中国药学杂志

No.	Place of origin(in Chinese)	Collection time	No.	Place of origin(in Chinese)	Collection time
S1	Yixian Hebei(河北易县)	2022-7	S16	Handan Hebei(河北邯郸)	2023-8
S2	Yixian, Hebei(河北易县)	2023-7	S17	Handan, Hebei(河北邯郸)	2022-8
S3	Handan, Hebei(河北邯郸)	2022-8	S18	Handan, Hebei(河北邯郸)	2023-8
S4	Handan, Hebei(河北邯郸)	2023-8	S19	Handan, Hebei(河北邯郸)	2022-8
S5	Handan, Hebei(河北邯郸)	2023-8	S20	Handan, Hebei(河北邯郸)	2023-8
S6	Handan, Hebei(河北邯郸)	2023-8	S21	Handan, Hebei(河北邯郸)	2022-8
S7	Handan, Hebei(河北邯郸)	2023-8	S22	Shijiazhuang, Hebei(河北石家庄)	2023-8
S8	Handan, Hebei(河北邯郸)	2023-8	S23	Shijiazhuang, Hebei(河北石家庄)	2022-8
S9	Handan, Hebei(河北邯郸)	2023-8	S24	Shijiazhuang, Hebei(河北石家庄)	2023-8
S10	Handan, Hebei(河北邯郸)	2023-8	S25	Shijiazhuang, Hebei(河北石家庄)	2023-8
S11	Handan, Hebei(河北邯郸)	2022-8	S26	Shijiazhuang, Hebei(河北石家庄)	2022-8
S12	Handan, Hebei(河北邯郸)	2023-8	S27	Shijiazhuang, Hebei(河北石家庄)	2023-8
S13	Handan, Hebei(河北邯郸)	2022-8	S28	Shijiazhuang, Hebei(河北石家庄)	2023-8
S14	Handan, Hebei(河北邯郸)	2023-8	S29	Handan, Hebei(河北邯郸)	2023-8
S15	Handan, Hebei(河北邯郸)	2022-8

No.	Place of origin(in Chinese)	Collection time	No.	Place of origin(in Chinese)	Collection time
S1	Yixian Hebei(河北易县)	2022-7	S16	Handan Hebei(河北邯郸)	2023-8
S2	Yixian, Hebei(河北易县)	2023-7	S17	Handan, Hebei(河北邯郸)	2022-8
S3	Handan, Hebei(河北邯郸)	2022-8	S18	Handan, Hebei(河北邯郸)	2023-8
S4	Handan, Hebei(河北邯郸)	2023-8	S19	Handan, Hebei(河北邯郸)	2022-8
S5	Handan, Hebei(河北邯郸)	2023-8	S20	Handan, Hebei(河北邯郸)	2023-8
S6	Handan, Hebei(河北邯郸)	2023-8	S21	Handan, Hebei(河北邯郸)	2022-8
S7	Handan, Hebei(河北邯郸)	2023-8	S22	Shijiazhuang, Hebei(河北石家庄)	2023-8
S8	Handan, Hebei(河北邯郸)	2023-8	S23	Shijiazhuang, Hebei(河北石家庄)	2022-8
S9	Handan, Hebei(河北邯郸)	2023-8	S24	Shijiazhuang, Hebei(河北石家庄)	2023-8
S10	Handan, Hebei(河北邯郸)	2023-8	S25	Shijiazhuang, Hebei(河北石家庄)	2023-8
S11	Handan, Hebei(河北邯郸)	2022-8	S26	Shijiazhuang, Hebei(河北石家庄)	2022-8
S12	Handan, Hebei(河北邯郸)	2023-8	S27	Shijiazhuang, Hebei(河北石家庄)	2023-8
S13	Handan, Hebei(河北邯郸)	2022-8	S28	Shijiazhuang, Hebei(河北石家庄)	2023-8
S14	Handan, Hebei(河北邯郸)	2023-8	S29	Handan, Hebei(河北邯郸)	2023-8
S15	Handan, Hebei(河北邯郸)	2022-8

Peak No.	Formula	Molecular ion peak	Observe mass (m/z)	δ ×10^-6	Predicted compounds	MS/MS fragments (m/z)	References
1	C₆H₈O₇	[M-H]^-	191.019 73	2.3	Citric acid	111.008 9[M-H-H₂O-CO₂-H₂O]^-; 87.008 8[M-H-H₂O-CO₂-C₂H₂O]^-	[27]
2	C₁₄H₂₄O₉	[M-H]^-	335.134 76	1.4	Rengynic acid-1'-O-β-D-glucoside	155.071 7[M-H-Glu-H₂O]^-	[25]
3	C₈H₁₄O₄	[M-H]^-	173.081 93	-0.2	Rengynic acid	111.047 1[M-H-H₂O-COO]^-	[25-26]
4	C₈H₁₆O₃	[M+H]⁺	161.117 22	-4.6	Rengyol	105.067 4[M+H-C₃H₄O]⁺; 91.053 9[M+H-C₃H₄O-CH₂]⁺; 77.038 4[M+H-C₃H₄O-2CH₂]⁺	[28]
5	C₂₀H₃₀O₁₃	[M-H]^-	477.161 37	-2	Forsythoside D	477.160 4[M-H]^-; 221.072 3[M-H-C₆H₁₀O₄-C₆H₆O₂]^-	[26]
6	C₁₆H₂₄O₁₀	[M-H]^-	375.129 67	2.7	Adoxosidic acid	213.014 2[M-H-Glu]^-; 169.087 1[M-H-Glu-CO₂]^-; 151.076 6[M-H-Glu-CO₂-H₂O]^-	[25]
7	C₁₆H₂₂O₁₁	[M-H]^-	389.108 94	0.8	Forsythide	345.116 9[M-H-COO]^-; 183.065 3[M-H-COO-Glu]^-; 165.055 0[M-H-COO-Glu-H₂O]^-	[25]
8	C₂₂H₂₆O₁₁	[M+H]⁺	467.154 79	0.3	Forsythenside B	245.080 7[M+H-C₁₁H₁₀O₅]⁺; 227.067 2[M+H-C₁₁H₁₀O₅-H₂O]⁺; 199.058 6[M+H-C₁₁H₁₀O₅-H₂O-CO]⁺	[26]
9	C₇H₆O₂	[M-H]^-	121.029 50	-3.8	4-Hydroxybenzaldehyde	121.022 9[M-H]^-; 93.031 7[M-H-CO]^-	[28]
10	C₂₆H₃₂O₁₁	[M+H]⁺	521.201 74	-4.4	Epipinoresinol-4'-O-β-D-glucoside	341.137 9[M+H-Glc-H₂O]⁺; 326.963 4[M+H-Glc-H₂O-CH₃]⁺	[26]
11	C₂₈H₃₄O₁₅	[M-H]^-	609.182 49	1	Forsythoside J	447.152 4[M-H-Glu]^-; 429.076 2[M-H-Glu-H₂O]^-	[25]
12¹⁾	C₂₉H₃₆O₁₅	[M-H]^-	623.198 14	1	Forsythoside A	461.167 1[M-H-C₆H₁₀O₅]^-; 443.156 3[M-H-C₆H₁₀O₅-H₂O]^-; 179.034 8[M-H-C₆H₁₀O₅-H₂O-C₁₃H₁₂O₆]^-; 161.024 2[M-H-C₆H₁₀O₅-C₁₃H₁₂O₆-2H₂O]^-	[25,27]
13	C₂₃H₂₆O₁₁	[M-H]^-	477.140 24	-0.2	Calcelarioside A	477.139 6[M-H]^-; 179.034 8[M-H-C₁₄H₁₈O₇]^-; 161.024 2[M-H-C₁₄H₁₈O₇-H₂O]^-; 133.029 0[M-H-C₁₄H₁₈O₇-H₂O-CO]^-	[26]
14¹⁾	C₂₇H₃₀O₁₆	[M-H]^-	609.146 11	1	Rutin	301.036 5[M-H-Rha-Glu]^-; 178.998 6[M-H-Rha-Glu-C₇H₇O₂]^-; 151.003 8[M-H-Rha-Glu-C₈H₆O₃]^-	[25]
15	C₂₁H₂₀O₁₂	[M+H]⁺	465.102 75	-1.2	Isoquercetin	303.049 3[M+H-C₆H₆O₅]⁺; 153.016 8[M+H-C₆H₆O₅-C₈H₆O₃]⁺	[25]
16	C₁₆H₁₂O₇	[M+H]⁺	317.065 58	-3.2	Rhamnetin	317.061 0[M+H]⁺; 273.033 9[M+H-2CO]⁺; 153.016 8[RDA frqgment]⁺	[28]
17	C₁₀H₁₀O₄	[M-H]^-	193.050 63	-1.1	Ferulic acid	133.029 0[M-H-CH₃-COOH]^-	[26]
18¹⁾	C₂₇H₃₄O₁₁	[M+H]⁺	535.217 39	-3.1	Forsythin	355.125 8[M+H-Glu-H₂O]⁺	[25-26]
19¹⁾	C₂₀H₂₂O₆	[M-H]^-	357.134 36	-1.5	Pinoresinol	189.262 0[M-H-C₉H₁₂O₃]^-; 163.040 3[M-H-C₉H₁₂O₃-C₂H₂]^-; 135.045 2[M-H-C₉H₁₂O₃-C₂H₂-CO]^-	[25]
20¹⁾	C₂₁H₂₄O₆	[M+H]⁺	373.164 57	-0.2	Forsythigenin	325.032 6[M+H-H₂O-2CH₃]⁺; 311.128 3[M+H-2OCH₃]⁺	[25-26]

Peak No.	Formula	Molecular ion peak	Observe mass (m/z)	δ ×10^-6	Predicted compounds	MS/MS fragments (m/z)	References
1	C₆H₈O₇	[M-H]^-	191.019 73	2.3	Citric acid	111.008 9[M-H-H₂O-CO₂-H₂O]^-; 87.008 8[M-H-H₂O-CO₂-C₂H₂O]^-	[27]
2	C₁₄H₂₄O₉	[M-H]^-	335.134 76	1.4	Rengynic acid-1'-O-β-D-glucoside	155.071 7[M-H-Glu-H₂O]^-	[25]
3	C₈H₁₄O₄	[M-H]^-	173.081 93	-0.2	Rengynic acid	111.047 1[M-H-H₂O-COO]^-	[25-26]
4	C₈H₁₆O₃	[M+H]⁺	161.117 22	-4.6	Rengyol	105.067 4[M+H-C₃H₄O]⁺; 91.053 9[M+H-C₃H₄O-CH₂]⁺; 77.038 4[M+H-C₃H₄O-2CH₂]⁺	[28]
5	C₂₀H₃₀O₁₃	[M-H]^-	477.161 37	-2	Forsythoside D	477.160 4[M-H]^-; 221.072 3[M-H-C₆H₁₀O₄-C₆H₆O₂]^-	[26]
6	C₁₆H₂₄O₁₀	[M-H]^-	375.129 67	2.7	Adoxosidic acid	213.014 2[M-H-Glu]^-; 169.087 1[M-H-Glu-CO₂]^-; 151.076 6[M-H-Glu-CO₂-H₂O]^-	[25]
7	C₁₆H₂₂O₁₁	[M-H]^-	389.108 94	0.8	Forsythide	345.116 9[M-H-COO]^-; 183.065 3[M-H-COO-Glu]^-; 165.055 0[M-H-COO-Glu-H₂O]^-	[25]
8	C₂₂H₂₆O₁₁	[M+H]⁺	467.154 79	0.3	Forsythenside B	245.080 7[M+H-C₁₁H₁₀O₅]⁺; 227.067 2[M+H-C₁₁H₁₀O₅-H₂O]⁺; 199.058 6[M+H-C₁₁H₁₀O₅-H₂O-CO]⁺	[26]
9	C₇H₆O₂	[M-H]^-	121.029 50	-3.8	4-Hydroxybenzaldehyde	121.022 9[M-H]^-; 93.031 7[M-H-CO]^-	[28]
10	C₂₆H₃₂O₁₁	[M+H]⁺	521.201 74	-4.4	Epipinoresinol-4'-O-β-D-glucoside	341.137 9[M+H-Glc-H₂O]⁺; 326.963 4[M+H-Glc-H₂O-CH₃]⁺	[26]
11	C₂₈H₃₄O₁₅	[M-H]^-	609.182 49	1	Forsythoside J	447.152 4[M-H-Glu]^-; 429.076 2[M-H-Glu-H₂O]^-	[25]
12¹⁾	C₂₉H₃₆O₁₅	[M-H]^-	623.198 14	1	Forsythoside A	461.167 1[M-H-C₆H₁₀O₅]^-; 443.156 3[M-H-C₆H₁₀O₅-H₂O]^-; 179.034 8[M-H-C₆H₁₀O₅-H₂O-C₁₃H₁₂O₆]^-; 161.024 2[M-H-C₆H₁₀O₅-C₁₃H₁₂O₆-2H₂O]^-	[25,27]
13	C₂₃H₂₆O₁₁	[M-H]^-	477.140 24	-0.2	Calcelarioside A	477.139 6[M-H]^-; 179.034 8[M-H-C₁₄H₁₈O₇]^-; 161.024 2[M-H-C₁₄H₁₈O₇-H₂O]^-; 133.029 0[M-H-C₁₄H₁₈O₇-H₂O-CO]^-	[26]
14¹⁾	C₂₇H₃₀O₁₆	[M-H]^-	609.146 11	1	Rutin	301.036 5[M-H-Rha-Glu]^-; 178.998 6[M-H-Rha-Glu-C₇H₇O₂]^-; 151.003 8[M-H-Rha-Glu-C₈H₆O₃]^-	[25]
15	C₂₁H₂₀O₁₂	[M+H]⁺	465.102 75	-1.2	Isoquercetin	303.049 3[M+H-C₆H₆O₅]⁺; 153.016 8[M+H-C₆H₆O₅-C₈H₆O₃]⁺	[25]
16	C₁₆H₁₂O₇	[M+H]⁺	317.065 58	-3.2	Rhamnetin	317.061 0[M+H]⁺; 273.033 9[M+H-2CO]⁺; 153.016 8[RDA frqgment]⁺	[28]
17	C₁₀H₁₀O₄	[M-H]^-	193.050 63	-1.1	Ferulic acid	133.029 0[M-H-CH₃-COOH]^-	[26]
18¹⁾	C₂₇H₃₄O₁₁	[M+H]⁺	535.217 39	-3.1	Forsythin	355.125 8[M+H-Glu-H₂O]⁺	[25-26]
19¹⁾	C₂₀H₂₂O₆	[M-H]^-	357.134 36	-1.5	Pinoresinol	189.262 0[M-H-C₉H₁₂O₃]^-; 163.040 3[M-H-C₉H₁₂O₃-C₂H₂]^-; 135.045 2[M-H-C₉H₁₂O₃-C₂H₂-CO]^-	[25]
20¹⁾	C₂₁H₂₄O₆	[M+H]⁺	373.164 57	-0.2	Forsythigenin	325.032 6[M+H-H₂O-2CH₃]⁺; 311.128 3[M+H-2OCH₃]⁺	[25-26]

Sample No.	DPPH radical clearance rate/%	ABTS radical clearance rate/%	Sample No.	DPPH radical clearance rate/%	ABTS radical clearance rate/%
S1	53.10	72.17	S16	53.73	70.58
S2	51.20	73.03	S17	47.40	71.49
S3	46.08	67.47	S18	50.73	76.76
S4	48.32	71.51	S19	46.27	71.09
S5	36.61	47.55	S20	51.64	74.45
S6	54.74	73.06	S21	55.51	70.85
S7	50.36	79.45	S22	51.23	72.29
S8	65.35	77.56	S23	47.09	71.00
S9	48.63	71.79	S24	46.49	72.84
S10	57.51	59.74	S25	53.64	67.04
S11	50.05	71.81	S26	52.09	65.96
S12	51.64	75.38	S27	56.51	74.54
S13	39.21	77.39	S28	46.54	53.99
S14	51.14	76.00	S29	55.15	58.45
S15	50.73	69.29

Sample No.	DPPH radical clearance rate/%	ABTS radical clearance rate/%	Sample No.	DPPH radical clearance rate/%	ABTS radical clearance rate/%
S1	53.10	72.17	S16	53.73	70.58
S2	51.20	73.03	S17	47.40	71.49
S3	46.08	67.47	S18	50.73	76.76
S4	48.32	71.51	S19	46.27	71.09
S5	36.61	47.55	S20	51.64	74.45
S6	54.74	73.06	S21	55.51	70.85
S7	50.36	79.45	S22	51.23	72.29
S8	65.35	77.56	S23	47.09	71.00
S9	48.63	71.79	S24	46.49	72.84
S10	57.51	59.74	S25	53.64	67.04
S11	50.05	71.81	S26	52.09	65.96
S12	51.64	75.38	S27	56.51	74.54
S13	39.21	77.39	S28	46.54	53.99
S14	51.14	76.00	S29	55.15	58.45
S15	50.73	69.29

Order	Peak No.	r (DPPH radical clearance rate)	Order	Peak No.	r (ABTS radical clearance rate)
1	P14	0.946	1	P5	0.941
2	P5	0.945	2	P14	0.939
3	P12	0.938	3	P15	0.931
4	P15	0.935	4	P12	0.930
5	P9	0.914	5	P1	0.918
6	P1	0.912	6	P9	0.906
7	P13	0.907	7	P13	0.905
8	P6	0.903	8	P6	0.903
9	P11	0.902	9	P10	0.896
10	P10	0.898	10	P17	0.892
11	P17	0.891	11	P11	0.887
12	P7	0.889	12	P7	0.885
13	P18	0.866	13	P18	0.873
14	P19	0.852	14	P19	0.863
15	P20	0.820	15	P20	0.830
16	P16	0.810	16	P16	0.821
17	P4	0.797	17	P4	0.800
18	P2	0.757	18	P2	0.760
19	P8	0.700	19	P8	0.707
20	P3	0.569	20	P3	0.571

Order	Peak No.	r (DPPH radical clearance rate)	Order	Peak No.	r (ABTS radical clearance rate)
1	P14	0.946	1	P5	0.941
2	P5	0.945	2	P14	0.939
3	P12	0.938	3	P15	0.931
4	P15	0.935	4	P12	0.930
5	P9	0.914	5	P1	0.918
6	P1	0.912	6	P9	0.906
7	P13	0.907	7	P13	0.905
8	P6	0.903	8	P6	0.903
9	P11	0.902	9	P10	0.896
10	P10	0.898	10	P17	0.892
11	P17	0.891	11	P11	0.887
12	P7	0.889	12	P7	0.885
13	P18	0.866	13	P18	0.873
14	P19	0.852	14	P19	0.863
15	P20	0.820	15	P20	0.830
16	P16	0.810	16	P16	0.821
17	P4	0.797	17	P4	0.800
18	P2	0.757	18	P2	0.760
19	P8	0.700	19	P8	0.707
20	P3	0.569	20	P3	0.571

冀产野生青翘UPLC指纹图谱的建立及其抗氧化谱效关系研究

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徐荣荣 , 段天华 , 李蕊 , 田伟 , 牛丽颖 , 王鑫国 ^* , 冯薇 ^*

中国药学杂志 | 论著 2025,60(6): 579-588

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中国药学杂志 | 论著 2025, 60(6): 579-588

冀产野生青翘UPLC指纹图谱的建立及其抗氧化谱效关系研究

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徐荣荣, 段天华, 李蕊, 田伟, 牛丽颖, 王鑫国^*, 冯薇^*

作者信息

河北中医药大学药学院, 河北省中药配方颗粒技术创新中心, 中药材品质评价与标准化河北省工程研究中心, 石家庄 050091

徐荣荣,女,硕士研究方向:中药药效物质基础

通讯作者:

^*冯薇,女,博士,教授,博士生导师研究方向:中药药效物质基础 Tel:(0311)85216828;

王鑫国,男,学士,教授研究方向:中药药效物质基础 Tel:(0311)89926208

Establishment of UPLC Fingerprint of Wild Qingqiao from Hebei Province and Study on Its Antioxidant Spectrum-Effect Relationship

Rongrong XU, Tianhua DUAN, Rui LI, Wei TIAN, Liying NIU, Xinguo WANG^*, Wei FENG^*

Affiliations

School of Pharmaceutical Sciences, Hebei University of Chinese Medicine, Hebei Traditional Chinese Medicine Formula Granule Technology Innovate Center, Quality Evaluation and Standardization Hebei Province Engineering Research Center of Traditional Chinese Medicine, Shijiazhuang 050091, China

出版时间: 2025-03-22 doi: 10.11669/cpj.2025.06.004

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摘要

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目的建立冀产野生青翘药材的超高效液相色谱(ultra-high-performance liquid chromatography, UPLC)指纹图谱,分析指纹图谱与抗氧化作用的关联性,初步确定其抗氧化活性物质,为控制冀产野生青翘药材的质量提供依据。方法采用UPLC法建立29批冀产野生青翘的指纹图谱,并采用超高液相色谱-四极杆-飞行时间质谱(ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry, UPLC-Q-TOF-MS)对共有峰进行成分鉴定。以1,1-二苯基-2-三硝基苯肼法(DPPH法)和2,2-联氮-二(3-乙基苯并噻唑-6-磺酸)二铵盐法(ABTS法)自由基清除率为抗氧化作用的指标,评价冀产野生青翘药材的抗氧化活性。采用灰色关联度法和偏最小二乘回归分析冀产野生青翘共有峰与抗氧化活性的谱效关系。结果建立了29批冀产野生青翘药材的UPLC指纹图谱,相似度均在0.950~0.999之间。共标定20个共有峰,利用对照品对比和UPLC-Q-TOF-MS解析指认出共有峰。通过清除DPPH和ABTS自由基实验发现,29批冀产野生青翘均具有抗氧化能力,谱效分析结果显示,枸橼酸、rengynic acid-1'-O-β-D-glucoside、连翘酸等9个成分与抗氧化能力呈正相关。综合两种统计模型,可推测连翘酯苷A、鼠李素、阿魏酸是冀产野生青翘发挥抗氧化活性的主要成分。结论本实验建立了基于化学成分和抗氧化活性的冀产野生青翘质量评价模式,可为冀产野生青翘的物质基础与质量控制研究提供参考。

关键词

青翘 / 指纹图谱 / 超高效液相色谱-四极杆-飞行时间质谱 / 抗氧化 / 谱效关系

Abstract

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OBJECTIVE To establish the UPLC fingerprint of wild Qingqiao from Hebei province, analyze the correlation between fingerprint and antioxidant effect, and preliminarily determine its antioxidant active substances, so as to provide a basis for controlling the quality of wild Qingqiao from Hebei province. METHODS The fingerprints of 29 batches of wild Qingqiao from Hebei province were established by UPLC, and the common peaks were identified by UPLC-Q-TOF-MS. The free radical scavenging rate of 1,1-diphenyl-2-picrylhydrazyl (DPPH) method and 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) method was used as the antioxidant index to evaluate the antioxidant activity of wild Qingqiao from Hebei province. The spectrum-effect relationship between common peaks and antioxidant activity of wild Qingqiao from Hebei province was analyzed by grey correlation method and partial least squares regression. RESULTS The UPLC fingerprints of 29 batches of wild Qingqiao from Hebei province were established, and the similarity was between 0.950 and 0.999. A total of 20 common peaks were identified. The common peaks were identified by comparison of standards and UPLC-Q-TOF-MS analysis as citric acid, rengynic acid-1'-O-β-D-glucoside, rengynic acid, rengyol, forsythoside D, adoxosidic acid, forsythide, forsythenside B, 4-hydroxybenzaldehyde, (+)-epipinoresinol-4'-O-β-D-glucoside, forsythoside J, forsythoside A, calcelarioside A, rutin, isoquercetin, rhamnetin, ferulic acid, forsythin, pinoresinol and forsythigenin. By scavenging DPPH and ABTS free radicals, it was found that 29 batches of wild Qingqiao from Hebei province had antioxidant capacity. The results of spectrum-effect analysis showed that 9 components such as citric acid, rengynic acid-1'-O-β-D-glucoside and rengynic acid were positively correlated with antioxidant capacity. Based on the two statistical models, it can be inferred that forsythoside A, rhamnetin and ferulic acid are the main components of the antioxidant activity of wild Qingqiao from Hebei province. CONCLUSION In this study, a quality evaluation model based on chemical composition and antioxidant activity was established, which could provide reference for the antioxidant active ingredients and quality control of wild Forsythiae Fructus from Hebei.

Key words

Hebei wild Qingqiao / fingerprint / UPLC-Q-TOF-MS / antioxidation / spectrum-effect relationship

引用本文

徐荣荣, 段天华, 李蕊, 田伟, 牛丽颖, 王鑫国, 冯薇. 冀产野生青翘UPLC指纹图谱的建立及其抗氧化谱效关系研究. 中国药学杂志, 2025 , 60 (6) : 579 -588 . DOI: 10.11669/cpj.2025.06.004

Rongrong XU, Tianhua DUAN, Rui LI, Wei TIAN, Liying NIU, Xinguo WANG, Wei FENG. Establishment of UPLC Fingerprint of Wild Qingqiao from Hebei Province and Study on Its Antioxidant Spectrum-Effect Relationship[J]. Chinese Pharmaceutical Journal, 2025 , 60 (6) : 579 -588 . DOI: 10.11669/cpj.2025.06.004

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,用于治疗风热感冒、温病初起、乳痈、丹毒、瘰疬等症。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”^[1]。连翘为我国40种常用大宗药材之一,年产销量约在5×10⁶ kg以上,目前连翘药材来源以野生为主^[2],河北省作为我国传统大宗药材连翘的三大主产区之一,野生资源蕴藏量大。有研究表明,青翘和老翘在很多方面存在差异,首先在化学成分方面,青翘中黄酮类、木脂素类、挥发油类等多种化学成分的含量均高于老翘^[3-4];其次在药效方面,青翘与老翘均有抗氧化、抗炎、抗肿瘤等作用,其中青翘的抗氧化、抗炎作用优于老翘^[5],且青翘的临床用药量远高于老翘^[6],因此冀产野生青翘成为市场流通中的主流产品。现代药理学研究表明,连翘具有抗菌^[7]、抗病毒^[8]、抗炎^[9]、抗氧化^[10]、止吐^[11]、抗肿瘤^[12]等作用,并具有肝保护^[13]、神经保护^[14-15]和心血管保护^[16]作用。其中,连翘保护线粒体、抗衰老、清除自由基、美白等药理活性的作用机制可能与抗氧化活性有关^[17-19]。连翘中含有连翘苷、连翘酯苷A、连翘脂素等多种化学成分,这些化合物具有较强的抗氧化、抗病毒等生物活性^[20]。目前针对连翘的研究多集中在连翘的化学成分、质量标准和药理作用等方面,而针对冀产野生青翘的指纹图谱和抗氧化谱效关系的研究报道较少。随着药理作用研究的不断深入,其药效物质基础的快速分析鉴定更是需要关注的方面^[21]。中药指纹图谱可以在整体水平上阐明中药的化学成分组成,可以作为评价中药材质量的方法之一,但不能直接对其功效进行评价,因此存在着一定的局限性^[22]。将中药指纹图谱与中药药效结果联系起来,建立具有实际意义的中药谱效关系,能为中药的质量控制和药效评价标准提供科学依据^[23]。

本实验采用超高效液相色谱(ultra-high-performance liquid chromatography, UPLC)法建立冀产野生青翘的指纹图谱,并对药材中的共有峰进行鉴定,利用1,1-苯基-2-三硝基苯肼法(DPPH法)和2,2-联氮-二(3-乙基苯并噻唑-6-磺酸)二铵盐法(ABTS法)检测体外抗氧化能力,结合灰色关联度分析和偏最小二乘回归分析,研究药材中共有峰与抗氧化活性的谱效关系^[24],初步确定其抗氧化活性物质,为冀产野生青翘的质量控制提供依据。

1 仪器与试药

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1.1 仪器

Waters H-Class型超高效液相色谱仪(PDA检测器,美国Waters公司);Triple-TOF^TM 6600⁺型四极杆串联飞行时间高分辨质谱仪(美国AB SCIEX公司);Analyst TF 1.8.1数据采集软件、PeakView 1.2数据处理软件(美国AB SCIEX公司);YB-150型多功能粉碎机(永康市速锋工贸有限公司);CPA225D型电子天平、BSA224S-CW型电子天平(德国赛多利斯有限公司);KQ-250E型超声波清洗器(昆山市超声仪器有限公司);H2050R高速离心机(湖南赛特湘仪离心机仪器有限公司);ELx800型酶标仪(美国Bio-Tek公司)。

1.2 试剂

连翘酯苷A、松脂醇对照品(成都普菲德生物技术有限公司,批号分别为22012104、20092410);连翘脂素、芦丁对照品(成都克洛玛生物科技有限公司,批号分别为CHB201204、CHB-L-062);连翘苷对照品(成都乐美天医药科技有限公司,批号为DSTDL004802);总抗氧化能力(DPPH法)试剂盒(苏州科铭生物技术有限公司);总抗氧化能力(ABTS法)试剂盒(苏州科铭生物技术有限公司)。乙腈、甲酸为色谱纯,其他试剂均为分析纯,实验用水为超纯水,由UPR-11-20L型优普系列超纯水机制备。

1.3 药材

所收集29批样品(安国市场直销的野生连翘商户),经河北中医药大学侯芳洁副教授鉴定为木犀科植物连翘[Forsythia suspensa (Thunb.) Vahl]的干燥果实,多不开裂,表面绿褐色,为青翘。样品信息见表1。

2 方法与结果

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2.1 连翘指纹图谱的建立

2.1.1 色谱条件

色谱柱为菲罗门Titank C₁₈色谱柱(2.1 mm×150 mm,1.8 μm);流动相为乙腈(A)-0.1%甲酸水溶液(B)梯度洗脱(0~4 min,5%~11%A;4~7 min,11%~19%A;7~14 min,19%A;14~16 min,19%~26%A;16~19 min,26%~48%A;19~24 min,48%~70%A;24~27 min,70%~85%A;27~29 min,85%~5%A);流速:0.3 mL·min^-1;柱温:30 ℃;进样量:0.4 μL。采用定时波长(0~13 min,265 nm;13~19 min,260 nm;19~21 min,270 nm;21~22 min,275 nm;22~29 min,280 nm)。

2.1.2 对照品溶液的制备

分别精密称定连翘酯苷A、芦丁、松脂醇、连翘苷、连翘脂素对照品适量,加甲醇溶解,分别制成单一对照品储备液。精密量取上述对照品储备液适量,加甲醇稀释并定容至2 mL,制成质量浓度分别为447.40、15.40、1.90、4.18、0.13 μg·mL^-1的混合对照品溶液。

2.1.3 供试品溶液的制备

取连翘药材粉末(过60目筛)1.0 g,精密称定,置于50 mL锥形瓶中,精密加入体积分数70%甲醇20 mL,称定质量,超声处理(功率250 W,频率40 kHz)30 min,取出,放至室温,再次称重,用体积分数70%甲醇补足减失的质量,摇匀,用0.22 μm微孔滤膜滤过,取续滤液即得。

2.2 指纹图谱方法学考察

2.2.1 精密度实验

取S6号连翘药材粉末,按“2.1.3”项下方法制备供试品溶液,按“2.1.1”项下色谱条件进样测定,连续进样6次,以12号色谱峰(连翘酯苷A)为参照峰,计算图谱中各共有峰的相对峰面积和相对保留时间的相对标准偏差(RSD)。结果显示,各共有峰相对保留时间的RSD为0.02%~0.30%,相对峰面积的RSD为1.28%~2.96%,表明仪器精密度良好。

2.2.2 重复性实验

取S6号连翘药材粉末6份,按“2.1.3”项下方法平行制备6份供试品溶液,按“2.1.1”项下色谱条件进样测定,以12号色谱峰(连翘酯苷A)为参照峰,计算图谱中各共有峰的相对峰面积和相对保留时间的RSD。结果显示,各共有峰相对保留时间的RSD为0.07%~0.36%,相对峰面积的RSD为1.80%~2.85%,表明该方法重复性良好。

2.2.3 稳定性实验

取S6号连翘药材粉末,按“2.1.3”项下方法制备供试品溶液,按“2.1.1”项下色谱条件进样测定,分别于0、2、4、8、12、24 h进样测定,以12号色谱峰(连翘酯苷A)为参照峰,计算图谱中各共有峰的相对峰面积和相对保留时间的RSD。结果显示,各共有峰相对保留时间的RSD为0.16%~0.84%,相对峰面积的RSD为2.03%~2.56%,表明供试品溶液在24 h内稳定性良好。

2.3 对照指纹图谱的生成与相似度评价

分别取29批冀产野生青翘药材粉末,按“2.1.3”项下方法制备供试品溶液,“2.1.1”项下色谱条件进样分析,记录色谱图。将所得29批药材的图谱文件导入中药色谱指纹图谱相似度评价系统(2012版),以S1为参照图谱,设置时间窗宽度为0.1,并基于中位数法生成对照指纹图谱,运用多点校正进行Mark色谱峰匹配,计算其相似度。结果表明,29批冀产野生青翘样品的相似度均在0.950~0.999之间。共标定20个共有峰,与对照品溶液色谱峰的保留时间进行对比,确定色谱峰12为连翘酯苷A,峰14为芦丁,峰18为连翘苷,峰19为松脂醇,峰20为连翘脂素。29批冀产野生青翘的UPLC指纹图谱见图1。29批冀产野生青翘对照指纹图谱见图2A,混合对照品溶液色谱图见图2B。

2.4 指纹图谱中共有峰的鉴定

2.4.1 质谱条件

离子模式:分别在正、负离子模式下分析检测样品。

仪器参数优化后设置如下:离子源(正离子模式):电喷雾离子源(ESI⁺);雾化电压(ISVF):+5 500 V;雾化温度(TEM):500 ℃;解簇电压(DP):+80 V;碰撞能量(CE):+45 eV;雾化气压力(Gas 1,N₂)、辅助气压力(Gas 2,N₂)、气帘气压力(Cur,N₂)分别为:50、55、35 MPa;高分辨质谱扫描质量范围设置为m/z 100~1 200,累积时间:250 ms;碎片离子扫描质量范围设置为50~1 200,累积时间:100 ms。碰撞能量扩展(CES):15 eV。信息依赖获取(IDA)方式开启。

离子源(负离子模式):ESI^-;雾化电压(ISVF):-4 500 V;TEM:500 ℃;DP:-80 V;CE:-45 eV;Gas 1、Gas 2、Cur分别为:50、55、35 MPa;其余各时间等参数同正离子模式保持一致。

2.4.2 共有峰鉴定

按“2.1.3”项下制备供试品溶液、“2.1.1”项下色谱条件和“2.4.1”项下质谱条件获得连翘的正、负离子流图,通过采用PeakView 1.2软件中“XIC Manager”功能进行数据分析,通过比对精确相对分子质量和特征碎片离子,鉴定出连翘指纹图谱中共有峰的结构^[25],结果见表2,冀产野生青翘的正、负离子全扫描离子流图见图3。连翘的主要化学成分有苯乙醇苷类和木脂素类,其中苯乙醇苷类成分的结构通常是由苯乙醇基和糖基构成,母核通常为β-葡萄糖,苯乙基和羟基被羟基或甲氧基取代,肉桂酰基被甲氧基取代,通过酯键或糖苷键形成天然糖苷类化合物^[26]。以连翘酯苷A(峰12)为例,在负离子模式下,其准分子离子峰为m/z 623.199 8[M-H]^-,其二级碎片离子有m/z 461.167 1[M-H-C₆H₁₀O₅]^-,m/z 443.156 3[M-H-C₆H₁₀O₅-H₂O]^-,m/z 179.035 4 [M-H-C₆H₁₀O₅-H₂O-C₁₃H₁₂O₆]^-,m/z 161.024 8[M-H-C₆H₁₀O₅-C₁₃H₁₂O₆-2H₂O]^-,结合裂解规律及对照品比对,推断其为连翘酯苷A,连翘酯苷A的二级质谱及裂解途径见图4^[27]。

2.5 体外抗氧化作用考察

2.5.1 DPPH自由基清除率

将“2.1.3”项下供试品溶液稀释40倍后,分别精密吸取20 μL至EP管中,加入DPPH试剂380 μL,充分混匀,室温避光孵育20 min,取200 μL置96孔板中,即得样品溶液。平行实验重复3次。取体积分数70%甲醇20 μL至EP管中,加入DPPH试剂380 μL,充分混匀,室温避光孵育20 min,取200 μL置96孔板中,即得空白溶液。酶标仪515 nm下测定吸光度值(A)。冀产野生青翘的总抗氧化能力以DPPH自由基清除率表示(公式1):

公式(1)DPPH自由基清除率(%)=[(A_空白-A_测定)÷A_空白]×100%

2.5.2 ABTS自由基清除率

按试剂盒说明书制备ABTS工作液,将“2.1.3”项下供试品溶液稀释40倍后,分别精密吸取10 μL至EP管中,加入ABTS工作液190 μL,充分混匀,取200 μL置96孔板中,即得样品溶液。平行实验重复3次。取体积分数70%甲醇10 μL至EP管中,加入ABTS工作液190 μL,充分混匀,取200 μL置96孔板中,即得空白溶液。酶标仪734 nm下测定吸光度值(A)。连翘的总抗氧化能力以ABTS自由基清除率表示(公式2):

公式(2)ABTS自由基清除率(%)=[(A_空白-A_测定)÷A_空白]×100%

抗氧化测定结果见表3。

2.6 谱效关系研究

2.6.1 灰色关联度分析

参考Li等^[29]的灰色关联度分析方法,对原始数据进行无量纲化处理,采用初值法,以29批冀产野生青翘药材的DPPH自由基清除率和ABTS自由基清除率作为参考序列,取对应批次连翘的UPLC图谱中20个共有峰面积作为比较序列,计算灰色关联系数(r)。

通过r可初步确定所对应的化学成分与抗氧化活性之间的联系。r越大,说明化学成分与抗氧化作用的关联性越大,反之则越小。从r结果可知,各峰所代表的化学成分对其抗氧化活性的影响程度有所差别,其中有16个共有峰清除DPPH和ABTS两种自由基活性的r均大于0.8(峰1、5、6、7、9、10、11、12、13、14、15、16、17、18、19、20),有较高的关联度,表明冀产野生青翘的抗氧化作用是通过多种成分共同发挥作用实现的。r计算结果见表4。

2.6.2 偏最小二乘回归分析(PLS)

偏最小二乘回归方程的建立:采用SIMCA-P 14.1软件中PLS模块对冀产野生青翘指纹图谱及其抗氧化能力进行相关性分析,以指纹图谱中各共有峰的峰面积为自变量X,分别以DPPH自由基清除率和ABTS自由基清除率为因变量Y,得到回归方程Y₁=0.000 43X₁+0.001 41X₂+0.000 54X₃-0.002 13X₄-0.000 64X₅-0.000 44X₆+0.006 22X₇+0.001 07X₈+0.001 74X₉+0.000 10X₁₀-0.003 60X₁₁+0.000 23X₁₂-0.001 20X₁₃-0.003 02X₁₄+0.000 53X₁₅+0.000 81X₁₆+0.000 55X₁₇-0.000 67X₁₈+0.002 70X₁₉-0.001 36X₂₀,Y₂=0.000 23X₁+0.001 18X₂+0.003 76X₃-0.002 33X₄+0.000 08X₅-0.000 51X₆-0.001 30X₇-0.000 69X₈+0.000 14X₉+0.001 03X₁₀+0.000 42X₁₁+0.000 94X₁₂-0.002 39X₁₃-0.000 03X₁₄+0.000 65X₁₅+0.001 54X₁₆+0.000 04X₁₇-0.000 47X₁₈-0.000 16X₁₉-0.000 19X₂₀。回归方程中Y₁,Y₂分别对应DPPH自由基清除率、ABTS自由基清除率,X₁~X₂₀分别对应1~20号共有峰的峰面积。在回归方程中,系数为正值的自变量与抗氧化能力呈正相关,系数为负值的自变量与抗氧化能力呈负相关。其中回归系数代表各自变量对抗氧化能力的贡献大小,回归系数越大,对药效贡献越大。DPPH自由基清除率回归系数见图5,ABTS自由基清除率回归系数见图6。结果表明,冀产野生青翘UPLC指纹图谱中的峰1、2、3、7、8、9、10、12、15、16、17、19与DPPH自由基清除能力呈正相关,贡献度依次为峰7>19>9>2>8>16>17>3>15>1>12>10,峰4、5、6、11、13、14、18、20与DPPH自由基清除能力呈负相关,贡献度依次为峰11>14>4>20>13>18>5>6;峰1、2、3、5、9、10、11、12、15、16、17与ABTS自由基清除能力呈正相关,贡献度依次为峰3>16>2>10>12>15>11>1>9>5>17,峰4、6、7、8、13、14、18、19、20与ABTS自由基清除能力呈负相关,贡献度依次为峰13>4>7>8>6>18>20>19>14。

变量重要性分析:在PLS中,VIP是反映自变量对因变量解释能力的重要指标,变量投影重要性指标(VIP值)越大,自变量X(指纹图谱中各共有峰的峰面积)的解释能力越强^[30]。一般认为当VIP>1时,自变量在解释因变量时具有显著重要性。将自变量X(各共有峰峰面积)与因变量Y(自由基清除率)导入SIMCA-P.14.1软件进行变量投影重要性分析,生成VIP,DPPH自由基清除率VIP值见图7,ABTS自由基清除率VIP值见图8。由图7、8可知,在以DPPH自由基清除能力为指标时,峰4、6、11、12、14、17、18、19的VIP值均大于1,依次为峰12>4>18>17>6>14>19>11;以ABTS自由基清除能力为指标时,峰4、11、12、16、17、18、19、20的VIP值均大于1,依次为峰16>12>19>17>4>18>20>11。

3 讨论

收起

本实验前期考察了提取溶剂(体积分数50%、70%、100%甲醇),料液体积比(1∶20、1∶30、1∶40),提取时间(20、30、40 min)对供试品溶液制备效果的影响,通过对比各样品色谱峰的数量和面积,最终确定冀产野生青翘药材的最优提取工艺为:甲醇体积分数为70%、料液比1∶20,提取时间为30 min。采用二极管阵列检测器(PDA检测器),对冀产野生青翘药材供试品溶液在210~400 nm内进行全波长扫描,检测波长为265 nm(0~13 min),260 nm(13~19 min),270 nm(19~21 min),275 nm(21~22 min),280 nm(22~29 min)时,色谱图出峰较多,待测成分的响应值较大。中药指纹图谱是一种可以全面反映中药化学成分种类和数量的定性分析方法,目前采用中药色谱指纹图谱相似度评价系统(2012版)中的相似度算法进行相似度计算。然而,此算法对化学成分的含量并不敏感,不能反映中药在含量上的变化情况。因此,相似度高仅表示不同批次的样品在成分上相似,而不能表明各成分含量相近^[31-32]。在指纹图谱相似度评价中,29批冀产野生青翘样品的相似度均大于0.95,说明冀产野生青翘各批次之间相似度较高,化学成分相似,质量较稳定。

抗氧化活性与测试体系密切相关,不同评价体系,反应原理不同,评价结果有时会有一定的差异,DPPH自由基和ABTS自由基经常用于衡量植物活性成分体外抗氧化的指标^[33]。29批冀产野生青翘DPPH自由基清除率的范围为36.61%~65.35%,ABTS自由基清除率的范围为47.55%~79.45%,两种检测方法得到的检测结果基本趋势一致,说明不同批次间的冀产野生青翘存在一定的差异,可能与采收时间、贮存环境等因素有关。以r作为化学评价手段,冀产野生青翘中峰3与DPPH以及ABTS的r分别为0.569、0.571,其余各共有峰与DPPH以及ABTS的r均大于0.7,表明各共有峰与抗氧化活性具有较大的相关性,揭示冀产野生青翘中抗氧化活性为多组分联合效应,并非单一物质主导。为进一步反映共有峰与抗氧化能力的正负相关性,结合PLS,其中峰1(枸橼酸)、峰2(rengynic acid-1'-O-β-D-glucoside)、峰3(连翘酸)、峰9(对羟基苯甲醛)、峰10[(+)-表松脂素-4'-O-B-D-葡萄糖苷]、峰12(连翘酯苷A)、峰15(异槲皮素)、峰16(鼠李素)、峰17(阿魏酸)与冀产野生青翘2种抗氧化活性均呈正相关,而峰4(连翘醇)、峰6(五福花苷酸)、峰13(木通苯乙醇苷A)、峰14(芦丁)、峰18(连翘苷)、峰20(连翘脂素)与冀产野生青翘2种抗氧化活性均呈负相关。综合上述2种统计模型,以r>0.7,PLS分析系数为正数(呈现正相关),且VIP值>1为筛选条件^[34],可推测出峰12(连翘酯苷A)、峰16(鼠李素)、峰17(阿魏酸)是冀产野生青翘发挥抗氧化活性的主要成分。

本实验将常规的化学分析方法(UPLC)与抗氧化活性测定相结合,进一步将化学信息的“谱”与活性信息的“效”进行关联度研究,即“谱效关系”研究,找到了与冀产野生青翘抗氧化作用有较大关联度的色谱峰,为冀产野生青翘药材的质量控制及药效物质的进一步阐明提供科学依据。

基金

收起

河北省省级科技计划项目资助(21372503D)

参考文献

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2025年第60卷第6期

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doi: 10.11669/cpj.2025.06.004

接收时间：2024-08-01
首发时间：2025-11-07
出版时间：2025-03-22

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收稿日期：2024-08-01

基金

河北省省级科技计划项目资助(21372503D)

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河北中医药大学药学院, 河北省中药配方颗粒技术创新中心, 中药材品质评价与标准化河北省工程研究中心, 石家庄 050091

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^*冯薇,女,博士,教授,博士生导师研究方向:中药药效物质基础 Tel:(0311)85216828;

王鑫国,男,学士,教授研究方向:中药药效物质基础 Tel:(0311)89926208

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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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No.	Place of origin(in Chinese)	Collection time	No.	Place of origin(in Chinese)	Collection time
S1	Yixian Hebei(河北易县)	2022-7	S16	Handan Hebei(河北邯郸)	2023-8
S2	Yixian, Hebei(河北易县)	2023-7	S17	Handan, Hebei(河北邯郸)	2022-8
S3	Handan, Hebei(河北邯郸)	2022-8	S18	Handan, Hebei(河北邯郸)	2023-8
S4	Handan, Hebei(河北邯郸)	2023-8	S19	Handan, Hebei(河北邯郸)	2022-8
S5	Handan, Hebei(河北邯郸)	2023-8	S20	Handan, Hebei(河北邯郸)	2023-8
S6	Handan, Hebei(河北邯郸)	2023-8	S21	Handan, Hebei(河北邯郸)	2022-8
S7	Handan, Hebei(河北邯郸)	2023-8	S22	Shijiazhuang, Hebei(河北石家庄)	2023-8
S8	Handan, Hebei(河北邯郸)	2023-8	S23	Shijiazhuang, Hebei(河北石家庄)	2022-8
S9	Handan, Hebei(河北邯郸)	2023-8	S24	Shijiazhuang, Hebei(河北石家庄)	2023-8
S10	Handan, Hebei(河北邯郸)	2023-8	S25	Shijiazhuang, Hebei(河北石家庄)	2023-8
S11	Handan, Hebei(河北邯郸)	2022-8	S26	Shijiazhuang, Hebei(河北石家庄)	2022-8
S12	Handan, Hebei(河北邯郸)	2023-8	S27	Shijiazhuang, Hebei(河北石家庄)	2023-8
S13	Handan, Hebei(河北邯郸)	2022-8	S28	Shijiazhuang, Hebei(河北石家庄)	2023-8
S14	Handan, Hebei(河北邯郸)	2023-8	S29	Handan, Hebei(河北邯郸)	2023-8
S15	Handan, Hebei(河北邯郸)	2022-8

No.

Place of origin(in Chinese)

Collection time

No.

Place of origin(in Chinese)

Collection time

Yixian Hebei(河北易县)

2022-7

S16

Handan Hebei(河北邯郸)

2023-8

Yixian, Hebei(河北易县)

2023-7

S17

Handan, Hebei(河北邯郸)

2022-8

Handan, Hebei(河北邯郸)

2022-8

S18

Handan, Hebei(河北邯郸)

2023-8

Handan, Hebei(河北邯郸)

2023-8

S19

Handan, Hebei(河北邯郸)

2022-8

Handan, Hebei(河北邯郸)

2023-8

S20

Handan, Hebei(河北邯郸)

2023-8

Handan, Hebei(河北邯郸)

2023-8

S21

Handan, Hebei(河北邯郸)

2022-8

Handan, Hebei(河北邯郸)

2023-8

S22

Shijiazhuang, Hebei(河北石家庄)

2023-8

Handan, Hebei(河北邯郸)

2023-8

S23

Shijiazhuang, Hebei(河北石家庄)

2022-8

Handan, Hebei(河北邯郸)

2023-8

S24

Shijiazhuang, Hebei(河北石家庄)

2023-8

S10

Handan, Hebei(河北邯郸)

2023-8

S25

Shijiazhuang, Hebei(河北石家庄)

2023-8

S11

Handan, Hebei(河北邯郸)

2022-8

S26

Shijiazhuang, Hebei(河北石家庄)

2022-8

S12

Handan, Hebei(河北邯郸)

2023-8

S27

Shijiazhuang, Hebei(河北石家庄)

2023-8

S13

Handan, Hebei(河北邯郸)

2022-8

S28

Shijiazhuang, Hebei(河北石家庄)

2023-8

S14

Handan, Hebei(河北邯郸)

2023-8

S29

Handan, Hebei(河北邯郸)

2023-8

S15

Handan, Hebei(河北邯郸)

2022-8

Peak No.	Formula	Molecular ion peak	Observe mass (m/z)	δ ×10^-6	Predicted compounds	MS/MS fragments (m/z)	References
1	C₆H₈O₇	[M-H]^-	191.019 73	2.3	Citric acid	111.008 9[M-H-H₂O-CO₂-H₂O]^-; 87.008 8[M-H-H₂O-CO₂-C₂H₂O]^-	[27]
2	C₁₄H₂₄O₉	[M-H]^-	335.134 76	1.4	Rengynic acid-1'-O-β-D-glucoside	155.071 7[M-H-Glu-H₂O]^-	[25]
3	C₈H₁₄O₄	[M-H]^-	173.081 93	-0.2	Rengynic acid	111.047 1[M-H-H₂O-COO]^-	[25-26]
4	C₈H₁₆O₃	[M+H]⁺	161.117 22	-4.6	Rengyol	105.067 4[M+H-C₃H₄O]⁺; 91.053 9[M+H-C₃H₄O-CH₂]⁺; 77.038 4[M+H-C₃H₄O-2CH₂]⁺	[28]
5	C₂₀H₃₀O₁₃	[M-H]^-	477.161 37	-2	Forsythoside D	477.160 4[M-H]^-; 221.072 3[M-H-C₆H₁₀O₄-C₆H₆O₂]^-	[26]
6	C₁₆H₂₄O₁₀	[M-H]^-	375.129 67	2.7	Adoxosidic acid	213.014 2[M-H-Glu]^-; 169.087 1[M-H-Glu-CO₂]^-; 151.076 6[M-H-Glu-CO₂-H₂O]^-	[25]
7	C₁₆H₂₂O₁₁	[M-H]^-	389.108 94	0.8	Forsythide	345.116 9[M-H-COO]^-; 183.065 3[M-H-COO-Glu]^-; 165.055 0[M-H-COO-Glu-H₂O]^-	[25]
8	C₂₂H₂₆O₁₁	[M+H]⁺	467.154 79	0.3	Forsythenside B	245.080 7[M+H-C₁₁H₁₀O₅]⁺; 227.067 2[M+H-C₁₁H₁₀O₅-H₂O]⁺; 199.058 6[M+H-C₁₁H₁₀O₅-H₂O-CO]⁺	[26]
9	C₇H₆O₂	[M-H]^-	121.029 50	-3.8	4-Hydroxybenzaldehyde	121.022 9[M-H]^-; 93.031 7[M-H-CO]^-	[28]
10	C₂₆H₃₂O₁₁	[M+H]⁺	521.201 74	-4.4	Epipinoresinol-4'-O-β-D-glucoside	341.137 9[M+H-Glc-H₂O]⁺; 326.963 4[M+H-Glc-H₂O-CH₃]⁺	[26]
11	C₂₈H₃₄O₁₅	[M-H]^-	609.182 49	1	Forsythoside J	447.152 4[M-H-Glu]^-; 429.076 2[M-H-Glu-H₂O]^-	[25]
12¹⁾	C₂₉H₃₆O₁₅	[M-H]^-	623.198 14	1	Forsythoside A	461.167 1[M-H-C₆H₁₀O₅]^-; 443.156 3[M-H-C₆H₁₀O₅-H₂O]^-; 179.034 8[M-H-C₆H₁₀O₅-H₂O-C₁₃H₁₂O₆]^-; 161.024 2[M-H-C₆H₁₀O₅-C₁₃H₁₂O₆-2H₂O]^-	[25,27]
13	C₂₃H₂₆O₁₁	[M-H]^-	477.140 24	-0.2	Calcelarioside A	477.139 6[M-H]^-; 179.034 8[M-H-C₁₄H₁₈O₇]^-; 161.024 2[M-H-C₁₄H₁₈O₇-H₂O]^-; 133.029 0[M-H-C₁₄H₁₈O₇-H₂O-CO]^-	[26]
14¹⁾	C₂₇H₃₀O₁₆	[M-H]^-	609.146 11	1	Rutin	301.036 5[M-H-Rha-Glu]^-; 178.998 6[M-H-Rha-Glu-C₇H₇O₂]^-; 151.003 8[M-H-Rha-Glu-C₈H₆O₃]^-	[25]
15	C₂₁H₂₀O₁₂	[M+H]⁺	465.102 75	-1.2	Isoquercetin	303.049 3[M+H-C₆H₆O₅]⁺; 153.016 8[M+H-C₆H₆O₅-C₈H₆O₃]⁺	[25]
16	C₁₆H₁₂O₇	[M+H]⁺	317.065 58	-3.2	Rhamnetin	317.061 0[M+H]⁺; 273.033 9[M+H-2CO]⁺; 153.016 8[RDA frqgment]⁺	[28]
17	C₁₀H₁₀O₄	[M-H]^-	193.050 63	-1.1	Ferulic acid	133.029 0[M-H-CH₃-COOH]^-	[26]
18¹⁾	C₂₇H₃₄O₁₁	[M+H]⁺	535.217 39	-3.1	Forsythin	355.125 8[M+H-Glu-H₂O]⁺	[25-26]
19¹⁾	C₂₀H₂₂O₆	[M-H]^-	357.134 36	-1.5	Pinoresinol	189.262 0[M-H-C₉H₁₂O₃]^-; 163.040 3[M-H-C₉H₁₂O₃-C₂H₂]^-; 135.045 2[M-H-C₉H₁₂O₃-C₂H₂-CO]^-	[25]
20¹⁾	C₂₁H₂₄O₆	[M+H]⁺	373.164 57	-0.2	Forsythigenin	325.032 6[M+H-H₂O-2CH₃]⁺; 311.128 3[M+H-2OCH₃]⁺	[25-26]

Peak
No.

Formula

Molecular
ion peak

Observe mass
(m/z)

δ
×10^-6

Predicted
compounds

MS/MS fragments
(m/z)

References

C₆H₈O₇

[M-H]^-

191.019 73

2.3

Citric acid

111.008 9[M-H-H₂O-CO₂-H₂O]^-;
87.008 8[M-H-H₂O-CO₂-C₂H₂O]^-

[27]

C₁₄H₂₄O₉

[M-H]^-

335.134 76

1.4

Rengynic acid-1'-O-β-D-glucoside

155.071 7[M-H-Glu-H₂O]^-

[25]

C₈H₁₄O₄

[M-H]^-

173.081 93

-0.2

Rengynic acid

111.047 1[M-H-H₂O-COO]^-

[25-26]

C₈H₁₆O₃

[M+H]⁺

161.117 22

-4.6

Rengyol

105.067 4[M+H-C₃H₄O]⁺;
91.053 9[M+H-C₃H₄O-CH₂]⁺;
77.038 4[M+H-C₃H₄O-2CH₂]⁺

[28]

C₂₀H₃₀O₁₃

[M-H]^-

477.161 37

-2

Forsythoside D

477.160 4[M-H]^-;
221.072 3[M-H-C₆H₁₀O₄-C₆H₆O₂]^-

[26]

C₁₆H₂₄O₁₀

[M-H]^-

375.129 67

2.7

Adoxosidic acid

213.014 2[M-H-Glu]^-;
169.087 1[M-H-Glu-CO₂]^-;
151.076 6[M-H-Glu-CO₂-H₂O]^-

[25]

C₁₆H₂₂O₁₁

[M-H]^-

389.108 94

0.8

Forsythide

345.116 9[M-H-COO]^-;
183.065 3[M-H-COO-Glu]^-;
165.055 0[M-H-COO-Glu-H₂O]^-

[25]

C₂₂H₂₆O₁₁

[M+H]⁺

467.154 79

0.3

Forsythenside B

245.080 7[M+H-C₁₁H₁₀O₅]⁺;
227.067 2[M+H-C₁₁H₁₀O₅-H₂O]⁺;
199.058 6[M+H-C₁₁H₁₀O₅-H₂O-CO]⁺

[26]

C₇H₆O₂

[M-H]^-

121.029 50

-3.8

4-Hydroxybenzaldehyde

121.022 9[M-H]^-;
93.031 7[M-H-CO]^-

[28]

C₂₆H₃₂O₁₁

[M+H]⁺

521.201 74

-4.4

Epipinoresinol-4'-O-β-D-glucoside

341.137 9[M+H-Glc-H₂O]⁺;
326.963 4[M+H-Glc-H₂O-CH₃]⁺

[26]

C₂₈H₃₄O₁₅

[M-H]^-

609.182 49

Forsythoside J

447.152 4[M-H-Glu]^-;
429.076 2[M-H-Glu-H₂O]^-

[25]

12¹⁾

C₂₉H₃₆O₁₅

[M-H]^-

623.198 14

Forsythoside A

461.167 1[M-H-C₆H₁₀O₅]^-;
443.156 3[M-H-C₆H₁₀O₅-H₂O]^-;
179.034 8[M-H-C₆H₁₀O₅-H₂O-C₁₃H₁₂O₆]^-;
161.024 2[M-H-C₆H₁₀O₅-C₁₃H₁₂O₆-2H₂O]^-

[25,27]

C₂₃H₂₆O₁₁

[M-H]^-

477.140 24

-0.2

Calcelarioside A

477.139 6[M-H]^-;
179.034 8[M-H-C₁₄H₁₈O₇]^-;
161.024 2[M-H-C₁₄H₁₈O₇-H₂O]^-;
133.029 0[M-H-C₁₄H₁₈O₇-H₂O-CO]^-

[26]

14¹⁾

C₂₇H₃₀O₁₆

[M-H]^-

609.146 11

Rutin

301.036 5[M-H-Rha-Glu]^-;
178.998 6[M-H-Rha-Glu-C₇H₇O₂]^-;
151.003 8[M-H-Rha-Glu-C₈H₆O₃]^-

[25]

C₂₁H₂₀O₁₂

[M+H]⁺

465.102 75

-1.2

Isoquercetin

303.049 3[M+H-C₆H₆O₅]⁺;
153.016 8[M+H-C₆H₆O₅-C₈H₆O₃]⁺

[25]

C₁₆H₁₂O₇

[M+H]⁺

317.065 58

-3.2

Rhamnetin

317.061 0[M+H]⁺;
273.033 9[M+H-2CO]⁺;
153.016 8[RDA frqgment]⁺

[28]

C₁₀H₁₀O₄

[M-H]^-

193.050 63

-1.1

Ferulic acid

133.029 0[M-H-CH₃-COOH]^-

[26]

18¹⁾

C₂₇H₃₄O₁₁

[M+H]⁺

535.217 39

-3.1

Forsythin

355.125 8[M+H-Glu-H₂O]⁺

[25-26]

19¹⁾

C₂₀H₂₂O₆

[M-H]^-

357.134 36

-1.5

Pinoresinol

189.262 0[M-H-C₉H₁₂O₃]^-;
163.040 3[M-H-C₉H₁₂O₃-C₂H₂]^-;
135.045 2[M-H-C₉H₁₂O₃-C₂H₂-CO]^-

[25]

20¹⁾

C₂₁H₂₄O₆

[M+H]⁺

373.164 57

-0.2

Forsythigenin

325.032 6[M+H-H₂O-2CH₃]⁺;
311.128 3[M+H-2OCH₃]⁺

[25-26]

Sample No.	DPPH radical clearance rate/%	ABTS radical clearance rate/%	Sample No.	DPPH radical clearance rate/%	ABTS radical clearance rate/%
S1	53.10	72.17	S16	53.73	70.58
S2	51.20	73.03	S17	47.40	71.49
S3	46.08	67.47	S18	50.73	76.76
S4	48.32	71.51	S19	46.27	71.09
S5	36.61	47.55	S20	51.64	74.45
S6	54.74	73.06	S21	55.51	70.85
S7	50.36	79.45	S22	51.23	72.29
S8	65.35	77.56	S23	47.09	71.00
S9	48.63	71.79	S24	46.49	72.84
S10	57.51	59.74	S25	53.64	67.04
S11	50.05	71.81	S26	52.09	65.96
S12	51.64	75.38	S27	56.51	74.54
S13	39.21	77.39	S28	46.54	53.99
S14	51.14	76.00	S29	55.15	58.45
S15	50.73	69.29

Sample No.

DPPH radical clearance rate/%

ABTS radical clearance rate/%

Sample No.

DPPH radical clearance rate/%

ABTS radical clearance rate/%

53.10

72.17

S16

53.73

70.58

51.20

73.03

S17

47.40

71.49

46.08

67.47

S18

50.73

76.76

48.32

71.51

S19

46.27

71.09

36.61

47.55

S20

51.64

74.45

54.74

73.06

S21

55.51

70.85

50.36

79.45

S22

51.23

72.29

65.35

77.56

S23

47.09

71.00

48.63

71.79

S24

46.49

72.84

S10

57.51

59.74

S25

53.64

67.04

S11

50.05

71.81

S26

52.09

65.96

S12

51.64

75.38

S27

56.51

74.54

S13

39.21

77.39

S28

46.54

53.99

S14

51.14

76.00

S29

55.15

58.45

S15

50.73

69.29

Order	Peak No.	r (DPPH radical clearance rate)	Order	Peak No.	r (ABTS radical clearance rate)
1	P14	0.946	1	P5	0.941
2	P5	0.945	2	P14	0.939
3	P12	0.938	3	P15	0.931
4	P15	0.935	4	P12	0.930
5	P9	0.914	5	P1	0.918
6	P1	0.912	6	P9	0.906
7	P13	0.907	7	P13	0.905
8	P6	0.903	8	P6	0.903
9	P11	0.902	9	P10	0.896
10	P10	0.898	10	P17	0.892
11	P17	0.891	11	P11	0.887
12	P7	0.889	12	P7	0.885
13	P18	0.866	13	P18	0.873
14	P19	0.852	14	P19	0.863
15	P20	0.820	15	P20	0.830
16	P16	0.810	16	P16	0.821
17	P4	0.797	17	P4	0.800
18	P2	0.757	18	P2	0.760
19	P8	0.700	19	P8	0.707
20	P3	0.569	20	P3	0.571

Order

Peak
No.

r (DPPH radical
clearance rate)

Order

Peak
No.

r (ABTS radical
clearance rate)

P14

0.946

0.941

0.945

P14

0.939

P12

0.938

P15

0.931

P15

0.935

P12

0.930

0.914

0.918

0.912

0.906

P13

0.907

P13

0.905

0.903

P11

0.902

P10

0.896

P10

0.898

P17

0.892

P17

0.891

P11

0.887

0.889

0.885

P18

0.866

P18

0.873

P19

0.852

P19

0.863

P20

0.820

P20

0.830

P16

0.810

P16

0.821

0.797

0.800

0.757

0.760

0.700

0.707

0.569

0.571