Abstract
The pharmacokinetics and distribution of natural compounds in living organisms are crucial to understanding their therapeutic potential and safety. In the study, ultraperformance liquid chromatography–mass spectrometry (UPLC-MS/MS) was employed to develop a method for the separation and analysis of seven saponins, namely, G-Rg1, G-Re, G-Rb1, G-Ro, PG-RT1, CS-IV, and CS-IVa, in plasma and tissue samples derived from Panacis Japonici Rhizoma (PJR). Additionally, the pharmacokinetic profiles and tissue distribution characteristics of these saponins in rats were systematically investigated. Following the oral administration of 2 g/kg of PJR extracts, UPLC-MS/MS was utilized to analyze the compounds’ presence in plasma and various tissues. Key findings indicate that G-Rb1 exhibited the highest T max, T 1/2, C max, and AUC0–t values, reflecting its slow and extended presence in the bloodstream. Dual and triple peak phenomena were observed in the plasma concentration–time curves for certain saponins, suggesting complex absorption dynamics. All seven saponins were detected across multiple tissues within 1 h postadministration and remained detectable for up to 10 h, with the lung and brain showing the highest and lowest concentrations, respectively. These results highlight the compounds’ broad distribution and prolonged elimination, providing a foundational understanding that is beneficial for future clinical applications of PJR.
1. Introduction
Panacis Japonici Rhizoma (PJR), commonly known as white Panax notoginseng, is derived from the dried rhizomes of Panax japonicus C. A. Mey., a member of the Araliaceae family. This herb is highly regarded in traditional Chinese medicine for its efficacy in dispersing blood stasis, halting bleeding, alleviating pain, clearing phlegm, fortifying health, and enhancing physical strength. Its application extends to the Tujia and Miao ethnic communities in China, where it is used in alcoholic infusions to treat rheumatoid arthritis (RA), showing notable effectiveness against immune and general inflammation. − PJR also features numerous Chinese medicinal prescriptions, including the compound Panax Japonicus tablet, aimed to combat RA.
Extensive phytochemical analyses have revealed that PJR is rich in saponins, polysaccharides, amino acids, volatile oils, inorganic elements, and nucleosides. Notably, its saponin compounds such as ginsenoside Rg1 (G-Rg1), ginsenoside Re (G-Re), ginsenoside Rb1 (G-Rb1), ginsenoside Ro (G-Ro), pseudoginsenoside RT1 (PG-RT1), chikusetsusaponin IV (CS-IV), and chikusetsusaponin IVa (CS-IVa) stand out as critical active ingredients. Previous research has demonstrated the anti-inflammatory, neuroprotective, and antiapoptotic properties of ginsenosides Rg1, Re, and Rb1, particularly in models of cerebral ischemia. − Additionally, G-Ro, PG-RT1, CS-IV, and CS-IVa have also exhibited potent anti-inflammatory activities, − further underscoring their potential utility in the management of RA.
Pharmacokinetics and tissue distribution are essential for understanding the in vivo behavior of drugs, which has attracted increasing interest in drug metabolism research. Previous studies have extensively explored the pharmacokinetics of single compounds and extracts from PJR in animal models. − However, few studies of comprehensive data on the pharmacokinetics and tissue distribution parameters of seven specific saponinsG-Rg1, G-Re, G-Rb1, G-Ro, PG-RT1, CS-IV, and CS-IVafollowing oral administration in rats have been conducted, particularly regarding its parameters in joint tissue.
The ultraperformance liquid chromatography–mass spectrometry (UPLC-MS/MS) assay, known for its rapid separation, high sensitivity, exceptional selectivity, and excellent measurement accuracy, has been successfully applied to multicomponent analysis in plasma. The present study aims to establish a method for the separation and analysis of these saponins and to fill the existing knowledge gaps by determining the dynamic concentrations of these saponins in the blood at different time points and in various tissues after rats are administered a single dose of PJR extract. This research is pivotal as it is the first report on investigation of the pharmacokinetics and tissue distribution for the seven saponins in this context. The findings are expected to provide crucial insights into further research and establish a theoretical foundation for component separation and clinical application of PJR, particularly for its anti-RA potential.
2. Materials and Methods
2.1. Chemicals and Reagents
PJR was sourced from Dongbei and authenticated by Associate Professor Limin Gong (Hunan University of Chinese Medicine, Hunan, China) according to the Chinese Pharmacopoeia (2020 edition). The saponins used in this study were procured with a purity of at least 98%: G-Rg1 (HG7162W2), G-Re (HR8526S1), G-Rb1 (HR191017B3), G-Ro (HS19115B2), PG-RT1 (HS191224B1), CS-IV (HS191225B1), and CS-IVa (HR5128B1) were all supplied by Baoji Herbest Biotechnology Co., Ltd. (Shanxi, China). Nimodipine, also with a purity of ≥98% (100270–201904), was used as an internal standard (IS) and was obtained from the National Institute for Food and Drug Control (Beijing, China). The structural details of these compounds are depicted in Figure . Acetonitrile and methanol, both of LC-MS grade, were purchased from Thermo Fisher Scientific (Waltham, MA, USA). LC-MS grade formic acid was obtained from Anaqua Chemicals Supply (Wilmington, Delaware, USA). Ultrapure water used in the experiments was prepared in the laboratory.
1.
Chemical structures of seven analytes and internal standard nimodipine.
2.2. Preparation of Extracts
The air-dried rhizomes of Panax japonicus (400 g) were cut into small pieces and subjected to two sequential reflux extractions with 70% ethanol, using solid-to-liquid ratios of 1:10 and 1:8, for 3 and 2 h, respectively. Following filtration, the extracts were evaporated under reduced pressure at 70 °C to remove the ethanol. The resulting concentrated extracts were further dried under vacuum to yield the final PJR extracts. The concentrations of the saponins G-Rg1, G-Re, G-Rb1, G-Ro, PG-RT1, CS-IV, and CS-IVa were quantified using UPLC, yielding values of 4.67 2.41, 4.72, 79.98, 33.09, 57.29, and 48.81 mg/g, respectively.
2.3. UPLC-MS/MS System and Analytical Conditions
Chromatographic analysis was performed by using a Waters ACQUITY series UPLC system (Waters, Milford, MA, USA). The separation was achieved on a Waters CORTECS UPLC T3 column (2.1 × 100 mm, 1.6 μm) at room temperature. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B), applied in a gradient as follows: 0–2 min, 21–33.7% B; 2–4 min, 33.7–34.4% B; 4–7.5 min, 34.4–34.5% B; 7.5–9.5 min, 34.5–100% B; and 9.5–10 min, 100% B. The flow rate was maintained at 0.3 mL/min, and the column temperature was held at 30 °C. Injection volumes for plasma and tissue supernatants were 1 and 3 μL, respectively.
Mass spectrometric detection was conducted using an AB SCIEX QTRAP 5500 system (AB SCIEX, Waltham, USA), employing both positive and negative electrospray ionization (ESI) sources in multiple-reaction monitoring (MRM) mode. The parameters are listed in Table S1 (Supporting Information). Key operational parameters included a curtain gas at 30 psi, a medium collision gas setting, ion spray voltages of 5500 and −4500 V, an auxiliary gas heating temperature of 500 °C, a nebulizer gas at 50 psi, and an auxiliary heating gas at 50 psi. The total run time was set to 10 min.
2.4. Animals
Specific Pathogen Free male Sprague–Dawley (SD) rats weighing 280–320 g were acquired from Beijing Huafukang Biotechnology Co., Ltd. The rats were housed at the Laboratory Animal Center of the Hunan University of Chinese Medicine (license no. SYXK [Hunan] 2019-0009) and maintained at 20–26 °C, relative humidity of 40–70%, and a 12 h light/dark cycle. All animals underwent a 5 day acclimatization period before experimentation. Ethical approval for the animal study was granted by the Institutional Animal Care and Use Committee of HNUCM (approval no. 202107090005).
2.5. Pharmacokinetic and Tissue Distribution Study
Five male SD rats were each administered a single oral dose of 2 g/kg of PJR extracts. Blood samples (0.3 mL) were collected at specified intervals (0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 4, 6, 9, 12, 24, 36, 48, 60, and 72 h postadministration) using heparin sodium anticoagulant tubes from the jugular veins. These samples were centrifuged at 3600 rpm for 15 min at 4 °C. The resulting plasma (upper layer) was transferred to a labeled centrifuge tube and stored at −80 °C for subsequent analysis. Additionally, 30 male SD rats were divided into six groups (n = 5) and similarly dosed with 2 g/kg of PJR extracts. These animals were euthanized at 1, 2, 4, 6, 12, and 24 h postdosing to collect tissue samples, including heart, liver, spleen, lung, kidney, brain, and ankle joints. Tissues were rinsed with 0.9% sodium chloride, patted dry, and stored at −80 °C until analysis. Tissue homogenization was performed by adding physiological saline at a weight/volume ratio 1:2, blending twice at 65 Hz for 2 min each. These homogenates were then centrifuged at 3600 rpm for 15 min. The resulting homogenates (upper layer) were stored at −80 °C for subsequent analysis.
2.6. Sample Processing
Plasma and tissue homogenate upper layer samples (50 μL) were mixed with 10 μL (30 μL for tissue samples) of Nimodipine (IS, 5 ng/mL) and 250 μL of acetonitrile and then vortexed for 1 min. The mixture was centrifuged at 12,000 rpm for 10 min to clarify. The supernatant was then dried at 40 °C for 1.5 h. The residue was reconstituted in 50 μL (150 μL for tissue samples) of 50% methanol, vortexed for 2 min, and centrifuged at 12,000 rpm for 10 min. The final clear supernatant was filtered through a 0.22 μm Millipore organic phase filter, and an appropriate volume was injected into the UPLC-MS/MS system for analysis.
2.7. Preparation of Standard Solution, Calibration Standard, and Quality Control (QC) Samples
Standard stock solutions for G-Rg1, G-Re, G-Rb1, G-Ro, PG-RT1, CS-IV, and CS-IVa (225.2, 229.2, 217.6, 200.0, 216.0, 218.0, and 205.6 μg/mL, respectively) and IS (210.0 μg/mL) were prepared in 50% methanol. These were further diluted to produce working solutions. Calibration standards were then prepared by spiking 10 μL of these working solutions into 50 μL of blank plasma along with 10 μL of IS solution to achieve concentration ranges from 1.13 to 1126.00 ng/mL for G-Rg1, 1.15 to 1146.00 ng/mL for G-Re, 10.88 to 1088.00 ng/mL for G-Rb1, 1.00 to 1000.00 ng/mL for G-Ro, 1.08 to 1080.00 ng/mL for PG-RT1, 1.09 to 1090.00 ng/mL for CS-IV, and 0.51 to 1028.00 ng/mL for CS-IVa. QC samples at low, medium, and high concentrations (2.25, 90.08, and 900.80 ng/mL for G-Rg1; 2.29, 91.68, and 916.80 ng/mL for G-Re; 21.76, 87.04, and 870.40 ng/mL for G-Rb1; 2.00, 80.00, and 800.00 ng/mL for G-Ro; 2.16, 86.40, and 864.00 ng/mL for PG-RT1; 2.18, 87.20, and 872.00 ng/mL for CS-IV′ 1.03, 82.24, and 822.40 ng/mL for CS-IVa) were prepared similarly. All solutions were stored at −4 °C until needed for analysis.
2.8. Method Validation
The UPLC-MS/MS method developed was rigorously validated for specificity, linearity, sensitivity, precision, accuracy, extraction recovery, matrix effect, and stability of the analytes.
2.8.1. Specificity
Specificity was evaluated by analyzing blank blood samples, blank blood samples spiked with standards, and IS and blood samples after oral administration of PJR extracts to confirm the absence of endogenous interference.
2.8.2. Linearity and Sensitivity
Standard curves were generated by spiking blank blood samples with varying concentrations of the working solutions. These curves were established using the peak area ratios of analyte to IS (Y) versus the standard concentrations (X), applying a weighted linear least-squares regression model (1/χ2). Sensitivity was assessed by determining the limit of detection (LOD) and limit of quantification (LOQ), established at signal-to-noise ratios of 3:1 and 10:1, respectively.
2.8.3. Precision and Accuracy
Precision and accuracy involved the analysis of three QC sample concentrations (low, medium, and high) in six replicates across 1 day and over 3 consecutive days. Precision was quantified as relative standard deviation (RSD %), and accuracy was reported as a relative error (RE %) across these days.
2.8.4. Extraction Recovery and Matrix Effect
Extraction recovery was assessed by comparing the peak areas of QC samples at three concentration levels (low, medium, and high) with those of blank samples spiked postextraction at similar concentrations across six replicates. The matrix effect was evaluated by comparing peak areas of analytes in spiked blank sample extracts to those in pure standard solutions with identical concentrations.
2.8.5. Stability
Stability tests were conducted on three-tier QC samples under various storage conditions: room temperature for 12 h, three freeze–thaw cycles (24 h each), and at −20 °C for 30 days. These stability assays were performed in six replicates.
2.9. Statistical Analysis
Pharmacokinetic data were analyzed using Phoenix WinNonlin software version 8.1 (Pharsight, Mountain View, CA, USA), which applied a noncompartmental model. Graphical representations were created with GraphPad Prism version 9. Data were expressed as means ± standard deviation (SD), and graphical data were presented as means ± standard error of the mean (SEM).
3. Results
3.1. Method Validation
3.1.1. Specificity
Representative chromatograms (Figure ) for blank plasma, plasma spiked with the seven analytes and IS, and plasma samples collected 1 h after administration of PJR extracts demonstrated successful separation of the analytes and IS with no significant endogenous interference at their retention times.
2.
Representative MRM chromatograms of seven analytes and IS in rat plasma. (A) Blank plasma sample, (B) blank plasma spiked with seven analytes and IS, and (C) plasma sample after a single oral administration of PJR extracts spiked with IS (1: G-Rg1; 2: G-Re; 3: G-Rb1; 4: G-Ro; 5: PG-RT1; 6: CS-IV; 7: CS-IVa; 8: IS+; 9: IS–).
3.1.2. Linearity and Sensitivity
As depicted in Table , all seven analytes exhibited strong linear relationships within their respective concentration ranges across plasma and tissue matrices. The correlation coefficients exceeded 0.9964 for plasma and 0.9959 for tissues. LOD and LOQ are listed in Table S2 (Supporting Information).
1. Linearity of Seven Analytes in Different Matrices Harvested from Rats.
| tissue | analytes | concentration range (ng/mL) | standard curve | correlation coefficient (r) |
|---|---|---|---|---|
| plasma | G-Rg1 | 1.13–1126.00 | y = 0.00970x + 0.00369 | 0.99774 |
| G-Re | 1.15–1146.00 | y = 0.00166x + 0.00050 | 0.99908 | |
| G-Rb1 | 10.88–1088.00 | y = 0.00098x + 0.00440 | 0.99642 | |
| G-Ro | 1.00–1000.00 | y = 0.01710x + 0.00685 | 0.99843 | |
| PG-RT1 | 1.08–1080.00 | y = 0.01548x + 0.00325 | 0.99991 | |
| CS-IV | 1.09–1090.00 | y = 0.04534x + 0.02204 | 0.99769 | |
| CS-IVa | 0.51–1028.00 | y = 0.03452x + 0.00275 | 0.99793 | |
| heart | G-Rg1 | 0.56–1126.00 | y = 0.01441x + 0.03269 | 0.99662 |
| G-Re | 0.57–573.00 | y = 0.00341x + 0.00339 | 0.99726 | |
| G-Rb1 | 0.44–544.00 | y = 0.00324x + 0.00362 | 0.99839 | |
| G-Ro | 0.50–2000.00 | y = 0.02056x + 0.09048 | 0.99854 | |
| PG-RT1 | 0.54–540.00 | y = 0.01762x + 0.02078 | 0.99784 | |
| CS-IV | 0.55–545.00 | y = 0.06770x + 0.11692 | 0.99784 | |
| CS-IVa | 0.51–514.00 | y = 0.06918x + 0.09793 | 0.99725 | |
| liver | G-Rg1 | 0.56–563.00 | y = 0.03221x + 0.04399 | 0.99798 |
| G-Re | 0.57–573.00 | y = 0.00678x + 0.00254 | 0.99627 | |
| G-Rb1 | 0.54–544.00 | y = 0.00923x + 0.00113 | 0.99708 | |
| G-Ro | 0.50–1500.00 | y = 0.03270x + 0.09993 | 0.99991 | |
| PG-RT1 | 1.08–1080.00 | y = 0.02809x + 0.02274 | 0.99936 | |
| CS-IV | 0.55–1090.00 | y = 0.06974x + 0.12198 | 0.99796 | |
| CS-IVa | 1.03–1028.00 | y = 0.06595x + 0.09887 | 0.99871 | |
| spleen | G-Rg1 | 0.45–563.00 | y = 0.00877x + 0.01791 | 0.99884 |
| G-Re | 0.57–573.00 | y = 0.00150x + 0.00197 | 0.99743 | |
| G-Rb1 | 0.54–1632.00 | y = 0.00199x + 0.00552 | 0.99700 | |
| G-Ro | 0.50–1000.00 | y = 0.01251x + 0.11313 | 0.99786 | |
| PG-RT1 | 0.54–540.00 | y = 0.01003x + 0.02392 | 0.99946 | |
| CS-IV | 0.55–1090.00 | y = 0.03535x + 0.19416 | 0.99943 | |
| CS-IVa | 0.51–1028.00 | y = 0.03749x + 0.12514 | 0.99919 | |
| lung | G-Rg1 | 1.13–2815.00 | y = 0.00723x + 0.00910 | 0.99727 |
| G-Re | 0.57–2865.00 | y = 0.00131x + 0.00738 | 0.99757 | |
| G-Rb1 | 1.09–544.00 | y = 0.00125x + 0.00255 | 0.99714 | |
| G-Ro | 0.50–2500.00 | y = 0.01117x + 0.05145 | 0.99987 | |
| PG-RT1 | 0.54–2700.00 | y = 0.00952x + 0.03659 | 0.99988 | |
| CS-IV | 0.55–2725.00 | y = 0.05078x + 0.23330 | 0.99955 | |
| CS-IVa | 0.51–2570.00 | y = 0.03682x + 0.19243 | 0.99954 | |
| kidney | G-Rg1 | 0.56–1689.00 | y = 0.02430x + 0.14456 | 0.99587 |
| G-Re | 0.46–343.80 | y = 0.00719x + 0.00214 | 0.99612 | |
| G-Rb1 | 0.44–544.00 | y = 0.00892x + 0.00470 | 0.99803 | |
| G-Ro | 1.00–1000.00 | y = 0.02637x + 0.06475 | 0.99931 | |
| PG-RT1 | 1.08–1080.00 | y = 0.02585x + 0.00367 | 0.99958 | |
| CS-IV | 1.09–1090.00 | y = 0.06140x + 0.05917 | 0.99875 | |
| CS-IVa | 0.51–514.00 | y = 0.05957x + 0.04303 | 0.99969 | |
| brain | G-Rg1 | 0.45–563.00 | y = 0.02147x + 0.02394 | 0.99912 |
| G-Re | 0.46–343.80 | y = 0.00518x + 0.00126 | 0.99721 | |
| G-Rb1 | 0.44–326.40 | y = 0.00692x + 0.00583 | 0.99829 | |
| G-Ro | 0.40–1500.00 | y = 0.02260x + 0.05539 | 0.99935 | |
| PG-RT1 | 0.43–540.00 | y = 0.01853x + 0.01283 | 0.99762 | |
| CS-IV | 0.44–545.00 | y = 0.04408x + 0.03822 | 0.99940 | |
| CS-IVa | 0.41–514.00 | y = 0.04157x + 0.03915 | 0.99914 | |
| ankle joints | G-Rg1 | 0.45–563.00 | y = 0.02531x + 0.03966 | 0.99826 |
| G-Re | 0.57–114.60 | y = 0.00627x + 0.00085 | 0.99735 | |
| G-Rb1 | 0.44–1632.00 | y = 0.00747x + 0.00497 | 0.99598 | |
| G-Ro | 2.50–500.00 | y = 0.02610x + 0.08250 | 0.99709 | |
| PG-RT1 | 0.43–540.00 | y = 0.01968x + 0.02441 | 0.99958 | |
| CS-IV | 1.09–545.00 | y = 0.04519x + 0.10723 | 0.99921 | |
| CS-IVa | 0.51–1028.00 | y = 0.04418x + 0.06545 | 0.99946 |
3.1.3. Precision and Accuracy
The data presented in Table show intra- and interday precision values ranging from 1.20 to 12.20% and accuracy levels between 91.74 and 103.50%. These metrics confirm the reliability and robustness of the analytical method for routine use in sample analysis.
2. Precision and Accuracy of Seven Analytes in Plasma Samples (mean ± SD, n = 6).
| intraday (n = 6) |
interday (n = 18) |
||||
|---|---|---|---|---|---|
| analytes | concentration (ng/mL) | precision RSD (%) | accuracy (%) | precision RSD (%) | accuracy (%) |
| G-Rg1 | 2.25 | 1.20 | 91.76 | 8.06 | 96.02 |
| 90.08 | 5.35 | 97.71 | 5.48 | 98.43 | |
| 900.80 | 7.77 | 99.80 | 7.39 | 99.97 | |
| G-Re | 2.29 | 11.50 | 101.08 | 10.42 | 102.10 |
| 91.68 | 3.68 | 98.78 | 7.33 | 103.27 | |
| 916.80 | 4.36 | 96.53 | 8.60 | 96.08 | |
| G-Rb1 | 21.76 | 7.41 | 103.10 | 6.36 | 101.21 |
| 87.04 | 6.38 | 95.49 | 6.88 | 101.24 | |
| 870.40 | 3.93 | 101.32 | 7.24 | 100.62 | |
| G-Ro | 2.00 | 8.30 | 101.68 | 7.44 | 101.90 |
| 80.00 | 6.82 | 92.04 | 9.84 | 100.65 | |
| 800.00 | 4.14 | 91.74 | 5.69 | 95.34 | |
| PG-RT1 | 2.16 | 11.98 | 101.90 | 9.95 | 103.50 |
| 86.40 | 2.38 | 95.75 | 6.43 | 101.05 | |
| 864.00 | 3.73 | 98.49 | 6.30 | 100.45 | |
| CS-IV | 2.18 | 3.24 | 97.79 | 8.15 | 100.78 |
| 87.2 | 4.46 | 93.07 | 6.65 | 96.06 | |
| 872.00 | 6.03 | 101.90 | 5.80 | 98.48 | |
| CS-IVa | 1.03 | 8.09 | 101.84 | 10.75 | 101.18 |
| 82.24 | 5.66 | 92.90 | 6.19 | 92.87 | |
| 822.40 | 4.64 | 98.71 | 5.05 | 95.10 | |
3.1.4. Extraction Recovery and Matrix Effect
The extraction efficiencies and matrix effects for the analytes across the three QC levels are summarized in Table . Recovery rates varied from 88.95 to 109.82%, while matrix effects ranged from 89.58 to 114.89%, indicating minimal influence from biological matrices and confirming the method’s efficacy for the analytes’ quantification.
3. Extraction Recovery and Matrix Effect of Seven Analytes in Plasma Samples (mean ± SD, n = 6).
| analytes | concentration (ng/mL) | extraction recovery (%) (mean ± SD, n = 6) | matrix effect (%) (mean ± SD, n = 6) |
|---|---|---|---|
| G-Rg1 | 2.25 | 97.11 ± 8.82 | 97.06 ± 4.87 |
| 90.08 | 89.70 ± 4.11 | 105.29 ± 8.92 | |
| 900.80 | 100.56 ± 6.51 | 103.29 ± 10.06 | |
| G-Re | 2.29 | 103.32 ± 9.14 | 101.71 ± 12.03 |
| 91.68 | 89.34 ± 3.15 | 110.12 ± 8.42 | |
| 916.80 | 99.30 ± 5.43 | 114.34 ± 6.72 | |
| G-Rb1 | 21.76 | 109.82 ± 6.72 | 89.58 ± 4.62 |
| 87.04 | 95.81 ± 8.78 | 99.13 ± 12.55 | |
| 870.40 | 104.90 ± 8.37 | 98.75 ± 11.73 | |
| G-Ro | 2.00 | 102.32 ± 6.77 | 95.99 ± 8.29 |
| 80.00 | 95.21 ± 8.39 | 102.13 ± 6.02 | |
| 800.00 | 97.13 ± 8.15 | 102.02 ± 9.91 | |
| PG-RT1 | 2.16 | 102.22 ± 5.16 | 103.95 ± 7.38 |
| 86.40 | 92.02 ± 5.60 | 98.28 ± 6.86 | |
| 864.00 | 93.18 ± 5.95 | 110.00 ± 8.27 | |
| CS-IV | 2.18 | 92.66 ± 5.76 | 105.53 ± 5.97 |
| 87.2 | 93.72 ± 8.14 | 98.99 ± 8.23 | |
| 872.00 | 89.39 ± 3.78 | 110.32 ± 7.97 | |
| CS-IVa | 1.03 | 96.79 ± 7.78 | 103.10 ± 7.07 |
| 82.24 | 92.10 ± 3.23 | 101.48 ± 5.12 | |
| 822.40 | 88.95 ± 3.40 | 114.89 ± 13.66 |
3.1.5. Stability
The stabilities of the seven components under various storage conditions are detailed in Table . The analytes remained stable at room temperature for 12 h (relative error, RE%, ranged from −11.05 to 7.70%; relative standard deviation, RSD, ≤ 11.32%) throughout three freeze–thaw cycles (RE from −9.58 to 7.15%; RSD ≤ 10.68%) and at −20 °C for 30 days (RE from −8.76 to 6.49%; RSD ≤ 10.98%), thereby demonstrating their stability under these conditions.
4. Stability of Seven Analytes in Plasma Samples (mean ± SD, n = 6).
| room temperature stability |
freeze–thaw stability |
long-term freezing stability |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| analytes | nominal concentration (ng/mL) | measured concentration (mean ± SD, ng/mL) | RSD precision (%) | accuracy (RE%) | measured concentration mean ± SD, ng/mL) | RSD precision(%) | accuracy (RE%) | measured concentration (mean ± SD, ng/mL) | RSD precision (%) | accuracy (RE%) |
| G-Rg1 | 2.25 | 2.35 ± 0.20 | 8.67 | 4.26 | 2.29 ± 0.19 | 8.29 | 1.96 | 2.40 ± 0.10 | 4.25 | 6.49 |
| 90.08 | 80.13 ± 2.84 | 3.54 | –11.05 | 87.40 ± 6.17 | 7.06 | –2.97 | 82.19 ± 6.24 | 7.60 | –8.76 | |
| 900.80 | 970.17 ± 70.87 | 7.30 | 7.70 | 944.82 ± 84.61 | 8.96 | 4.89 | 893.22 ± 98.12 | 10.98 | –0.84 | |
| G-Re | 2.29 | 2.32 ± 0.22 | 9.65 | 1.26 | 2.35 ± 0.23 | 9.69 | 2.82 | 2.29 ± 0.11 | 4.98 | 0.20 |
| 91.68 | 88.78 ± 7.79 | 8.78 | –3.16 | 90.75 ± 9.69 | 10.68 | –1.01 | 92.64 ± 4.26 | 4.59 | 1.05 | |
| 916.80 | 916.60 ± 91.14 | 9.94 | –0.02 | 941.97 ± 40.64 | 4.31 | 2.75 | 912.32 ± 52.29 | 5.73 | –0.49 | |
| G-Rb1 | 21.76 | 22.77 ± 1.25 | 5.50 | 4.63 | 21.64 ± 0.67 | 3.11 | –0.54 | 22.44 ± 1.22 | 5.46 | 3.12 |
| 87.04 | 83.63 ± 7.36 | 8.80 | –3.91 | 87.12 ± 7.95 | 9.12 | 0.10 | 83.36 ± 6.03 | 7.23 | –4.22 | |
| 870.40 | 890.03 ± 82.54 | 9.27 | 2.26 | 873.50 ± 78.45 | 8.98 | 0.36 | 878.43 ± 95.93 | 10.92 | 0.92 | |
| G-Ro | 2.00 | 1.99 ± 0.18 | 9.16 | –0.50 | 1.98 ± 0.17 | 8.80 | –1.03 | 1.98 ± 0.19 | 9.36 | –1.05 |
| 80.00 | 76.47 ± 5.96 | 7.79 | –4.41 | 77.91 ± 4.52 | 5.80 | –2.61 | 73.32 ± 4.36 | 5.94 | –8.35 | |
| 800.00 | 801.17 ± 37.13 | 4.63 | 0.15 | 728.63 ± 32.84 | 4.51 | –8.92 | 749.63 ± 49.85 | 6.65 | –6.30 | |
| PG-RT1 | 2.16 | 2.28 ± 0.08 | 3.61 | 5.42 | 2.31 ± 0.11 | 4.54 | 7.15 | 2.19 ± 0.21 | 9.50 | 1.19 |
| 86.40 | 81.29 ± 5.09 | 6.26 | –5.91 | 78.13 ± 3.44 | 4.41 | –9.58 | 82.15 ± 2.83 | 3.44 | –4.92 | |
| 864.00 | 848.30 ± 33.26 | 3.92 | –1.82 | 822.60 ± 30.97 | 3.76 | –4.79 | 844.15 ± 51.06 | 6.05 | –2.30 | |
| CS-IV | 2.18 | 2.09 ± 0.24 | 11.32 | –4.04 | 2.25 ± 0.12 | 5.39 | 3.33 | 2.18 ± 0.15 | 6.68 | –0.11 |
| 87.2 | 83.62 ± 7.63 | 9.13 | –4.11 | 81.93 ± 4.68 | 5.71 | –6.05 | 82.43 ± 6.78 | 8.23 | –5.47 | |
| 872.00 | 866.82 ± 25.89 | 2.99 | –0.59 | 848.13 ± 26.29 | 3.10 | –2.74 | 868.92 ± 25.15 | 2.89 | –0.35 | |
| CS-IVa | 1.03 | 1.02 ± 0.08 | 7.91 | –0.64 | 0.97 ± 0.06 | 6.68 | –5.85 | 1.08 ± 0.06 | 5.38 | 4.61 |
| 82.24 | 79.13 ± 7.35 | 9.28 | –3.78 | 78.38 ± 3.90 | 4.97 | –4.69 | 76.33 ± 6.46 | 8.47 | –7.19 | |
| 822.40 | 802.40 ± 13.92 | 1.74 | –2.43 | 827.77 ± 24.48 | 2.96 | 0.65 | 838.45 ± 18.36 | 2.19 | 1.95 | |
3.2. Pharmacokinetic and Tissue Distribution Study
The UPLC-MS/MS method was effectively employed to investigate the pharmacokinetics and tissue distribution of PJR extracts in rats administered orally at a dose of 2 g/kg. Figure illustrates the mean plasma concentration–time profiles of the seven saponins, with the primary noncompartmental pharmacokinetic parameters detailed in Table .
3.
Mean plasma concentration–time curves of seven analytes after oral administration of PJR extracts (2 g/kg, n = 5).
5. Pharmacokinetic Parameters of Seven Analytes after Oral Administration of 2 g/kg PJR Extracts in Rats (mean ± SD, n = 5).
| analytes | AUC(0–t) (h*ng/mL) | AUC(0‑∞)(h*ng/mL) | MRT(0–t) (h) | t 1/2 (h) | T max (h) | Vd/F(mL/kg) | CL/F(mL/h/kg) | C max (ng/mL) |
|---|---|---|---|---|---|---|---|---|
| G-Rg1 | 54.40 ± 13.95 | 84.92 ± 21.44 | 11.79 ± 0.61 | 13.69 ± 4.07 | 1.20 ± 0.27 | 4,455,735.00 ± 1,467,420.65 | 230,784.53 ± 59,445.89 | 5.26 ± 1.89 |
| G-Re | 56.32 ± 28.56 | 93.56 ± 23.33 | 4.93 ± 1.63 | 8.70 ± 6.59 | 0.90 ± 0.45 | 1,248,131.40 ± 80,2571.13 | 105,169.34 ± 31,300.87 | 19.10 ± 13.60 |
| G-Rb1 | 5122.23 ± 1090.58 | 6314.59 ± 2048.25 | 30.05 ± 4.03 | 26.22 ± 11.22 | 19.20 ± 6.57 | 149,884.33 ± 27,453.35 | 4390.10 ± 1514.30 | 136.44 ± 21.60 |
| G-Ro | 369.77 ± 60.85 | 392.42 ± 64.27 | 11.76 ± 2.36 | 7.39 ± 1.99 | 0.65 ± 0.34 | 8,754,863.56 ± 2,407,501.68 | 834,751.02 ± 127,674.20 | 54.60 ± 23.07 |
| PG-RT1 | 211.09 ± 30.53 | 301.43 ± 89.17 | 10.14 ± 1.36 | 11.62 ± 5.19 | 0.65 ± 0.34 | 7,433,641.82 ± 2,063,011.10 | 489,052.81 ± 139,409.13 | 32.14 ± 16.36 |
| CS-IV | 641.27 ± 95.06 | 665.63 ± 87.57 | 12.46 ± 2.48 | 5.97 ± 2.04 | 0.65 ± 0.34 | 2,840,793.46 ± 1,205,136.58 | 327,250.28 ± 42,300.65 | 62.86 ± 28.34 |
| CS-IVa | 57.76 ± 15.97 | 75.36 ± 20.19 | 14.08 ± 3.60 | 15.43 ± 6.27 | 1.15 ± 0.55 | 64,304,499.60 ± 32,122,989.54 | 2,962,851.54 ± 832,591.12 | 11.18 ± 6.62 |
The time to peak concentration (T max) of plasma varied among the saponins: G-Rg1 at 1.20 ± 0.27 h, G-Re at 0.90 ± 0.45 h, G-Rb1 at 19.20 ± 6.57 h, G-Ro, PG-RT1, and CS-IV at both 0.65 ± 0.34 h, and CS-IVa at 1.15 ± 0.55 h. These results suggest slower absorption rates for G-Rg1, G-Rb1, and CS-IVa in plasma. The elimination half-life (T 1/2) was 13.69 ± 4.07 h for G-Rg1, 8.70 ± 6.59 h for G-Re, 26.22 ± 11.22 h for G-Rb1, 7.39 ± 1.99 h for G-Ro, 11.62 ± 5.19 h for PG-RT1, 5.97 ± 2.04 h for CS-IV, and 15.43 ± 6.27 h for CS-IVa. In the same way, the mean residence time (MRT) of G-Rb1 was longer than those of the others. The results of T 1/2 and MRT demonstrated that the elimination of G-Rb1 may be slow because its T 1/2 and MRT were the longest among the seven components. The probability of low elimination of G-Rb1 was confirmed by the clearance rate/bioavailability (CL/F) of G-Rb1 listed in Table . The maximum concentrations (C max) of G-Rg1, G-Re, G-Rb1, G-Ro, PG-RT1, CS-IV, and CS-IVa were 5.26 ± 1.89, 19.10 ± 13.60, 136.44 ± 21.60, 54.60 ± 23.07, 32.14 ± 16.36, 62.86 ± 28.34, and 11.18 ± 6.62 ng/mL, respectively, showing that the C max of G-Rb1 was higher than those of other six components. The area under the concentration–time curve (AUC0–t ) of G-Rb1, G-Ro, PG-RT1, and CS-IV was much higher than those of G-Rg1, G-Re, and CS-IVa in Table . Especially, G-Rb1 showed the highest AUC0–t with 5122.23 ± 1090.58 h*ng/mL.
The distribution profiles of seven components in tested tissues (heart, liver, spleen, lung, kidney, brain, and ankle joints) in rats at 1, 2, 4, 6, 12, and 24 h after oral administration of PJR extracts are shown in Figure . G-Rg1, G-Re, G-Rb1, G-Ro, PG-RT1, CS-IV, and CS-IVa were detected in the heart, liver, spleen, lung, kidney, brain, and ankle joints at 1 h and still exited in the detected tissues within 10 h after oral administration of PJR extracts. The results showed that the seven components were widely distributed in various tissues and their eliminations were slow. The concentrations of the detected components reached a peak level at 1 h in the heart, liver, spleen and kidney and at 2 h in the lung, brain, and ankle joints. The results indicated that the seven components could be rapidly distributed to the tissues with abundant blood supply. All components were detected in the kidney at 24 h, and the concentration showed an upward trend compared to 12 h except CS-IVa, which indicated that the seven components were excreted through the kidney. In the ankle joints, the analyzed components also had a high concentration at 24 h. The AUC0–t of the seven components in tissues is displayed in Figure . As shown in Figure , the lung and brain displayed the highest and lowest concentrations among all examined tissues, respectively. The orders of concentrations of G-Rg1 and G-Re in the tissues were lung > liver > kidney > ankle joints > heart > spleen > brain. G-Rb1 concentrations in the analyzed tissues were found to decrease in the order of lung > liver > ankle joints > kidney > heart > spleen > brain. G-Ro, PG-RT1, and CS-IVa in the tissues varied in the order of lung > liver > spleen > kidney > ankle joints > heart > brain, and CS-IV concentration in the analyzed tissues decreased in the order of lung > liver > kidney > spleen > ankle joints > heart > brain.
4.
Tissue concentration–time curves of seven analytes after oral administration of PJR extracts.
5.
AUC0–t of seven analytes in tissues after oral administration of PJR extracts.
These findings illustrate the saponins’ broad distribution and prolonged presence in rat tissues, underscoring their potential for extensive therapeutic use. The comprehensive distribution and kinetic profiles provided here lay a robust foundation for further exploration of PJR’s clinical applications, particularly in anti-inflammatory treatments.
4. Discussion
In this research, we successfully established a robust UPLC-MS/MS methodology to quantify the concentrations of G-Rg1, G-Re, G-Rb1, G-Ro, PG-RT1, CS-IV, and CS-IVa in the blood and tissue samples of rats following the oral administration of PJR extracts. A critical aspect of our method development was the reduction of interference from endogenous substances within the samples, which can significantly impede the accuracy of mass spectrometric analysis.
Our investigations focused on the efficacy of different protein precipitation techniques using methanol, acetonitrile, and liquid–liquid extraction using n-butanol to clarify the plasma and tissue homogenates. We observed that protein precipitation with acetonitrile was particularly effective, providing cleaner mass spectra and less interference from endogenous substances than with the other solvents tested. The optimal ratio of acetonitrile to homogenate was 5:1. At this ratio, the precipitation of proteins was nearly complete and the response for each of the saponins under investigation was optimal. Consequently, we adopted a consistent approach of using a 5:1 acetonitrile/plasma/tissue homogenate ratio for sample preparation across all experiments. This methodological consistency ensured the reliability of our results, facilitating a clearer understanding of the saponins’ pharmacokinetics and tissue distribution. This technique could be applied to similar pharmacokinetic studies involving complex biological matrices, thereby broadening the utility of our findings in pharmacological research.
Developing and researching PJR is crucial, particularly in understanding its pharmacokinetics and tissue distribution. Currently, simultaneous studies on the pharmacokinetic characteristics of the seven saponins contained in PJR are sparse. Our study highlighted distinct pharmacokinetic behaviors among these saponins; notably, the T max for ginsenoside Rb1 (G-Rb1) was significantly slower than those for the other saponins. This delayed T max can be attributed to G-Rb1’s high molecular weight and polarity, factors that arise from the presence of glucose at C20 in its structure. These characteristics likely hinder its permeation through the intestinal membrane, affecting its absorption rate. , Previous research by Zhou et al. supports this finding, indicating that the number of sugar substituents in saponins correlates with their absorption rates. Specifically, G-Rb1, classified as a damarane-type tetra glycoside, possesses more glycosidic connections, resulting in a longer T max than that of its counterparts. This particular behavior of G-Rb1 emphasizes the complexity of saponin absorption and the impact of structural differences on its pharmacokinetic profiles. Such insights are indispensable for designing therapeutic agents from these compounds, suggesting that modifications in saponin structures could potentially enhance their bioavailability and efficacy.
The mean plasma concentration–time curves of G-Rg1, Re, Ro, PG-RT1, and CS-IV exhibited a double-peak phenomenon, while CS-IVa displayed a three-peak phenomenon. Multiple factors might contribute to these multipeak absorption profiles observed in the pharmacokinetics of these saponins in rats. First, recirculation processes within or between organs could produce repeated peaks in plasma concentration–time curves. The oral bioavailability of the seven saponins was relatively low, potentially due to their large molecular weights, numerous sugar groups, and multiple hydrogen bonds, which impede the membrane permeability of most ginsenosides. This structural complexity can restrict absorption by the gastrointestinal tract in a single pass, possibly leading to reabsorption after initial absorption and metabolism, thereby promoting hepatointestinal circulation. Second, the initial high concentrations of saponins before reaching equilibrium might be sequestered by fat cells, with subsequent release back into the circulation as plasma levels decline, leading to secondary peaks. This process illustrates the dynamic distribution and redistribution of saponins within the body. Third, the potential for metabolic transformation of saponins within the body could further complicate their pharmacokinetic profiles, Oxygenation and disaccharidation are primary metabolic pathways for saponins in rats, with structural transformations significantly affecting their bioactivity and pharmacokinetics. For example, the gastrointestinal microbiota may play a crucial role in the preabsorption and deglycosylation of saponins, which is more extensive than the mild liver deglycosylation typically mediated by β-glucosaccharase. Notably, the transformation of G-Ro into CS-IVa through glycosylation suggests an interconversion among saponins that could influence their detected plasma concentrations and tissue distributions.
Understanding these complex absorption and metabolic processes is critical for developing effective therapeutic strategies involving saponins and underscores the need for detailed studies to elucidate the full spectrum of their pharmacokinetic behaviors.
In our tissue distribution study, the highest concentrations of all analyzed saponins were observed in the lung, followed by the liver. Importantly, given the ankle joint’s role as a primary therapeutic target for RA treatments, we also monitored the drug concentrations in this location. At 24 h postadministration of PJR extracts, high concentrations of all seven saponin components were detectable in the ankle joints. The area under the curve (AUC0–t ) for G-Rb1, G-Ro, PG-RT1, and CS-IVa ranked second highest, while G-Rg1, G-Re, and CS-IV ranked third among all tissues examined. These findings support previous reports of PJR’s significant antiarthritis effects. , The contents of seven components in brain tissue could be detected, which suggested that they could penetrate the blood–brain barrier and carry out their brain protection effects.
The present study developed a straightforward, reliable, and sensitive UPLC-MS/MS method for simultaneous quantification of G-Rg1, G-Re, G-Rb1, G-Ro, PG-RT1, CS-IV, and CS-IVa in biological samples. This method has been rigorously validated and effectively applied to the pharmacokinetic and tissue distribution assessment of these analytes in rats following oral administration of PJR extracts. The pharmacokinetic data revealed rapid distribution of the saponins to plasma and tissues, achieving high concentrations in the ankle joints, which are relevant for RA treatment. This study is the first to quantify these seven saponins in the ankle joints of rats, providing a valuable experimental foundation for the clinical use of PJR, especially in the management of RA.
Supplementary Material
Acknowledgments
This work was supported by Hunan Provincial Natural Science Foundation of China (2025JJ90119), The Scientific Research Fund of Hunan Provincial Department of Education (21A0169, 22A0242), Key Discipline Project on Chinese Pharmacology of Hunan University of Chinese Medicine (202302), and Open Fund Project of Traditional Chinese Medicine Processing Technology Inheritance Base (2022ZYPZ06; 2022ZYPZ12).
Glossary
Abbreviations Used
- PJR
Panacis Japonici Rhizoma
- RA
rheumatoid arthritis
- IS
internal standard
- SD
Sprague–Dawley
- UPLC-MS/MS
ultraperformance liquid chromatography–mass spectrometry
- AUC
area under the concentration–time curve
- C max
maximum concentrations
- T 1/2
elimination half-life
- MRT
mean residence time
- T max
time-to-peak concentration
- CL/F
clearance rate/bioavailability
- QC
quality control
- LOD
limit of detection
- LOQ
limit of quantification
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c06902.
Table S1: Mass spectrometric parameters of seven analytes and IS; Table S2: LOD and LOQ of seven analytes in different matrices harvested from rats (PDF)
#.
Y.Z. and Y.W. are listed as cofirst authors.
The authors declare no competing financial interest.
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