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Deep eutectic solvent: Improve the stability of constituents in Gentianae radix et rhizoma (GR) extract and oral absorption in rats
* Corresponding authors: E-mail addresses: ndyfy09692@ncu.edu.cn (C Feng); 359861672@qq.com (J Zhou)
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Received: ,
Accepted: ,
Abstract
Gentianae radix et rhizoma (GR) is generally applied in clinical practice for the treatment of liver disease as a traditional Chinese medicine. This research examined the effects of a Deep Eutectic Solvent (DES), composed of lactic acid and choline chloride in a 4:1 ratio, on the stability and pharmacokinetics of the chemical constituents of GR extract. The stability of four compounds, namely 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin, was assessed in GR extract dissolved in water and 15% DES (v/v). Additionally, we developed and validated a sensitive ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-MS/MS) method for our study, capable of simultaneously quantifying 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin in rat plasma following oral administration of GR extract. The findings indicated that the stability of these components in GR extract, which was dissolved with 15% DES (v/v), was significantly improved. The devised approach was effectively utilized in the pharmacokinetic study of these four analytes from GR extract in rats. The pharmacokinetic characteristics, including Cmax, T1/2, AUC(0-T), and AUC(0-∞), of GR extract dissolved in 15% DES (v/v) via oral administration were superior to those dissolved in water. The results revealed that DES may enhance the absorption of components in GR extract, extend the duration and concentration of chemical components in vivo, and possibly augment the therapeutic efficacy.
Keywords
Deep eutectic solvent
Gentianae radix et rhizome
Infrared spectrum
Pharmacokinetics
Stability
1. Introduction
Gentianae radix et rhizoma (GR) was initially documented in the Shen Nong Ben Cao Jing, a renowned classic of traditional Chinese medicine. It has been regarded as a drug for over 2000 years. The herb of GR is widely distributed in provinces including Liaoning, Heilongjiang, Jilin, Zhejiang, and Inner Mongolia in northern China, and Guangxi in southern China [1-3]. Contemporary studies have demonstrated that GR exhibits a large quantity of pharmacological effects, such as hepatoprotection [4], anti-inflammatory properties [5], antipyretic effects [6], analgesic capabilities [7], antioxidant activity [8], antibacterial properties [9], and immune modulation [10], among others. These pharmacological effects can be ascribed to the diverse active substances present in GR. Iridoids, triterpenoids, and flavonoids have been extracted from GR [11-13], with iridoids being of special significance. The principal iridoid chemicals in GR comprise 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin [14-16]. Modern pharmacological research has validated the diverse biological properties of iridoid compounds in GR, including anti-tumor, antibacterial, antiviral, antioxidant, immune-enhancing, liver-protective, spasmolytic, analgesic, antidiabetic, and lipid-lowering effects [17-19]. Although existing studies have shown that iridoid compounds possess a broad spectrum of clinical applications, they also indicate that GR extracts have poor stability and fat solubility. The apparent oil-water partition coefficient (log P) of gentiopicroside, a GR extract, is -1.21 at 23°C, indicating its strong hydrophilicity and limited lipophilicity, which may impede its absorption through oral administration [20]. Consequently, enhancing the stability and oral bioavailability of iridoid components in GR extracts is essential.
Deep eutectic solvents (DES) were introduced by Abbott in 2003. They are created by combining hydrogen bond acceptors and donors in precise proportions [21]. DES exhibit remarkable physical and chemical properties, including low toxicity, optimal viscosity, biodegradability, appropriate polarity, and negligible environmental impact, rendering them suitable for extensive uses in electrochemistry, organic synthesis, and the development of novel materials [22-26]. In recent years, DES has been utilized to extract active ingredients from traditional Chinese medicinal herbs with remarkable efficacy. DES can augment connections between target compounds through intermolecular hydrogen bonding, thereby improving extraction efficiency. It may also increase the stability of target substances via intermolecular interactions [27-30].
This research group effectively employed DES to extract gentiopicroside and loganic acid from GR, yielding promising results [31]. The study investigated the stability effects of DES on 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin in GR extract. Additionally, ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was developed to analyze the amounts of the four aforementioned GR extracts in rat plasma. The IS and chemical structures of 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin have been shown in Figure 1. The GR extracts were dissolved in various solvents, and the blood concentrations of these components were measured following oral administration to the rats. The primary pharmacokinetic parameters were obtained to evaluate the impact of DES on the absorption of these components. This research serves as a significant reference for the future expansion of the applications of DES in subsequent studies.
- The chemical structures of the four analytes and IS.
2. Methods and Materials
2.1. Reagents and chemicals
The materials applied in the study were loganic acid (LOT: M07HB177364; purity ≥ 98%), swertiamarin (LOT: M31HB181437; purity ≥ 98%), gentiopicroside (LOT: O11IB228424; purity ≥ 98%), 6’-O-β-D-glucosylgentiopicroside (LOT: D01HB202760; purity ≥ 98%), and caffeine (LOT: DSTDK004902; purity ≥ 98%). They were acquired from Shanghai Yuanye Biotechnology Co., Ltd. Figure 1 illustrates the internal standard (IS) and chemical structures of 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin.
2.2. Plant materials
GR (Batch number: 202308012, shown in Figure 2) was purchased from Dasenlin Pharmacy (Nanchang, China) and authenticated by Professor Jian Zhou from the Department of Pharmacy, the First Affiliated Hospital, Jiangxi Medical College, Nanchang University. The specimen of GR was the desiccated root of the plant Gentiana manshurica Kitag, a member of the Gentianaceae family. Prior to extraction, the medicinal material is sectioned into approximately 1 cm pieces and subsequently dried to a consistent weight in a hot air-drying cabinet at 70°C.
- Image of Gentianae radix et rhizoma.
2.3. Experimental animal
Twelve male Sprague-Dawley (SD) rats were used in the research. The body weight of SD rats should range from 180 to 220 g. Hunan Slack Jingda Experimental Animal Co., Ltd. supplied these rats. The license number was SYXK (Xiang) 2019-0004.
2.4. Preparation of DES
The DES adopted in this study was formulated following a prior methodology. Nonetheless, we implemented minor alterations to it [32,33]. Lactic acid and choline chloride were accurately measured in a 4:1 ratio and placed in a 100 mL beaker. The solution was continuously heated in a water bath at 80°C, with constant stirring necessary until a transparent and homogeneous liquid was obtained.
2.5. Preparation of GR extract
To prepare the GR extract, 500 g of GR was accurately weighed, and 10 L of 70% EtOH (v/v) was introduced for reflux extraction. This procedure was conducted three times, each lasting 2 h. The combined extract was condensed through evaporation at diminished pressure [34]. The GR extract obtained from the preceding step was subsequently ground and stored. The concentrations of swertiamarin, gentiopicroside, 6’-O-β-D-glucosylgentiopicroside, and loganic acid in the GR extract were 4.37%, 55.8%, 3.96%, and 17.54%, respectively.
2.6. Stability of chemical constituents in GR extract
GR extracts were precisely weighed and transferred to 50 mL volumetric flasks, then individually dissolved in water and 15% DES (v/v). The combination volumes were adjusted to the scale, and the solutions were stored shielded from light [35,36]. The contents of 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin in the GR extracts were quantified using high-performance liquid chromatography (HPLC) on days 0, 5, 10, and 15 to assess the stability of each component.
2.7. Instruments and conditions
2.7.1. HPLC conditions
A Shimadzu LC-20A system was employed for HPLC analysis to identify components in GR extracts. The system comprised an SPD-20A detector, a CTO-20AC column oven, an SIL-20A automatic sampler, and an LC-20AT pump.
2.7.2. UHPLC-MS/MS conditions
The instrument utilized for the UHPLC-MS/MS analysis was a Waters XEVO-TQS Cronos. An XBridge BEH C18 column was employed for chromatographic separation. The column specifications were 2.5 μm, 4.6 mm × 75 mm, with a temperature of 35°C. The mobile phase consisted of 0.1% formic acid (solvent A) and MeOH (solvent B). The gradient elution was executed according to the subsequent steps: 0–3 min: 90–70% B, 3–7 min: 70–45% B, 7.01–10 min: 45–90% B, at a flow rate of 0.3 mL/min and an injection volume of 2 μL.
2.8. Infrared spectrum
Dried gentiopicroside, DES, and gentiopicroside solubilized in DES were mixed with potassium bromide powder in a weight ratio of 2:100. The compounds were pulverized and fashioned into thin slices. Infrared detection was conducted at a scanning frequency of 32 scans per second, with a scanning range of 400-4000 cm-1 [37,38].
2.9. Preparation of calibration standards, quality control (QC) samples, and standard solutions
Calibration solutions were prepared by adding specific quantities of the mixed standard solution and 10 μL of IS to 200 μL of pure rat plasma. The ultimate concentrations were as follows: Loganic acid: 0.3, 0.5, 1.3, 2.7, 5.8, and 13.1 μg/mL; Gentiopicroside: 0.2, 0.4, 1.0, 2.0, 4.3, and 10.7 μg/mL; Swertiamarin: 0.3, 0.6, 1.4, 2.9, 6.3, and 15.1 μg/mL; 6’-O-β-D-glucosylgentiopicroside: 1.4, 3.4, 8.3, 17.4, 34.9, and 78.3 μg/mL. The QC samples were prepared at three concentrations: high, medium, and low, using the same procedure. The preparation of standard solutions involved the precise weighing of each of the four analytes-6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin-along with the IS, caffeine, followed by dissolution in MeOH. The solution concentration was 1.0 mg/mL.
2.10. Preparation of plasma samples
The used plasma samples were maintained at room temperature and thawed. After the addition of 10 μL of IS solution (caffeine, 10 ng/mL) to each sample, these samples were swirled for 60 s to achieve uniform mixing. Subsequently, the samples were swirled for another 60 s following the addition of 800 μL of MeOH. The mixed samples underwent centrifugation at 12,000 rpm for 10 min. A 5 mL centrifuge tube was utilized to contain the acquired supernatant. The products from the aforementioned steps were dried using a sample concentrator with nitrogen flow, and 200 μL of MeOH was precisely dripped into samples. Following a 60-s vortex to dissolve the residue, the mixtures were centrifuged under identical conditions (12,000 rpm, 10 min), after which the supernatant was transferred to suitable containers for detection.
2.11. Validation of method
The validation of the UHPLC-MS/MS method developed by our team was implemented in accordance with the guidelines established by the United States Food and Drug Administration (FDA) and the European Medicines Agency. All requisite evaluative parameters, encompassing linearity, precision, specificity, recovery, accuracy, stability, and matrix effects [39,40].
2.11.1. Specificity
Six pre-dose plasma samples were collected from the 12 SD rats to assess the method’s specificity. The aforesaid samples were detected to determine potential chromatographic interference from endogenous plasma constituents by comparing the retention times of the four substances with the internal standard.
2.11.2. Linearity
The calibration curves were generated by plotting the peak area ratio of the analyte to the IS on the Y-axis against the nominal concentration on the X-axis. The analyst conducted linear regression using the acquired data. The correlation coefficient (r2) of the calibration curves should exceed 0.995. The detector’s response at the lowest concentration point on the calibration curve was at least 5 times higher than that of the blank, thereby designating this point as the lower limit of quantitation (LLOQ). The acceptance criterion for back-calculated standard concentration values should not exceed ±15% of the standard deviation (SD) from the nominal value. The acceptance threshold for the LLOQ was within ±20%.
2.11.3. Accuracy and precision
Six QC samples in replicate were detected at four concentration levels (high quality control (HQC), medium quality control (MQC), low quality control (LQC), and LLOQ) across four different days to evaluate inter- and intra-assay accuracy and precision. The U.S. FDA stipulates that accuracy is accepted if the deviation is within ±15% of the relative error (RE) and precision is accepted if it is within ±15% of the relative standard deviation (RSD). The LLOQ criterion must be within ±20% of the SD.
2.11.4. Recovery and matrix effect
The responses of four analytes extracted from QC samples in replicate were compared to those of the analytes from post-extraction plasma standards at equivalent concentrations, thus allowing for the assessment of recovery. The matrix effect, which assesses the influence of co-eluted and undetected endogenous compounds on the efficiency of droplet formation or evaporation (and consequently on the number of charged ions that may reach the detector), was evaluated. The matrix effect can be evaluated through comparing the responses of QC samples from post-extraction plasma with those of blank samples at equivalent concentrations.
2.11.5. Stability
The stability of the analytes was evaluated through repeated freezing and thawing, while the sample conditions for short-term and long-term storage were tested via low and high QC samples. To assess sample stability, three freeze-thaw cycles were repeated, wherein the samples were stored at −80°C for 24 h for freezing and subsequently thawed completely at room temperature. Storage for 4 h at room temperature before sample preparation, followed by 12 h in the autosampler post-preparation, was adopted to evaluate short-term stability, whereas long-term stability was assessed through 30 days of storage at −80°C.
2.11.6. Pharmacokinetic investigation
All animal experiments received approval from the Institutional Animal Ethics Committee of the First Affiliated Hospital of Nanchang University. The approval number was CDYFY-IACUC-202303QR018. Our developed method was validated and successfully determined the concentrations of 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin in the plasma of SD rats (180-220 g). The rats were nourished under standard conditions. A twelve-hour light/dark cycle was established, with the laboratory temperature maintained at 25 ± 5°C and relative humidity at 50 ± 15%. The rats were provided with standard rat pellet food and water.
The 12 rats used for this study were randomly assigned to two groups. One group of rats was administered GR extract dissolved in water (62 mg/mL), while the other group received GR extract dissolved in a 15% DES (62 mg/mL). Both were administered via the oral route. Blood samples (about 0.3 mL) were collected from the orbital venous plexus at the following time points: 0, 0.08, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h post-oral administration. The samples were afterwards transferred to microcentrifuge tubes and centrifuged at 4,000 rpm for 10 min. Subsequently, the plasma samples were preserved at -80°C before further determination.
2.12. Data analysis
All data obtained from the test were processed with DAS 2.0 software (Chinese Pharmacological Society) to compute the pharmacokinetic parameters.
3. Results and Discussion
3.1. Optimization of HPLC conditions
This work details the separation of 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid and swertiamarin using a Dikma ODS2 C18 column. The column specifications were 5 μm, 4.6 mm × 250 mm. The mobile phase utilized for the experiment consisted of 0.4% phosphoric acid (solvent A), MeOH (solvent B), and water. The operational gradient elution protocol was as follows: 0-10 min: 10% B to 28% B; 10-20 min: 28% B to 29% B; 20-25 min: 29% B to 28% B. The column oven was maintained at 35°C, while the flow rate was regulated at 1.0 mL/min. The study of the analytes was completed with a photodiode array detector (PAD) at a wavelength of 240 nm. The chromatogram has been shown in Figure 3.
- HPLC Chromatograms (a) was substances chromatograms; (b) was GR extracts sample chromatograms. 1: loganic acid; 2: swertiamarin; 3:6’-O-β-D-glucosylgentiopicroside ; 4:gentiopicroside).
3.2. Optimization of UHPLC-MS/MS conditions
The mass spectrometry parameters influencing ion response in UHPLC-MS/MS settings were optimized. The multiple reaction monitoring (MRM) mode was selected, allowing for the simultaneous analysis of both positive and negative ionizations. The instrument parameters were as follows: desolation temperature at 550°C and ion source temperature at 150°C. The quantitative parameters for the IS and the four aforesaid analytes have been presented in Table 1. The analytes and IS were eluted without any interfering peaks, as illustrated in Figure 4.
| Component | Quantitative ion pair | Ion mode | cone-hole voltage(V) | collision energy (eV) |
|---|---|---|---|---|
| Loganic acid | 375.2>113.1 | positive | 32 | 25,17 |
| Swertiamarin | 419.2>179.1 | negative | 14 | 26,22 |
| 6’-O-β-D-glucosylgentiopicroside | 563.3>179.1 | negative | 36 | 13,15 |
| Gentiopicroside | 401.2>179.1 | negative | 12 | 18,8 |
| Caffeine (IS) | 195>138.1 | negative | 14 | 12,16 |
- MRM chromatograms of four analytes and IS. (a) Blank plasma sample spiked with IS; (b) Blank plasma spiked with four analytes and IS; (c) Plasma samples after oral administration of GR extract dissolved with water; (d) Plasma samples after oral administration of GR extract dissolved with 15% DES (v/v). 1. Caffeine (IS), 2. Loganic acid, 3. Gentiopicroside, 4. Swertiamarin, 5. 6’-O-β-D-Glucosylgentiopicroside.
3.3. Stability of chemical composition
Previous studies have reported that the chemical constituents of GR extract deteriorate when dissolved in water. This study evaluated the stability of GR extract dissolved in water and 15% DES (v/v) for 0, 5, 10, and 15 days. The results, presented in Table 2, indicate that 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin in the water-dissolved GR extract degraded over time. Among these, 6’-O-β-D-glucosylgentiopicroside exhibited the fastest degradation, achieving 100% degradation by the 15th day, aligning with previously documented literature. When GR extract was dissolved in 15% DES (v/v), the degradation rates of 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin were significantly reduced, implying that DES may influence the retardation of degradation of these chemical components. To explore the mechanism by which DES delays degradation, the interaction between DES and gentiopicroside was investigated using near-infrared (IR) spectroscopy. Figure 5 depicts the infrared spectrum of the complex formed between the major components in the GR extract and DES. Notably, the complex of gentiopicroside with DES exhibits distinct variations in peak position and shape within the 600-500 cm⁻1 range of the sample’s IR spectrum. Concurrently, the O-H peak in the high-frequency region (3650-2500 cm⁻1) shows shifts in both position and width, suggesting that chloride ions in DES may form hydrogen bonds with gentiopicroside. A similar phenomenon is observed in the complex of 6’-O-β-D-glucosylgentiopicroside with DES. For the complex of loganic acid and DES, the IR spectrum reveals significant discrepancies around 2600 cm⁻1, potentially indicating hydrogen bonding between the hydroxyl groups of loganic acid and the amino groups of choline chloride. The IR spectra of swertiamarin-DES complexes further exhibit notable differences at 2600 cm⁻1 and 500 cm⁻1, implying the co-occurrence of the above hydrogen-bonding mechanisms.
| Time/Day | Water | 15% DES (v/v) | ||||||
|---|---|---|---|---|---|---|---|---|
| Loganic acid | Swertiamarin | 6’-O-β-D- glucosylgentiopicroside | Gentiopicroside | Loganic acid | Swertiamarin | 6’-O-β-D- glucosylgentiopicroside | Gentiopicroside | |
| 0 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| 5 | 70.87±12.36 | 82.08±7.69 | 64.34±11.32 | 71.22±8.61 | 97.95±8.82 | 98.56±8.45 | 82.08±6.14 | 97.57±6.49 |
| 10 | 67.48±9.65 | 79.21±9.94 | 26.81±9.32 | 68.06±9.33 | 95.47±7.52 | 93.65±7.94 | 71.22±5.58 | 89.47±8.52 |
| 15 | 66.89±10.32 | 67.45±6.65 | / | 67.18±8.34 | 94.95±8.13 | 89.54±5.66 | 69.62±7.81 | 85.95±7.94 |
- The infrared spectrum: (a) Loganic acid, DES and complexes formed by Loganic acid with DES, (b) Swertiamarin, DES and complexes formed by Swertiamarin with DES, (c) 6’-O-β-D-Glucosylgentiopicroside, DES and complexes formed by 6’-O-β-D-Glucosylgentiopicroside with DES.
- The infrared spectrum: (d) Gentiopicroside, DES and complexes formed by Gentiopicroside with DES.
3.4. Method validation
3.4.1. Specificity
Figure 3 indicates that no abnormal peaks were observed in blank plasma at the retention durations corresponding to 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, swertiamarin, and caffeine. The approach has been validated as sufficiently specific for the quantification of these substances in rat plasma.
3.4.2. Linearity and LLOQ
The correlation coefficients, linear ranges, regression equations, and LLOQs for the four analytes of 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin have been provided in Table 3. The calibration curves demonstrated excellent linearity across the concentration ranges for all four above-mentioned substances, with r values exceeding 0.9982. The LLOQs for the four substances were 0.7, 0.2, 0.2, and 0.1 μg/mL, respectively.
| Compounds | Calibration curves | Linear range (μg/mL) | r | LLOQ |
|---|---|---|---|---|
| Loganic acid | y=0.00242803x + 7.89155×10-5 | 0.3-13.1 | 0.9987 | 0.2 |
| Swertiamarin | y=0.0376017x - 6.06406×10-4 | 0.3-15.1 | 0.9993 | 0.1 |
| 6’-O-β-D- glucosylgentiopicroside | y=0.0183338x + 1.11116×10-2 | 1.4-78.3 | 0.9982 | 0.7 |
| Gentiopicroside | y=0.0227796x - 2.45134×10-4 | 0.2-10.7 | 0.9996 | 0.1 |
Note: r: Correlation coefficients; LLOQ: Lower limit of quantitation.
3.4.3. Accuracy and precision
Table 4 displays the statistics regarding inter- and intra-day accuracy and precision. The back-calculated concentrations exhibited exceptional accuracy and precision within acceptable criteria, confirming that the proposed approach is dependable for accurate quantification of the analytes.
| Compounds | Spiked Concentration (μg/mL) | Intra-day | Inter-day | ||||
|---|---|---|---|---|---|---|---|
| Measured (μg/mL) | RE (%) | RSD (%) | Measured (μg/mL) | RE (%) | RSD (%) | ||
| Loganic acid | 0.3 | 0.28±0.16 | 9.86 | 8.33 | 0.31±0.05 | 8.63 | 6.84 |
| 3.0 | 3.14±0.27 | -4.12 | 4.62 | 2.98±0.17 | 6.36 | 3.38 | |
| 12.6 | 13.2±1.28 | 2.69 | 5.16 | 12.93±0.48 | -3.17 | 7.25 | |
| Swertiamarin | 0.5 | 0.46±0.08 | 10.08 | 6.27 | 0.51±0.03 | -9.96 | 8.67 |
| 6.0 | 6.12±0.23 | 5.32 | 3.69 | 5.85±0.36 | 3.29 | 5.56 | |
| 13.8 | 13.4±0.56 | 3.06 | 5.24 | 14.16±0.44 | 5.02 | 2.27 | |
| 6’-O-β-D-glucosylgentiopicroside | 2.8 | 3.06±0.26 | -8.89 | 7.73 | 2.95±0.17 | 10.24 | 5.98 |
| 32.0 | 33.6±2.54 | 4.71 | 4.14 | 31.68±1.19 | -4.36 | 3.12 | |
| 68.4 | 65.85±4.61 | 2.39 | 4.98 | 70.31±3.38 | 2.21 | 6.01 | |
| Gentiopicroside | 0.5 | 0.49±0.05 | 9.75 | 9.36 | 0.52±0.04 | 9.44 | 8.17 |
| 4.0 | 4.17±0.25 | -3.58 | 5.75 | 4.12±0.36 | 2.52 | 4.25 | |
| 10.0 | 9.86±0.24 | 3.06 | 6.52 | 10.38±0.68 | -4.69 | 6.11 | |
Note: RE is relative error; RSD is relative standard deviation.
3.4.4. Matrix effect and recovery
Table 5 summarizes the data regarding the matrix effect and recovery of the four substances. The recovery values for the four analytes at three different concentration levels of rat plasma varied from 80.92% to 91.17%. Matrix effects ranged from 81.17% to 90.85%. These findings demonstrate that both the matrix effects and recovery data are within acceptable parameters, signifying reliable sample preparation and analysis.
| Compounds | Spiked concentration (μg/mL) | Extraction recovery (%) | RSD (%) | Matrix effect (%) | RSD (%) |
|---|---|---|---|---|---|
| Loganic acid | 0.3 | 82.46±5.86 | 9.54 | 87.74±6.53 | 11.32 |
| 3.0 | 86.61±4.33 | 3.86 | 84.47±5.11 | 6.57 | |
| 12.6 | 85.32±6.61 | 5.17 | 85.24±3.68 | 3.38 | |
| Swertiamarin | 0.5 | 80.92±8.62 | 8.87 | 85.72±5.17 | 8.74 |
| 6.0 | 84.51±4.31 | 5.31 | 89.98±3.15 | 2.69 | |
| 13.8 | 89.31±2.64 | 6.74 | 90.85±2.38 | 5.17 | |
| 6’-O-β-D-glucosylgentiopicroside | 2.8 | 81.36±5.21 | 9.25 | 83.54±4.08 | 9.08 |
| 32.0 | 88.31±4.62 | 4.61 | 81.17±5.06 | 2.26 | |
| 68.4 | 91.17±5.32 | 2.27 | 88.39±3.17 | 2.37 | |
| Gentiopicroside | 0.5 | 83.62±6.62 | 7.93 | 84.47±3.54 | 8.75 |
| 4.0 | 90.38±4.52 | 4.25 | 82.35±4.52 | 3.04 | |
| 10.0 | 86.51±5.56 | 5.08 | 85.28±2.76 | 4.46 |
Note: RE is relative error; RSD is relative standard deviation.
3.4.5. Stability
Table 6, which summarizes the stability data, indicates that the four analytes maintained stability throughout diverse circumstances. The samples remained stable at room temperature for 4 h, thereafter held in the auto-sampler for 12 h, subjected to three freeze-thaw cycles, or stored at -80°C for 30 days. The RSD values were all below 9.92%, confirming that the analytes were stable under these conditions.
| Compounds | Spiked Concentration (μg/mL) | Room temperature for 4 h | Autosampler for 12 h | Three freeze-thaw cycles | -80°C for 30 days | ||||
|---|---|---|---|---|---|---|---|---|---|
| Measured (μg/mL) | RSD (%) | Measured (μg/mL) | RSD (%) | Measured (μg/mL) | RSD (%) | Measured (μg/mL) | RSD (%) | ||
| Loganic acid | 0.3 | 0.32±0.03 | 5.86 | 0.28±0.05 | 6.31 | 0.30±0.03 | 6.64 | 0.28±0.03 | 7.56 |
| 3.0 | 3.15±0.19 | 3.69 | 3.08±0.11 | 4.09 | 2.95±0.11 | 3.86 | 2.88±0.18 | 4.32 | |
| 12.6 | 12.55±0.24 | 4.17 | 12.46±0.18 | 4.35 | 12.87±0.25 | 5.91 | 13.12±0.31 | 5.77 | |
| Swertiamarin | 0.5 | 0.48±0.06 | 6.62 | 0.53±0.06 | 8.32 | 0.49±0.04 | 8.64 | 0.47±0.06 | 9.92 |
| 6.0 | 5.98±0.17 | 2.73 | 6.12±0.25 | 5.57 | 6.16±0.25 | 3.18 | 5.96±0.15 | 3.27 | |
| 13.8 | 13.92±0.24 | 3.92 | 13.96±0.31 | 6.28 | 14.27±3.85 | 4.65 | 14.05±0.35 | 4.17 | |
| 6’-O-β-D- glucosylgentiopicroside | 2.8 | 2.86±0.08 | 5.56 | 3.04±0.10 | 5.43 | 3.10±0.33 | 8.36 | 3.02±0.19 | 6.32 |
| 32.0 | 33.65±2.31 | 9.38 | 32.68±1.86 | 7.69 | 31.74±0.46 | 2.27 | 33.24±2.39 | 8.15 | |
| 68.4 | 65.78±4.39 | 4.61 | 66.41±3.38 | 2.17 | 70.06±4.17 | 4.38 | 71.16±4.72 | 3.65 | |
| Gentiopicroside | 0.5 | 0.52±0.04 | 8.24 | 0.49±0.03 | 3.38 | 0.45±0.08 | 4.65 | 0.46±0.06 | 7.14 |
| 4.0 | 3.98±0.12 | 5.81 | 4.08±0.16 | 5.63 | 4.11±0.16 | 5.61 | 3.99±0.13 | 4.08 | |
| 10.0 | 9.96±0.27 | 4.74 | 9.82±0.22 | 2.09 | 9.92±0.27 | 3.39 | 10.08±0.11 | 2.94 | |
3.4.6. Pharmacokinetic study
An appropriate UHPLC-MS/MS method was developed to measure the concentrations of 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin in rat plasma following oral administration of GR extract dissolved in either water or 15% DES (v/v). Upon dissolution of the GR extract in 15% DES (v/v) and subsequent oral administration, the four compounds exhibited satisfactory absorption. Conversely, when the GR extract was dissolved in water and administered, the plasma concentrations of loganic acid, swertiamarin, and gentiopicroside were markedly diminished, while the plasma concentration of 6’-O-β-D-glucosylgentiopicroside was insufficient to establish a comprehensive pharmacokinetic profile. The average concentration-time curves of the plasma for each component in both rat groups have been shown in Figure 6. The main pharmacokinetic parameters acquired from the study have been presented in Table 7.
- Mean plasma concentration-time curves of (a) loganic acid, (b) swertiamarin, (c) 6’-O-β-D-glucosylgentiopicroside, and (d) gentiopicroside after orally administration of GR extract were dissolved with water and 15% (v/v) DES, respectively (n = 6).
| Group | Compounds | Tmax (h) | Cmax (μg/mL) | T1/2 (h) | AUC(0-t) (h*μg/L) | AUC(0-∞) (h*μg/L) | CLz/F (L/h/kg) |
|---|---|---|---|---|---|---|---|
| 15% DES (v/v) | loganic acid | 0.25±0 | 0.94±0.23 | 9.97±2.52 | 2.85±0.75 | 3.44±1.08 | 14524.79±3386.52 |
| swertiamarin | 0.25±0 | 0.21±0.08 | 6.84±1.76 | 0.79±0.31 | 0.85±0.25 | 23.56±6.65 | |
| 6’-O-β-D-glucosylgentiopicroside | 0.25±0 | 0.23±0.12 | 16.77±4.58 | 0.66±0.15 | 1.46±0.37 | 13.68±3.78 | |
| gentiopicroside | 0.25±0 | 4.65±0.35 | 31.64±6.92 | 10.89±3.38 | 19.70±4.61 | 2537.88±568.39 | |
| Water | loganic acid | 2±0 | 0.24±0.09 | 2.44±0.98 | 1.02±0.29 | 1.07±0.27 | 18.77±2.84 |
| swertiamarin | 2±0 | 0.04±0.02 | 4.35±1.17 | 0.24±0.07 | 0.29±0.06 | 69.87±22.31 | |
| gentiopicroside | 2±0 | 0.86±0.27 | 8.03±2.06 | 4.15±1.32 | 5.64±1.48 | 3.55±0.68 |
According to Table 7, the AUC(0-∞) and AUC(0-t) for 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin were comparable, indicating that the monitoring period was appropriate. The Tmax values of gentiopicroside, swertiamarin, 6’-O-β-D-glucosylgentiopicroside, and loganic acid were reduced when GR extract was dissolved in 15% DES (v/v) compared to water, suggesting that DES facilitates accelerated absorption of the components into the bloodstream. The Cmax values of loganic acid, swertiamarin, and gentiopicroside in rat plasma were elevated when the GR extract was dissolved in 15% DES (v/v), indicating that DES promotes the gastrointestinal absorption of these components, leading to higher plasma concentrations. The T1/2 values for 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin in rat plasma after administration of GR extract dissolved in 15% DES (v/v) were (9.97±2.52), (6.84±1.76), (16.77±4.58), and (31.64±6.92) h, respectively. The T1/2 values for loganic acid, swertiamarin, and gentiopicroside after oral administration of GR extract dissolved in water were 2.44±0.98, 4.35±1.17, and 8.03±2.06 h, respectively. These results indicate that DES promotes the absorption of GR extracts and extends the retention of analytes in the body when utilized as a solvent, resulting in a prolonged impact.
3.5. Discussion
In this paper, gentiopicroside, loganin, and other compounds are iridoid glycosides. Their structures contain hemiacetal structures, so they have poor stability and are prone to hydrolysis, rearrangement, and other changes [41]. Our previous research found that gentiopicroside is relatively stable in a slightly acidic environment, but its stability significantly decreases as the alkalinity increases. In this paper, we compared the stability of the main compounds in GR extract in water and DESs. The results showed that the stability of gentiopicroside and other compounds was significantly improved in DESs, laying a foundation for the design of GR extract preparations. Da Silva reported that natural DESs can enhance the bioavailability of phenolic compounds in blueberry extracts [42]. Gao found that natural DESs can significantly increase the oral bioavailability and anti-obesity effects of hydroxysafflor yellow A [43]. This study found that the DESs can enhance the absorption of compounds in the GR extract. The oral bioavailability was significantly higher than that of the aqueous solution of the GR extract, indicating that DESs have great potential in promoting the absorption of compounds.
4. Conclusions
This research explored the effects of DES on the stability and pharmacokinetics of essential chemical components in GR extract. The findings demonstrated that the stability of 6’-O-β-D-glucosylgentiopicroside, gentiopicroside, loganic acid, and swertiamarin was significantly improved when GR extracts were dissolved in 15% DES (v/v). The researchers developed and validated a precise UHPLC-MS/MS method for measuring the concentrations of four substances in rat plasma. The pharmacokinetic parameters, including T1/2, Cmax, AUC(0-∞), and AUC(0-T), were elevated when GR extracts were dissolved in 15% DES (v/v) compared to those dissolved in water. These findings suggest that DES can improve the absorption of GR extract constituents, extend in vivo exposure, mitigate drug degradation, and consequently may improve the therapeutic efficacy of GR.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (No.82160737; No.81960703); Science and technology project of Jiangxi Provincial Administration of Traditional Chinese Medicine (No.2023A0387; No.2019A155); The First Affiliated Hospital of Nanchang University young talents research and cultivation project (No.YFYPY202263); Project of Science and Technology Department of Jiangxi Province (No.20203BBGL73217; No.20242BAB25568); Academic and Technical leaders (Youth) in major disciplines in Jiangxi Province (20212BCJ23026); Jiangxi Provincial Administration of Traditional Chinese Medicine key research office construction project (KP202203007); 2023 National Training Program for Inheritance of Characteristic Techniques of Traditional Chinese Medicine (T20234832005).
CRediT authorship contribution statement
Chuanhua Feng: Software Development, Methodological Framework, Conceptualization, Review, Editing. Xiaolin Tang: Authorship, Initial Manuscript Preparation, Data Management. Jian Zhou: Visualization, Investigation. Huiling Guo: Visualization, Investigation. Jinfang Hu: Supervision, Securing Funding. Guosong Zhang: Supervision, Securing Funding.
Declaration of competing interest
The authors declare that they possess no recognized competing financial interests or personal affiliations that may have seemingly affected the work presented in this paper.
Declaration of Generative AI and AI-assisted technologies in the writing process
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
References
- Effect of postharvest processing on quality traits of Radix Gentianae Macrophyllae: A integrative analysis of metabolomics and proteomics. Plant Physiology and Biochemistry : PPB. 2023;204:108099. https://doi.org/10.1016/j.plaphy.2023.108099
- [Google Scholar]
- Hepatoprotective effect and metabonomics studies of radix gentianae in rats with acute liver injury. Pharmaceutical Biology. 2021;59:1172-1180. https://doi.org/10.1080/13880209.2021.1969414
- [Google Scholar]
- Isolation of gentiopicroside from Gentianae radix and its pharmacokinetics on liver ischemia/reperfusion rats. Journal of Ethnopharmacology. 2012;141:668-673. https://doi.org/10.1016/j.jep.2011.08.001
- [Google Scholar]
- Gentiopicroside prevents alcoholic liver damage by improving mitochondrial dysfunction in the rat model. Phytotherapy Research : PTR. 2021;35:2230-2251. https://doi.org/10.1002/ptr.6981
- [Google Scholar]
- Gentiopicroside ameliorated ductular reaction and inflammatory response in DDC-induced murine cholangiopathies model. Current Molecular Pharmacology 2023 2023 Oct 19. https://doi.org/10.2174/0118761429251911231011092145
- [Google Scholar]
- Network pharmacology integrated with molecular docking reveals the common experiment-validated antipyretic mechanism of bitter-cold herbs. Journal of Ethnopharmacology. 2021;274:114042. https://doi.org/10.1016/j.jep.2021.114042
- [Google Scholar]
- Establishment and application of a new HPLC qualitative and quantitative assay for Gentiana Macrophyllae radix based on characteristic constituents of anofinic acid and its derivatives. Biomedical Chromatography : BMC. 2018;32:e4341. https://doi.org/10.1002/bmc.4341
- [Google Scholar]
- Preparative separation and purification of four glycosides from Gentianae radix by high-speed counter-current chromatography and comparison of their Anti-NO production effects. Molecules (Basel, Switzerland). 2017;22:2002. https://doi.org/10.3390/molecules22112002
- [Google Scholar]
- A study of Gentianae radix et Rhizoma class differences based on chemical composition and core efficacy. Molecules (Basel, Switzerland). 2023;28:7132. https://doi.org/10.3390/molecules28207132
- [Google Scholar]
- Comparative analysis of phytochemical profile and antioxidant and anti-inflammatory activity of four Gentiana species from the qinghai-tibet plateau. Journal of Ethnopharmacology. 2024;326:117926. https://doi.org/10.1016/j.jep.2024.117926
- [Google Scholar]
- A review on the ethnomedicinal usage, phytochemistry, and pharmacological properties of Gentianeae (Gentianaceae) in tibetan medicine. Plants (Basel, Switzerland). 2021;10:2383. https://doi.org/10.3390/plants10112383
- [Google Scholar]
- Botany, traditional use, phytochemistry, pharmacology, quality control, and authentication of radix Gentianae Macrophyllae-A traditional medicine: A review. Phytomedicine : International Journal of Phytotherapy and Phytopharmacology. 2018;46:142-163. https://doi.org/10.1016/j.phymed.2018.04.020
- [Google Scholar]
- Phytochemical characterization and anti-inflammatory activity of a water extract of Gentiana purpurea roots. Journal of Ethnopharmacology. 2023;301:115818. https://doi.org/10.1016/j.jep.2022.115818
- [Google Scholar]
- Seco-iridoid glycosides from the Gentiana olivieri Griseb and their bioactivities. Phytochemistry. 2023;215:113839. https://doi.org/10.1016/j.phytochem.2023.113839
- [Google Scholar]
- Iridoid derivatives as anticancer agents: An updated review from 1970-2022. Cancers. 2023;15:770. https://doi.org/10.3390/cancers15030770
- [Google Scholar]
- Pharmacokinetic Analysis of four bioactive iridoid and Secoiridoid glycoside components of radix Gentianae Macrophyllae and their synergistic excretion by HPLC-DAD combined with second-order calibration. Natural Products and Bioprospecting. 2017;7:445-459. https://doi.org/10.1007/s13659-017-0145-7
- [Google Scholar]
- Exploring the multitarget potential of iridoids: Advances and applications. Current Topics in Medicinal Chemistry. 2023;23:371-388. https://doi.org/10.2174/1568026623666221222142217
- [Google Scholar]
- Assessment of iridoids and their similar structures as antineoplastic drugs by in silico approach. Journal of Biomolecular Structure & Dynamics 2024:1-16. https://doi.org/10.1080/07391102.2024.2314262
- [Google Scholar]
- A comprehensive review of phytochemistry, pharmacology and clinical application of Gentianae Macrophyllae radix. Natural Product Research. 2024;38:4446-4467. https://doi.org/10.1080/14786419.2023.2298724
- [Google Scholar]
- Design, synthesis and biological evaluation of gentiopicroside derivatives as potential antiviral inhibitors. European Journal of Medicinal Chemistry. 2017;130:308-319. https://doi.org/10.1016/j.ejmech.2017.02.028
- [Google Scholar]
- Novel solvent properties of choline chloride/urea mixtures. Chemical Communications (Cambridge, England) 2003:70-71. https://doi.org/10.1039/b210714g
- [Google Scholar]
- Deep eutectic solvent as green solvent in extraction of biological macromolecules: A review. International Journal of Molecular Sciences. 2022;23:3381. https://doi.org/10.3390/ijms23063381
- [Google Scholar]
- Deep eutectic solvent-based dispersive liquid-liquid micro-extraction of pesticides in food samples. Food Chemistry. 2021;342:127943. https://doi.org/10.1016/j.foodchem.2020.127943
- [Google Scholar]
- Development of deep eutectic solvent-based aqueous biphasic system for the extraction of lysozyme. Talanta. 2019;202:1-10. https://doi.org/10.1016/j.talanta.2019.04.053
- [Google Scholar]
- Decomposition of deep eutectic solvent aids metals extraction in lithium-ion batteries recycling. ChemSusChem. 2022;15:e202200966. https://doi.org/10.1002/cssc.202200966
- [Google Scholar]
- Ultrasonic extraction and purification of scutellarin from Erigerontis Herba using deep eutectic solvent. Ultrasonics Sonochemistry. 2023;99:106560. https://doi.org/10.1016/j.ultsonch.2023.106560
- [Google Scholar]
- A deep eutectic solvent modified magnetic β-cyclodextrin particle for solid-phase extraction of trypsin. Analytica Chimica Acta. 2020;1137:125-135. https://doi.org/10.1016/j.aca.2020.09.005
- [Google Scholar]
- Enhanced enzymatic saccharification of sorghum straw by effective delignification via combined pretreatment with alkali extraction and deep eutectic solvent soaking. Bioresource Technology. 2021;340:125695. https://doi.org/10.1016/j.biortech.2021.125695
- [Google Scholar]
- Optimization of green extraction process with natural deep eutectic solvent and comparative in vivo pharmacokinetics of bioactive compounds from Astragalus-Safflower pair. Phytomedicine : International Journal of Phytotherapy and Phytopharmacology. 2023;114:154814. https://doi.org/10.1016/j.phymed.2023.154814
- [Google Scholar]
- Vortex-assisted dispersive liquid-liquid microextraction based on the hydrophobic deep eutectic solvent-based ferrofluid for extraction and detection of myclobutanil. Mikrochimica Acta. 2023;190:352. https://doi.org/10.1007/s00604-023-05884-y
- [Google Scholar]
- Extraction efficiency of loganic acid and gentiopicroside from Gentianae radix et rhizoma by deep eutectic solvent-based ultrasound-assisted extraction. Results in Chemistry 2024:101740. https://doi.org/10.1016/j.rechem.2024.101740
- [Google Scholar]
- Composition and antioxidant activity of anthocyanins from Aronia melanocarpa extracted using an ultrasonic-microwave-assisted natural deep eutectic solvent extraction method. Ultrasonics Sonochemistry. 2022;89:106102. https://doi.org/10.1016/j.ultsonch.2022.106102
- [Google Scholar]
- Green extraction of bioactive compounds from apple pomace by ultrasound assisted natural deep eutectic solvent extraction: Optimisation, comparison and bioactivity. Food Chemistry. 2023;398:133871. https://doi.org/10.1016/j.foodchem.2022.133871
- [Google Scholar]
- An ultra-rapid and eco-friendly method for determination of loganic acid and gentiopicroside from Gentianae Macrophyllae radix by vortex-assisted matrix solid-phase dispersion extraction and LC-MS. Journal of Pharmaceutical and Biomedical Analysis. 2023;222:115085. https://doi.org/10.1016/j.jpba.2022.115085
- [Google Scholar]
- Analysis and stability of the constituents of curcuma longa and Harpagophytum procumbens tinctures by HPLC-DAD and HPLC-ESI-MS. Journal of Pharmaceutical and Biomedical Analysis. 2011;55:479-486. https://doi.org/10.1016/j.jpba.2011.02.029
- [Google Scholar]
- Stability of the constituents of Calendula, milk-thistle and passionflower tinctures by LC-DAD and LC-MS. Journal of Pharmaceutical and Biomedical Analysis. 2002;30:613-624. https://doi.org/10.1016/s0731-7085(02)00352-7
- [Google Scholar]
- Molecular dynamics simulation study of the far-infrared spectrum of a deep eutectic solvent. The Journal of Physical Chemistry. B. 2022;126:5695-5705. https://doi.org/10.1021/acs.jpcb.2c03277
- [Google Scholar]
- The effect of ZIF-67 nanoparticles on the desulfurization performance of deep eutectic solvent based nanofluid system. Journal of Hazardous Materials. 2022;426:128098. https://doi.org/10.1016/j.jhazmat.2021.128098
- [Google Scholar]
- Pharmacokinetics, tissue distribution and excretion of peimisine in rats assessed by liquid chromatography-tandem mass spectrometry. Archives of pharmacal research. 2015;38:1138-1146. https://doi.org/10.1007/s12272-014-0434-1
- [Google Scholar]
- Matrix effect in bio-analysis of illicit drugs with LC-MS/MS: Influence of ionization type, sample preparation, and biofluid. Journal of the American Society for Mass Spectrometry. 2003;14:1290-1294. https://doi.org/10.1016/s1044-0305(03)00574-9
- [Google Scholar]
- Iridoids: Research advances in their phytochemistry, biological activities, and pharmacokinetics. Molecules (Basel, Switzerland). 2020;25:287. https://doi.org/10.3390/molecules25020287
- [Google Scholar]
- Natural deep eutectic solvent (NADES): A strategy to improve the bioavailability of blueberry phenolic compounds in a ready-to-use extract. Food Chemistry. 2021;364:130370. https://doi.org/10.1016/j.foodchem.2021.130370
- [Google Scholar]
- Oral bioavailability-enhancing and anti-obesity effects of hydroxysafflor yellow A in natural deep eutectic solvent. ACS Omega. 2022;7:19225-19234. https://doi.org/10.1021/acsomega.2c00457
- [Google Scholar]