Screening anti-fatigue components of American ginseng saponin by analyzing spectrum–effect relationship coupled with UPLC-Q-TOF-MS

Lin, Meiyu; Hu, Shaiping; Zeng, Qi; Xiao, Bixia; He, Yao

doi:10.2478/abm-2023-0057

Figures & Tables

The HPLC chromatogram of AGS. The total content of ginsenosides Rg1, Re, and Rb1 was measured as 2.3% using HPLC. According to the provisions of Chinese Pharmacopoeia 2015, the total amount of ginsenosides Rg1, Re, and Rb1 should not be less than 2.0%. The content was up to the standards, indicating that the samples were qualified: 1, ginsenoside Rg1; 2, ginsenoside Re; and 3, ginsenoside Rb1. AGS, American ginseng saponin.

The flow chart of the study. Rats were randomized into three groups: blank group, negative model group, and test group. The rats in the test group were orally administrated with AGS at a portion of 50 mg/kg once per day and successively for 30 d; meanwhile, the rats in the blank and negative model groups were given water instead. The anti-fatigue effect of AGS was measured based on the swimming time, as well as on the contents of BUN, HG, and LA in rats. Blood specimens were taken from the retinal venous plexus from the rats in the test group both before and after administration of AGS. To identify the components of AGS that had undergone absorption in serum, measurements were made after administration, and the measured values were compared with the corresponding values obtained before administration. The relationship between the peak area values from rat serum and pharmacodynamic parameters of AGS was established using PLSR and gray correlation method. AGS, American ginseng saponin; BUN, blood urea nitrogen; HG, hepatic glycogen; LA, lactic acid.

TIC of AGS. UPLC-Q-TOF-MS in negative ESI mode was utilized to analyze the extract of AGS: 1, ginsenoside Rg1; 2, ginsenoside Re; 3, ginsenoside Malonyl-Rg1; 4, ginsenoside Malonyl-Re; 5, pseudoginsenoside-F11; 6, 20(S)-ginsenoside Rg2; 7, ginsenoside Rb1; 8, malonylginsenoside Rb1; 9, ginsenoside Rc; 10, malonylginsenoside Rb1 isomer; 11, malonylginsenoside Ra2; 12, ginsenoside Rb2; 13, ginsenoside Rb3; 14, malonylginsenoside Rb2 isomer; 15, malonylginsenoside Rb3 isomer; 16, Zingibroside R1; 17, ginsenoside Rd; 18, malonylginsenoside Rd; 19, gypenosid XVII; 20, 20(S)-ginsenoside Rg3; 21, Chikusetsu saponin IVa; 22, 20(R)-ginsenoside Rg3. AGS, American ginseng saponin.

TIC of blank serum. UPLC-Q-TOF-MS in negative ESI mode was utilized to analyze blank serum from rat before administration of AGS. AGS, American ginseng saponin.

TIC of serum containing drug. UPLC-Q-TOF-MS in negative ESI mode was utilized to analyze serum containing drug from rat after administration of AGS: 2, ginsenoside Re; 5, pseudoginsenoside-F11; 7, ginsenoside Rb1; 9, ginsenoside Rc; 12, ginsenoside Rb2; 13, ginsenoside Rb3; 17, ginsenoside Rd; and 19, gypenosid XVII. AGS, American ginseng saponin.

The regression coefficients plot of pharmacological results. The coefficient value before X reflects the degree of contribution of X to Y. (A) Using the regression equation coefficients, the graphs of regression coefficients of swimming time have been drawn. (B) Using the regression equation coefficients, the graphs of regression coefficients of the content of BUN have been drawn. (C) Using the regression equation coefficients, the graphs of regression coefficients of the content of HG have been drawn. (D) Using the regression equation coefficients, the graphs of regression coefficients of the content of LA have been drawn. Values are shown as mean (n = 10); error bars indicate standard deviation. BUN, blood urea nitrogen; HG, hepatic glycogen; LA, lactic acid.

Anti-fatigue effect of AGS

Group (n = 10)	Swimming time (min)	Content of BUN (mmol/L)	Content of HG (mg/g)	Content of LA (mmol/L)
Blank group	57.8 ± 8.6	8.57 ± 1.03	7.21 ± 1.05	7.62 ± 0.75
Negative model group	65.7 ± 9.5	8.28 ± 1.25	7.31 ± 1.24	7.39 ± 0.90
Test group	122.3 ± 18.9^***	7.21 ± 1.14^*	12.81 ± 1.58^***	6.42 ± 0.75^*

Correlation between 8 common peak areas and efficacy

No.	Swimming time	Content of BUN	Content of HG	Content of LA
2	0.91 ± 0.04†	0.90 ± 0.06	0.94 ± 0.05	0.95 ± 0.04
5	0.70 ± 0.22	0.80 ± 0.13	0.70 ± 0.21	0.70 ± 0.21
7	0.95 ± 0.05	0.93 ± 0.05	0.95 ± 0.06	0.93 ± 0.06
9	0.64 ± 0.24	0.72 ± 0.17	0.63 ± 0.22	0.63 ± 0.22
12	0.86 ± 0.10	0.85 ± 0.11	0.89 ± 0.11	0.88 ± 0.12
13	0.86 ± 0.10	0.69 ± 0.22	0.89 ± 0.10	0.87 ± 0.11
17	0.83 ± 0.10	0.62 ± 0.22	0.85 ± 0.13	0.84 ± 0.13
19	0.75 ± 0.19	0.83 ± 0.10	0.74 ± 0.17	0.74 ± 0.17

Identification of composition of AGS using UPLC-Q-TOF-MS

No.	t_R/min	Molecular formula	Mean measured mass	Theoretical exact mass	δ	[M − H + HCOOH]⁻	Identity	Fragments
1	7.33	C₄₂H₇₂O₁₄	799.4908	799.4849	7.38	845.5096	Ginsenoside Rg1	637.4366 [M–Glc–H]⁻475.3792 [M–2Glc–H]⁻
2	7.55	C₄₈H₈₂O₁₈	945.5429	945.5428	0.11	991.5691	Ginsenoside Re	799.4850 [M–Rha–H]⁻783.4913 [M–Glc–H]⁻637.4314 [M–Rha–Glc–H]⁻
3	9.48	C₄₅H₇₄O₁₇	885.4820	885.4853	−3.72	–	Ginsenoside Malonyl-Rg1	841.4949 [M–CO₂–H]⁻
4	10.14	C₅₁H₈₄O₂₁	1031.5400	1031.5432	−3.10	–	Ginsenoside Malonyl-Re	987.5555 [M–CO₂–H]⁻
5	15.87	C₄₂H₇₂O₁₄	799.4850	799.4844	0.75	845.4918	Pseudoginsenoside-F11	653.4298 [M–Rha–H]⁻
6	18.80	C₄₂H₇₂O₁₃	783.4913	783.4900	1.66	829.4960	20 (S)-ginsenoside Rg2	637.4314 [M–Xyl–H]⁻475.3792 [M–Xyl–Glc–H]⁻
7	20.58	C₅₄H₉₂O₂₃	1107.5995	1107.5957	3.43	1153.6097	Ginsenoside Rb1	945.5552 [M–Glc–H] −783.4336 [M–2Glc–H]⁻
8	21.32	C₅₇H₉₄O₂₆	1193.6053	1193.5961	7.71	–	Malonylginsenoside Rb1	1149.6067 [M–CO₂–H]⁻
9	21.67	C₅₃H₉₀O₂₂	1077.5851	1077.5845	0.56	1123.5918	Ginsenoside Rc	945.5429 [M–Araf–H]⁻783.4913 [M–Araf–Glc–H]⁻
10	22.02	C₅₇H₉₄O₂₆	1193.5983	1193.5961	1.84	–	Malonylginsenoside Rb1 isomer	1149.6136 [M–CO₂–H]⁻
11	22.34	C₅₆H₉₂O₂₅	1163.5919	1163.5855	5.50	–	Malonylginsenoside Ra2	1119.5933 [M–CO₂–H]⁻
12	22.79	C₅₃H₉₀O₂₂	1077.5851	1077.5845	0.56	1123.5918	Ginsenoside Rb2	945.5429 [M–Arap–H]⁻915.5326 [M–Glc–H]⁻783.4913 [M–2Glc–H]⁻
13	23.18	C₅₃H₉₀O₂₂	1077.5851	1077.5845	0.56	1123.5918	Ginsenoside Rb3	945.5429 [M–Xyl–H]⁻915.5326 [M–Glc–H]⁻783.49133 [M–2Glc–H]⁻
14	23.41	C₅₆H₉₂O₂₅	1163.5850	1163.5849	0.086	–	Malonylginsenoside Rb2 isomer	1119.6001 [M–CO₂–H]⁻
15	23.72	C₅₆H₉₂O₂₅	1163.5919	1163.5849	6.02	–	Malonylginsenoside Rb3 isomer	1119.6069 [M–CO₂–H]⁻
16	24.20	C₄₂H₆₅O₁₄	793.4396	793.4374	2.8	–	Zingibroside R1	631.38606 [M–Glc–H]⁻
17	24.56	C₄₈H₈₂O₁₈	945.5429	945.5428	0.11	991.5626	Ginsenoside Rd	783.4913 [M–Glc–H]⁻621.4373 [M–2Glc–H]⁻
18	24.81	C₅₁H₈₄O₂₁	1031.5466	1031.5432	0.58	–	Malonylginsenoside Rd	987.5684 [M–CO₂–H]⁻
19	25.29	C₄₈H₈₂O₁₈	945.5429	945.5423	0.63	991.5626	Gypenosid XVII	783.4913 [M–Glc–H]⁻621.4373 [M–2Glc–H]⁻
20	26.87	C₄₂H₇₂O₁₃	783.4913	783.4900	1.66	829.4960	20 (S)-ginsenoside Rg3	621.4375 [M–Glc–H]⁻459.4088 [M–2Glc–H]⁻
21	27.12	C₄₂H₆₆O₁₄	793.4396	793.4380	2.02	–	Chikusetsu saponin IVa	613.3718 [M–Glc–H]⁻
22	27.69	C₄₂H₇₂O₁₃	783.4913	783.4900	1.66	829.5065	20 (R)-ginsenoside Rg3	621.4373 [M–Glc–H]⁻

Screening anti-fatigue components of American ginseng saponin by analyzing spectrum–effect relationship coupled with UPLC-Q-TOF-MS

Figures & Tables

Figure 1.

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Figure 6.

Anti-fatigue effect of AGS

Correlation between 8 common peak areas and efficacy

Identification of composition of AGS using UPLC-Q-TOF-MS

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