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

Meiyu Lin
Meiyu Lin
, 
Shaiping Hu
Shaiping Hu
, 
Qi Zeng
Qi Zeng
, 
Bixia Xiao
Bixia Xiao
 et 
Yao He
Yao He
   | 18 oct. 2023
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Article Category: Original article
Publié en ligne: 18 oct. 2023
Pages: 163 - 172
DOI: https://doi.org/10.2478/abm-2023-0057
Mots clés
© 2023 Meiyu Lin et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1.

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 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.

Figure 2.

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.
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.

Figure 3.

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 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.

Figure 4.

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 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.

Figure 5.

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.
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.

Figure 6.

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.
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. tR/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]−
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