQA

-OH

499.124 1

499.128 1

8.01

Neg

113.024 6 (9), 285.041 0 (100), 417.083 3 (22)

M21

11.095

1, 5-CQA

+Me

529.134 6

529.137 7

5.86

Neg

254.072 5 (42), 371.148 5 (100), 411.184 8 (11)

M22

10.248

1, 5-CQA

+H₂

517.134 7

517.135 1

0.77

Neg

137.060 8 (4), 181.050 8 (100), 365.087 9 (20)

M23

1.982

1, 5-CQA

191.055 6

0.00

Neg

79.957 1 (51), 109.029 9 (11), 125.024 1 (100)

M24

2.338

MD-2H-A

-Me

313.071 3

313.075 2

12.46

Neg

79.957 3 (100), 96.960 5 (41), 124.007 4 (24)

M25

2.695

MLP-A

-OH

323.092 0

323.090 7

-4.02

Neg

59.013 4 (27), 79.957 4 (100), 124.007 5 (48)

M26

2.371

MLP-2H-B

-OH

311.128 4

311.129 3

2.89

Neg

79.957 4 (100), 106.980 8 (9), 135.045 4 (16)

M27

2.818

MLP-2H-B

-Me-OH

297.112 7

297.113 7

3.37

Neg

59.013 5 (100), 71.013 5 (43), 143.107 6 (31)

M28

3.322

MLP-2H-B

+Me

341.138 9

341.137 6

-3.81

Neg

79.957 1 (100), 124.007 2 (24), 153.129 1 (8)

M29

1.783

MD-D

-Me

355.081 9

355.083 3

3.94

Neg

59.013 0 (14), 135.044 7 (100), 179.033 9 (9)

M30

10.849

RO-D

+Me

867.474 2

867.470 8

-3.92

Neg

61.987 5 (11), 112.985 1 (100), 783.487 2 (3)

M31

7.836

RO-C

-OH

879.474 3

879.470 5

-4.32

Neg

61.987 6 (10), 112.985 1 (100), 391.284 2 (4)

M32

10.548

RO-C

+H₂

897.484 8

897.481 2

-4.01

Neg

61.987 8 (5), 112.985 2 (100), 130.943 0 (21)

M33

5.245

Rg1

-OH

783.489 5

783.489 0

-0.64

Neg

165.055 5 (100), 287.164 2 (23), 405.262 7 (29)

M34

11.489

Rg1

+Me

813.500 0

813.498 4

-1.97

Neg

61.988 0 (1), 89.024 1 (6), 637.431 2 (100)

M35

10.242

-Me

931.526 7

931.526 8

0.11

Neg

61.987 7 (46), 885.521 2 (100), 915.530 3 (6)

M36

11.390

20S-Rg2

-Me-OH

753.478 9

753.477 6

-1.73

Neg

89.023 7 (100), 149.045 1 (58), 283.264 1 (21)

M37

11.062

20R-Rh2

+GlcA

797.468 8

797.467 2

-2.01

Neg

61.987 5 (100), 89.024 3 (94), 149.045 3 (45)

M38

2.055

DSZ-A

-OMe

387.217 1

387.220 4

8.52

Pos

61.039 7 (14), 83.085 5 (11), 316.311 6 (100)

M39

1.860

DSZ-A

+OH

433.222 6

433.224 7

4.85

Pos

88.111 8 (57), 100.111 7 (100), 226.107 0 (9)

M40

3.448

DSZ-B

+OH

417.191 3

417.189 6

-4.07

Pos

81.069 8 (59), 109.101 2 (100), 123.117 2 (48)

M41

3.036

SS-A

+OH

449.217 5

449.213 1

-9.79

Pos

85.028 4 (43), 146.027 0 (100), 204.123 6 (43)

M42

2.286

SS-A

+2O-2H

463.196 8

463.195 1

-3.67

Pos

70.064 9 (6), 114.065 9 (12), 285.074 9 (100)

M43

4.908

SS-A

-OMe

403.212 0

403.210 9

-2.73

Pos

145.101 1 (16), 302.114 7 (55), 413.266 2 (100)

M44

5.479

SS-A

-OH

417.227 7

417.226 8

-2.16

Pos

357.278 6 (2), 403.283 4 (5), 415.281 7 (100),

M45

5.205

SS-A

+GlcA

609.254 7

609.258 1

5.58

Pos

59.059 8 (11), 114.065 7 (100), 149.132 8 (15)

M46

3.727

SS-B

-Me

403.175 7

403.173 6

-5.21

Pos

57.069 3 (100), 114.065 5 (46), 234.148 7 (71)

M47

3.497

SS-B

+OH

433.186 2

433.184 9

-3.00

Pos

84.080 7 (26), 125.096 0 (52), 203.110 3 (100)

M48

5.254

SS-B

-OMe

387.180 7

387.179 3

-3.62

Pos

72.080 9 (100), 111.080 4 (35), 350.305 5 (28)

M49

5.081

SS-B

-OH

401.196 4

401.195 5

-2.24

Pos

242.093 3 (20), 302.114 8 (100), 355.262 1 (32)

M50

4.074

SS-B

-OH

499.233 2

499.230 2

-6.01

Pos

76.075 0 (41), 114.065 7 (100), 136.047 0 (20)

M51

4.202

GMS-D

-OH

515.228 1

515.225 3

-5.43

Pos

60.080 3 (23), 85.028 4 (100), 162.112 5 (85)