Qualitative and quantitative analyses of chemical constituents in vitro and in vivo and systematic evaluation of the pharmacological effects of Tibetan medicine Zhixue Zhentong capsules

Introduction: Zhixue Zhentong capsules (ZXZTCs) are a Tibetan medicine preparation solely composed of Lamiophlomis rotata (Benth.) Kudo. L. rotata is the only species of the genus Laniophlomis (family Lamiaceae) that has medicinal constituents derived from the grass or root and rhizome. L. rotata is one of the most extensively used folk medicines by Tibetan, Mongolian, Naxi, and other ethnic groups in China and has been listed as a first-class endangered Tibetan medicine. The biological effects of the plant include hemostasis, analgesia, and the removal of blood stasis and swelling. Purpose: This study aimed to profile the overall metabolites of ZXZTCs and those entering the blood. Moreover, the contents of six metabolites were measured and the hemostatic, analgesic, and anti-inflammatory effects of ZXZTCs were explored. Methods: Ultra-performance liquid chromatography–tandem quadrupole time-of-flight high-resolution mass spectrometry (UPLC-Q-TOF-MS) was employed for qualitative analysis of the metabolites of ZXZTCs and those entering the blood. Six metabolites of ZXZTCs were quantitatively determined via high-performance liquid chromatography The hemostatic, analgesic, and anti-inflammatory effects of ZXZTCs were evaluated in various animal models. Results: A total of 36 metabolites of ZXZTCs were identified, including 13 iridoid glycosides, 9 flavonoids, 9 phenylethanol glycosides, 4 phenylpropanoids, and 1 other metabolite. Overall, 11 metabolites of ZXZTCs entered the blood of normal rats. Quantitative analysis of the six main metabolites, shanzhiside methyl ester, chlorogenic acid, 8-O-acetyl shanzhiside methyl ester, forsythin B, luteoloside, and verbascoside, was extensively performed. ZXZTCs exerted hemostatic effects by reducing platelet aggregation and thrombosis and shortening bleeding time. Additionally, ZXZTCs clearly had an analgesic effect, as observed through the prolongation of the latency of writhing, reduction in writhing, and increase in the pain threshold of experimental rats. Furthermore, significant anti-inflammatory effects of ZXZTCs were observed, including a reduction in capillary permeability, the inhibition of foot swelling, and a reduction in the proliferation of granulation tissue. Conclusion: Speculative identification of the overall metabolites of ZXZTCs and those entering the blood can provide a foundation for determining its biologically active constituents. The established method is simple and reproducible and can help improve the quality control level of ZXZTCs as a medicinal product. Evaluating the hemostatic, analgesic, and anti-inflammatory activities of ZXZTCs can help reveal its mechanism.

representative iridoid metabolites that exert analgesic, anti-inflammatory, and hemostatic effects (Zhu et al., 2012). Forsythin B and verbascoside are phenylethanol glycosides that display analgesic activity (Bai et al., 2015). Chlorogenic acid is a phenylpropanoid that acts to reduce the production of inflammatory factors and free radicals to suppress the inflammatory response Zhou et al., 2021).
The exertion of a curative effect is dependent on the constituents, highlighting the importance of characterizing the metabolite components of medicinal plants. Luteoloside, shanzhiside methyl ester, 8-O-acetyl shanzhiside methyl ester, forsythin B, verbascoside, and chlorogenic acid are key potential metabolites of choice for determining activity indicators in L. rotata. Wang. et al. (2018) identified 51 metabolites in L. rotata via ultra-performance liquid chromatography (UPLC)-quadrupole time-of-flight (Q-TOF)-high-resolution mass spectrometry (MS). Wu et al. (2016) used LC-Q-TOF/MS technology to identify 42 metabolites in L. rotata. In similar studies, La et al. (2015) identified 48 metabolites and Zan et al. (2018) uncovered 30 metabolites in L. rotata via LC-TOF/MS analysis. Although the metabolites of L. rotata have been extensively investigated, no studies have focused on the metabolic profiles of the medicinal preparations of L. rotata, such as ZXZTCs.
Based on the results of in vitro studies, we analyzed the metabolites of ZXZTCs that enter the blood for the first time in this study, providing a reference for follow-up investigations of the effective constituents and their therapeutic mechanisms of action. LC-MS technology is an effective tool for qualitative analysis. In view of its simple, rapid, and accurate characteristics, UPLC-Q-TOF-MS was used in this study. Using high-resolution UPLC and MS, critical information, such as retention time, can be calculated quickly and accurately, and the molecular mass and fragment ions can be detected and collected precisely (Wu et al., 2016).
The contents of various metabolites (luteoloside, shanzhiside methyl ester, 8-O-acetyl shanzhiside methyl ester, forsythin B, verbascoside, chlorogenic acid, sesamoside, rutin, quercetin, and ergosterol glycoside) of Duyiwei capsules and Lamiophlomis otate have been determined via high-performance liquid chromatography (HPLC) in earlier reports (Zhong et al., 2014;Gao et al., 2015;Zhou et al., 2021;Guo et al., 2017). However, to our knowledge, no studies have focused on determining the content of the Tibetan medicinal preparation of L. rotata, ZXZTCs. To comprehensively and accurately reflect the quality of ZXZTCs as a medicinal preparation, the contents of six active metabolites were determined via HPLC for the first time, including two iridoid glycosides (shanzhiside methyl ester and 8-O-acetyl shanzhiside methyl ester), two phenylethanol glycosides (forsythin B and verbascoside), one flavone (luteoloside), and one phenylpropanoid (chlorogenic acid). The data obtained should aid in addressing the gaps in existing research, further improve the quality control standard of ZXZTCs, and provide a scientific basis for the optimal utilization of the preparation.
Several research groups, including our own, have conducted pharmacological research on ZXZTCs. Li et al (1992a); Li et al. (1992b) verified the hemostatic effect of ZXZTCs based on a series of experiments on rat, mouse, and rabbit models, demonstrating that ZXZTCs reduce tail bleeding time in mice, increase the number of platelets in rats, and shorten coagulation time. The total effective rate of ZXZTCs in the clinical treatment of patients with thrombocytopenia is 87.8%, which is indicative of a better therapeutic effect (Han et al., 1995). In another study, ZXZTCs combined with remifentanil were used to treat 55 patients with postpartum pain after cesarean section. Notably, the levels of serum cortisol, HBV, FIB, LBV, plasma viscosity, serum IL-6, CRP, TNF-α, 5-HT, and PRL were decreased to a significant extent and the total effective rate was higher in the experimental group after treatment compared than in the control group. In recent experiments by Li et al. (2021), the degree of reduction in lower limb motor nerve block and analgesic effect were more obvious in the experimental group, supporting a curative effect of ZXZTCs. Moreover, ZXZTCs were used to effectively treat endometrial hyperplasia, primarily inducing a reduction in endometrial thickness and the alleviation of uterine tissue edema in rats (Fu et al., 2019). ZXZTCs protect the ovary and uterus by increasing the release of ovarian estrogen and improving uterine lesions, promoting significant reductions in the uterine coefficient, transparency, and disorder of myometrial smooth muscle cells and interstitial hyperplasia of ovarian tissue, and marked increases in PROG concentration in serum and VEGF protein expression in uterine tissue (Xiong et al., 2020a). Additionally, ZXZTCs show efficacy in inhibiting the contraction of isolated rat uterine smooth muscle primarily through reducing the average muscle tension (Xiong et al., 2020b). Further recent studies have demonstrated that ZXZTCs regulate functional uterine bleeding, reduce uterine bleeding time, improve the hormone level, and promote the residual excretion of uterine villi and decidual cells. During this process, estradiol and progesterone content is significantly increased (Huang et al., 2020). The present study comprehensively explored the hemostatic, analgesic, anti-inflammatory, and swelling effects of ZXZTCs and revealed a 'dose-effect' relationship to confirm and expand its existing and potential pharmacological activities, with the aim of providing guidance for rational clinical applications of this medicinal plant preparation.
Animals were maintained in the Science and Technology 2.4 UPLC-Q-TOF-MS/HPLC test conditions and sample preparation 2.4.1 Equipment parameters and sample preparation of UPLC-Q-TOF-MS Equipment parameters: For chromatography, the column was an ACQUITY BEH C18 column (2.1 mm × 150 mm, 1.7 μm), the mobile phases were acetonitrile solution (B)-0.1% formic acid aqueous solution (A) at a column temperature of 30°C, the flow rate was 0.4 mL min -1 , and the injection volume was 2 μL. The gradient elution was 95%-20%, B at 0-20 min and 20%-5% B at 20-30 min. The mass spectrum conditions were as follows: electrospray ionization in the positive and negative modes, nitrogen flow rate of 600 L h -1 , desolvation temperature of 350°C, capillary voltage of 3.0 kV, cone voltage of 30 kV, collision energy of 15-45 eV, ion source temperature of 120°C, and scanning range of m/z 50-1500.
Preparation of test and standard solutions: A volume of 10 mL of methanol was added to 0.1 g of ZXZTCs (four samples in parallel). Following ultrasonic treatment for 30 min, the liquid was passed through a 0.22 μm microporous filter and stored at 4°C as the test solution. Appropriate amounts of chlorogenic acid, forsythin B, shanzhiside methyl ester, 8-O acetyl shanzhiside methyl ester, verbascoside, loganin, and luteoloside were accurately weighed and placed in a 10 mL brown volumetric flask, dissolved in methanol, and diluted to the required volume to prepare a mixed standard solution with concentrations of 83, 104.75, 79.5, 115.13, 103.88, 75.25, and 101.38 μg/mL, respectively. The solution (2 mL) was passed through a 0.22 μm microporous filter and stored at 4°C as a mixed standard solution for testing.
Blood serum sample preparation: Twenty rats (10 male and 10 female) were divided into four batches (two batches of females and two batches of males, with five animals in each batch). Five rats were divided into two groups, specifically, ZXZTCs (four rats) and blank control (one rat). The ZXZTCs group was gavaged with the maximum dose (10.50 g/kg, 200 times the daily dose of 0.0525 g/kg for clinical adults) of ZXZTCs solution. For the maximum dose, ZXZTCs were dissolved in normal saline until the solution could be successfully extracted and administered to rats by gavage. At slightly higher concentrations, the solution could not be successfully gavaged. The blank control group was gavaged with the same dose of normal saline (20 mL/kg). At 30, 60, 90, and 120 min after gavage in the ZXZTCs group, blood was collected from the abdominal aorta of one animal at each time point. Blood was collected from the abdominal aorta directly after the gavage of normal saline in the blank control group. Subsequent batches were treated as above. After blood stasis for 10 min, the upper liquid layer was centrifuged at 3,500 r/min for 10 min. Blood sera were separated from both the blank control and ZXZTCs groups and stored at −80°C. During the test, 50 μL of the drug-containing serum solution at each of the four time points was precisely mixed Frontiers in Pharmacology frontiersin.org 04 to obtain 200 μL of drug-containing serum mixture, which was placed in a 2.0 mL EP tube. Next, 1,000 μL of acetonitrile precipitate was added, vortexed for 2 min for mixing, and centrifuged at 13,000 r/min and 4°C for 10 min. The supernatant was pipetted into a 2 mL EP tube, blow-dried at 37°C with a nitrogen blower, reconstituted with 200 μL of methanol, vortexed for 2 min, sonicated for 10 min, thoroughly mixed, and re-centrifuged at 13,000 r/min (4°C for 10 min). The collected supernatant was passed through a 0.22 μm microporous filter. An aliquot of blank serum (200 μL) was precisely pipetted into a 2.0 mL EP tube and processed in a similar manner as above.
Data processing and analysis: After UPLC-Q-TOF-MS analysis of the test solution, standard solution, blank serum, and drug-containing serum, the relevant mass spectrometric data were analyzed with MassLynx V4.2 software. The relative molecular mass of the compound was determined according to the quasi-molecular ion peak detected in the mass spectrum corresponding to the base peak ion flow chromatographic peak. Total metabolites and metabolites entered into the blood of ZXZTCs were identified by further comparison and speculation of cleavage fragment ion information from firstand second-stage mass spectra combined with data from the literature and standard solution experiments.
Preparation of test and standard solutions: ZXZTCs powder (0.5 g) was accurately weighed with a balance, placed in a 50 mL conical flask, and mixed with 25 mL of 70% methanol solution. The mixture was weighed, subjected to ultrasonic treatment (power, 200 W; frequency, 40 kHz) for 30 min, allowed to cool, and reweighed with a balance. After the addition of 70% methanol to reduce weight loss, the mixture was shaken well. The filtrate was collected and subsequently passed through a 0.45 μm microporous filter membrane. The supernatant obtained was stored at 4°C as the test solution. Appropriate amounts of shanzhiside methyl ester, chlorogenic acid, 8-O acetyl shanzhiside methyl ester, forsythin B, luteoloside, and verbascoside chemicals were accurately weighed, placed in a 10 mL brown volumetric flask and dissolved in 10 mL methanol. After dilution to the required volume, the mixture was used to prepare a mixed standard solution with concentrations of 48.2, 64.9, 57.3, 172.8, 111, and 132 μg/mL, respectively, of the above six constituents, and stored as a mixed standard solution for testing.
For the evaluation of anti-inflammatory activity, four animal experiments were conducted. In experiment 1, the ear swelling model induced by xylene in mice was established (Gu et al., 2016) (1 h after the final administration, 50 µL of xylene was applied evenly on the inside and outside of the right ear contour of each mouse). Mice were divided into model control (normal saline), prednisone acetate control (Feng et al., 2013) (0.01 g kg -1 d -1 ), low-dose ZXZTCs (0.2625 g kg -1 d -1 ), mediumdose ZXZTCs (0.5250 g kg -1 d -1 ), and high-dose ZXZTCs (1.0500 g kg -1 d -1 ) groups. The dosage volume was 20 mL/kg, with 10 animals per group (five male and five female) fed in separate cages. In experiment 2, the foot swelling model induced by carrageenan in rats was established (Yan et al., 2004;Wang. et al., 2018) (1 h after the final administration, 0.1 mL of 1% carrageenan was injected into the foot pad of the right hind leg of rats in each group to induce inflammation). Rats were divided into model control (normal saline), prednisone acetate control (Lin et al., 2010) (0.005 g kg -1 d -1 ), low-dose ZXZTCs (0.1313 g kg -1 d -1 ), medium-dose ZXZTCs (0.2625 g kg -1 d -1 ), and high-dose ZXZTCs (0.5250 g kg -1 d -1 ) groups. The dosage volume was 10 mL/kg, with 10 animals per group (all males) maintained in separate cages. In experiment 3, the model of peritoneal permeability induced by acetic acid in mice was established (Feng et al., 2013;Ouyang et al., 2014) (1 h after the final administration, the mice in each group were injected with 1% Evans blue normal saline solution through the tail vein according to standard (0.1 mL/10 g) body weight, followed by immediate intraperitoneal injection of 0.2 mL of 0.6% acetic acid). Mice were divided into model control (normal saline), chlorphenamine maleate tablets control  (0.002 g kg -1 d -1 ), low-dose ZXZTCs (0.2625 g kg -1 d -1 ), mediumdose ZXZTCs (0.5250 g kg -1 d -1 ), and high-dose ZXZTCs (1.0500 g kg -1 d -1 ) groups. The dosage volume was 20 mL/kg, with 10 animals per group (all males) maintained in a single cage. In experiment 4, the cotton ball-induced granuloma model in rats was established (Yang et al., 2009;Efimova et al., 2010;Zelenin et al., 2011;Wang et al., 2016) (rats were anesthetised via intraperitoneal injection of 20% urethane solution (0.6 mL/100 g), a 0.5 cm incision made in the abdomen, and a 20 mg sterile cotton ball implanted under the skin of the right groin, following which the incision was sutured and disinfected). Rats were divided into model control (normal saline), prednisone acetate control  (0.005 g kg -1 d -1 ), low-dose ZXZTCs (0.1313 g kg -1 d -1 ), mediumdose ZXZTCs (0.2625 g kg -1 d -1 ), and high-dose ZXZTCs (0.5250 g kg -1 d -1 ) groups. The dosage volume was 10 mL/kg, with 10 animals per group (all males) maintained in separate cages. Treatments were administered by gavage once a day for 7 days.
In mice experiments, the low, medium, and high doses of ZXZTCs were used at 5, 10, and 20 times of the adult daily dose (0.0525 g/kg) and the positive control dose was 10 times that of the adult daily dose.
In rat experiments, the low, medium, and high doses of ZXZTCs were used 2.5, 5, and 10 times of the adult daily dose (0.0525 g/kg) and the positive control dose was 5 times that of the adult daily dose.

Anesthetisation and handling of animals
After samples were collected or the relevant indicators were tested, animals were anesthetized via intraperitoneal injection of 20% urethane solution (0.6 mL/100 g) and sacrificed through excessive blood loss from the abdominal aorta.

Statistical analysis
Data are expressed as mean ± standard deviation ( x± s). The independent sample t-test or one-way ANOVA provided by SPSS 17.0 for Windows software was employed to assess the significance of mean differences between two or more groups of data. Levene's test for homogeneity of variance was performed. At p values > 0.05, variance was homogeneous. p values were    Indicates the metabolites have been reported previously in L. rotata.

Qualitative analysis of metabolites in ZXZTCs via UPLC-Q-TOF-MS
ZXZTCs were analyzed by UPLC-Q-TOF-MS in both positive and negative ion modes. According to the standard substance atlas (Figures 1C, E) and published metabolite data for L. rotata (Yi et al., 1992;Afifi et al., 1993;Li et al., 1999;Tripetch et al., 2002;Zhang, 2008b;Xue et al., 2009;Wu et al., 2013;Pan, 2015;Wu et al., 2016;Pan et al., 2018;Wang et al., 2018;Zan et al., 2018), a total of 36 metabolites from 5 categories were detected in the base peak ion (BPI) chromatogram of ZXZTCs ( Figure 1A). By comparing the retention times and relative molecular masses of these metabolites with the standard substance ( Figure 1) and references, we deduced the presence of 13 iridoid glycosides, nine flavonoids, nine phenylethanol glycosides, four phenylpropanoids, and one other metabolite (Table 1).  (Pan, 2015), the identity of the metabolite was deduced as phloyoside I with a molecular formula of C 17 H 26 O 13 . The mass spectrum, structural formula, and fragmentation process are shown in Figure 2A.

Representative metabolites of iridoid glycosides
Lamiophlomiol C: The retention time in the UPLC system was 3.67 min. In the ESI negative ion mode, a quasi-molecular ion peak at m/z 257 [M-H]-was obtained. Additionally, a molecular ion peak at m/ z 515 [2M-H]appeared in the spectrum. In the ESI positive ion mode, a molecular ion peak at m/z 539 [2M + Na] + was detected. The molecular mass of the metabolite was confirmed as 258. Based on the present data in combination with earlier literature (Wu et al., 2016), the identity of the metabolite was deduced as lamiophlomiol C with a molecular formula of C 11 H 14 O 7 . The mass spectrum, structural formula (Yi et al., 1992), and fragmentation process are shown in Figure 2B.
Shanzhiside methyl ester: The retention time in the UPLC system was 4.33 min. In the ESI negative ion mode, a quasimolecular ion peak at m/z 405 [M-H]was obtained. Additionally, a fragment ion of a molecular ion peak at m/z 451 [M-H + HCOOH]appeared in the spectrum. In the ESI positive ion mode, molecular ion peaks at m/z 813 [2M + H]+and 429 [M + Na]+were detected, along with fragment ions at m/z 209 [M + H-C 6 H 10 O 5 -2H 2 O] + . The molecular mass of the metabolite was confirmed as 406. Combined with earlier literature (La et al., 2015;Pan, 2015;Wu et al., 2016) and data from comparisons with the standard substance, our findings inferred that the metabolite was shanzhiside methyl ester with a molecular formula of C 17 H 26 O 11 . The mass spectrum, structural formula (Pan, 2015) and fragmentation process are shown in Figure 2C. The mass spectrum of the standard substance is shown in Figure 1.
Loganin: The retention time in the UPLC system was 7.31 min. In the ESI negative ion mode, fragment ions of a molecular ion peak at m/z 435 [M-H + HCOOH]were obtained. In the ESI positive ion mode, a molecular ion peak at m/z 413 [M + Na] + was detected. Moreover, fragment ions at m/z 211 [M + H-C 6 H 10 O 5 -H 2 O] + appeared. The molecular mass of the metabolite was determined as 390. Combined with earlier literature (Pan, 2015) and data from a comparison with the standard substance, our findings inferred that the metabolite was loganin with a molecular formula of C 17 H 26 O 10 . The mass spectrum, structural formula (Pan, 2015), and fragmentation process are shown in Figure 2D. The mass spectrum of the standard substance is shown in Figure 1.  (Pan, 2015) and data from a comparison with the standard substance, our findings inferred that the metabolite was 8-O-acetyl shanzhiside methyl ester with a molecular formula of C 19 H 28 O 12 . The mass spectrum, structural formula (Pan, 2015), and fragmentation process are shown in Figure 2E. The mass spectrum of the standard substance is shown in Figure 1.

Representative metabolites of flavonoids
Hyperoside: The retention time in the UPLC system was 7.72 min. In the ESI negative ion mode, a quasi-molecular ion peak at m/z 463 [M-H]was obtained. In the ESI positive ion mode, a quasi-molecular ion peak at m/z 465 [M + H] + was obtained. The molecular mass of the metabolite was confirmed as 464. Based on the present data in combination with earlier literature (Wu et al., 2016), the identity of the metabolite was deduced as hyperoside with a molecular formula of C 21 H 20 O 12 . The mass spectrum, structural formula, and fragmentation process are shown in Figure 3A.
Luteoloside: The retention time in the UPLC system was 9.66 min. In the ESI negative ion mode, a quasi-molecular ion peak at m/z 447 [M-H]and molecular ion peak at m/z 895 [2M-H]were obtained. In the ESI positive ion mode, a quasi-molecular ion peak at m/z 449 [M + H] + was detected, along with fragment ions of a molecular ion peak at m/z 287 [M + H-C 6 H 10 O 5 ] + . The molecular mass of the metabolite was confirmed as 448. Combined with earlier literature (Pan, 2015;Zan et al., 2018) and data from comparisons with the standard substance, the identity of the metabolite was inferred as luteoloside with a molecular formula of C 21 H 20 O 11 . The mass spectrum, structural formula (Pan, 2015), and fragmentation process are shown in Figure 3B. The mass spectrum of the standard substance is shown in Figure 1.
Luteolin: The retention time in the UPLC system was 14.57 min. In the ESI negative ion mode, a quasi-molecular ion peak at m/z 285 [M-H]was obtained. In the ESI positive ion mode, a quasimolecular ion peak at m/z 287 [M + H] + appeared. The molecular mass of the metabolite was confirmed as 286. Based on the present data in combination with earlier literature (Zan et al., 2018), the identity of the metabolite was inferred as luteolin with a molecular formula of C 15 H 10 O 6 . The mass spectrum, structural formula, and fragmentation process are shown in Figure 3C. Apigenin: The retention time in the UPLC system was 18.10 min. In the ESI negative ion mode, a quasi-molecular ion peak at m/z 269 [M-H]was obtained. The molecular mass of the metabolite was confirmed as 270. Based on the present data in combination with earlier literature (Zan et al., 2018), the identity of the metabolite was inferred as apigenin with a molecular formula of C 15 H 10 O 5 . The mass spectrum, structural formula, and fragmentation process are shown in Figure 3D.

Representative metabolites of phenylethanol glycosides
Forsythin B: The retention time in the UPLC system was 9.97 min. In the ESI negative ion mode, a quasi-molecular ion at peak m/z 755 [M-H]was obtained. In the ESI positive ion mode, a molecular ion peak at m/z 779 [M + Na] + was obtained. The molecular mass of the metabolite was confirmed as 756. Combined with earlier literature (Pan, 2015) and data from a comparison with the standard substance, the identity of the metabolite was deduced as forsythin B with a molecular formula of C 34 H 44 O 19 . The mass spectrum, structural formula (Pan, 2015), and fragmentation process are shown in Figure 4A. The mass spectrum of the standard substance is shown in Figure 1.
Verbascoside: The retention time in the UPLC system was 10.69 min. In the ESI negative ion mode, a quasi-molecular ion peak at m/z 623 [M-H]was obtained. In the ESI positive ion mode, a molecular ion peak at m/z 647 [M + Na] + was detected. The molecular mass of the metabolite was confirmed as 624. Combined with earlier literature (Pan, 2015;Zan et al., 2018) and data from comparisons with the standard substance, the identity of the metabolite was deduced as verbascoside with a molecular formula Frontiers in Pharmacology frontiersin.org of C 29 H 36 O 15 . The mass spectrum, structural formula (Pan, 2015), and fragmentation process are shown in Figure 4B. The mass spectrum of the standard substance is shown in Figure 1. Lamiophlomioside A: The retention time in the UPLC system was 13.64 min. In the ESI negative ion mode, a quasi-molecular ion peak at m/z 783 [M-H]was obtained, in addition to a molecular ion peak at m/z 829 [M-H + HCOOH] + . In the ESI positive ion mode, molecular ion peaks at m/z 802 [M + NH 4 ] + and 807 [M + Na] + were detected. The molecular mass of the metabolite was confirmed as 784. In combination with earlier literature (Wu et al., 2016), we inferred that the metabolite was lamiophnomioside A with a molecular formula of C 36 H 48 O 19 . The mass spectrum, structural formula, and fragmentation process are shown in Figure 4C.
Leucosceptoside B: The retention time in the UPLC system was 13.97 min. In the ESI negative ion mode, a quasi-molecular ion peak at m/z 783 [M-H]was obtained. In the ESI positive ion mode, a molecular ion peak at m/z 807 [M + Na] + was obtained. The molecular mass of the metabolite was confirmed as 784. In combination with earlier literature (Pan, 2015), our findings inferred that the metabolite was leucosceptoside B with a molecular formula of C 36 H 48 O 19 . The mass spectrum, structural formula, and fragmentation process are shown in Figure 4D.
Rossicaside C: The retention time in the UPLC system was 19.79 min. In the ESI negative ion mode, a quasi-molecular ion peak at m/z 619 [M-H]was obtained. In the ESI positive ion mode, a quasi-molecular ion peak at m/z 621 [M + H] + was obtained. In addition, a molecular ion peak at m/z 643 [M + Na] + and another fragment ion at m/z 603 [M + H-H 2 O] + appeared in the spectrum. The molecular mass of the metabolite was confirmed as 620. In combination with earlier literature (Wang et al., 2018), our findings inferred that the metabolite was rossicaside C with a molecular formula of C 30 H 36 O 14 . The mass spectrum, structural formula (Wu et al., 2013), and fragmentation process are shown in Figure 4E.

Representative metabolites of phenylpropanoids
Markhamioside A: The retention time in the UPLC system was 3.79 min. In the ESI negative ion mode, a quasi-molecular ion peak at m/z 593 [M-H]was obtained. In the ESI positive ion mode, molecular ion peaks at m/z 612 [M + NH 4 ] + and 649 [M + H+3H 2 O] + were obtained. The molecular mass of the metabolite was confirmed as 594. Based on the present data in combination with earlier literature (Pan et al., 2018), the identity of the metabolite was deduced as markhamioside A with a molecular formula of C 25 H 38 O 16 . The mass spectrum, structural formula (Xue et al., 2009), and fragmentation process are shown in Figure 5A.
Chlorogenic acid: The retention time in the UPLC system was 4.00 min. In the ESI negative ion mode, a quasi-molecular ion peak at m/ z 353 [M-H]and a molecular ion peak at m/z 707 [2M-H]appeared in the spectrum. In the ESI positive ion mode, a quasi-molecular ion peak at m/z 355 [M + H] + was obtained. Fragment ions of m/z 283 [M + H-4H 2 O] + were additionally detected. The molecular mass of the metabolite was confirmed as 354. Combined with earlier literature (Zan et al., 2018) and data from a comparison with the standard substance, our findings inferred that the metabolite was chlorogenic acid with a molecular formula of C 16 H 18 O 9 . The mass spectrum, structural formula, and fragmentation process are presented in Figure 5B. The mass spectrum of the standard substance is shown in Figure 1.
6-β-D-Apioufarnosyl cistanoside C: The retention time in the UPLC system was 11.90 min. In the ESI negative ion mode, a quasimolecular ion peak at m/z 769 [M-H]was obtained. In the ESI positive ion mode, a molecular ion peak at m/z 793 [M + Na] + appeared. The molecular mass of the metabolite was confirmed as 770. In combination with earlier literature (Pan, 2015), our findings inferred that the metabolite was 6-β-D-apioufarnosyl cistanoside C with a molecular formula of   (Pan, 2015), and fragmentation process are shown in Figure 5C. Alyssonoside: The retention time in the UPLC system was 13.13 min. In the ESI negative ion mode, a quasi-molecular ion peak at m/z 637 [M-H]was obtained. In the ESI positive ion mode, a molecular ion peak at m/z 661 [M + Na] + was obtained. The molecular mass of the metabolite was confirmed as 638. In combination with earlier literature (Pan, 2015), our findings inferred that the metabolite was alyssonoside with a molecular formula of C 30 H 38 O 15 . The mass spectrum, structural formula (Pan, 2015), and fragmentation process are shown in Figure 5D.

Other metabolites
6β-n-butoxy-7,8-dehydropenstemonoside: The retention time in the UPLC system was 17.88 min. In the ESI negative ion mode, a quasi-molecular ion peak at m/z 443 [M-H]was obtained. In the ESI positive ion mode, a quasi-molecular ion peak at m/z 445 [M + H] + and a molecular ion peak at m/z 467 [M + Na] + were detected. The molecular mass of the metabolite was confirmed as 444. In combination with earlier literature (La et al., 2015), we inferred that the metabolite was 6β-n-butoxy-7,8-dehydropentemonoside with a molecular formula of C 21 H 32 O 10 . The mass spectrum is shown in Figure 1D. Frontiers in Pharmacology frontiersin.org 3.2 Qualitative UPLC-Q-TOF-MS analysis of ZXZTCs metabolites that entered the blood A total of 11 metabolites from three categories were detected in the base peak ion (BPI) chromatogram of the drug-containing serum of ZXZTCs ( Figure 1B). Based on the retention times and relative molecular masses of these metabolites, data from the literature (Luo et al., 2003;La et al., 2015;Pan, 2015;Wu et al., 2016;Zan et al., 2018), and comparisons with the standard substance, we identified five iridoid glycosides, five flavonoids, and one phenylethanol glycoside metabolites that entered the blood. The details are presented in Table 2. The metabolites detected included iridoid glycosides (shanzhiside, shanzhiside methyl ester, 8-O-acetyl shanzhiside methyl ester, 7deoxyloganic acid, and notohamosin B), flavonoids (hyperoside, luteoloside, eugenyl-β-D-glucopyranoside, 7methoxyapigenin, and apigenin-7-O-β-D-glucoside), and a phenylethanol glycoside (betonyoside A). The analytical features of the five metabolites of ZXZTCs detected in blood are listed below.
Eugenyl-β-D-glucopyranoside: The retention time in the UPLC system was 5.51 min. In the ESI negative ion mode, a quasimolecular ion peak at m/z 325 [M-H]and molecular ion peak at m/z 651 [2M-H]were detected near molecular ions 324, 322, 650, and 648. In the ESI positive ion mode, a quasi-molecular ion peak at m/z 327 [M + H] + appeared, along with a molecular ion peak at m/z 349 [M + Na] + . The molecular mass of the metabolite was confirmed as 326. Based on the present findings in combination with earlier literature (La et al., 2015), the metabolite was inferred as eugenyl-β-D-glucopyranoside with a molecular formula of C 16 H 22 O 7 . The mass spectrum, structural formula, and fragmentation process are shown in Figure 6A.
7-Deoxyloganic acid: The retention time in the UPLC system was 8.47 min. In the ESI negative ion mode, a quasi-molecular ion peak at m/z 359 [M-H]was obtained. The molecular mass of the metabolite was confirmed as 360. Based on the present findings in combination with earlier literature (Wu et al., 2016), the metabolite was inferred as 7-deoxyloganic acid with a molecular formula of C 16 H 24 O 9 . The mass spectrum, structural formula, and fragmentation process are shown in Figure 6B.
Shanzhiside: The retention time in the UPLC system was 16.53 min. In the ESI negative ion mode, a quasi-molecular ion peak at m/z 391 [M-H]was obtained. The molecular mass of the metabolite was confirmed as 392. Based on the present findings in combination with earlier literature (Wu et al., 2016), the metabolite was inferred as shanzhiside with a molecular formula of C 16 H 24 O 11 . The mass spectrum, structural formula, and fragmentation process are shown in Figure 6C.
Apigenin-7-β-D-glucoside: The retention time in the UPLC system was 18.00 min. In the ESI negative ion mode, a quasimolecular ion peak m/z 431 [M-H]was obtained. In the ESI positive ion mode, fragment ions of a molecular ion peak at m/z 387 [M + H-CO-H 2 O] + were obtained. The molecular mass of the metabolite was confirmed as 432. Based on the present findings in combination with earlier literature (Pan, 2015), the metabolite was inferred as apigenin-7-O-β-D-glucoside with a molecular formula of C 21 H 20 O 10 . The mass spectrum, structural formula, and fragmentation process are shown in Figure 6D.  , the metabolite was inferred as notohamosin B with a molecular formula of C 29 H 46 O 4 . The mass spectrum, structural formula (Luo et al., 2003), and fragmentation process are shown in Figure 6E.

HPLC determination of the six metabolites of ZXZTCs
The batch numbers of the six batches of ZXZTCs tested in this experiment were 180902, 200801, 200802, 190101, 201101, and 190201.

Methodological investigation
We prepared 0.625, 1.25, 2.5, and 5 mL of mixed standard solution for HPLC as described in the 'Preparation of the test and standard solutions' section. Solutions were placed in a 10 mL volumetric flask and diluted to the required volume with methanol to obtain a series of concentrations of mixed standard solution. Taking the mixed standard solution of each series of concentrations and the mixed standard solution for HPLC as described in the 'Preparation of test and standard solutions' section, 10 μL aliquots were injected into the liquid chromatography system, and peak areas were measured. The concentration was taken as the abscissa and the peak area was taken as the ordinate to obtain the standard curve equation for each component (Kongkiatpaiboon et al., 2017). The peak area of each standard substance was linearly related to the concentration (Supplementary Table S1). A 10 μL aliquot of the mixed standard solution was precisely prepared for HPLC, as described in the 'Preparation of test and standard solutions' section, and was injected six times continuously within 1 day. The peak area of each component was recorded (Alfredo et al., 2019). The results showed that the relative standard deviation of each standard substance was <3%, indicating good intraday precision of the instrument, as shown in Supplementary Table S1. Approximately 0.5 g of ZXZTCs powder, a total of 6 parts, were weighed accurately, and 10 μL of test solution of each ZXZTCs was accurately sucked according to the preparation method described in the 'Preparation of test and standard solutions' section for HPLC. Solutions were injected into the liquid chromatography system, the peak areas were measured, and RSD values were calculated. Notably, the RSD of each standard substance was <3%, indicating good repeatability of the method, as shown in Supplementary Table S1. Approximately 0.5 g of ZXZTCs powder, 10 μL of test solution of ZXZTCs, was accurately sucked according to the preparation method described in the 'Preparation of test and standard solutions' section for HPLC. Solutions were injected into the liquid chromatography system at 0, 2, 4, 8, 12, and 24 h, the peak areas were recorded, and RSD values were calculated. The RSD of each standard substance was <3%, indicating that the test solution was stable within the 24 h experimental period, as shown in Supplementary Table S1.

Content determination
Approximately 0.5 g of powder (capsule content) from each of six batches (batch numbers 180902, 200801, 200802, 190101, 201101, and 190201) was accurately weighed. Three parallel experiments were performed for each batch. Samples were accurately weighed and prepared according to the method for HPLC described in the 'Preparation of test and standard solutions' section. A precise 10 μL aliquot of the test solution was injected and the chromatography peak area of each component was recorded. The contents were measured, and average values were calculated (Table 3). The metabolite contents from high to low levels were as follows: forsythin B, 8-O-acetyl shanzhiside methyl ester, luteoloside, verbascoside, shanzhiside methyl ester, and chlorogenic acid. The content chromatograms for each batch are shown in Figure 1F.

Effects of ZXZTCs on the bleeding and coagulation times of severed tails in mice
Details are presented in Table 4. Compared with the blank control group, the tail bleeding times of mice in each drug treatment group were very significantly shortened (p < 0.01), whereas no significant changes were observed in coagulation time (p > 0.05). Compared with the carbazochrome control group, the bleeding and coagulation times of mice in each drug treatment group showed no significant changes (p > 0.05). Our findings indicate that ZXZTCs stop bleeding by shortening the bleeding time. The action strength (from strong to weak) of the preparation was in the following order: medium dose, high dose, and low dose.

Effects of ZXZTCs on blood agglutination parameters and plasma recalcification time in rats
Details are presented in Table 4. Compared with the blank control group, the thrombin time of rats in the low-dose group of ZXZTCs was significantly prolonged (p < 0.05); the thrombin times of animals in the Yunnan Baiyao capsules control group and high-dose ZXZTCs group were prolonged to a more significant extent (p < 0.01); the plasma recalcification time of rats in each drug group was very markedly decreased (p < 0.01). Compared with the Yunnan Baiyao capsules control group, the plasma recalcification time of rats in the low-dose ZXZTCs group was very significantly prolonged (p < 0.01); the thrombin time of rats in the medium-dose ZXZTCs group was decreased to a highly significant extent (p < 0.01); the plasma recalcification time of rats in the high-dose ZXZTCs group was significantly prolonged (p < 0.05). Based on the results, we speculate that ZXZTCs has a two-way regulatory effect, promoting either coagulation or anti-coagulation. On the one hand, all three doses of ZXZTCs shortened plasma recalcification time in rats, which is indicative of a potential procoagulant effect. On the other hand, low and high doses of ZXZTCs prolonged the thrombin time of rats, signifying a potential anticoagulant effect.

Effects of ZXZTCs on platelets and aggregation rates in rats
Details are presented in Table 5. Compared with the blank control group, the numbers of leukocytes, lymphocytes, and monocytes in the low-dose ZXZTCs group were significantly increased (p < 0.05); the platelet aggregation rates of rats in low-Frontiers in Pharmacology frontiersin.org   and medium-dose ZXZTCs groups was significantly decreased (p < 0.05). Our results show that ZXZTCs reduce platelet aggregation but have no significant effect on the number of platelets. The action strength of the preparation (from strong to weak) was in the following order: low dose, medium dose, and high dose.

Effects of ZXZTCs on the model of thrombocytopenia induced by cytarabine in mice
Details are presented in Table 6. Compared with the blank control group, the number of platelets in the model control group was significantly increased (p < 0.05), concomitant with a highly significant increase in hematocrit levels (p < 0.01). Compared with the model control group, the number of erythrocytes and hematocrit levels in the prednisone acetate control group were significantly increased (p < 0.05); the average erythrocyte hemoglobin content was significantly decreased (p < 0.05), the number of monocytes was significantly increased (p < 0.05), and the percentage of monocytes was highly significantly increased (p < 0.01) in the low-dose ZXZTCs group; the number of monocytes in the medium-dose ZXZTCs group was significantly increased (p < 0.05), while average erythrocyte volume and erythrocyte hemoglobin content were highly significantly decreased (p < 0.01); the number of leukocytes and percentage of monocytes in the high-dose ZXZTCs group were significantly increased (p < 0.05), along with a highly significant increase in the number of monocytes (p < 0.01); the organ coefficients of spleen and thymus in each drug group showed no significant changes (p > 0.05). These findings showed that there were no noteworthy effects of ZXZTCs on the cytarabine-induced thrombocytopenia model in mice.

Effects of ZXZTCs on tail thrombosis induced by carrageenan in mice
Details are presented in Table 7. Compared with the model control group, at 24, 48, and 72 h, the tail thrombosis rates of the aspirin entericcoated tablets control group and medium-dose ZXZTCs group were significantly decreased (p < 0.05). Compared with the aspirin entericcoated tablets control group, the tail thrombosis rates of mice in lowand high-dose ZXZTCs groups were significantly higher at 24, 48, and 72 h (p < 0.05); at all three time points, there were no significant differences in the tail thrombosis rates of mice in the medium-dose ZXZTCs group (p > 0.05), suggesting similar strengths of action of the two groups (the medium-dose group of ZXZTCs and the aspirin enteric-coated tablet control group). This finding indicates that ZXZTCs can reduce tail thrombosis in mice. The effect strength of the medium dose was the greatest, equivalent to that of aspirin entericcoated tablets, followed by low and high doses of ZXZTCs.

Effects of ZXZTCs on the chandler thrombus model in vitro of rats
Details are presented in Table 7. Compared with the blank control group, the length and dry weight of thrombi in the aspirin enteric-coated tablets control group were significantly decreased (p < 0.05), along with a highly significant decrease in the wet weight of thrombus (p < 0.01); thrombus length and thrombus dry and wet weights in the high-dose ZXZTCs group were significantly decreased (p < 0.05). Compared with the aspirin enteric-coated tablets control group, thrombus length of rats in the low-dose ZXZTCs group was significantly higher (p < 0.05); moreover, the length, dry weight, and Note: mice tail thrombosis induced by carrageenan: compared with the model control group, *p < 0.05, **p < 0.01; Compared with aspirin enteric-coated tablets control group, # p < 0.05, ## p < 0.01. In vitro rat Chandler thrombus model: Compared with the model control group, *p < 0.05, **p < 0.01; compared with the aspirin enteric-coated tablets control group, # p < 0.05, ## p < 0.01.
Frontiers in Pharmacology frontiersin.org 22 Rotundine tablets control group    Note: writhing response in mice induced by acetic acid: compared with the model control group, *p < 0.05, **p < 0.01; compared with the aspirin enteric-coated tablets control group, # p < 0.05, ## p < 0.01. Foot pain in mice induced by a hot plate: compared with pain threshold before administration, # p < 0.05, ## p < 0.01; compared with the model control group, *p < 0.05, **p < 0.01; compared with the aspirin enteric-coated tablets control group, $ p < 0.05, $$ p < 0.01. Foot tenderness in rats induced by mechanical stimuli: compared with pain threshold before administration, # p < 0.05, ## p < 0.01; compared with the model control group, *p < 0.05, ** p < 0.01; compared with the rotundine tablets control group, $ p < 0.05, $$ p < 0.01. Tail flick response in rats induced by photothermal stimulation: compared with pain threshold before administration, # p <0.05, ## p < 0.01; compared with the model control group, *p < 0.05, **p < 0.01; compared with the rotundine tablets control group, $ p < 0.05, $$ p < 0.01. Dysmenorrhea model in mice induced by oxytocin: compared with the model control group, *p < 0.05, **p < 0.01; compared with the ibuprofen capsules control group, # p < 0.05, ## p < 0.01. wet weight of thrombus of rats in the medium-dose ZXZTCs group were significantly increased (p < 0.05); no marked differences were observed in thrombus length, thrombus dry weight, and thrombus wet weight in the high-dose ZXZTCs group of (p > 0.05), suggesting similar action strengths of the two treatments (the high-dose ZXZTCs group and the aspirin enteric-coated tablets control group). Our results indicate that ZXZTCs can effectively reduce thrombosis in rats. The effect strength of the high dose was the greatest, equivalent to that of aspirin enteric-coated tablets, followed by low and medium doses.

Effects of ZXZTCs on writhing response induced by acetic acid in mice
Details are presented in Table 8. Compared with the model control group, the latency of writhing was significantly prolonged (p < 0.05), and writhing was significantly reduced in the low-dose ZXZTCs group (p < 0.05); prolongation of the latency of writhing was highly significant (p < 0.01) in the aspirin enteric-coated tablets control group and medium-and high-dose ZXZTCs groups, along with very markedly reduced writhing (p < 0.01). Compared with the aspirin enteric-coated tablets control group, the latency of writhing in mice in the low-dose ZXZTCs group was very significantly shortened (p < 0.01); writhing of mice in the low-, medium-, and high-dose ZXZTCs groups was highly significantly increased (p < 0.01). Overall, ZXZTCs could prolong the latency of writhing, reduce writhing, and produce analgesic effects in mice. The effect strength of the high dose was the greatest, followed by medium and low doses. The effect strength of the medium and high doses of ZXZTCs in terms of prolonging latency of writhing in mice was equivalent to that of aspirin enteric-coated tablets.

Effects of ZXZTCs on foot pain induced by a hot plate in mice
Details are presented in Table 8. The pain threshold of aspirin enteric-coated tablets control group was significantly increased 30 min (p < 0.05) after drug administration; the pain thresholds of mice in the medium-and high-dose ZXZTCs groups were significantly increased at 120 min (p < 0.05).
Relative to the model control group, the increase in pain threshold of the aspirin enteric-coated tablets control group was highly significant at 30 min (p < 0.01), and the percentage of pain threshold was highly significantly increased at 30 min (p < 0.01); the pain threshold of mice in the medium-dose ZXZTCs group was significantly increased at 120 min (p < 0.05) and was significantly increased at 30 and 120 min (p < 0.05) in the high-dose ZXZTCs group. Compared with the control aspirin enteric-coated tablets group, the percentage of pain threshold increase in the low-and medium-dose ZXZTCs groups was significantly lower at 30 min (p < 0.05); moreover, no significant changes were observed in the percentage of pain threshold increase at other time points and at all time points for the other groups (p > 0.05).
Our results showed that ZXZTCs can increase the threshold of foot pain in mice caused by a hot plate and subsequently achieve the effect of analgesia. The effect strength of the high dose was the greatest, followed by medium and low doses. The effect strength of high-dose ZXZTCs in terms of percentage increase in pain threshold was equivalent to that of aspirin enteric-coated tablets.

Effects of ZXZTCs on foot tenderness induced by mechanical stimulation in rats
Details are presented in Table 8. The pain threshold of rats in the rotundine tablets control group was significantly increased 30 and 90-120 min after drug administration (p < 0.05) and highly significantly increased 60 min (p < 0.01) after drug administration; the increase in the pain threshold of rats in the medium-dose ZXZTCs group was highly significant during the 60-90 min period (p < 0.01) and the pain threshold of rats in the high-dose ZXZTCs group was significantly increased during the 30-60 min period (p < 0.05).
After drug administration, compared with the model control group, the pain threshold of the rotundine tablets control group was significantly greater at 30 and 90 min (p < 0.05) and highly significantly greater at 60 min (p < 0.01), along with the percentage of pain threshold increase at 60 min (p < 0.05); the pain threshold of rats in the medium-dose ZXZTCs group was significantly increased during the 60-90 min period (p < 0.05) and the pain threshold of rats in the high-dose ZXZTCs group was significantly increased at 60 min (p < 0.05). Compared with the rotundine tablets control group, the percentage of pain threshold increase in rats in low-, medium-, and high-dose ZXZTCs groups was not markedly different over 30-120 min (p > 0.05), suggesting that the action strength of all three doses of ZXZTCs was equivalent to that of the rotundine tablets.
Our results showed that ZXZTCs can increase the pain threshold of foot tenderness induced by mechanical stimulation and subsequently achieve the effect of analgesia. The effect strength of the high dose was the greatest, followed by medium and low doses. Moreover, the effect strength of the ZXZTCs preparation in increasing pain threshold parameters was equivalent to that of the rotundine tablets.

Effects of ZXZTCs on tail flick reaction induced by photothermal stimulation in rats
Details are presented in Table 8. The pain threshold of the rotundine tablets control group was very significantly increased 30-120 min after drug administration (p < 0.01); the increase in pain threshold of rats in the high-dose ZXZTCs group was significant at 30 min (p < 0.05) and very significantly increased at 60-90 min (p < 0.01). Compared with the model control group, the increase in pain threshold and percentage of pain threshold in the rotundine tablets control group was highly significant during the 30-120 min period (p < 0.01); the pain threshold of rats in the medium-dose ZXZTCs group was significantly increased at 60 min (p < 0.05); the pain threshold of rats in the high-dose ZXZTCs group was significantly increased at 30 min (p < 0.05) and highly significantly increased at 60 min (p < 0.01), and the increase in the percentage of pain threshold was significant at 30 and 120 min (p < 0.05) and highly significant at 60 min (p < 0.01). Compared with the rotundine tablets control group, the percentage of pain threshold increase of rats in low-, medium-, and high-dose ZXZTCs groups was significantly lower at 30 min (p < 0.05) and was highly significantly lower 60-120 min (p < 0.01).
Our results showed that ZXZTCs could increase the pain threshold of tail flick induced by photothermal stimulation in rats and subsequently achieve the effect of analgesia. The strength of action of the high dose was the greatest, followed by medium and low doses.  Table 8. Compared with the model control group, the latency of writhing in the ibuprofen capsules control group and low-, medium-and high-dose ZXZTCs groups was highly significantly increased (p < 0.01), whereas writhing in the ibuprofen capsule control group and medium and high-dose ZXZTCs groups decreased to a highly significant extent (p < 0.01). Compared with the ibuprofen capsule control group, the latency of writhing of mice in the low-, medium-, and high-dose ZXZTCs groups was highly significantly shortened (p < 0.01); the writhing of mice was highly significantly increased (p < 0.01) in the low-and medium-dose ZXZTCs groups and significantly increased in the high-dose ZXZTCs group (p < 0.05).
Our results showed that ZXZTCs can prolong the latency of writhing, reduce the writhing, and subsequently achieve an analgesic effect in mice. The strength of action was the greatest for the high dose, followed by medium and low doses.
3.6 Anti-inflammatory and anti-swelling effects of ZXZTCs 3.6.1 Effects of ZXZTCs on ear swelling induced by xylene in mice Details are presented in Table 9. Compared with the model control group, ear swelling of mice in the prednisone acetate tablets control group was significantly reduced (p < 0.05). Our results indicated no significant effects of ZXZTCs on ear swelling induced by xylene in mice, although a trend of inhibition was observed. The dose-effect relationship (from strong to weak) was in the following order: low dose, high dose, and medium dose.

Effects of ZXZTCs on paw swelling induced by carrageenan in rats
Details are presented in Table 9. Compared with the model control group, paw swelling of rats in the prednisone acetate tablets control group was significantly reduced at 1 h (p < 0.05), and swelling at 2, 4, and 6 h was highly significantly reduced (p < 0.01). The degree of swelling was significantly reduced at 2 h (p < 0.05) and highly significantly reduced (p < 0.01) at 4 and 6 h in the low-dose ZXZTCs group. Paw swelling of rats in the highdose ZXZTCs group was significantly reduced at 4 and 6 h (p < 0.05). Our results indicate that ZXZTCs effectively reduce the degree of paw swelling induced by carrageenan in rats and exert clear anti-inflammatory effects. The dose-effect relationship (from strong to weak) was in the following order: low dose, high dose, and medium dose.

Effects of ZXZTCs on increase in peritoneal permeability induced by acetic acid in mice
Details are presented in Table 9. Compared with the model control group, absorbance values of the chlorphenamine control group and the medium-and high-dose ZXZTCs groups were highly significantly decreased (p < 0.01). Our findings indicate that ZXZTCs effectively inhibit the increase in capillary permeability induced by acetic acid in mice. The dose-effect relationship (from strong to weak) was in the following order: high dose, medium dose, and low dose.

Effects of ZXZTCs on granuloma induced by cotton pellets in rats
Details are presented in Table 9. Compared with the model control group, granulomas in the prednisone acetate tablets control group and medium-and high-dose ZXZTCs groups were significantly reduced (p < 0.05). Our results showed that ZXZTCs could attenuate granulation tissue hyperplasia induced by cotton pellets in rats, specifically inducing inhibitory effects on the pathological changes of granulation hyperplasia in the late stage of inflammation. The dose-effect relationship (from strong to weak) was in the following order: high dose, medium dose, and low dose.

Discussion
The only medicinal material composition of ZXZTCs is L. rotata. In this study, UPLC-Q-TOF-MS technology was used to analyze 36 metabolites in ZXZTCs, all of which have been reported for L. rotata previously. Among the documented studies, La et al. (2015) identified 48 metabolites, Wang et al. (2018) identified 51 metabolites, Wu et al. (2016) identified 42 metabolites, and Zan et al. (2018) identified 30 metabolites in L. rotata. The common metabolite uncovered by different research groups and the present study was shanzhiside methyl ester. The comprehensive data indicated that shanzhiside methyl ester content is high in L. rotata and would not be lost when it is processed into ZXZTCs. A total of 11 metabolites of ZXZTCs were detected in the blood circulation of normal rats in our experiments. Among these, the main metabolites of iridoid glycosides in the blood were shanzhiside, shanzhiside methyl ester, 8-O-acetyl shanzhiside methyl ester, 7-deoxyloganic acid, and notohamosin B. Studies have shown that iridoid glycosides in L. rotata have good hemostatic (Feng, 2011) and analgesic (Yan, 2013) effects. Feng (2011) and Li et al. (2005) compared the hemostatic effects of various components via tail cutting and the examination of capillary coagulation after intragastric administration into mice. The results revealed markedly shortened thrombin times and significantly increased fibrinogen content in the presence of iridoid glycosides. Du et al. (2014) further demonstrated that iridoid glycosides could significantly shorten bleeding time. Examination of the clinical effects by Qu (2018) revealed that iridoid glycosides had strong analgesic activity and were effective constituents of analgesic prescriptions. Among them, shanzhiside methyl ester and 8-O-acetyl shanzhiside methyl ester were representative effective analgesic constituents of iridoid glycosides and their main action sites were in the spinal cord (Xue et al., 2009;Zhu, 2013). He (2011) showed that 8-O-acetyl shanzhiside methyl ester had good procoagulant activity, which could significantly increase plasma fibrin content and inhibit fibrinolytic activity in experimental mice. In addition to iridoid glycosides, L. rotata also contains flavonoids. The flavonoids entering into the blood circulation primarily included eugenylβ-D-glucopyranoside, 7-methoxyapigenin, hyperoside, luteoloside, and apigenin-7-O-β-D-glucoside. The analgesic effect of flavonoids was not as significant as that of iridoid glycosides (Zhu et al., 2012;Zheng et al., 2015). However, Zhou (2009) revealed a potential dose-dependent association of the analgesic effect of L. rotata with total flavonoid content, indicating that the pharmacological value of flavonoids in this plant requires further research. Another previous study (Meng et al., 2009) showed that the flavonoid and iridoid glycoside metabolites of L. rotata exert a certain proliferative effect on bone marrow granulocytic progenitors. Additionally, phenylethanoid glycosides are among the main metabolite constituents of L. rotata. Among the metabolites detected in the blood, betonyoside A belongs to phenylethanoid glycosides, which have a wide range of pharmacological properties, including antibacterial, anti-inflammatory, and immune regulation activities (Jing et al., 2006). Although the specific activity of betonyoside A in L. rotata has not been reported, the above effects were clearly observed. Its efficacy may be attributable to combined effects with other active constituents. In summary, metabolites of ZXZTCs entering the blood may serve as potential active constituents of L. rotata, which have therapeutic effects on various hemorrhages, trauma (such as fracture and soft tissue injury), postoperative analgesia, congestion headache, and inflammation conditions (Zhao, 2004). In this study, an effective method for determining the contents of the six major metabolites (shanzhiside methyl ester, chlorogenic acid, 8-O acetyl shanzhiside methyl ester, forsythin B, luteoloside, and verbascoside) of ZXZTCs was established and provided a more reliable basis for the quality control and regulation of medicinal ZXZTCs. Compared with earlier literature, the average contents of the corresponding metabolites in ZXZTCs (shanzhiside methyl ester, 1.81 mg/g; chlorogenic acid, 1.43 mg/g; 8-O-acetyl shanzhiside methyl ester, 3.39 mg/g; forsythin B, 5.46 mg/g; luteoside, 2.30 mg/g; and verbascoside 1.90 mg/g) were between the lowest and highest values reported for L. rotata (slightly closer to the lowest reported contents). For example, Zhong et al. (2014) reported contents of 1.369-11.265 mg/g for shanzhiside methyl ester, 0.000-8.487 mg/g for chlorogenic acid, 0.000-14.898 mg/g for 8-O-acetyl shanzhiside methyl ester, 2.484-23.140 mg/g for forsythin B, 3.544-28.143 mg/g for verbascoside, and 0.000-10.757 mg/g for luteoside in L. rotata; Yi et al. (2016) documented a shanzhiside methyl ester content of 5.42-5.69 mg/g and Jin et al. (2021) reported a content of Frontiers in Pharmacology frontiersin.org 0.50-0.58 mg/g in L. rotata. He et al. (2013) determined the contents of forsythin B, verbascoside, and luteoside in Lamiophlomis rotate as 0.430-6.782 mg/g, 0.661-8.600 mg/g, and 1.320-6.877 mg/g, respectively. These differences in the metabolite contents of L. rotata are mainly attributable to differences in origin, variety, and medicinal parts across studies. Therefore, it is more reliable to use the same L. rotata medicinal materials under the same preparation conditions for comparative analysis, which will provide a direction for future research on the quality of ZXZTCs. Compared with Duyiwei capsules, the corresponding metabolite contents of ZXZTCs are relatively low. Gao et al. (2015) determined the average contents of shanzhiside methyl ester, 8-O-acetyl shanzhiside methyl ester, luteoside, and verbascoside in three batches of Duyiwei soft capsules as 3.51 mg/g, 10.40 mg/g, 3.41 mg/g, and 3.60 mg/g, respectively. Overall, the quality control level of ZXZTCs needs to be improved, particularly its processing technology, on the premise of ensuring consistency of content and efficacy of the constituents.
In the present pharmacodynamic experiments, ZXZTCs have a clear hemostatic effect in different animal model states and diseases, which is manifested by shortened bleeding time, reduced platelet aggregation, and thrombosis. The mechanisms underlying the shortening of bleeding time may be related to the effects on the functions of platelets and capillaries, such as increasing the number of platelets, promoting the release of procoagulant substances from platelets, constricting local blood vessels, and decreasing capillary permeability (Lu et al., 2016). In addition, potential pharmacological activity was observed, specifically in terms of promoting the blood circulation effect. We speculate that ZXZTCs contain two main constituent types that exert procoagulant or anticoagulant activity. Shortened plasma recalcification time and prolonged thrombin time are representative of potential procoagulant and anticoagulant activities, respectively. However, the final coagulation effect of ZXZCTs was not examined in this study, and the mechanisms and metabolites that affect the coagulation system need to be further explored. We further hypothesize that thrombin time is primarily related to the coagulation, anticoagulation, and fibrinolytic system functions, and the plasma recalcification time predominantly determines the effect of drugs on the internal coagulation system. The effects of drugs on any one of the coagulation factors may influence the time of blood coagulation. In view of the theory of traditional Chinese medicine of 'pass without pain', the chemical constituents with anticoagulant activity may primarily be flavonoids, such as luteoloside, which exert analgesic effects by promoting blood circulation. The current study also demonstrated that the analgesic effect of ZXZTCs is mainly manifested in the response to several physical, chemical, photothermal, and other stimuli, whereas the anti-inflammatory effect is exerted primarily following exposure to acute and chronic inflammatory physical and chemical stimuli.

Conclusion
Based on previous literature on data mining, UPLC-Q-TOF-MS was used to analyze 36 metabolites in ZXZTCs, including 13 iridoid glycosides, nine flavonoids, nine phenylethanol glycosides, four phenylpropanoids, and one other metabolite. A total of 11 main metabolites of ZXZTCs were detected in the blood of normal rats, including five iridoid glycosides, five flavonoids, and one phenylethanol glycoside. Quantitative analysis of the six main metabolites (shanzhiside methyl ester, chlorogenic acid, 8-O-acetyl shanzhiside methyl ester, forsythin B, luteoloside, and verbascoside) in ZXZTCs was further conducted using HPLC. The method established in this study was simple, accurate, convenient, and reproducible, laying a foundation for improving the quality standard of ZXZTCs. Furthermore, our results on the hemostasis, analgesia, anti-inflammation, and anti-swelling effects of ZXZTCs can provide a valuable reference for its rational clinical application.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Ethics statement
The animal study was reviewed and approved by Experimental Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine.