1. Introduction
Abnormal uterine bleeding (AUB) is a common gynecological disease, usually manifesting in excessive menstruation, prolonged menstruation, and irregular vaginal bleeding, which seriously affects the work efficiency and quality of life of patients. Modern medicine holds that AUB is caused by neuroendocrine disorders regulated by the hypothalamic–pituitary–ovary axis (HPOA) [
1]. At present, estrogen and progesterone drugs are often used in clinical treatment. Although hormone therapy has certain benefits, it has side effects such as weight gain, gastrointestinal reactions and increased liver burden, and AUB may recur after stopping taking the drugs [
2]. Due to its complicated etiology and pathogenesis, and the limitations of existing therapeutic drugs, it is very important to seek a better treatment scheme to improve curative effects and reduce recurrence. In recent years, many clinical studies have shown that traditional Chinese medicine (TCM) has a good effect on AUB and a low recurrence rate, and is easily tolerated by patients [
3].
Baoyin Jian (BYJ) is a classic prescription for the treatment of hemorrhagic disorders in gynecology. It originated in
Jingyue Quanshu, written by Zhang Jingyue in the Ming Dynasty, and consists of Rehmanniae Radix; Rehmanniae Radix Praeparata; Paeoniae Radix Alba; Phellodendri Chinensis Cortex; Scutellariae Radix; Dipsaci Radix; Dioscoreae Rhizoma; and Glycyrrhizae Radlx et Rhizoma. It has the effects of nourishing yin deficiency, nourishing blood, and stopping bleeding [
4]. In modern clinical practice, BYJ is mainly used to treat abnormal uterine bleeding, threatened abortion, menorrhagia, and other gynecological bleeding-related diseases [
5]. While most of the studies on BYJ focused on clinical efficacy evaluation and identification of chemical components
in vitro, there is a lack of research on quality markers (Q-Markers) related to clinical efficacy, which makes it difficult to investigate the basic characteristics of BYJ in the treatment of AUB and limits the assurance of its clinical efficacy, which also hinders the clinical promotion of BYJ [
6].
The quality of TCM is the basis to ensure its clinical efficacy and safety. However, due to the complex components of TCM prescription, the existing quality control standards for Chinese medicines are not sufficient to objectively evaluate their quality, and it is difficult to guarantee the safety and efficacy of their clinical use [
7]. The Q-marker concept, combined with the characteristics of TCM and its use in clinical practice, links efficacy, effective constituents, and quality, and improves the deficiencies of the existing quality standards of TCM. Chinmedomics refers to the discovery of biomarkers for TCM syndromes through metabolomics technology, the application of the TCM serum pharmacochemical method to analyze active ingredients in the body under the effective condition of TCM prescriptions, the correlation between these and biomarkers, and the selection of drug ingredients highly associated with biomarker trajectory changes. Thus, the theoretical system of biological evaluation of TCM efficacy, pharmacodynamic material basis, and mechanism of action was elucidated. Chinmedomics is an effective way to investigate the effectiveness, traceability, and compatibility of prescriptions of Q-markers. In recent years, Q-markers were successfully discovered in various single herbs and prescriptions, such as American ginseng, Yinchenhao Decoction, ZhibaiDihuang Pill and Wenxin Formula, etc., by employing Chinmedomics [
8,
9,
10,
11]. The successful application of Chinmedomics helps to comprehensively and accurately identify the Q-markers of TCM and provides a powerful means of quality control of TCM.
We aimed to elucidate the potential efficacy and mechanism of action of BYJ in the treatment of AUB and to identify its Q-markers, which can help improve the quality standards of Chinese medicine. In the present study, based on an AUB rat model, histopathological and biochemical indices were used to evaluate the efficacy of BYJ in the treatment of AUB. Then, under the guidance of Chinmedomics, we evaluated the changes to the overall metabolic profile of AUB rats after treatment with BYJ and investigated the active components closely related to the efficacy of BYJ based on the “Five Principles” of Q-markers to identify the potential Q-markers of BYJ for the treatment of AUB. This study provides technical guidance for the development and utilization of the famous classical formulae and the further development of quality control standards.
3. Discussion
Numerous women experience physical, social, and emotional distress due to AUB. Previous studies have indicated that endometrial vascular damage and uterine tissue inflammation are the main clinical features of AUB [
12]. Hormonal imbalance is also a major pathological mechanism for uterine bleeding [
13,
14]. Research has demonstrated that an increase in pro-angiogenic factors and a decrease in anti-angiogenic factors can impair vascular maturation, leading to more fragile blood vessels and consequently AUB [
15]. Therefore, promoting endometrial and vascular repair and regulating immune responses may help to suppress the occurrence of AUB. BYJ is a classic prescription for treating AUB, with the effects of nourishing
yin, clearing heat, and hemostasis. However, the specific mechanism action of BYJ is largely unknown, and its Q-markers are not yet clear, which has hindered the development of new drugs based on BYJ.
In this study, the AUB model was simulated by incomplete medical abortion, reflecting the characteristics of metrorrhagia in TCM syndromes. After incomplete medication abortion, the physiological function of HPOA in rats was disordered, and the gonadotropin-releasing hormone (GnRH) secreted by hypothalamus was reduced, which in turn led to the decrease of FSH and LH secreted by pituitary and the decrease of Pro and E2 secreted by ovary; additionally, the coagulation system of the body was activated, PT, APTT and TT were significantly prolonged, and FIB was significantly decreased. By studying the changes to pathological tissue, uterine bleeding, coagulation function, and hormone levels in rats after drug administration, it was confirmed that BYJ can obviously increase the hormone levels of FSH, LH, E2 and Pro, thus adjusting the physiological function of the HPOA axis, adjusting the disorder of coagulation system caused by incomplete medical abortion, obviously shortening PT, APTT and TT, and increasing FIB level, thus reducing the uterine bleeding volume in rats and showing a good hemostatic effect.
Metabolomic techniques can better reveal the pathological characteristics of diseases, help to discover the targets of drugs, and explain their overall mechanisms of action. Here, metabolomics techniques were used to identify biomarkers of AUB, and the metabolic pathways and pathogenesis of AUB, as well as the hemostatic mechanism of BYJ, were preliminarily elucidated. In this study, we identified 32 potential biomarkers of AUB; after treatment with BYJ, 16 abnormal metabolic products were significantly reversed, mainly involving lipid metabolism, amino acid metabolism, and carbohydrate metabolism.
Arachidonic acid (AA) metabolism is a key pathway in the development of AUB, and it plays an important role in various physiological and pathological processes such as coagulation balance and inflammatory response. AA is an unsaturated fatty acid that plays an important role in the structure and function of cell membranes in the human body. It can be converted into various metabolites such as prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETEs) through the action of cyclooxygenase and lipoxygenase [
16]. TXA
2 and PGI
2 are vasoactive substances, which are endogenous metabolites closely related to the formation mechanism of AUB and regulate hemodynamics in the endothelium. TXA
2 has a strong vasoconstrictive effect and promotes platelet aggregation and release [
17], while PGI
2 is synthesized by the endothelial cells of blood vessels and has vasodilatory and platelet-inhibiting effects. Under normal conditions, TXA
2 and PGI
2 are in a dynamic balance, jointly regulating vasoconstriction and dilation and participating in the regulation of blood clotting [
18]. In patients with AUB, the release of AA and the secretion of TXA
2 are reduced, while the content of PGI
2 increases significantly, resulting in an imbalance of the TXA
2/PGI
2 ratio, leading to vasodilation, local blood flow increase, and increased local bleeding. On the other hand, AA is converted into leukotrienes (including LTA
4 and LTB
4) under the action of 5-lipoxygenase. Leukotrienes are strong inflammatory factors that can participate in smooth muscle contraction and mediate inflammatory reactions. In addition, AA metabolized through the CYP pathway produces hydroxyeicosatetraenoic acids (HETEs), including 19-HETE and 20-HETE. These C20 fatty acids have important regulatory effects on blood rheology, vascular elasticity, leukocyte function, and platelet activation. They can promote vasoconstriction and control or alter the vascular delivery system [
19,
20]. In our study, it was found that the content of PGI
2 and LTB
4 in the AUB model group of rats was significantly increased, while the content of AA, TXA
2, LTA
4, 19-HETE, and 20-HETE was significantly decreased. This indicates that AA metabolism is disrupted, leading to an increase in PG synthesis and a decrease in TX and HETE synthesis, which inhibit platelet aggregation, uterine smooth muscle contraction, and vasoconstriction, resulting in increased bleeding and prolonged bleeding time. On the other hand, the increase in LTB
4 leads to increased inflammation. Compared with the model group, the BYJ-dosed group could significantly modulate the disordered status of arachidonic acid, TXA
2, PGI
2, LTB
4 and 20-HETE in rats with AUB, regulate platelet aggregation and release, promote the contraction of blood vessels and uterine smooth muscle, and thus exert the effect of blood clotting.
In our study, we found that the underlying mechanism of AUB is closely related to the synthesis of steroid hormones, including the metabolism products of common steroids such as pregnenolone, progesterone, 17α-hydroxyprogesterone, and dihydrocortisol. Steroids are a class of biologically active substances derived from cholesterol, which is catalyzed by cholesterol side-chain cleavage enzyme (CYP11A1) to produce pregnenolone, a precursor to most steroid hormones [
21]. Pregnenolone is then converted to progesterone by the action of 3beta-hydroxy-Delta5-steroid dehydrogenase, which has an inhibitory effect on uterine smooth muscle contraction [
22]. Progesterone is further converted to 17α-hydroxyprogesterone by 17α-monooxygenase. In the rat model of AUB, the normal physiological function of the HPOA is disrupted after incomplete abortion, leading to reduced secretion of uterine endometrial steroid hormones and lysosomal enzymes, and decreased uterine smooth muscle contraction, resulting in uterine bleeding. Our study found that pregnenolone, progesterone, 17α-hydroxyprogesterone, cortisol, dihydrocortisol, and testosterone were disturbed, and the concentrations of these markers were significantly lower in the model group compared to the control group. BYJ might therefore increase the secretion of uterine endometrial steroid hormones and lysosomal enzymes and promote uterine smooth muscle contraction, which is beneficial for hemostasis and repair of the endometrium.
Glycerophospholipid metabolism mainly involves metabolites such as lysophosphatidic acid (LysoPA), lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamine (LysoPE), and lysophosphatidylinositol (LysoPI). These lysophospholipids are widely present in cell membranes of organisms and mainly participate in the synthesis of various phospholipids. Among them, lysophosphatidic acid (LysoPA), which is a cell-signaling molecule closely related to the occurrence and development of vascular endothelial smooth muscle-related diseases, is the simplest structure of phospholipids. LysoPA can cause a series of vascular reactions, including platelet aggregation, smooth muscle contraction, and stimulation of smooth muscle cell proliferation [
23]. Phosphatidylcholine is a phospholipid mediator with multiple biological activities and which performs important biological functions in vascular smooth muscle diseases. Phosphatidylcholine can be hydrolyzed into lysophosphatidylcholine by phospholipase A, thereby promoting platelet aggregation and stimulating smooth muscle cell proliferation [
24,
25]. In this study, these markers were disrupted in the model group, consistent with literature reports, and can be used as diagnostic markers for AUB. Moreover, BYJ significantly reversed the disorder of LysoPC (18:1(9Z)), Glycerophosphocholine, and LysoPA (0:0/16:0) in the rat model of AUB, ultimately achieving a therapeutic effect in stopping bleeding.
α-Linolenic acid is an essential fatty acid in the human body with important physiological activities. It competes with cyclooxygenase and lipoxygenase in cell membrane phospholipids, inhibits the production of TXA
2, and generates TXA
3 and PGI, thus inhibiting platelet aggregation and dilating blood vessels [
26]. In this study, the content of α-linolenic acid in the model group increased significantly, and compared with the model group, BYJ can significantly reduce the content of α-linolenic acid and restore the production of TXA
2, thus regulating the disorder of arachidonic acid, TXA
2, PGI
2, LTB
4 and 20-HETE in rats with AUB and regulating platelet aggregation and release.
In addition, tryptophan is an essential amino acid required for cell proliferation and differentiation in animals. 5-Hydroxy-L-tryptophan (5-HT) is an important messenger and neuromodulator in the body that promotes platelet aggregation, smooth muscle cell proliferation, and enhances the vasoconstrictive effect of vasoconstrictors, thereby playing a role in hemostasis. 5-Hydroxyindoleacetic acid is a metabolite of the 5-HT pathway, and studies have shown that indole and its derivatives are cytotoxic metabolites that can cause vascular dysfunction [
27,
28]. In our study, the content of 5-HT and 5-Hydroxyindoleacetic acid in the model group rats was significantly reduced. Compared with the model group, the BYJ-dosed group significantly adjusted the contents of 5-HT and 5- hydroxyindoleacetic acid, which promotes the contraction of blood vessels and uterine smooth muscle and plays a role in coagulation.
A total of 59 compounds that directly act on the body for the treatment of AUB were identified in BYJ by using the serum pharmacochemistry technology of TCM. Moreover, 13 compounds highly correlated with potential biomarkers of AUB rats were selected by analyzing the correlation between blood components and potential biomarkers. These 13 compounds were catalpol, gallic acid, rehmannioside D, chlorogenic acid, phellodendrine, paeoniflorin, liquiritin, Baicalin, liquiritin+O−H
2+C
6H
8O
6, berberine, asperosaponinVI, baicalin+H
2+C
2H
2O, and glycyrrhetinic acid. However, gallic acid and chlorogenic acid are not unique components of a single herb and do not meet the “Five Principles” of quality markers. Liquiritin+O−H
2+C
6H
8O
6 is a metabolite of liquiritin, glycyrrhetinic acid is a metabolite of glycyrrhizic acid, and baicalin+H
2+C
2H
2O is a metabolite of baicalin. Therefore, based on the “Five Principles” of Q-markers [
29], 9 of these compounds were ultimately determined to be quality markers for the treatment of AUB with BYJ, namely catalpol, rehmannioside D, phellodendrine, paeoniflorin, liquiritin, baicalin, berberine, asperosaponinVI, and glycyrrhizic acid.
Catalpol is a highly promising substance for regulating the treatment of vascular inflammatory diseases. It has strong anti-inflammatory and hemostatic functions, promotes angiogenesis, improves barrier permeability, and repairs damage to vascular endothelial cells (Zhang et al., 2020; Zhang et al., 2022). Rehmannioside D has the effect of nourishing yin and replenishing blood. Paeoniflorin has immunomodulatory and anti-inflammatory effects and can inhibit PDGF-BB-induced vascular smooth muscle cell (VSMC) proliferation through the ROS-mediated ERK1/2 and p38 signaling pathways [
30]. Berberine hydrochloride reduces lipopolysaccharide-induced endometritis in mice by suppressing activation of the NF-κB signal pathway [
31]. Berberine hydrochloride also has the potential to treat various chronic inflammatory diseases by activating NF-κB in mononuclear cells to inhibit the inflammatory response [
32]. Baicalin protects against endometritis by inhibiting the NF-κB and JNK signaling pathways and pro-inflammatory cytokines, reducing inflammatory cell infiltration, congestion, bleeding, and epithelial cell shedding. It also protects against decidual cell damage in LPS-induced miscarriage mice [
33,
34]. Saponin components can inhibit vascular smooth muscle proliferation and platelet aggregation. Asperosaponin VI has a progesterone-like effect and promotes decidualization by regulating the expression of key targets (JUN, CASP3, STAT3, SRC, and PTGS2) in decidual cells through the activation of PR expression and the Notch signaling pathway, thereby treating recurrent spontaneous abortion [
35,
36]. Liquiritin significantly activates the Keap1/Nrf2/HO-1 signaling pathway, thereby inhibiting cell oxidative stress and inflammatory reactions and effectively protecting the endometrium [
37]. Glycyrrhizic acid significantly affects rat uterine tissue contraction by regulating the biosynthesis of steroids and reducing uterine smooth muscle contraction [
38].
4. Materials and Methods
4.1. Reagents and Materials
HPLC-grade methanol and acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA, USA), and formic acid and leucine enkephalin were purchased from Sigma-Aldrich (Shanghai, China). APCI Positive/Negative Calibration (AB SCIEX, Framingham, MA, USA) and distilled water were obtained from Watsons (Guangzhou, China). Other analytical grade reagents were purchased from Beijing Institute of Chemical Reagents Co., Ltd.). ELISA kits including follicle-stimulating hormone (FSH, Lot number: H101-1-2), luteinizing hormone (LH, Lot number: H206-1-2), estrogen (E2, Lot number: H102-1-2), and progesterone (Pro, Lot number: H089-1-2) were provided by Nanjing Jiancheng Bioengineering Research Institute.
Mifepristone and misoprostol were obtained from Zizhu Pharmaceutical Co. (Peking, China). The Duanxueliu pills used were produced by Anqing Huiyinbi Pharmaceutical Co., Ltd., (Anqing, China). The following were provided by Shenwei Pharmaceutical Co. Ltd. (Shijiazhuang, China): Rehmanniae Radix (Lot number: HNJZ20191201); Rehmanniae Radix Praeparata (Lot number: HNJZ20191203); Paeoniae Radix Alba (Lot number: SCZ20201013); Phellodendri Chinensis Cortex (Lot number: HBL2011091); Scutellariae Radix (Lot number: SXSL20191101); Dipsaci Radix (Lot number: SCYY2011091); Dioscoreae Rhizoma (Lot number: 20201101); and Glycyrrhizae Radlx et Rhizoma. (Lot number: NMHJQ20191102); these were authenticated by Professor Xijun Wang in the Department of Pharmacognosy, Heilongjiang University of Chinese Medicine.
4.2. Preparation of Medicinal Drugs
The decoction method recorded in Jingyue Quanshu was used to prepare BYJ samples. According to metrological verification, the modern formulation of BYJ was as follows: Rehmanniae Radix: 7.46 g; Rehmanniae Radix Praeparata: 7.46 g; Paeoniae Radix Alba: 7.46 g; Phellodendri Chinensis Cortex: 5.60 g; Scutellariae Radix: 5.60 g; Dipsaci Radix: 5.60 g; Dioscoreae Rhizoma: 5.60 g; and Glycyrrhizae Radlx et Rhizoma: 3.73 g. The mixture of above herbs were combined with 400 mL of water and boiled to 140 mL. We collected the filtrate through 140-mesh gauze and dried it into lyophilized powder. The average powder yield was 29.57% (n = 15). The powder was stored at room temperature in a desiccator, and an appropriate amount of freeze-dried powder was dissolved in distilled water (0.6 g/mL) for later use.
The content of paeoniflorin, berberine, baicalin, asperosaponinVI, and glycyrrhizic acid and their fingerprints were used as indicators of the quality of the above extracts. The RSD values of the above components in five batches of freeze-dried powder were 1.23%, 1.80%, 1.30%, 1.26%, and 1.66% respectively (
Figure S6,
Table S5). The similarity of the fingerprints of 15 batches of BYJ is greater than 0.9 (
Figure S7,
Table S6). This indicates that the prepared samples are stable and controllable.
4.3. Animal Handling
Sprague–Dawley (SD) female rats (body weight: 250 ± 10 g) were purchased from the Experimental Animal Center of Heilongjiang University of Traditional Chinese Medicine (Harbin, China). The SD rats were raised in an environment at 23 ± 2 °C with 50 ± 5% relative humidity and a 12 h/12 h light/dark cycle with free access to food and water. All the rats were acclimated for seven days prior to experimentation.
Female rats in proestrus or estrus were mated with male rats in a separate cage at a ratio of 2:1 overnight, and the vaginal secretions of females were collected for vaginal smear examination the next morning [
39,
40,
41]. As soon as sperm and keratoepithelial cells were found, it was considered the first day of pregnancy (
Figure 10). Pregnant rats were randomly divided into three groups (
n = 6): the AUB model group, positive control group with Duanxueliu solution (DXL), and treatment group (BYJ). On day 7 of pregnancy, each rat was orally administered mifepristone (8.5 mg/kg, 8:00 am) and misoprostol (0.1 mg/kg, 6:00 pm) to establish an AUB model of early-pregnancy incomplete medical abortion. An additional six non-pregnant normal rats were used as the control group.
Concurrently, each group of rats received the corresponding drugs by gavage on the first day after modeling. For a period of 7 days, the control and model groups were given equal volumes of normal saline, the positive control group received oral administration of DXL at a dose of 0.91 mg/kg/d, and the treatment group was administered BYJ at a dose of 13.97 mg/kg/d. The experimental protocol was reviewed and approved by the Ethics Committee of Heilongjiang University of Chinese Medicine.
4.4. Pharmacodynamic Evaluation of BYJ on AUB Rats
4.4.1. Morphological and Pathological Analysis
The fresh uterine tissue was fixed in 4% paraformaldehyde for 48 h after photographing its complete morphology, and hematoxylin and eosin (HE) staining was performed to observe pathological changes in the uterine endometrium under a microscope.
4.4.2. Uterine Bleeding Volume Evaluation
After modeling, a quantitative cotton ball (60 ± 5 mg) was placed in the vagina of each rat to absorb uterine bleeding, and the cotton ball was wrapped with plastic film to prevent blood leakage. The cotton ball was removed from the vagina and placed in a sealed EP tube for refrigeration at 8:00 a.m. and 6:00 p.m. of each day. The amount of vaginal bleeding was observed and recorded, and a new cotton ball was replaced in the vagina; this procedure continued until the 14th day.
On the 7th day after administration, 0.02 mL of blood was collected from each rat’s tail vein and added to 4 mL of 5% NaOH solution (V
1), mixed and diluted for use. Each collected vaginal cotton ball from the rats was placed in a beaker and washed with an appropriate volume of 5% NaOH solution according to the amount of uterine bleeding in the rats. The above procedure was repeated 1–2 times until the cotton ball was washed to a white color, and the total volume of 5% NaOH solution (V
2) used was recorded. A total of 5 mL of the extracted solution from the washed cotton ball and 4 mL of 5% NaOH solution containing rat tail vein blood were taken and compared to the blank control with 5% NaOH solution. The absorbance value (A) was detected using a microplate reader (546 nm wavelength) [
41]. The formula for the volume of uterine bleeding was as follows:
4.4.3. Coagulation Function Test
This study used biochemical indexes of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB) to evaluate the coagulation function of AUB rats. On the 7th day after administration, the rats were terminated, and blood was collected. After standing at room temperature for 1.5 h, the serum was obtained by centrifugation at 3000 rpm for 10 min, and the above indexes were measured by fully automated hemagglutination analyzer.
4.4.4. Hormone Levels Assessment
The blood was collected and placed at room temperature for 1.5 h and centrifuged at 3000 rpm for 10 min to obtain the serum, and FSH, LH, E2, and Pro were determined according to the operating instructions of the corresponding kits.
4.5. Metabolomics Analysis
4.5.1. Sample Collection and Preparation
Urine samples were collected from rats placed in metabolic cages for 12 h overnight and the urine samples were centrifuged for 10 min (4 °C, 13,000 rpm). The supernatant was diluted 2 times with HPLC-grade ultrapure water, vortexed for 1 min, then filtered through a 0.22 μm membrane prior to AB SCIEX Triple TOFTM 5600+ analysis.
4.5.2. Metabolomics Analysis Conditions
The Waters ACQUITY UPLCTM system was used for chromatographic separation, and a HSS T3 (2.1 mm × 100 mm, 1.8 μm) chromatography column was maintained at a temperature of 40 °C and a flow rate of 0.4 mL/min. The injection sample volume was 4 μL. The mobile phase consisted of 0.1% formic acid in acetonitrile (solvent A) and 0.1% formic acid in water (solvent B), and the gradient eluting conditions were stated as follows: 0–2 min, 1–11% A; 2–2.3 min, 11–21% A; 2.3–5 min, 21–40% A; 5–8 min, 40–100% A.
The mass spectrometry analysis was performed using an AB SCIEX Triple TOFTM 5600+ analysis system with electrospray ionization (ESI). The ion spray voltage was 5500 V in positive ion mode and 4000 V in negative ion mode. The nebulizer gas was set to 55 psi, and the desolvation gas was set to 55 psi. The ion source temperature was 600 °C. The MS scan declustering potential (DP) was 100 V/−100 V, and the collision energy (CE) was 10 Ev with an accumulation time of 100 ms. The IDA scan CE was set at 35 V/−35 V with a collision energy spread (CES) of 15 and an accumulation time of 50 ms. The mass scan range was set at 50–1200 Da. The automatic calibration delivery system (CDS) used APCI and an external standard calibration method for automatic adjustment and calibration of MS and MS/MS.
4.6. Serum Pharmacochemistry Analysis
4.6.1. Serum Sample Preparation
The serum pharmacochemistry analysis was performed on samples from the model and BYJ groups. Phosphoric acid of 40 μL was added to 2 mL of serum, and the mixture was sonicated for 1 min and vortexed for 30 s. The sample was loaded onto the activated HLB solid-phase extraction (SPE) column, washed with 2 mL water, and eluted with 2 mL methanol. The methanol eluate was collected and dried with nitrogen at 37 °C, and the residue was dissolved in 60% methanol (200 μL) by sonication. After centrifugation for 10 min (4 °C, 13,000 rpm), the supernatant was collected for UPLC-MS analysis.
4.6.2. Constituents Analysis Conditions
Chromatographic analysis was conducted on Waters AcquityTM UPLC System, and a Waters AcquityTM UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) (Waters Corp., Milford, MA, USA) chromatography column was used at a temperature of 50 °C and a flow rate of 0.4 mL/min. The injection sample volume was 5 μL. The mobile phase consisted of 0.1% formic acid in acetonitrile (solvent A) and 0.1% formic acid in water (solvent B), and the gradient eluting conditions were as follows: 0–2 min, 5–13% A; 2–11 min, 13–25% A; 11–18 min, 25–45% A; 18–20 min, 45–70% A;20–23 min, 70–100% A.
The Waters SynaptTM G2-Si MSE mass spectrometry system (Waters Corporation, Milford, MA, USA) was used to collect data in both positive and negative ion modes (ESI+, ESI−). The optimized conditions were as follows: ion source temperature 110 °C, desolvation gas temperature 350 °C, cone gas flow rate 50 L/h. In positive ion detection mode, the capillary voltage was 2.8 kV, the sample cone voltage was 20 V, the extraction cone voltage was 4.0 V, and the desolvation gas flow rate 800 L/h. In the negative ion detection mode, the capillary voltage was 2.2 kV, the sample cone voltage was 40 V, the extraction cone voltage was 4.0 V, and the desolvation gas flow was 600 L/h. Leucine enkephalin solution was used as the mass lock solution. The data acquisition interval was 0.2 s/scan, and the mass scanning range was 50–1200 Da.
4.6.3. Constituent Analysis Method In Vivo
High-resolution UPLC-Q-TOF-MSE combined with UNIFI software (Waters, the US) was used to analyze the prototypical components and metabolites of the blood. The MSE data of the model serum samples, drug-containing serum samples, and BYJ in vitro samples were imported into UNIFI software, and 3D data-processing mode was adopted to reduce false positives and effectively match high and low energy spectra. The secondary fragments and cleavage pathways of the blood components were analyzed by using the MassFragmentTM module for structural confirmation.
4.7. Data Processing and Multivariate Statistical Analysis
The raw data were imported into Waters Progenesis QI software for peak alignment, matching, extraction, and normalization, resulting in a data matrix of ion retention time–charge ratio–peak intensity. Normalized data were imported into SIMCA 13.0 (Umetrics AB, Umea, Sweden) for multivariate statistical analysis. Principal component analysis (PCA) was used to reveal the overall differences between the groups, and orthogonal partial least squares–discriminant analysis (OPLS-DA) was used to further distinguish the characteristic variables among the groups and validate the model. Potential biomarker sets were selected based on variable importance in projection (VIP) > 1, t-test of intergroup changes (p < 0.05), and fold change (FC) > 1.2.
4.8. Biomarker Identification and Metabolic Pathway Analysis of AUB Model
The retention time-m/z data of potential biomarkers were matched with the compound identifications table obtained from the Progenesis QI software to obtain possible compound molecular formulas within the measurement error range. The chemical structures of biomarkers were determined by searching for their molecular formulas and molecular weights in metabolomics databases such as HMDB and KEGG, combined with MS/MS data information. Furthermore, advanced pathway analysis programs in the MetaboAnalyst platform were used to conduct pathway analysis of the identified potential biomarkers of AUB model.
4.9. Correlation Analysis between Biomarkers and Absorbed Constituents
The Pearson correlation analysis platform was used to calculate the correlation coefficient between in vivo active ingredients and biomarkers of the AUB model. The larger the value of correlation coefficient, the stronger the correlation between the two variables. In this experiment, the correlation coefficient of r = 0.7 was used as the critical value to screen highly correlated ingredients; components with 5 or more highly correlated numbers of biomarkers were considered the potential pharmacodynamic material basis of BYJ for the treatment of AUB in rats.
5. Conclusions
In summary, the present study adopts the Chinmedomics strategy to evaluate the therapeutic effect of BYJ on AUB in rats. Based on the effective treatment of BYJ on the AUB rat model, correlation analysis was performed between the absorbed components and the biological markers of the model, and the pharmacological material basis and mechanism of BYJ in treating AUB were clarified for the first time. The study confirms that BYJ can improve coagulation function and sex hormone levels in abnormal uterine bleeding and regulate metabolic profiles, biomarkers, and pathways. It mainly regulates arachidonic acid metabolism, steroid hormone biosynthesis, glycerol phospholipid metabolism, and tryptophan metabolism through catalpol, gallic acid, rehmannioside D, chlorogenic acid, phellodendrine, paeoniflorin, liquiritin, Baicalin, liquiritin+O−H2+C6H8O6, berberine, asperosaponinVI, baicalin+H2+C2H2O, and glycyrrhetinic acid. Expression of the biomarkers arachidonic acid, progesterone, lysophospholipid(0:0/16:0), and L-tryptophan were reduced to promote platelet aggregation and release and to enhance contraction of vessels and uterine smooth muscle. Moreover, using the combined “Five Principles” of Q-markers, nine components (namely catalpol, rehmannioside D, phellodendrine, paeoniflorin, liquiritin, baicalin, berberine, asperosaponin VI, and glycyrrhetinic acid) were selected as the Q-markers of BYJ for the treatment of AUB, providing a theoretical basis for the quality control of BYJ in the future. This study is helpful to further improve the internal relationship between classical prescription, pharmacodynamic material basis, and quality markers.