Identification of Protosappanoside D from Caesalpinia decapetala and Evaluation of Its Pharmacokinetic, Metabolism and Pharmacological Activity

Protosappanoside D (PTD) is a new component isolated from the extract of Caesalpinia decapetala for the first time. Its structure was identified as protosappanin B-3-O-β-D-glucoside by 1H-NMR, 13C-NMR, 2D-NMR and MS techniques. To date, the pharmacological activities, metabolism or pharmacokinetics of PTD has not been reported. Therefore, this research to study the anti-inflammatory activity of PTD was investigated via the LPS-induced RAW264.7 cells model. At the same time, we also used the UHPLC/Q Exactive Plus MS and UPLC-MS/MS methods to study the metabolites and pharmacokinetics of PTD, to calculate its bioavailability for the first time. The results showed that PTD could downregulate secretion of the pro-inflammatory cytokines. In the metabolic study, four metabolites were identified, and the primary degradative pathways in vivo involved the desaturation, oxidation, methylation, alkylation, dehydration, degradation and desugarization. In the pharmacokinetic study, PTD and its main metabolite protosappanin B (PTB) were measured after oral and intravenous administration. After oral administration of PTD, its Tmax was 0.49 h, t1/2z and MRT(0–t) were 3.47 ± 0.78 h and 3.06 ± 0.63 h, respectively. It shows that PTD was quickly absorbed into plasma and it may be eliminated quickly in the body, and its bioavailability is about 0.65%.


Introduction
Caesalpinia decapeta (C. decapetala) (Latin name: Caesalpinia decapetala (Roth) Alston) is a traditional medicinal plant of the Miao people. It belongs in the Fabaceae family and has been used to treat colds, fever, rheumatic pain, etc. for many years [1,2]. In previous studies, it was found that the extract of C. decapetala mainly contains compounds such as phenolics, flavonoids and terpenoids, which has good anti-inflammatory, antiviral and antitumor properties [3][4][5]. For example, gallic acid has a good proapoptotic effect on A-489 renal cancer cells [6], and can also inhibit the secretion of inflammatory factors induced by LPS [7]; sappanchalcones can significantly reduce the secretion of tumor necrosis factor alpha (TNFα), interleukin-1β (IL-1β) and interleukin-6 (IL-6) to improve the joint destruction and surface erosion claws of CIA mice [8,9]; luteolin can play an anti-inflammatory role by inhibiting NF-κB pathway [10], and inhibiting the STAT3 pathway, showing a strong ability to inhibit liver fibrosis [11]; protosappanin B (PTB) has obvious pharmacological activities such as antitumor, antioxidant and anti-inflammatory activities [12,13]. Protosappanoside D (PTD), a novel dibenzoxocin derivative, has been discovered in the extract of C. decapetala by the 1 H-NMR, 13 C-NMR, 2D-NMR and MS methods for the first time. The structure of PTD was very similar to that of PTB. Therefore, it is speculated that PTD may have good activity. Further studies on PTD are warranted.
Pharmacokinetic studies of the absorption, distribution, metabolism and excretion of drugs in the body are important to the research and development of new drugs [14]. In some cases, natural products may act as prodrugs, they are converted by metabolic activation into an active pharmacologic agent, which exerts pharmacological effects [15]. For example, baicalin and baicalein are the main chemical components of Scutellaria baicalensis (Georgi) [16], baicalin may undergo removal of its glycosidic bonds and thus be converted into baicalein, which has good pharmacological activity and bioavailability through enzyme-catalyzed reactions and ultrasonic extraction in previous studies [17,18]. After identification, PTD can be transformed into PTB after the removal of its glycosidic bonds, and PTB has obvious pharmacological activities. Therefore, it is important to study the metabolites and processes of PTD in vivo.
An effective analytical method must be utilized to analyze the complex chemical mixtures found in biological samples. Therefore, in the present study, ultra-high performance liquid chromatography-quadrupole/electrostatic field orbital trap high resolution mass spectrometry (UHPLC/Q Exactive Plus MS) technology was implemented to study the metabolism of PTD, and the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was used for the quantitative study of PTD in rat blood plasma [19][20][21]. At the same time, we also used an in vitro model of inflammation using lipopolysaccharide (LPS) induced inflammatory response in RAW264.7 cells to study the anti-inflammatory activity of PTD. Through the above experiments, this study will report the pharmacological activity, pharmacokinetics and metabolism of PTD for the first time, so as to provide a scientific basis for in-depth research and drug development of PTD.

Structural Qualification of PTD
PTD was isolated as white solid. The molecular formula was established by HR-QTOF-MS, 1 H-NMR and 13 13 C-NMR spectra data of PTD, it was found that it is basically consistent with the mixture of protosapanoside A and isoprotosapanoside A was reported in the literature [22]. However, the positions of sugar substituents in the compound are different, and the compound is substituted at carbon 3, so the compound is also a mixture of two dibenzoxyoctyl glycosides. 1 H-NMR spectrum (600 MHz, CD 3 OD) ( Figure 1A) showed the signals for two 1,2,4trisubstituted benzene ring systems in the low field region: δ H 7.10, 7.10 (1H, 1H, d, J = 8.5 Hz, H-1), 6.89, 6.84 (1H, 1H, d, J = 8.5 Hz, H-2 ), 6.84, 6.78 (1H, 1H, d, J = 2.5 Hz, H-4); two 1,2,4,5-tetrasubstituted benzene ring systems: δ H 6.74, 6.69 (1H, 1H, s, H-12), 6.75, 6 Figure 1C), H-1 is related to C-3, H-2 is related to C-4/C-12b, H-4 is related to C-12b/C-3/C-4a, H-6 is related to C-4a/C-7/C-8, H-8 is related to C-6/C-7/C-7a/C-9/C-8a/C-12a, H-9 is related to C-8/C-11, H-12 is related to C-10/C-8a/C-12b, and H-1 is related to C-3, indicating that the glucose group is connected to C-3. sonal carbon signals(δC 73.2, 72.9). The other signals are the carbon signals on the benzene ring (δC 160. 4, 159.5, 159.3, 159.3, 145.2, 145.2, 145.1, 145.1, 132.8, 132.1, 132.1, 131.6, 128.3, 127.9, 127.6, 127.0, 120.1, 119.1, 117.9, 117.6, 113.1, 112.6, 110.6, 110.0). According to the HMBC spectrum ( Figure 1C), H-1 is related to C-3, H-2 is related to C-4/C-12b, H-4 is related to C-12b/C-3/C-4a, H-6 is related to C-4a/C-7/C-8, H-8 is related to C-6/C-7/C-7a/C-9/C-8a/C-12a, H-9 is related to C-8/C-11, H-12 is related to C-10/C-8a/C-12b, and H-1′ is related to C-3, indicating that the glucose group is connected to C-3. All hydrocarbon signals of PTD were accurately assigned by integrating MS and NMR data (Table 1). After searching, PTD was identified as a pair of new compounds, and its chemical structure was protosappanin B-3-O-β-D-glucoside, named protosapanoside D (1a) and isoprotosapanoside D (1b) as shown in Figure 2.  All hydrocarbon signals of PTD were accurately assigned by integrating MS and NMR data (Table 1). After searching, PTD was identified as a pair of new compounds, and its  The results of MTS assay ( Figure 3) showed that there was no cytotoxicity to RAW264.7 cells by PTD treatment below 200 μmol/L and PTB treatment below 300 μmol/L. Subsequent experiments were conducted with 200 μmol/L PTD and PTB.   Many proinflammatory factors are secreted during inflammation, but excessive secretion of inflammatory factors will aggravate inflammation, so inhibiting the secretion of inflammatory factors is of great significance for the treatment of inflammation. The results are shown in Figure 4, the release of IL-6, TNF-α, IL-1β and NO in the supernatant of RAW264.7 cells were significantly induced by LPS. Compared to the model group, PTD and PTB significantly reduced the secretion of TNF-α, IL-1β and IL-6, and the efficacy of the two drugs was similar to that of dexamethasone, which proves that they have good anti-inflammatory effects. Many proinflammatory factors are secreted during inflammation, but excessive secretion of inflammatory factors will aggravate inflammation, so inhibiting the secretion of inflammatory factors is of great significance for the treatment of inflammation. The results are shown in Figure 4, the release of IL-6, TNF-α, IL-1β and NO in the supernatant of RAW264.7 cells were significantly induced by LPS. Compared to the model group, PTD and PTB significantly reduced the secretion of TNF-α, IL-1β and IL-6, and the efficacy of the two drugs was similar to that of dexamethasone, which proves that they have good anti-inflammatory effects.

Mass Spectrometric Analysis of PTD
To identify the metabolites, a comprehensive understanding of the characteristic product ions and fragmentation pathways of the parent compounds is necessary. In the present study, chromatographic and mass spectrometry conditions were optimized with PTD.
PTD showed a HPLC profile with a retention time of 4.32 min. In negative scan mode ( Figure 5A    − ). The proposed mechanism for fragment ion generation was shown in Figure 5B. The fingerprint product ions could serve well in the screening and identification of metabolites derived from PTD [23].

Metabolites Identification
Metabolites were detected and identified in blood plasma samples. Full scans and product ion scans in positive ion mode were conducted for all samples. As Figure S1 shows, four metabolites were identified by comparison between blank and administered samples. The metabolic messages of PTD are shown in Table 2 and the predicted metabolic pathways are depicted in Figure 6. It was found that the deprotonated molecule [M-H] − ion at m/z 303.0874 was PTB by comparing the chromatograms of the reference stand-

Metabolites Identification
Metabolites were detected and identified in blood plasma samples. Full scans and product ion scans in positive ion mode were conducted for all samples. As Figure S1 shows, four metabolites were identified by comparison between blank and administered samples. The metabolic messages of PTD are shown in Table 2 and the predicted metabolic pathways are depicted in Figure 6. It was found that the deprotonated molecule [M-H] − ion at m/z 303.0874 was PTB by comparing the chromatograms of the reference standards, blank plasma, rat administered plasma and deducted blank plasma (Figure 7), and its response was high. At present, studies have reported that PTB has antitumor and anti-inflammatory activities, so it was necessary to study the pharmacokinetics of PTD and its metabolite PTB.

Specificity
The MRM chromatograms of blank plasma, blank plasma spiked with standard solutions and IS, and a pharmacokinetic sample at 30 min after oral PTD administration are shown in Figure 8. The retention times of PTD, PTB, and IS were 0.74, 1.46 and 1.42 min, respectively. No endogenous interference was observed in MRM mode for any analyte.

Pharmacokinetic Study 2.4.1. UPLC-MS/MS Method Validation Specificity
The MRM chromatograms of blank plasma, blank plasma spiked with standard solutions and IS, and a pharmacokinetic sample at 30 min after oral PTD administration are shown in Figure 8. The retention times of PTD, PTB, and IS were 0.74, 1.46 and 1.42 min, respectively. No endogenous interference was observed in MRM mode for any analyte.

Linearity and Lower Limit of Quantification
Typical calibration curves, linearity ranges, coefficients of correlation, and LLOQ for PTD and PTB are shown in Table 3. The two analytes exhibited good linearity, with correlation coefficients varying from 0.9958-0.9998.

Specificity
The MRM chromatograms of blank plasma, blank plasma spiked with standard solutions and IS, and a pharmacokinetic sample at 30 min after oral PTD administration are shown in Figure 8. The retention times of PTD, PTB, and IS were 0.74, 1.46 and 1.42 min, respectively. No endogenous interference was observed in MRM mode for any analyte.

Linearity and Lower Limit of Quantification
Typical calibration curves, linearity ranges, coefficients of correlation, and LLOQ for PTD and PTB are shown in Table 3. The two analytes exhibited good linearity, with correlation coefficients varying from 0.9958-0.9998.

Precision and Accuracy
The results for intra-day and inter-day precision and accuracy for QC samples at three concentrations are shown in Table 4. The results showed that the RSD of intra-and inter-day measurements was less than 15%, and the accuracy range was approximately ranging from 86.06-109.38%. These results indicate that the proposed method is precise and accurate.

Extraction Recovery and Matrix Effect
The results of the matrix effect and extraction recovery of PTD and PTB are summarized in Table 5. The extraction recoveries of the two analytes were within the range of 87.65-103.25%, indicating that the recovery of analytes was consistent and reproducible. The matrix effects ranged from 88.83-105.08%, suggesting an insignificant matrix effect on the quantification of PTD and PTB.

Stability
The sample stability results (n = 5) are summarized in Table 6. The results showed that all three analytes were stable under all testing conditions, including at room temperature for 24 h, refrigeration at 4 • C for 24 h, and three freeze-thaw cycles. The RSD of all the samples under the above conditions were both within 15%.

Pharmacokinetic Study
The method established above was applied to determine the pharmacokinetics of PTD in rats. After oral administration (27.3 mg/kg) and intravenous administration (9.1 mg/kg) of PTD, the plasma concentration versus time profiles were shown in Figures 9 and 10, and the pharmacokinetic parameters were listed in Table 7. Both administration methods could detect PTD and its metabolite PTB. PTD reached a peak concentration rapidly at about 0.49 h after oral administration. The t 1/2z of oral and intravenous administration were 3.47 ± 0.78 h and 3.65 ± 1.26 h, respectively, whereas MRT (0-t) was 3.06 ± 0.63 h and 1.80 ± 0.50 h, respectively. The result suggested that it has a fast absorption rate with a short residence time. Thus, it can be quickly absorbed into blood circulation and eliminated rapidly from the body. The studies had shown that the exposure of PTD in vivo after intravenous injection is significantly higher than the exposure after oral absorption, and its bioavailability was about 0.65%. and 10, and the pharmacokinetic parameters were listed in Table 7. Both administration methods could detect PTD and its metabolite PTB. PTD reached a peak concentration rapidly at about 0.49 h after oral administration. The t1/2z of oral and intravenous administration were 3.47 ± 0.78 h and 3.65 ± 1.26 h, respectively, whereas MRT(0-t) was 3.06 ± 0.63 h and 1.80 ± 0.50 h, respectively. The result suggested that it has a fast absorption rate with a short residence time. Thus, it can be quickly absorbed into blood circulation and eliminated rapidly from the body. The studies had shown that the exposure of PTD in vivo after intravenous injection is significantly higher than the exposure after oral absorption, and its bioavailability was about 0.65%.

Discussion
PTD was identified in the extract of C. decapetala for the first time. Through NMR and MS identification methodology, the chemical structure of the mother nucleus was found to be very similar to that of PTB (refer Figure 2), of which are the tricyclic mother nuclei of dibenzo oxygen-containing eight-membered heterocycles. According to prior studies, PTB has a good anti-inflammatory effect [24]. Based on structure-function similarities, we speculated that PTD might also have good anti-inflammatory activity.
Based on these similarities and assumptions, we chose the in vitro inflammatory

Discussion
PTD was identified in the extract of C. decapetala for the first time. Through NMR and MS identification methodology, the chemical structure of the mother nucleus was found to be very similar to that of PTB (refer Figure 2), of which are the tricyclic mother nuclei of dibenzo oxygen-containing eight-membered heterocycles. According to prior studies, PTB has a good anti-inflammatory effect [24]. Based on structure-function similarities, we speculated that PTD might also have good anti-inflammatory activity.
Based on these similarities and assumptions, we chose the in vitro inflammatory model utilizing RAW264.7 cells, induced by LPS to detect the efficacy of PTD and PTB as anti-inflammatory agents [25,26]. The experimental results showed that PTD and PTB both significantly downregulated the secretion of TNF-α, IL-1β, and IL-6 by the RAW264.7 cells, compared to the model group, and the anti-inflammatory effects of PTB were similar to those previously reported. A comparison of the anti-inflammatory activities of PTD and PTB showed similar activity for both compounds.
Due to the high similarity between the parent heterocyclic nuclei of PTD and PTB, it was speculated that they may be prototypes or metabolites of each other. Prior studies had found that PTB and PTD in the extract of C. decapetala were able to enter the bloodstream. To eliminate any interference of PTB in the extract, PTD was administered orally and intravenously to explore the dynamic and transformation processes of PTD in vivo.
Studies of the degradative metabolism of these compounds proved that the primary metabolic pathways in vivo involved the organic chemical desaturation, oxidation, methylation, alkylation, dehydration, degradation, and desugarization. Previous studies showed that the in vivo efficacy of the bioactive components depends on their blood concentration and the activity of their metabolites. Therefore, it is of great significance to study the metabolites of PTD. Preliminary experiments were performed to compare the in vivo pharmacokinetic processes of these metabolites. The results showed that PTB was the main active metabolite of PTD, not only because its content was high, but also because only PTB had a complete in vivo kinetic process. PTB is a metabolite of PTD, which has great utilization value because of its anti-inflammatory, anti-cancer, antioxidant and other activities. Thus, PTD can be used as a prodrug to exploit its value through structural modification and transformation [27,28].
We further investigated the kinetics of PTD and its main metabolite PTB in vivo. A rapid, reliable and sensitive UPLC-MS/MS method was established and validated for simultaneous determination of PTD and its main metabolite PTB in rat plasma for the first time in pharmacokinetic study. The positive and negative modes were scanned simultaneously, and the scanning mode was selective ion monitoring (MRM) and the analysis time was 3 min, which considerably improved the experimental efficiency. The results of the experimental methodology met the requirements for its validation for biological sample analysis and determination, and were simple, selective and sensitive. This method was successfully used to study the pharmacokinetic processes of PTD and PTB, and relevant pharmacokinetic parameters were obtained. The experimental results showed that the T max , t 1/2z , and MRT (0-t) of PTD administered orally in rats were 0.49 h, 3.47 h and 3.06 h, respectively, indicating that PTD can be absorbed into the blood circulation rapidly and transformed rapidly into PTB and other metabolites with a short residence time. Bioavailability is a measure of drug absorption in organisms, where the bioavailability of drugs is higher. The results showed that the AUC (0-t) of PTD after intravenous administration was significantly higher than that oral administration. Its absolute bioavailability was low (approximately 0.65%), which indicates that the total amount of PTD entering the systemic circulation was significantly reduced after oral administration. It is speculated that some drugs are not absorbed due to the first-pass effect, resulting in their low absolute bioavailability. However, if a drug has low bioavailability but high therapeutic exposure, it may also have high developmental significance for future drug development, and other methods should be used to modify or transform them to make full use of their value. Based on formula, CL = kV, t 1/2 = 0.693/k. The t 1/2 of PTD after oral and intravenous injection is close, indicating that k is close after two kinds of administration, which proves that CL z/F is mainly affected by V z/F . The CL z/F and V z/F after oral PTD were much greater than those after intravenous injection. But that V = X0/C (X0: in vivo dose; C: plasma concentration). Therefore, we speculated that after oral PTD, intestinal flora or some metabolic enzymes make PTD metabolize rapidly in the gastrointestinal tract, resulting in low plasma drug concentration of PTD. Thus, the CL z/F and V z/F of oral administration were higher than those of intravenous administration. After oral administration, the AUC (0-t) of PTD was 382.17 ng/mL·h, and the AUC (0-t) of PTB was 179.33 ng/mL·h. The PTB content in vivo was approximately half of that of PTD, indicating that PTD was mainly transformed into PTB. It was speculated that the efficacy of PTD in vivo might be attributed to the role of PTB and other metabolites. This hypothesis needs to be verified through further experiments [29][30][31].
This study had provided the structural identification, determined the anti-inflammatory efficacy, and in vivo pharmacokinetic processes of PTD, which can be used as the basic research before clinical application of PTD and is of great significance to the development and utilization of PTD as a therapeutic agent.

Drug and Reagents
Caesalpinia decapeta was purchased from the medicinal materials market Wandongqiao, Guiyang City, Guizhou Province, China (2019 harvest), and it was identified as the dried root of Caesalpinia decapetala (Roxb.) Alston by Associate Professor Chunhua Liu (School of Pharmacy, Guizhou Medical University). Protosappanoside D (PTD, purity ≥ 99%, it was isolated from the extract of Caesalpinia decapeta, and its extraction and separation data are shown in the results Section 2.1), protosappanin B (PTB, purity ≥ 98%; Chengdu AIFA Biotechnology Co., Ltd., Chengdu, China) and puerarin (internal standard, IS) standards were purchased from the China Food and Drug Administration Research Institute; lipopolysaccharide (LPS) and dexamethasone (Dex) were obtained from Sigma-Aldrich (St Louis, MO, USA), and the nitric oxide assay kit was obtained from Nanjing Jiancheng Biotechnology Co., Ltd. (Nanjing, China). Tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) ELISA kits were purchased from Proteintech Group Inc. HPLC-grade acetonitrile, methanol and formic acid were obtained from Merck (Darmstadt, Germany). Distilled water was supplied by Watsons Group Co., Ltd. (Hong Kong, China). All other solvents used in the present study were of analytical grade and commercially available.

Extraction and Separation of PTD
The crude powder of Caesalpinia decapeta was weighed at 15 kg, which was then cold soaked and extracted with 8 volumes of 80% ethanol for 5 d, filtered to obtain the extract, and concentrated to 20 L. This was separated with D101-8 macroporous resin, eluted with 70 L of water, 20 L of 50% ethanol and 30 L of 90% ethanol; further, 50% ethanol eluent was collected and concentrated to 2.5 L. The concentrated solution was again separated with D101-8 macroporous resin and washed, in which 90 L with water, 25 L with 50% ethanol, and 15 L with 20% ethanol. Then, 50% ethanol eluent was collected and concentrated to 250 mL. Purification at medium pressure and gradient elution with water, 5% acetonitrile, 10% acetonitrile, 25% acetonitrile, 35% acetonitrile, 50% acetonitrile and 90% acetonitrile were conducted; further, 25% acetonitrile were collected and concentrated to a small volume. We added 50% methanol water to 100 mL, and the Welch Uitimate XB-C 18 column (50 × 250 mm, 10 µm) was washed with 7% acetonitrile water at a flow rate of 70 mL/min for high-pressure preparation and collection of the target stream. The organic solvent was concentrated at 50 • C and lyophilised to obtain a 1.0 g sample. This was dissolved in 30 mL pure water. A Kromasil C4 column (30 × 250 mm, 5 µm) was used. The extract was washed with 9% acetonitrile water at a flow rate of 40 mL/min for secondary high-pressure preparation, and the target stream was collected. The organic solvent was concentrated at 50 • C and lyophilised to obtain PTD. Cell viability was determined using the MTS cell viability assay kit. RAW264.7 cells were cultured at a density of 1 × 10 5 cells per well in 96-well plates by the treatment with various concentrations of PTD and PTB, respectively. After 24 h, 5 µL MTS was added to each well and incubated for 3 h. The plates were then read at 490 nm using a microplate reader [32]

Animal Studies
Specific pathogen-free Sprague-Dawley rats (230 ± 10 g) were purchased from Changsha Tianqin Biotechnology Co., Ltd. (Changsha, China; certificate no. SCXK (Xiang) 2014-0010). Before starting the experiments, the rats were allowed one week of acclimatization in the animal quarters under air conditioning (25 ± 1 • C) and an automatically controlled photoperiod of 12 h of light daily. They were fed with standard rodent chow and tap water ad libitum. Rats were fasted for 12 h before the start of the experiment but were free to drink water. All experiments were approved by the Animal Ethics Committee of Guizhou Medical University and conducted in accordance with the guidelines of the Committee on the Care and Use of Laboratory Animals in China.
To investigate the PTD metabolites, Sprague-Dawley rats were randomly divided into two groups with six rats in each group. PTD was continuously administered by gavage twice daily for 3 d at a dose of 27.3 mg/kg. Blood was collected from the rat femoral artery 2 h after the last administration, centrifuged at 6000 rpm at 4 • C for 6 min, and 100 µL of plasma was separated and frozen at −20 • C until analysis.
For the pharmacokinetic study, 12 rats were randomly divided into two groups, with 6 rats in each group. In the oral administration group, PTD was intragastrically administered to the rats at a dose of 27.3 mg/kg. For intravenous administration, PTD was injected at a dose of 9.1 mg/kg via the tail vein. Blood samples (0.3 mL) were collected directly from the caudal vein using anticoagulant centrifugal tubes at 0.083, 0.167, 0.33, 0.5, 0.75, 1, 1.5, 2, 4, 6, 9, 12 and 24 h after intravenous and oral administration, respectively. The blood sample was centrifuged at 6000 rpm for 6 min, and the supernatant plasma was transferred into a new clean tube and stored at −80 • C prior to further use.

UPLC-MS/MS Conditions
The UPLC-MS/MS system consisted of an ACQUITY I-Class UPLC system and an XEVO TQ-S triple-quadrupole tandem mass spectrometer (Waters Corp., Milford, MA, USA) equipped with an ESI source. Chromatographic separation was performed using a UPLC BEN C 18 column (2.1 × 50 mm, 1.7 m; Waters Corp., Milford, MA, USA) at 40 • C. The mobile phase consisted of water containing 0.2% formic acid (B) and acetonitrile containing 0.2% formic acid (A) delivered at 0.3 mL/min as a gradient as follows: 0-0.5 min, 10% A; 0.5-3 min, 90% A; 3-4 min, 90% A; 4-4.5 min, 10% A; 4.5-5 min, 10% A. Waters XEVO TQ-S triple-quadrupole mass spectrometer equipped with an ESI source was used for the mass analysis and detection. All analytes were quantified in the MRM mode, and the optimised MS parameters are listed in Table 8. The main working parameters were set as follows: source temperature, 120 • C; desolvation temperature, 350 • C; and nebuliser gas (N 2 ), 650 L/h. The scan time for all analytes was set at 0.2 s and acquisition and processing were conducted on a Micromass Masslynx4.1. A 600 µL aliquot of each plasma sample was transferred into a 5 mL centrifuge tube, 300 µL of 2% formic acid solution and 2.4 mL of methanol was added before vortexing for 2 min. This was followed by sonication in an ultrasonic machine for 10 min, and centrifugation at 13,225× g for 10 min. The supernatant was then placed in a centrifuge tube and evaporated to dryness under a stream of nitrogen at 37 • C. The residues were dissolved in 200 µL of 50% methanol and centrifuged at 13,225× g for 10 min, and 1 µL of the solution was injected into the QE system.

Pharmacokinetic Study
A 100 µL aliquot of each plasma sample was transferred into a 1.5 mL centrifuge tube, and 50 µL of 2% formic acid solution was added before vortexing for 30 s. Subsequently, the internal standard (IS) (50 µL of 20 ng/mL puerarin) and 400 µL of methanol were added and vortexed for 1 min. For subsequent treatment methods were the same as mentioned in the above section (4.7.1); finally, 1 µL of the solution was injected into the UPLC-MS/MS system.

Preparation of Standards and Quality Control (QC) Samples
Stock solutions of PTD, PTB and IS were separately weighed and dissolved in methanol to obtain final concentrations of 1.002, 1.004 and 0.495 mg/mL, respectively. Four sets of working standards were obtained by mixing and diluting the stock solutions in methanol. An appropriate amount of IS was dissolved in 50% methanol and diluted to 20 ng/mL. A mixture of working standard solutions (100 µL) and IS solutions (50 µL) was added to blank rat plasma (100 µL) to prepare the calibration standard solutions at final concentrations of 1-30,000, 1-1000 and 4 ng/mL for PTD, PTB and IS, respectively. QC plasma samples containing PTD (5.01, 250.5, 15,030 ng/mL) and PTB (2.51, 20.08, 251 ng/mL) were prepared in the same manner.

Method Validation
The analytical method was fully validated for specificity, precision, accuracy, extraction recovery, matrix effect, linearity, lower limit of quantification (LLOQ) and stability, according to the nonclinical drug pharmacokinetic study technical guidelines (China Food and Drug Administration 2014) and the Bioanalytical Method Validation Guideline (Chinese Pharmacopoeia 2015, Vol. 4).

Specificity
The specificity of the method was assessed by comparing the chromatograms of blank plasma from two different rats and blank plasma spiked with PTD, PTB and the IS. This test determines where there is potential interference from endogenous substances with the analytes and the IS.

Linearity and LLOQ
The linearity of the calibration curves for PTD and PTB was evaluated using nine appropriate concentrations and by plotting the area ratio of analytes to IS (y) versus the nominal concentration of analytes (x) using 1/x 2 weighted least-squares linear regression. The LLOQ was the concentration of the lowest calibration standard with a signal-to-noise ratio that exceeded 10, along with an accuracy and precision of ±20%.

Precision and Accuracy
Intra-and inter-day precision and accuracy were assessed by analyzing QC samples at low, medium and high concentrations, on three successive days. Precision was expressed as RSD.

Extraction Recovery and Matrix Effect
The extraction recovery of PTD and PTB at low, medium and high QC levels was detected by comparing the peak areas of analytes obtained from rat plasma samples with the average peak areas of the same analytes spiked before and after extraction. Matrix effects were determined by comparing the peak areas of the analytes in the spiked postextraction samples with those of the same analytes dissolved in 50% methanol at the same concentrations. Similarly, the extraction recovery and matrix effect of IS were determined. All QC sample analyses were performed in five replicates.

Stability
The stability of analytes in rat plasma was assessed using QC samples at low, medium and high concentrations (n = 5 for each concentration level) under three conditions: (1) at room temperature for 24 h; (2) refrigerated at 4 • C for 24 h; and (3) three freeze-thaw cycles.

Data Analysis
Compound discoverer 3.2 was used for the analysis of metabolites of PTD; WinNonlin was used for the analysis of pharmacokinetic parameters, and Graphpad 8.2 was used for the illustrations. Data are expressed as mean ± SD. Statistical analysis was performed using the Statistical Product and Service Solutions software package (SPSS 22.0, SPSS Inc., Chicago, IL, USA). The results were analyzed using one-way ANOVA and bioavailability (F) was calculated according to the equation reported in He et al. [33].

Conclusions
PTD is a compound found in the extract of C. decapetala, which was isolated for the first time. Its structure was identified as protosappanin B-3-O-β-D-glucoside. In this study, we found that PTD could downregulate the secretion of TNF-α, IL-1β and IL-6, demonstrating its efficacy as an anti-inflammatory agent, and its efficacy is similar to that of PTB in anti-inflammatory potency. In the metabolic study, four metabolites were identified, and the primary degradative metabolic pathways in vivo involved desaturation, oxidation, methylation, alkylation, dehydration, degradation and desugarization. Pharmacokinetic studies showed that PTD was quickly absorbed and transformed rapidly in vivo, mainly into PTB, and its bioavailability in vivo was 0.65%. This was the first report of the pharmacological activity and pharmacokinetics of PTD, which will be of great significance for its development and utilization as a therapeutic agent in the future.

Data Availability Statement:
The authors declare that all data supporting the findings of this study are available within the article.