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

The AGS was processed according to the Chinese Pharmacopoeia 2015 [8]. The decoction pieces of American ginseng were boiled for 2 times, 2 h for the first time, and for 1.5 h for the second time. Water 10 times the weight of American ginseng was added each time. The filtrates of the two times were combined. The filtrate was eluted with water till it became colorless through D101 macroporous adsorption resin column, and then eluted with 60% ethanol. The 60% ethanol was concentrated into the extract, after which it was dried and crushed. The content of AGS was measured using vanillin–glacial acetic acid–perchloric acid in a UV-2550 ultraviolet spectrophotometer (Shimadzu) [9]. The total saponin content of American ginseng was 81%. The total content of ginsenosides Rg1, Re, and Rb1 was determined using HPLC (e2695, Waters) [8]. Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B. The gradient elution condition was performed, as follows: 0–25 min, 19%–20% A; 25–60 min, 20%–40% A; 60–90 min, 40%–55% A; 90–100 min, 55%–60% A. The detection wavelength was 203 nm. The column temperature was 40 ºC. The total content of ginsenosides Rg1, Re, and Rb1 was 2.3%, which was up to the Chinese Pharmacopoeia 2015 standards. The HPLC chromatogram is shown in Figure 1. Accurately weighed AGS sample powders (20 mg) were dissolved in 25 mL methanol.

Figure 1.

A certain amount of standards was dissolved in methanol in numerous instances to produce various standard stock solutions. Adding different amounts of the above-mentioned standard stock solutions makes the mixed standard solution obtain final concentrations of about 1.50 μg/mL for each standard substance.

All the solutions were kept in reserve at 4 ºC in the pre-analytical phase. Before UPLC-Q-TOF-MS analysis, all the solutions were filtered through a 0.22 μm microporous membrane.

Chromatographic analysis was conducted with the application of a Waters ACQUITY UPLC I-Class system, provided with an autosampler, a thermostated column compartment, and a binary solvent delivery system. ACQUITY BEH C18 (2.1 mm × 100 mm, 1.7 μm, Waters) was for chromatographic fractionation. The mobile phase is composed of 0.1% formic acid in Milli-Q water (A) and acetonitrile (B). The current velocity of it stood at 0.3 mL/min. The UPLC elution condition is: 0–3 min, 20%–21% B; 3–5 min, 20%–21% B; 5–7 min, 22%–23% B; 7–10 min, 23%–25% B; 10–11 min, 25%–28% B; 11–17 min, 28%–30% B;17–22 min, 30%–35% B; 22–27 min, 35%–60% B; 27–31 min, 60%–90% B; 31–32 min, 90%–100% B; 32–36 min, 100% B; 36–42 min, 100%–20% B. The column temperature was 35 ºC, and the injection volume was 5 μL.

A Waters Xevo G2-XS Q-TOF system was offered for the MS detection. An electrospray ionization source (Waters) was available. All the operation and data analyses were conducted using Masslynx V4.1 software (Waters). The mass spectrometry was performed in the negative mode, and the optimized mass parameters were as below: capillary voltage, 2.5 kV; extraction cone voltage, 4 V; sample cone voltage, 40 V; source temperature, 120 ºC; cone gas flow rates, 50 L/h; desolvation gas flow rates, 500 L/h; and desolvation temperature, 600 ºC. Data were gathered in continuum mode. The width is 100–1200 Da. A lock-mass of leucine enkephalin was used in mass spectrometry analysis to ensure accuracy.

Liquid chromatography (LC) and mass spectrometry (MS) data were preprocessed with MarkerLynx V4.1 software for peak detection and peak alignment. Researchers retrieved the data by performing detailed searches in databases. The National Medical Library PubMed and the National Institutes of Health, SciFinder scholars of the American Chemical Society, and China National Knowledge Infrastructure (CNKI) of Tsinghua University were reported in the literature from a review of Panax species in Microsoft Office Excel. The sheet established a data bank that contains the name, molecular formula, chemical structure, and literature for each chemistry. The above information was imported into the UNIFI Scientific Information System (Waters) in the form of a database. UNIFI can identify compounds through an automated matching database. The “Search Database” in the “Identify Compounds” in UNIFI was used for qualitative analysis of chromatographic peaks.

Rats were randomized into two groups; blank group and fatigue model group. Model group rats were further randomized to negative model group and test group. The rats in test group were orally administrated with AGS at a potion of 50 mg/kg once per day and successively for 30 d; meanwhile, the rats in blank and negative model group were given water instead. There were 10 rats in each group. The rats in the fatigue model group were exposed to weight-loaded swimming every day. On the final day, all the rats were forced to swim after administration. It was determined that the rats were exhausted when they lacked normal coordination, and failed to surface in 10 s [10]. Researchers kept track of exhaustive swimming time at once. The next day, blood was collected in the second hour after administration. Serum samples were prepared for the BUN and LA determination and the livers for HG determination. All biochemical parameters were measured using commercial kits, and the whole of the process was performed in accordance with the instruction manual. All empirical data were represented as mean ± standard deviation and T test was applied. The data were analyzed for frequency and tested for significance using the statistical software SPSS20.0 (International Business Machines Corporation, IBM) computer package.

Blood specimens were taken from the retinal venous plexus from the rats in the test group before the administration of AGS. The blood samples, together with those collected after the animal experiments, were kept for 30 min and then centrifuged at 4,000 rpm for 10 min. Next, 400 μL methanol was added to the 100 μL serum specimens, followed by thorough mixing for 5 min, and being centrifuged at 12,000 rpm at 4 ºC for 10 min. The supernatant was evaporated to dryness with a frozen dryer, re-dissolved in 100 μL methanol, and centrifuged at 12,000 rpm for 10 min at 4 ºC; thereafter, the supernatant was injected into the LC-MS system for research purpose.

The partial least squares regressions (PLSR) and gray correlation method were carried out using MATLAB 2017b (MathWorks Inc.) to analyze the correlation between the values of peak areas in LC-MS fingerprints and pharmacological results from the anti-fatigue experiment. The flow chart of this study is as shown in Figure 2.

Figure 2.

The results obtained from carrying out a Total ion chromatography (TIC) of AGS using UPLC-Q-TOF-MS in negative electrospray ionization (ESI) mode are displayed in Figure 3. The compositions of AGS were well-separated and identified by analyzing the precise relative molecular mass of primary mass spectrometry, the cleavage fragments of MS/MS and reported paper [11,12,13]. Twenty-two peaks were identified, and the results are shown in Table 1.

Figure 3.

Eight components were detected in the serum samples after administration, compared with the serum collected before administration. The TIC of blank serum and serum containing drug, obtained with the use of UPLC-Q-TOF-MS in negative ESI mode, are, respectively, displayed in Figures 4 and 5. Through comparisons with the standard as well as with the literature, they were identified as ginsenoside Re, pseudoginsenoside-F11, ginsenosides Rb1, Rc, Rb2, Rb3, and Rd, and gypenosid XVII. They are all prototype components.

Figure 4.

Figure 5.

The rats were weighed daily and were given appropriate medication. No adverse events occurred in any of the groups. As shown in Table 2, swimming time and content of HG in the test group outclassed those in the negative model group; meanwhile, the contents of BUN and LA were well below those in the negative model group.

The eight common peak areas were marked as independent variables (X). Swimming time, contents of HG, BUN, and LA were marked as dependent variables (Y). Regression and correlation analyses were constituted using PLSA analysis. Employing these values, we derived correlation coefficients. The results of the data were as follows:

Swimming time

Y1 = 0.02 × 2–0.03 × 5 + 0.05 × 7–0.05 × 9 + 0.03 × 12 + 0.18 × 13 + 0.19 × 17-0.03 × 19

The coefficient value before X reflects the degree of contribution of X to Y. Using the regression equation coefficients, the graphs of regression coefficients of physiological activity have been drawn (Figure 6).

Figure 6.

Gray correlation theory modeling was used to analyze the correlation degree of eight common peak areas with swimming time and contents of HG, BUN, and LA. The results are shown in Table 3.