Development and Validation of an UHPLC-MS/MS Method for Quantification of DMAG in Rat Plasma and Its Application in a Preliminary Pharmacokinetic Study in Thrombocytopenia Rats

Li, Yan; Wang, Yuqing; Zhao, Zhiqiang; Li, Yunxia; Peng, Cheng; Wu, Jianming

doi:https://doi.org/10.1155/2023/9746087

Journal of Chemistry

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Abstract Introduction Materials and Methods Results and Discussion Conclusions Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 9746087 | https://doi.org/10.1155/2023/9746087

Development and Validation of an UHPLC-MS/MS Method for Quantification of DMAG in Rat Plasma and Its Application in a Preliminary Pharmacokinetic Study in Thrombocytopenia Rats

Yan Li,^1,2Yuqing Wang,²Zhiqiang Zhao,^2,3Yunxia Li,¹Cheng Peng,¹and Jianming Wu²

Academic Editor: Damião Pergentino de Sousa

Received28 Feb 2023

Revised07 Aug 2023

Accepted19 Aug 2023

Published02 Sept 2023

Abstract

A preliminary study has shown that 3, 8-Di-O-methylellagic acid 2-O-glucoside (DMAG), an ellagic tannin from Sanguisorba officinalis L., has the potential in relieving thrombocytopenia. However, there is a lack of information on the pharmacokinetics of DMAG in thrombocytopenia rats. Therefore, we aimed to establish a simple, rapid, and sensitive UHPLC-MS/MS method for quantifying DMAG and study its pharmacokinetic behavior in this study. DMAG and hispidulin (internal standard, IS) were separated on an Acquity Shim-pack GIST column using 0.1% formic acid in water and acetonitrile as the mobile phase with a total run time of 5 min and gradient elution at a flow rate of 0.3 mL/min. The recovery and matrix effects of DMAG were within 94.85%–100.40% and 94.55%–102.46%, respectively. The intraday RSD and interday RSD were between 3.31% and 13.19%, accuracy RE was ≤6.69%, and stability RSD changes were 3.38%–8.78%. As for intragastric administration, with shortened T_max (3.00 vs. 2.16 h), C_max (25.67 vs. 35.38 ng/mL) was added for a 2 mg/kg dose after the establishment of the thrombocytopenia rat model. Relative to normal rats treated with 4 mg/kg, in thrombocytopenia rats treated with the same dose, C_max (49.13 vs. 67.78 ng/mL), AUC_(0–t) (234.60 vs. 318.17 ng·h/mL), and AUC_(0–∞) (322.74 vs. 498.57 ng h/mL) increased, MRT (18.15 vs. 26.32 h) prolonged, T_max (3.00 vs. 2.33 h) shortened, and CL/F (12746.50 vs. 8093.50 mL/h/kg) reduced. As for intravenous administration, C_max (1679.54 ng/mL), AUC_(0–t) (589.02 g·h/mL), and AUC_(0–∞) (605.58 g·h/mL) were significantly increased in thrombocytopenia rats than that in normal rats (743.76 ng/mL for C_max, 242.46 g·h/mL for AUC_(0–t), and 245.19 g·h/mL for AUC_(0–∞)). V_z/F and CL/F were remarkably decreased from 343196.86 to 194659.43 mL/kg for V_z/F and 8236.18 to 3326.01 mL/h/kg for CL/F with the model establishment, respectively. Overall, we successfully developed a reliable UHPLC-MS/MS method for determining DMAG levels in rat plasma. The pharmacokinetic difference could be attributed to the pathological state of the thrombocytopenia rats, which may affect the absorption, distribution, metabolism, and excretion of DMAG. These findings lay the foundation for further evaluating the clinical efficiency and safety of DMAG in the treatment of thrombocytopenia.

1. Introduction

Sanguisorba officinalis L. is an herbal medicine from the Rosaceae family that can be found in both north and south China. It has been reported to have various beneficial effects, such as hemostatic, antitumor, antioxidative, antibacterial, and anti-inflammatory [1]. One of the most common clinical applications of Sanguisorba officinalis L. is as a hemostatic agent. It has been used to treat various conditions, such as upper gastrointestinal hemorrhages, ulcer hemorrhages, hematochezia, metrorrhagia, and metrostaxis. Moreover, it has been studied for its potential in the treatment of leukemia, hepatic and gastric carcinoma, and scalds [2]. Our research group has primarily focused on studying the mechanism of hematopoiesis induced by Sanguisorba officinalis L. for a considerable period of time. Previous studies conducted by our group have demonstrated that the extract of Sanguisorba officinalis L. can stimulate bone marrow hematopoiesis by promoting the proliferation and differentiation of hematopoietic stem and progenitor cells, ultimately leading to an increase in the number of leukocytes, erythrocytes, and platelets [3, 4].

Tannins, flavonoids, and saponins are primary chemical components in Sanguisorba officinalis L. [5, 6]. Tannins, one of the primary components, can be divided into three categories: hydrolyzable, condensed, and complex tannins formed by glucoside bond formation between condensed and hydrolyzable tannins. Hydrolyzable tannins can be further categorized into gallic acid tannins and ellagitannins based on the type of esterified polyols and phenolic acids produced during the hydrolysis process. DMAG, a typical ellagic tannin, exhibits a wide range of effects, including antitumor, antioxidation, antisepsis, and immune regulation [7, 8]. Our previous research has demonstrated that it can enhance megakaryocyte differentiation, platelet production, aggregation, and adhesion, resulting in a reduction in tail bleeding time. The underlying mechanism of these effects may involve the upregulation of p-Akt, VWF, PLEK, ITGB3, and ITGA2B expression [9]. Therefore, DMAG is a potential candidate for the treatment of thrombocytopenia. However, the pharmacokinetic characteristics of DMAG in the plasma of normal and thrombocytopenic rats have not been reported at present.

Currently, various analytical methods have been employed to determine the active components in Sanguisorba officinalis L. For instance, Xu et al. identified 3, 3′-di-O-methylellagic acid and 3, 3′, 4′-trimethylellagic acid in Polygonum runcinatum using HPLC [10]. Xiong and Yang employed HPLC to investigate the changes in pharmacokinetic characteristics of catechin, epicatechin, and ziyuglycoside I in rats after compatibility with different tannins and saponins. When the compatibility proportion reached 8 : 1, the three components exhibited favorable pharmacokinetic behavior in rats [11]. Sun et al. employed HPLC-ELSD to detect triterpenoids and phenolic acids in Sanguisorba officinalis L. [12]. Tang et al. used HPLC-MS/MS to simultaneously determine the pharmacokinetic characteristics of ziyuglycoside I and II in rats [13]. Fan et al. conducted a study using HPLC-MS/MS to detect and analyze thirteen phenolic acids and six triterpenoids in the n-butanol extract of Sanguisorba officinalis L. [14]. They also compared the pharmacokinetic differences of these components between normal rats and rats with leukopenia. In a separate study, Wu et al. utilized UHPLC-MS/MS to analyze six triterpenoids [15]. Wei et al. used UHPLC-MS/MS to measure the pharmacokinetic parameters of three compounds in rats after they were orally administered Sanguisorba officinalis L. extract [16]. Zhu et al. employed UHPLC-MS/MS to detect ziyuglycoside I and compared its pharmacokinetic behavior in normal and leukopenia rats [17]. However, to the best of our knowledge, there is currently no reported analytical method specifically designed to detect DMAG in thrombocytopenia rats.

This present study aims to establish an UHPLC-MS/MS method that is simple, rapid, and sensitive for determining DMAG. The pharmacokinetic characteristics of DMAG will be studied, and the difference in pharmacokinetics between normal and thrombocytopenic rats will be compared. The findings of this study will provide reference information for the development of innovative drugs for treating thrombocytopenia.

2. Materials and Methods

2.1. Chemicals and Reagents

DMAG (98% purity), IS (98% purity), and cyclophosphamide (98% purity) were purchased from BioBioPha Co., Ltd. (Yunnan, China), Chengdu Mansite Pharmaceutical Co., Ltd. (Chengdu, China), and Meryer Chemical Technology Co., Ltd. (Shanghai, China), respectively. The chemical structures of DMAG and IS are shown in Figure 1. Quick Start Bradford 1x Dye Kit and TIANamp Genomic DNA Kit were purchased from Bio-Rad Laboratories (California, USA) and Tiangen Biotech Co., Ltd. (Beijing, China), respectively. Formic acid and acetonitrile (LC/MS grade) were acquired from Thermo Fisher Scientific Co., Ltd. (USA). Distilled deionized water was prepared using a Plus Milli-Q water purification system (Bedford, MA, USA). Ethyl acetate was of analytical grade supplied by Aladdin Reagents Co., Ltd. (Shanghai, China).

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2.2. Animals and Model Preparation

Female and male specific pathogen-free Sprague Dawley rats, weighing 180–220 g (Certificate no. SCXK 2020-030), were provided by Da-suo Biotechnology Co., Ltd. (Chengdu, China). These rats were housed for one week in a standard breeding room (temperature 22–25°C, humidity 55 ± 10%, and 12 h light/12 h dark cycle) and allowed free access to food and water. All animal experiments were conducted following US guidelines [18] and approved by the Ethical Committee on Use and Care of Animals of Chengdu University of Traditional Chinese Medicine (No. 2015-0727-03). Before the experiment, the rats were fasted for 12 h with free access to water.

The rats were then randomly divided into normal and model groups. A rat model for thrombocytopenia induced by cyclophosphamide was established in our previous study [19]. The blood of six normal rats and six model rats was collected for platelet count. Bone marrow cells were extracted from rat femur to calculate the bone marrow cell count with an automatic blood cell analyzer and protein content and DNA content according to kit instructions. In the meantime, the heart, liver, spleen, lung, kidney, and thymus indices of the normal and model groups were collected and determined.

2.3. Chromatographic Condition

A UHPLC-MS/MS system (Shimadzu Corp., Tokyo, Japan) equipped with an LC-MS triple quadrupole mass spectrometer was used to quantify DMAG and IS. Chromatographic separation was achieved on an Acquity Shim-pack GIST column (2.0 × 75 mm, 2.0 μm; Shimadzu Corp., Japan) at 40°C. The mobile phase was composed of 0.1% formic acid in water (A) and acetonitrile (B) at a flow rate of 0.3 mL/min: 0–2.00 min, 40% to 60% B; 2.01–3.00 min, 60% to 40% B; 3.01–5.00 min, and the ratio of B maintained at 40%.

The mass spectrometer with an ESI ion source was operated in the negative ion mode. The MRM fragmentation transitions were m/z 491.35⟶313.10 for DMAG and m/z 299.05⟶284.10 for IS (Figure 2). The parameters of mass spectrometry were optimized: flow rates of atomizing gas, 2.0 L/min; heating gas, 10.0 L/min; drying gas, 10.0 L/min; interface voltage, 4 kV; interface temperature, 300°C; desolvation temperature, 250°C; heating block temperature, 400°C; and detector voltage, 2.24 kV. The collision energies of DMAG and IS were 35 eV and 19 eV, respectively.

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2.4. Preparation of Standard Solutions, Calibration, and Quality Control (QC) Samples

Stock solutions of DMAG and IS were prepared with acetonitrile at 0.20 and 25 mg/mL, respectively. The calibration curve working solution of DMAG was prepared by diluting the stock solution with 0.1% formic acid aqueous: acetonitrile = 60 : 40 (mobile phase) to achieve a series of concentrations: 0.5, 4, 20, 50, 100, 200, 500, and 1000 ng/mL. IS stock solutions were diluted into 400 ng/mL with the mobile phase. The calibration standard was prepared by spiking 90 μL blank rat plasma with 10 μL working standard solutions. QC samples were prepared at 0.5, 20, 100, and 500 ng/mL with the same operation.

2.5. Plasma Sample Preparation

A 10 μL aliquot of IS was added to 100 μL rat plasma. After mixing for 30 s, 3.5 mL of ethyl acetate was added for drug extraction. Then, the mixture was vortexed for 5 min and centrifuged at 8000 g for 10 min at 4°C. The supernatant was collected, dried under nitrogen flow at 37°C, and reconstituted with 100 μL mobile phase. A 10 μL aliquot was injected immediately into the UHPLC-MS/MS spectrometer for analysis.

2.6. Method Validation

According to the FDA guidelines [20–22], the bioanalytical method by UHPLC-MS/MS technique was developed, and specificity, recovery, matrix effect, accuracy, precision, linearity, and stability of the method should be validated.

2.6.1. Specificity

The specificity of the method was assessed by analyzing the chromatograms of six different batches of blank rat plasma, plasma samples with DMAG and IS, and plasma samples after DMAG administration.

2.6.2. Linearity

For the calibration curve, a series of concentrations (0.05, 0.4, 2, 5, 10, 20, 50, and 100 ng/mL) were prepared for DMAG in rat plasma. Also, the concentration of IS was 40 ng/mL in rat plasma. To evaluate the linearity, the calibration curve of DMAG was constructed by plotting the peak area ratios (y) of DMAG to IS against the theoretical concentrations (x) and fitted to linear regression analysis. The lower limit of quantification (LLOQ) was sufficient for the pharmacokinetic study of DMAG in rat plasma with a signal-to-noise ratio (SNR) ≥10. In the meantime, the accuracy (relative error, RE) and precision (relative standard deviation, RSD) were permitted to be ≤20%.

2.6.3. Matrix Effect and Recovery

The matrix effect and recovery of rat plasma samples were determined at quality control (QC) concentrations (2, 10, and 50 ng/mL, n = 6) by comparing the following samples: (A) DMAG and IS were added into plasma before extraction, (B) both DMAG and IS were added into the residue after extracting blank plasma, and (C) DMAG and IS were directly added into acetonitrile. Matrix effects were evaluated by calculating the peak area ratio of DMAG and IS with or without plasma after extraction at three QC concentrations (group B/group C). The recovery was assessed by comparing the peak area ratio of DMAG and IS before and after treatment with ethyl acetate (group A/group B).

2.6.4. Accuracy and Precision

Accuracy and precision were evaluated using QC samples of LLOQ, low, medium, and high concentration on one day and three consecutive days. Accuracy was presented as the relative error (RE, %) between measured concentrations and actual concentrations. And precision was exhibited as relative standard deviation (RSD, %). Accuracy and precision for low, medium, and high concentrations should be less than 15%, and LLOQ not exceed 20%.

2.6.5. Stability

The stability of the analyte under various storage and handling conditions was calculated using three concentrations of QC samples, which included the short-term (12 h, 25°C), long-term (30 d, −80°C), postpreparative (24 h, 4°C), and three freeze-thaw cycles (−80°C) stabilities. Then, the stability of the analyte in rat plasma was assessed by analyzing six replicates.

2.7. Pharmacokinetics

Twelve normal rats and twelve model rats were randomly divided into low- and high-dose groups (three female and three male rats in each group). The low- and high-dose groups were orally administered with 2 and 4 mg/kg DMAG, respectively. In addition, six normal rats and six model rats (three female and three male rats in each group) were intravenously administered one dose of 2 mg/kg DMAG. A series of blood samples were acquired before and after oral administration at time points of 0.08, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h. Also, blood samples were centrifuged at 1500 g for 10 min to obtain plasma samples and then kept at −80°C.

2.8. Data Analysis

Pharmacokinetic parameters, including AUC_0–t, AUC_0–∞, MRT, T_max, V_z/F, CL/F, and C_max, were calculated with DAS 3.0 software. Simultaneously, experimental data were analyzed using GraphPad Prism 8.0 or SPSS 22.0. When the two groups of samples followed the normal distribution and homogeneity of variance, a t-test was used; otherwise, nonparametric tests were used. or was considered statistically significant.

3. Results and Discussion

3.1. Thrombocytopenia Rat Model Validation

Cyclophosphamide is a potent alkylating agent that can form covalent bonds with DNA molecules, leading to the inhibition of DNA synthesis in marrow hematopoietic cells. This disruption of DNA synthesis affects the balance between cell proliferation and maturation of megakaryocytes, and platelet senescence. As a result, cyclophosphamide can cause a decrease in platelet levels in the peripheral blood, a condition known as thrombocytopenia [23]. Consequently, cyclophosphamide is commonly used to establish rat models of thrombocytopenia. Compared with normal rats, DNA content, protein content, platelet count, bone marrow cell count, spleen index, and thymus index in model rats were all reduced (Supplementary Figure 1S).

3.2. Method Development and Validation

Pharmacokinetics is the scientific study of how drugs are absorbed, distributed, metabolized, and eliminated by the body. It focuses on understanding the movement of drugs within the body over time, including factors such as the drug’s absorption into the bloodstream, its distribution to various tissues and organs, its metabolism by enzymes, and its elimination from the body through urine or feces [24]. By studying pharmacokinetics, researchers can gain insights into how drugs are processed by the body, which can help optimize drug dosing regimens and improve drug safety and efficacy. To judge the application prospects of a drug, especially its marketability, it not only has to have strong curative effects, minimal toxicity, and side effects but also excellent pharmacokinetic properties. The most typical examples are peptide drugs. Many bioactive peptides in the body, such as endorphins, which are high efficiency and low toxicity, are unstable in the body with poor absorption [25]. Pharmacokinetic studies in the early stages of drug design and development will help obtain safe and effective therapeutic drugs and reduce the elimination rate of candidate drugs. Nevertheless, no pharmacokinetic study of DMAG in rats has been reported till date. In the present study, a novel, accurate, sensitive, and efficient UHPLC-MS/MS method was established to determine the pharmacokinetics of DMAG in rats in vivo. To improve the ionization efficiency and peak shape of DMAG, an aqueous solution containing 0.1% formic acid and acetonitrile was selected as the mobile phase in gradient elution for analysis. Liquid-liquid extraction with ethyl acetate was adopted because of its excellent recovery efficiency for DMAG.

Specificity was assessed by comparing the MRM chromatograms of blank plasma samples, blank plasma samples with DMAG and IS, and plasma samples after the administration of DMAG (Figure 3). As shown in the figure, the retention times of DMAG and IS were 1.068 and 2.000 min, respectively. No endogenous substances interfered with DMAG and IS detection, which proved its high specificity. As shown in Table 1, recovery and matrix effects of DMAG were within the ranges of 94.85%–100.40% and 94.55%–102.46%, respectively. The intra- and interday precision and accuracy of DMAG at QC concentrations are shown in Table 2. The intraday RSD and interday RSD were between 3.31% and 13.19%, and the accuracy RE was ≤6.69%, which proved that the analytical method was accurate and reproducible. A calibration curve was established by plotting the peak area ratio of DMAG and IS to the concentration. Linearity was good in the concentration range of 0.05–100 ng/mL. The regression equation for DMAG was y = 0.00600871x + 0.00011248 (r² = 0.9994). The lower limit of quantitation (LLOQ) for DMAG was 0.05 ng/mL. The stability of DMAG in rat plasma under different storage conditions is shown in Table 3, with stability RSD changing in the range of 3.38%–8.78%. These results indicated that DMAG was sufficiently stable under the above analytical conditions.

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3.3. Pharmacokinetic Study

The mean plasma concentration-time profiles of DMAG in rat plasma after intragastric administration are shown in Figure 4. Pharmacokinetic parameters of DMAG in rats after intragastric administration in the four groups are presented in Table 4. For normal and model rats, C_max, AUC_(0–t), and AUC_(0–∞) increased between the two dose groups. After establishing the thrombocytopenia rat model, T_max was reduced (3.00 vs. 2.16 h), and C_max was increased (25.67 vs. 35.38 ng/mL) at a dose of 2 mg/kg. Relative to normal rats, in thrombocytopenia rats treated with 4 mg/kg, C_max (49.13 vs. 67.78 ng/mL), AUC_(0–t) (234.60 vs. 318.17 ng h/mL), and AUC_(0–∞) (322.74 vs. 498.57 ng h/mL) were increased, MRT (18.15 vs. 26.32 h) was prolonged, T_max (3.00 vs. 2.33 h) was clearly shortened, and CL/F (12746.50 vs. 8093.50 mL/h/kg) was reduced. Furthermore, mean plasma concentration-time profiles of normal and thrombocytopenia rats after intravenous administration are exhibited in Figure 5. Pharmacokinetic parameters of DMAG in rats after intravenous administration are manifested in Table 5. Although no difference can be observed for MRT and t_1/2 between two groups, C_max (1679.54 ng/mL), AUC_(0–t) (589.02 g·h/mL), and AUC_(0–∞) (605.58 g·h/mL) were significantly increased in thrombocytopenia rats than that in normal rats (743.76 ng/mL for C_max, 242.46 g·h/mL for AUC_(0–t), and 245.19 g·h/mL for AUC_(0–∞)). V_z/F and CL/F were remarkably decreased from 343196.86 to 194659.43 mL/kg for V_z/F and 8236.18 to 3326.01 mL/h/kg for CL/F with the model establishment, respectively. The results showed that the absorption of DMAG was increased, T_max advanced, and elimination slowed in thrombocytopenia rats, signifying that the pharmacokinetic behavior of DMAG may be affected by the pathological state. Also, it is consistent with the relevant literature report. For example, the AUC, C_max, and t_1/2 of protosappanin B and Brazilian were increased in diabetic rats when compared to normal rats after oral administration of the extract from Sappan Lignum [26]. Compared to the normal group, the exposure to paeoniflorin, albiflorin, and plumbagin in liver injury rats was enhanced, while their elimination was slowed down [27]. With decreased CL, AUC, C_max, and t_1/2 of aloe-emodin, rhein, emodin, and chrysophanol almost doubled after administration of the extract of Rheum palmatum L. in thrombotic focal cerebral ischemia rats compared to normal rats [28].

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The pharmacokinetic alteration of DMAG may be also influenced by the presence of intestinal flora. These bacteria, specifically Anaerococcus hydrogenalis, Clostridium asparagiforme, and Clostridium hathewayi, are capable of producing trimethylamine, which can induce the concentration of endogenous trimethylamine oxide (TMAO) [29]. When platelets are directly exposed to TMAO, it can increase the release of stored calcium ions, leading to the activation of platelets in response to stimuli [30]. In addition, intestinal flora can promote platelet adhesion, aggregation, and activation through stimulation of Toll-like receptor 2, regulation of von Willebrand factor. Intestinal flora can also facilitate the production of intestinal endotoxins, which stimulate the secretion of endothelial cytokine VIII and are associated with the occurrence and development of thrombocytopenia [31, 32]. Furthermore, studies have shown that ellagic tannins can increase the diversity of intestinal flora in normal rats and alter the composition of the flora by increasing the proportion of Bacteroidales S24-7, Lachnospiraceae NK4A136, and Bacteroides [33]. Gallic acid tannins, when administered tannins from Sanguisorba officinalis L., can be hydrolyzed by intestinal flora to produce gallic acid, which can further be metabolized to pyrogallol. However, due to their large molecular weight and high polarity, ellagic tannins have poor absorption and low bioavailability in the gastrointestinal tract of mammals, making it difficult for them to reach bioactive concentrations in tissues and plasma. Some of the unabsorbed ellagellin is hydrolyzed to ellagic tannins, which then lose a lactone ring under the action of intestinal flora. These tannins are gradually dehydroxylated or methylated into more liposoluble and absorbable urolithins. Urolithins have high biological activity and may be the active components in vivo [34, 35]. In summary, there is an interaction between ellagic tannins and intestinal flora, and the presence of intestinal flora may cause pharmacokinetic changes in DMAG. However, further research is needed to confirm whether DMAG can be absorbed by intestinal flora and converted to urolithin during intestinal metabolism, as well as how it regulates the intestinal flora in thrombocytopenia rats.

In addition, while establishing the pathological model of thrombocytopenia, we also considered the drug-drug interactions between cyclophosphamide and DMAG. This interaction could be related to the changes in the pharmacokinetics of DMAG. Therefore, it is important to evaluate the potential drug-drug interaction between cyclophosphamide and DMAG. Previous research has shown that both cyclophosphamide and tannin are substrates for CYP3A4 [36–38], which could explain the decreased clearance and extended mean residence time of DMAG. Other studies have also found that the levels of Lactobacillus and Bifidobacterium were reduced, while Enterococcus and Escherichia coli were increased in the intestinal tract of mice treated with cyclophosphamide. This could be one of the reasons for the variations in the absorption of DMAG [39, 40].

Tumors are neoplasms formed by the excessive proliferation of local tissue cells in the body under the effect of various tumorigenic factors. Tumor cells can adapt to change in the metabolic environment by transforming between oxidative phosphorylation (OXPHOS) and glycolysis, making them extremely difficult to eliminate. Traditional therapies for tumors include surgery, radiation, and chemotherapy. However, the current most commonly used means of tumor treatment, chemoradiotherapy, not only kills cancer cells but also normal cells. This can result in bone marrow suppression and severe side effects such as leukopenia, erythrocytopenia, and thrombocytopenia, thus limiting its scope and efficacy [41]. Among these side effects, thrombocytopenia is a common cause of clinical bleeding with spontaneous and intracranial hemorrhage, which is life-threatening. The pathogenesis is complex, has not been fully elucidated, and is a major problem that has puzzled clinicians for a long time. Therefore, in the process of chemoradiotherapy, it is of paramount importance to protect the hematopoietic function of the bone marrow and improve the platelet count in the peripheral blood [42]. A relevant study has shown that tannins from Sanguisorba officinalis can inhibit proliferation and promote apoptosis of SMMC-7721 and HepG2 cells [43, 44]. Qin et al. found that the survival rate of human cancer cell lines such as A549, SGC-7901, and BEL7402 gradually decreased with increasing concentration of total saponins from Sanguisorba officinalis L. [45]. Bastow et al. reported that Sanguiin H-6 had a strong inhibitory effect on DNA topoisomerase, with IC50 of 1 μmol/L and 0.01 μmol/L for topoisomerase I and II in vitro, respectively. The IC50 of Sanguiin H-6 on HeLa25 cells was 12 μmol/L and showed a dose-response relationship [46]. Consequently, Sanguisorba officinalis L. can be simultaneously used to treat tumors and thrombocytopenia. DMAG, a typical ellagic tannin from Sanguisorba officinalis L., is expected to be developed as an innovative candidate with this dual effect, which requires further experimental verification.

4. Conclusions

This study presents the first bioanalytical method for quantifying and studying the pharmacokinetics of DMAG in rat plasma using UHPLC-MS/MS. Additionally, significant differences in the pharmacokinetic characteristics of normal and thrombocytopenia rats were observed after oral and intravenous administration, potentially due to the pathological state. These findings provide valuable insights into the pharmacokinetic behavior of DMAG and contribute to our understanding of its effectiveness and toxicity.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Li and Wang contributed equally to this paper.

Acknowledgments

This study was supported by the National Major Science and Technology Project of the Ministry of Science and Technology of China (Grant no. 2018ZX09721004-006-004), National Natural Science Foundation of China (Grant nos. 81774013, 81804221, and 82074129), Science and Technology Planning Project of Sichuan Province, China (Grant no. 2022NSFSC1451), Science and Technology Program of Luzhou, China (Grant nos. 2019LZXNYDJ11 and 2018-JYJ-34), Xinglin Scholar Research Premotion Project of Chengdu University of Traditional Chinese Medicine (Grant no. MPRC2022018), and Science and Technology Programs of Southwest Medical University (Grant no. 2019ZZD006).

Supplementary Materials

Supplementary Figure 1S: DNA content, protein content, platelet count, bone marrow cell count, spleen index, and thymus index of normal and model rats. (Supplementary Materials)

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