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. 2021 Oct 13;9(12):6596–6609. doi: 10.1002/fsn3.2605

Metabolomics study of Angelica sinensis (Oliv.) Diels on the abnormal uterine bleeding rats by ultra‐performance liquid chromatography–quadrupole–time‐of‐flight mass spectrometry analysis

Ting‐Ting Chen 1,2, Liang Zou 3, Di Wang 4, Wei Li 5, Yong Yang 5, Xiao‐Min Liu 2, Xin Cao 5, Jia‐Rong Chen 5, Yan Zhang 5,✉, Jia Fu 1,✉
PMCID: PMC8645739  PMID: 34925789

Abstract

The objective of this study was to explore the effects and underlying intervention mechanisms of Angelica water extract (AWE) on abnormal uterine bleeding (AUB) based on serum metabolomics. Firstly, the concentration of main active substances in AWE was determined and the chemical components were identified by UPLC‐Q‐Exactive Orbitrap‐MS/MS. A drug‐induced abortion model was established by mifepristone and misoprostol. After administration AWE (2.16 g/kg) for 7 days, the coagulation function, serum hormone levels, H&E staining, and immunohistochemistry observation of uterus were detected. In addition, serum metabolites profiles were performed on ultra‐performance liquid chromatography–quadrupole–time‐of‐flight mass spectrometry (UPLC‐Q‐TOF‐MS). The contents of ferulic acid, senkyunolide A, and ligustilide in AWE were 0.7276, 0.0868, and 1.9908 mg/g, respectively. Twenty‐six compounds were identified in AWE. It was found that AWE was effective in regulation of coagulation function and promoting endometrial recovery. Meanwhile, the levels of E2, Pg, and HCG and the expression of ERα, Erβ, and PR were down‐regulated in AUB model and up‐regulated by the treatment of AWE. Twenty‐one potential biomarkers were eventually identified by multivariate statistical analysis. Study indicated that glycerophospholipid, sphingolipid, amino acids, retinol metabolism and primary bile acid biosynthesis were the main related metabolic pathways involved for the treatment of AUB by AWE. The results showed that AWE has potential therapeutic effect on AUB by altering the metabolic aberrations.

Keywords: abnormal uterine bleeding, Angelica water extract, metabolomics, UPLC‐Q‐TOF‐MS

Angelica water extract could reduce alleviate pathological injury and regulate the coagulation function to promote recovery of the endometrium. Angelica water extract could increase the levels of E2, Pg, and HCG and the expression of ERα, Erβ, and PR. Angelica water extract could regulate the metabolic pathways which include the glycerophospholipid metabolism, sphingolipid metabolism, glycine, serine and threonine metabolism, and retinol metabolism.

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1. INTRODUCTION

In the early 1970s and 1980s, medical abortion became an alternative way for early termination of pregnancy (Regina et al., 2011). Mifepristone combined with misoprostol is the preferred clinical approach for the induction of abortion (Klaira & Paul, 2020), but the severe side effects of incomplete medical abortion still reached 15% (Ma et al., 2016). Abnormal uterine bleeding (AUB) is a common characteristic in incomplete medical abortion, with an incidence of approximately 3%–30% among reproductive‐aged women (Munro et al., 2018). The use of estrogen, tranexamic acid, multi‐dose compound contraceptive, and multi‐dose progesterone regimen are common clinically available non‐surgical options for AUB (Bradley & Gueye, 2015), but these treatments may cause multiple side effects (Yujie et al., 2018).

Angelica sinensis (Oliv.) Diels (Danggui) was widely used as a functional food or a dietary supplement in Asia, Europe, and America. It is also a famous traditional Chinese medicine for the treatment of anemia, dysmenorrhea, premenstrual, menopausal syndrome, and other gynecological diseases (Ma et al., 2015). Previous research indicates that polysaccharides, volatile oils, and organic acids are the main bioactive ingredients of A. sinensis. (Jin et al., 2016; Wei et al. 2016). The results of pharmacological studies indicated that A. sinensis can replenish and invigorate blood, prevent pain, and moisten the intestines (Ma et al., 2016). In addition, A. sinensis also have anti‐arrhythmic effects, enhanced immune function, cardioprotective effects, anti‐atherosclerotic effects, and inhibiting platelet aggregation (Gu et al., 2016). However, few reports focus on the underlying therapeutic effects and mechanisms of A. sinensis for AUB.

Metabolomics is a technique to study the metabolites and their dynamic changes before and after being stimulated or disturbed for the biological system (e.g., after a specific gene variation or environmental change). Metabolomics has been widely used in plant molecular phenotype (Showkat et al., 2019), drug safety (Chen et al., 2021; Yu et al., 2018), molecular pathology (Yang & Lao, 2019), mechanism of drug action (Su et al., 2020), and disease diagnosis (Karakioulaki & Stolz, 2019). In addition, it was also used to evaluate the impact of storage environment on food quality (Guo et al., 2019). Metabolomics is considered to be a useful strategy to explain the underlying mechanisms of TCM for the treatment of diseases. It emphasizes the study objects (humans or animals) as a unified whole, which is in accordance with the principle of integrity and dynamics of Traditional Chinese Medicine (TCM). The analysis methods of metabolomics mainly include nuclear magnetic resonance spectroscopy (NMR), gas chromatography‐mass spectrometry (GC‐MS), and liquid chromatography‐mass spectrometry (LC‐MS). Among all of the analytical technologies, UPLC‐Q‐TOF‐MS is becoming a key technology in biomarker discovery (Gika et al., 2014).

In the present study, the concentration of main active substances in AWE was determined and the chemical components were identified by UPLC‐Q‐Exactive Orbitrap‐MS/MS. The coagulation function, serum hormone levels, H&E staining, and immunohistochemistry observation of uterus were detected after the intervention of AWE. An UPLC‐Q‐TOF‐MS method and multivariate analysis were applied to identify the potentially authentic biomarkers. The purpose of this study was to reveal the underlying mechanisms of AWE for the treatment of AUB and to provide a theoretical basis for clinical application.

2. MATERIALS AND METHODS

2.1. Chemicals and reagents

Angelica sinensis was harvested in Longxi County, Gansu Province in July 2019, and identified by Prof. Ying Liu (School of preclinical medicine, Chengdu University). Ferulic acid (batch No. MUST‐20060511) was brought from Chengdu Mansite Biotechnology Co., Ltd. Senkyunolide A (batch No. wkq20050703) was brought from Sichuan Weikeqi Co., Ltd. Ligustilide (batch No. G01001909022) was brought from Chengdu Ruifensi Biotechnology Co., Ltd. Mifepristone and Misoprostol were brought from Zizhu Pharmaceutical Co. (Peking, China). Pg ELISA kit (batch No. VE3AZJHQ1W) and E2 ELISA kit (batch No. 44IBV1FHE5) were brought from Elabscience Biotechnology Co., Ltd (Wuhan, China). HCG ELISA kit (batch No. 11/2019) was brought from Shanghai MLBIO Biotechnology Co., Ltd. (Shanghai, China). Antibody against ERα (batch No. 00,046,360), Antibody against ERβ (batch No. 00,053,760), and Antibody against PR (batch No. 00,235,360) were brought from Abcam Co., Ltd. (Shanghai, China). HRP (batch No. 20,200,528) was brought from Bioss Co., Ltd. (Peking, China). PBS (batch No. 20,190,307) was brought from Zsbio Co., Ltd. (Peking, China). DAB (batch No. 07,062,019) was brought from Baso Co., Ltd. (Zhuhai, China).

2.2. Preparation of the water extract of Angelica sinensis

The water extract of A. sinensis (AWE) was extracted by heating reflux method. Thirty grams of crude herbal drugs was added to purified water ten times and extracted for twice (v/w), each extraction time was 30 min. The concentration of AWE was 0.6 g/ml after filtration (expressed by the weight per mL of crude drugs).

2.3. UPLC‐MS analysis of AWE

The analysis was performed in a Thermo Fisher Vanquish UPLC system with a Thermo Fisher Q Exactive (Iowa, USA). The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B). The elution program was as follows: 0–35 min, 95%–5% A; 35–35.01 min, 5%–95% A; and 35.01–40 min, 5% A. C18 column (4.6 × 100 mm, 2.7 µm) was maintained with the temperature of 30℃; flow rate, 0.4 ml/min; injection volume, 1 µl.

The MS operating parameters were as follows: the ion mode was positive; ion spray voltages, 3.5 kV; turbo spray temperature, 320℃; and m/z range, 100–1000. The main chemical constituents of A. sinensis were identified according to the exact molecular mass, the cleavage fragments of MS2, the mz cloud, mzVault 2.0 MS database, and literature review.

2.4. Determination of ferulic acid, senkyunolide A, and ligustilide in AWE by UPLC‐MS

The AWE was mixed with 50% methanol (1:1) and filtered through a 0.22 µm membrane filter. The concentration of AWE was 0.022 g/ml after filtration (expressed by the weight per mL of crude drugs).

The analysis was performed using an Vanquish UPLC system with a TSQ Fortis triple quadrupole mass spectrometer (Thermo Fisher, USA), Accucore™ C18 column (2.1mm × 100mm, 2.6 µm, Thermo Fisher, USA). The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B). The UPLC elution program was as follows: 0–5 min, 85%‐60% A; 5–10 min, 60%‐55% A; 10–18 min, 55%‐30% A; 18–18.01 min, 30%‐85% A; and 18.01–23 min, 85% A, and Injection volume, 10 µl; flow rate, 0.2 ml/min; column temperature, 35 ℃. The Mass operating parameters were as follows: The ion mode was positive; scan type, SRM; sheath gas flow rate, 35 arb; aux gas flow rate, 15 arb; aux gas heater temp, 350℃; spray voltage, 3.5 kV; and capillary temp, 350℃.

2.5. Animal experiments

Female Sprague–Dawley (SD) rats of specific pathogen‐free (SPF) status, weighing 200–220 g; and male SD rats of SPF status, weighing 250–300 g (Certificate No. SCXK (Chuan) 2020‐030) were brought from the Chengdu Dossy Experimental Animals CO.LTD. (Chengdu, China). The all animals were kept under the same conditions. All experimental protocols were approved by the Animal Ethics Committee of the Chengdu University (20191209‐lxsz003).

The AUB rat model was established by mifepristone and misoprostol according to the method of previous literature (Zuo et al., 2019). The pregnancy control group (P) and AUB model group (M) were given with sterile saline, and the AUB + AWE group was administrated with dosage of 2.16 g/kg AWE once a day for 7 days.

2.6. Histopathological examination

The uterine tissues were immediately dissected after the experiment, removed fat and connective tissue, and fixed in 4% paraformaldehyde solution. Then, the uterine tissues were dehydrated at 4℃ for 24–48 hr, conventionally paraffin embedded, sectioned at 4 µm, and stained with hematoxylin–eosin (HE). Pathological changes of the endometrium were observed and photographed under a microscope.

2.7. Measurement of serum hormone levels

The serum levels of progesterone (Pg), estradiol (E2), and human chorionic gonadotrophin (HCG) were measured according to the instructions of manufacturer of the ELISA kits, respectively.

2.8. Detection of plasma coagulation function

Collected blood (3 ml) from the abdominal aorta with sodium citrate at a mass concentration of 3.8 g/L (anticoagulant: blood = 1:9) was centrifuged to obtain plasma. Prothrombin time (PT), thrombin time (TT), activated partial thrombin activity time (APTT), and fibrinogen (FIB) were determined by a hemagglutination analyzer.

2.9. Protein distribution analyses by immunohistochemistry

The uterine tissues were dehydrated, defatted, and conventionally paraffin embedded. Then, the uterine tissues were sectioned at 4 µm, deparaffinized, and rehydrated. After that, the tissue sections were incubated with 3% H2O2, repaired in antigen recovery solution, and then sealed at room temperature for 20 min after drip‐adding normal goat serum blocking solution. Then, the samples were incubated at 4 ℃ overnight with primary inhibitors (dilution 1:200): estrogen receptor α (ERα), estrogen receptor β (ERβ), and progesterone receptor (PR). One day after incubation, the cells were washed with PBS three times for 5 min each time, and then, drip‐added and incubated the secondary antibody at room temperature for 1 hr. SABC was added and incubated at 37℃ for 1 hr. Stained the proteins to dark‐brown by immersion in diaminobenzidine. The slices were rinsed with deionized water for 10 min. Hematoxylin was used for counterstaining, and hydrochloric acid alcohol was differentiated. Routine dehydration, transparentizing, sealing, and microscopy were performed.

2.10. Serum sample preparation

Added 400 µl anhydrous acetonitrile containing internal standard into 100 µl serum sample and vortex mixed for 3 min. Then, centrifuged the mixture at 12,000 rpm for 10 min to obtain the supernatant and putted it into a sampling vial.

2.11. UPLC‐Q‐TOF‐MS analysis conditions

An UPLC‐Q‐TOF‐MS system (Agilent, USA) was used for analysis with a BEH C18 column (2.1 mm × 100 mm, 1.7 µm). 0.1% formic acid in water (A) and acetonitrile (B) was used as a mobile phase with the following elution program: 0–3 min, 10%–30% B, 3–25 min, and 30%–95% B. Column temperature, 35℃; flow rate, 0.35 ml/min. The full scan range was 50 to 1,200 m/z; sheath gas temperature, 320℃; sheath gas flow, 12 L/min; drying gas temperature, 300℃; drying gas flow, 6 L/min; capillary voltage, 3.5 kV; and nebulizer pressure, 1.0 bar.

2.12. Data processing

The partial least‐squares discriminant analysis (PLS‐DA) and orthogonal partial least‐squares discriminant analysis (OPLS‐DA) were used for data analysis. The database used to identify the potential biomarkers was as follows: https://hmdb.ca/, http://www.lipidmaps.org/, http://www.genome.jp/kegg/, http://metlin.scripps.edu/. All results were described as the mean ± standard deviation (SD). One‐way analysis of variance (ANOVA) was used to analyze study data for significance comparison.

3. RESULTS

3.1. Identification of major compounds in AWE

The representative chromatography was shown in Figure 1. Twenty‐six constituents were identified by the accurate mass and relative ion abundance of the target peaks. The main constituents in AWE were γ‐Aminobutyric acid (GABA), Nystose, l‐Valine, Nicotinic acid, 4‐Oxoproline, Guanosine, Succinic acid, l‐Phenylalanine, dl‐Tryptophan, 2‐Anisic acid, Isophthalic acid, Caffeic acid, l‐Histidine, N‐Acetyl‐d‐alloisoleucine, Vanillin, Isofraxidin, Ferulic acid, Azelaic acid, Coniferyl aldehyde, Berberine, Jatrorrhizine, Coptisine chloride, Palmatine, Ligustilide, Senkyunolide A, and Levistilide A. The area percentage of the constituents is shown in Table 1.

FIGURE 1.

FIGURE 1

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The total ion chromatograms of samples by UPLC‐MS

TABLE 1.

The information of compounds identified in AWE by UPLC‐MS

Peak Constituent Formula Molecular Weight RT (min) Area(Max.) M/Z Fragments
1 γ‐Aminobutyric acid (GABA) C4H9NO2 103.0639 2.500 1.12E+09 104.0714 87.04505
2 Nystose C24H42O21 666.2216 2.563 1.51E+08 711.2197 665.21375,485.15158,341.10703,221.06606,179.05527,89.02335
3 L‐Valine C5H11NO2 117.0795 2.734 4.18E+08 118.0867 72.08173,55.05518
4 Nicotinic acid C6H5NO2 123.0328 3.298 2.58E+08 124.0395 96.05189,80.05059
5 4‐Oxoproline C5H7NO3 129.0417 3.427 3.28E+08 128.0345 82.02883
6 Guanosine C10H13N5O5 283.0923 4.155 1.59E+09 284.0998 152.05748
7 Succinic acid C4H6O4 118.0258 4.250 8.39E+07 117.0185 73.02842
8 L‐Phenylalanine C9H11NO2 148.0530 5.404 1.48E+09 166.0869 120.08154,103.05509
9 DL‐Tryptophan C11H12N2O2 204.0919 6.996 3.04E+09 188.0725 146.06157,118.06660
10 2‐Anisic acid C8H8O3 152.0467 7.592 2.45E+08 151.0394 123.04420,107.04922,93.03354
11 Isophthalic acid C8H6O4 166.0260 8.380 1.24E+09 165.0186 121.02848
12 Caffeic acid C9H8O4 180.0420 9.202 8.76E+08 179.0347 135.04422,71.01277
13 L‐Histidine C6H9N3O2 155.0688 9.876 7.35E+07 154.0616 137.03474,110.07134,93.04478
14 N‐Acetyl‐D‐alloisoleucine C8H15NO3 173.1048 10.273 1.52E+07 172.0974 130.08641
15 Vanillin C8H8O3 152.0467 11.346 9.69E+07 151.0394 136.01569
16 Isofraxidin C11H10O5 222.0529 11.967 3.23E+07 221.0455 206.02150,190.99799,163.00291,135.00803
17 Ferulic acid C10H10O4 194.0574 12.226 7.56E+07 193.0503 178.02640,134.03628
18 Azelaic acid C9H16O4 188.1044 13.153 3.51E+08 187.0971 125.09621,97.06487
19 Coniferyl aldehyde C10H10O3 178.0626 13.573 8.28E+07 177.0553 162.03142
20 Berberine C20H17NO4 335.1177 13.627 4.55E+07 336.1250 320.09439,292.09912
21 Jatrorrhizine C20H19NO4 337.1354 13.691 9.45E+08 338.1427 322.11090,308.09509,294.11551,280.09985
22 Coptisine chloride C19H14ClNO4 319.0885 13.998 8.28E+07 320.0958 292.09979
23 Palmatine C21H21NO4 351.1500 15.186 1.86E+08 352.1573 336.12640,308.13129
24 Ligustilide C12H14O2 190.0996 22.968 1.80E+08 191.1065 173.09673,145.10168,117.07053,91.05499
25 Senkyunolide A C12H16O2 192.1150 23.990 2.08E+08 193.1222 175.11226,147.11722,137.06015,105.07059,91.05498
26 Levistilide A C24H28O4 380.1986 32.075 4.40E+08 381.2062 191.10716
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3.2. Determination of ferulic acid, senkyunolide A, and ligustilide in AWE

The representative chromatography was shown in Figure 2. Ferulic acid was linear over a concentration range of 3.10–154.90 µg/ml, senkyunolide A was linear at 1.33–40.00 µg/ml, and ligustilide was linear at 2.04–254.65 µg/ml. The concentration of the mixed reference solution was taken as the abscisic coordinate, and the peak area was taken as the ordinate for linear regression. Typical equation of calibration curve for ferulic acid was y = 14333x + 63,294 (r = 0.9970); the curve of senkyunolide A was y = 343389x−340450 (r = 0.9978); and that of ligustilide was y = 88,686x + 876,354 (r = 0.9953). The contents of ferulic acid, senkyunolide A, and ligustilide were 0.7276, 0.0868, and 1.9908 mg/g, respectively.

FIGURE 2.

FIGURE 2

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The chromatogram of mixed reference solution and AWE by UPLC‐MS. (a) Mixed reference solution, (b) AWE. 1. Ferulic acid, 2. Senkyunolide A, 3. Ligustilide

3.3. AWE improved the histopathological damage

Microscopic examination explored that the endometrium of the P group was significantly thickened, the uterine cavity was small, the glands and blood vessels in the lamina propria were rich and dilated, and some of them were hyperemic (Figure 3a). Compared with the P group, the endometrium was thin, with local defects, and the lamina propria was mainly showed densely distributed blood vessels with slight congestion in the M group (Figure 3b). The endometrium in the AWE group was rich in blood vessels and loose in the stroma, but the symptoms were less severe than those in the model group (Figure 3c).

FIGURE 3.

FIGURE 3

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Effects of AWE on pathological changes of endometrium in rats by HE Staining (200×). (a) Pregnant group, (b) AUB model group, (c) AWE group

3.4. Measurement of serum hormone levels

Comparing with the P group, the E2, Pg, and HCG levels were significantly decreased in the M group (p < .01). The E2 and Pg levels were significantly increased with the treatment of AWE (p < .05) (Figure 4a–c).

FIGURE 4.

FIGURE 4

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The E2, Pg, and HCG Levels in each group. (a) E2, (b) Pg, (c) HCG. The results were presented as the mean ± SD, n = 6. **p < .01 AUB model group versus pregnant group, #p < .05 AWE group versus AUB model group, ##p < .01 AWE group versus medical aborting group

3.5. Effects of AWE on blood coagulation function in rats

Compared with the P group, the APTT and TT levels were significantly longer and FIB level was significantly lower in the M group (p < .05). The APTT and TT levels were significantly lower, and FIB level was significantly longer with the treatment of AWE (p < .05). (Figure 5).

FIGURE 5.

FIGURE 5

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The four coagulation indices of each experimental group. The results were presented as the mean ± SD, n = 6. *p < .05 AUB model group versus pregnant group, #p < .05 AWE group versus AUB model group, ##p < .01 AWE group versus AUB model group

3.6. Effects of AWE on ERα, Erβ, and PR levels in rats

Expression of ERα, Erβ, and PR was reduced in the M group when compared to the P group (p < .01). The expression of ERα and ERβ was increased with the treatment of AWE (p < .05). (Figure 6a–c).

FIGURE 6.

FIGURE 6

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The ERα, Erβ, and PR Levels in each group. (a) ERα, (b) ERβ, (c) PR. The results were presented as the mean ± SD, n = 6. **p < .01 AUB model group versus pregnant group, #p < .05 AWE group versus AUB model group

3.7. Data quality assurance of UPLC‐Q‐TOF‐MS

The typical total ion current chromatograms of each group of serum samples were shown in Figure 7.

FIGURE 7.

FIGURE 7

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The typical total ion chromatography of all serum samples in positive mode. P: Pregnant group, M: AUB model group, AWE: AWE group

3.8. Differential metabolites between the AUB and the pregnancy rats

The PLS‐DA and OPLS‐DA analyses explored that there was an obvious separation between the P, M, and AWE groups (Figure 8a,b). The S‐plots indicated the contribution of different metabolites variables between the P and M groups (Figure 8c). Fourteen significantly differential metabolites were shown in Table 2. LysoPC (20:5), Glycine, N‐Acetyl‐leukotriene E4, PC (18:1(9Z)/18:1(9Z)), LysoPC (18:3), LysoPC (18:0/0:0), Leukotriene D5, 20‐Oxo‐leukotriene E4, LysoPC (17:0), Sphinganine, LysoPC (18:2), LysoPC (16:0), L‐Valine, and N‐Lactoylleucine were significantly lower in the M group compared with the P group (Figure 9). The metabolism pathways of glycine, serine and threonine, glyoxylate and dicarboxylate, glycerophospholipid, primary bile acid biosynthesis, glutathione, and sphingolipid were significantly altered in the M group (Figure 10).

FIGURE 8.

FIGURE 8

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The multivariate statistical analysis. (a) PLS‐DA; (b) OPLS‐DA; (c) OPLS‐DA s‐plot

TABLE 2.

14 Differential metabolites in the serum of P and M groups

Compound Formula Metabolites M/Z Rt (min) VIP
A1 C28H48NO7P LysoPC (20:5) 542.3176 9.604 4.6232
A2 C26H43NO9S Glycine 546.3472 10.749 3.5524
A3 C25H39NO6S N‐Acetyl‐leukotriene E4 482.3185 11.836 1.7062
A4 C44H84NO8P PC (18:1(9Z)/18:1(9Z)) 786.5852 16.334 2.3486
A5 C26H48NO7P LysoPC (18:3) 518.3189 9.676 3.8540
A6 C26H54NO7P LysoPC (18:0/0:0) 524.3638 12.447 1.2658
A7 C25H38N2O6S Leukotriene D5 495.3220 9.898 2.3342
A8 C23H35NO6S 20‐Oxo‐leukotriene E4 454.2870 10.300 1.2371
A9 C25H52NO7P LysoPC (17:0) 510.3476 11.330 1.8070
A10 C18H39NO2 Sphinganine 302.2703 8.897 1.3561
A11 C26H50NO7P LysoPC (18:2) 520.3303 11.049 3.1960
A12 C24H50NO7P LysoPC (16:0) 496.3338 10.884 10.9197
A13 C5H11NO2 L‐Valine 118.0849 0.937 2.9892
A14 C9H17NO4 N‐Lactoylleucine 204.1198 1.003 5.7701
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FIGURE 9.

FIGURE 9

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The relative intensities of the examined metabolites obtained from P and M groups. The results were presented as the mean ± SD, n = 6. *p < .05 versus pregnant group, ** p < .01 versus pregnant group

FIGURE 10.

FIGURE 10

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Summary of pathway analysis of P and M groups

3.9. Differential metabolites between AUB and AWE treatment rats

There was a significant separation between the M and AWE groups in the OPLS‐DA model (Figure 11a), indicating that AWE had an effect on the metabolic profile of AUB rats. The S‐plots indicated the contribution of different metabolites variables between the M and AWE groups (Figure 11b). Twenty‐one significantly differential metabolites were shown in Table 3, and there showed that the specific changes of the relative content in these specific metabolites (Figure 12). LysoPC (20:5), Glycine, N‐Acetyl‐leukotriene E4, PC (18:1(9Z)/18:1(9Z)), LysoPC (18:3), LysoPC (18:0/0:00), Leukotriene D5, 3‐Hydroxybutyric acid, 20‐Oxo‐leukotriene E4, Hippuric acid, LysoPC (17:0), D‐Leucine, and L‐Valine were significantly higher in the AWE group compared with the M group. However, the other metabolites, including D‐Glucuronic acid, All‐trans‐Retinoic acid, and 25‐Hydroxyvitamin D3, were significantly lower in the AWE group compared with the M group. The metabolism pathways of primary bile acid biosynthesis, pentose and glucuronate interconversions, glycerophospholipid, glutathione, glyoxylate and dicarboxylate, sphingolipid, glycine, serine and threonine, retinol, ascorbate, and aldarate were significantly altered in the M group (Figure 13).

FIGURE 11.

FIGURE 11

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The results of multivariate statistical analysis from the serum samples. LC‐MS positive (a and b), (a) OPLS‐DA; (b) OPLS‐DA s‐plot

TABLE 3.

21 Differential metabolites in the serum of M and AWE groups

Compound Formula Metabolites M/Z Rt (min) VIP
B1 C28H48NO7P LysoPC (20:5) 542.3176 9.604 1.7704
B2 C26H43NO9S Glycine 546.3472 10.749 1.6321
B3 C25H39NO6S N‐Acetyl‐leukotriene E4 482.3185 11.836 1.3480
B4 C44H84NO8P PC (18:1(9Z)/18:1(9Z)) 785.5852 16.334 1.7397
B5 C26H48NO7P LysoPC (18:3) 518.3189 9.676 1.1027
B6 C26H54NO7P LysoPC (18:0/0:0) 524.3638 12.447 11.0291
B7 C25H38N2O6S Leukotriene D5 495.3220 9.898 1.1269
B8 C4H8O3 3‐Hydroxybutyric acid 105.0338 4.681 1.0865
B9 C23H35NO6S 20‐Oxo‐leukotriene E4 454.2870 10.300 1.2019
B10 C9H9NO3 Hippuric acid 180.0650 4.681 1.2399
B11 C25H52NO7P LysoPC(17:0) 510.3476 11.330 1.3592
B12 C18H39NO2 Sphinganine 302.2703 8.897 1.4056
B13 C26H50NO7P LysoPC(18:2) 520.3303 11.049 2.0366
B14 C9H17NO4 N‐Lactoylleucine 204.1198 1.003 1.6609
B15 C18H34O2 Oleic acid 283.1748 5.656 2.7529
B16 C6H10O7 D‐Glucuronic acid 195.1221 1.815 4.0168
B17 C20H28O2 all‐trans‐Retinoic acid 301.2044 5.656 4.2104
B18 C24H50NO7P LysoPC(16:0) 496.3338 10.884 2.4304
B19 C27H44O2 25‐Hydroxyvitamin D3 401.3184 7.223 8.3933
B20 C6H13NO2 D‐Leucine 132.1005 0.927 3.4918
B21 C5H11NO2 L‐Valine 118.0849 0.937 4.1642
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FIGURE 12.

FIGURE 12

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The relative intensities of the examined metabolites obtained from M and AWE groups. The results were presented as the mean ± SD, n = 6. #p < .05 versus. AUB model group, ## p < .01 versus AUB model group

FIGURE 13.

FIGURE 13

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Summary of pathway analysis of M and AWE groups

4. DISCUSSION

Angelica sinensis, as a medicinal food, has widely been used for the treatment of amenorrhea, dysmenorrheal, and premenstrual syndrome of gynecological disorders (Li et al., 2012). Studies have shown A. sinensis contains ferulic acid, senkyunolide A, ligustilide, etc., so it was speculated that AWE had a certain influence on the anti‐inflammatory (Fang et al., 2020), blood replenishing (Tao et al., 2016), liver lipid accumulation, and fatty regeneration (Ma et al., 2020). Peng Cao et al. found Angelica sinensis polysaccharide as a kind of “tonic foods,” which has potential to be used as a hepatoprotective agent for Acetaminophen‐induced hepatic damage (Cao et al., 2018). Yong li Hua et al. found that A. sinensis can promote hematopoiesis, enhance antiapoptotic effects, and regulate energy metabolism (Hua et al., 2017). Qin Fan et al. found that ferulic acid could scavenge PPH‐ and ABTS‐free radicals, while ligustilide exhibited scavenging capacity for ABTS‐free radicals (Fan et al., 2020). Zi‐wen Yuan et al. found that A. sinensis intervention could significantly relieve blood stasis syndrome in rats (Yuan et al., 2019). In this study, APTT and TT levels were significantly lower in AWE group (p < .05), the APTT and TT levels were significantly lower, and FIB level was significantly longer with the treatment of AWE, indicating that A. sinensis can regulate blood coagulation function of AUB rats.

Metabolomics is a large‐scale research technology that uses modern analytical method to assess the creature physiological status in different conditions (Wei et al., 2020). The metabolic profiles reflect an individual's function state at a certain point, which is consistent with the integrality and systematicness of traditional Chinese medicine (Bao et al., 2017). This technique has been used to study the effects of Chinese medicine syndrome patterns (Fengxia et al., 2010; Li et al., 2016). As an important part of the human body, blood contains abundant information and is often used as the matrix for metabolomics research. Therefore, the UPLC‐Q‐TOF‐MS metabolomics platform and multivariate statistical analysis method were used to assess the rats’ serum to reveal the mechanism of AWE in AUB caused by incomplete abortion. In our study, we identified 21 potential biomarkers that were directly or indirectly correlated for the therapeutic effects of AWE in AUB caused by incomplete abortion, mainly including amino acids, retinol, fatty acids, and lysophospholipids. We have found that the serum of the incomplete medical abortion rats showed altered metabolism mainly in amino acid, retinol, lipid metabolism and primary bile acid biosynthesis pathways.

Amino acids are necessary for embryonic growth and development. In our study, we found that the level of some amino acids including glycine, d‐leucine, and l‐valine was abnormal in the AWE group. Glycine was closely associated with inflammation among these amino metabolites (Angélica et al., 2014). Glycine promotes myelin phagocytosis and the production NO and TNF‐α, which may affect immunological processes of inflammatory diseases (Carmans et al., 2010). In the inflammation, the TNF‐α induces protein decomposition and catabolism to up‐regulate the urea synthesis (Louise et al., 2013). Yong‐li Hua et al. found that volatile oil from A. sinensis can inhibit inflammation through down‐regulating the synthesis of glycine, arachidonic acid, L‐glutamate, pyruvate, and succinate (Angélica et al., 2014). D‐Leucine and L‐valine are branched chain amino acids (BCAAs), especially leucine, and can enhance protein synthesis through the mTOR signaling pathway to regulate energy balance, which plays a vital role in blastocyst development. Banerjee et al. found that the metabolites, including lysine, L‐arginine, glutamine, threonine, histidine, phenylalanine, and tyrosine, were significantly increased in patients with idiopathic recurrent spontaneous miscarriage, which may be involved in vascular dysfunction associated with poor endometrial receptivity and excessive inflammatory reactions (Priyanka et al., 2014). Houghton et al. used reversed‐phase high‐performance liquid chromatography (RHPLC) and found that the content of leucine was significantly reduced after in vitro fertilization when the embryo developed into the cyst embryo culture medium, while the contents of valine and isoleucine were significantly reduced in the embryo culture medium that did not develop into the blastocyst. Valine and isoleucine have a definite influence on embryo development (Houghton et al., 2002). Zhang et al. found that BCAAs are closely involved in pregnancy outcome. The elevated BCAAs can clearly impair the development of diploids cells to the morula and blastocyst stages (Thuan et al., 2002). Xiao‐yao Miao et al. used metabolomic‐based gas chromatography‐mass spectrometry and pattern recognition to study the potential mechanism of Danggui Buxue Tang's anti‐fatigue effect on weight‐bearing forced swimming mice and found that Danggui Buxue Tang had a good anti‐fatigue effect (Miao et al., 2018). In our study, we found that the glycine, D‐leucine, and L‐valine serum concentrations in the AWE group were higher than those in the M group, which may indicate that Angelica sinensis may affect the inflammatory process in AUB rats.

Lipid metabolism mainly consists of linoleic acid, glycerophospholipid, and sphingolipid metabolism. Oleic acid, which is closely related to leukotriene synthesis, is considered to be a key factor in tissue injury, inflammation, and vasoconstriction. Kim et al. found that the water extract of Angelica sinensis has an anti‐inflammatory effect via the NO‐burst/calcium‐mediated JAK‐STAT pathway (Young‐Jin et al., 2018). Yao et al. found that the anti‐inflammatory activity of the volatile oil of Angelica sinensis mainly through regulating glycine and arachidonic acid metabolic disorders (Yao et al., 2015). And in our study, after AWE intervention, the AWE group showed significant upregulation of N‐Acetyl‐leukotriene E4, 20‐Oxo‐leukotriene E4, and Leukotriene D5, which indicated that the effect of AWE on rats with AUB may involve the regulation of arachidonic acid metabolic network disorders. Lyso PC, a phospholipid, can regulate vascular tone and induce endothelial dysfunction. It is reported that saturated fatty acids can induce the expression of cyclooxygenase (Akito et al., 2009) and promote the synthesis of PGF2α (Helliwell et al., ). In this study, LysoPC (16:0), LysoPC (17:0), PC (18:1(9Z)/18:1(9Z)), LysoPC (18:2), LysoPC (18:3), LysoPC (20:5), and LysoPC (18:0/0:00) were up‐regulated in the AWE group. These results indicate that glycerophospholipid metabolism was abnormal.

Sphinganine is a sphingolipid that is involved in the formation of cell membranes. It can be phosphorylated under the catalysis of sphingosine kinase to produce a potent signaling lipid molecule, sphingosine‐1‐phosphate (S1P). S1P can regulate the physiological functions of cell survival, growth, proliferation, and apoptosis from the extracellular receptor pathway and the intracellular second messengers. Roth et al. indicated that sphingosine‐1‐phosphate can promote the maturation of bovine oocytes and enhance the development of embryos (Roth & Hansen, 2004). Hannoun et al. indicated that the fragmentation rate of human preimplantation embryos medium with S1P was significantly lower and the embryos quality was better (Antoine et al., 2010). In our study, we found that the serum concentration of sphingosine in the AWE group was higher than that in the M group, which may be because AWE has a regulatory effect on sphingosine.

All‐trans‐retinoic acid belongs to vitamin A (retinol), which is a basic nutrient required for mammalian reproduction. This molecule plays a vital role in promoting the normal development of embryos, regulating cell proliferation and differentiation, and maintaining normal cell differentiation and immune system function integrity. Ueda et al. showed that chemical signaling induced by the use of retinoic acid can promote the differentiation of embryonic stem cells into neurons (Ueda et al., 2018). In our study, we found that the level of all‐trans‐retinoic acid was down‐regulated in the AWE group. In summary, AWE could adjust the abnormal metabolism state of amino acid, retinol, and lipid metabolism with AUB induced by incomplete medical abortion.

5. CONCLUSIONS

An UPLC‐Q‐TOF‐MS‐based serum metabolomic approach was applied to investigate the mechanisms of AWE for the treatment of AUB. Twenty‐one potential biomarkers were eventually identified, which may be involved in the intervention mechanism of AWE for the treatment of AUB. Some metabolic pathways including primary bile acid biosynthesis, glycerophospholipid metabolism, pentose and glucuronate interconversions, glutathione metabolism, glyoxylate and dicarboxylate metabolism, sphingolipid metabolism, glycine, serine and threonine metabolism, retinol metabolism, ascorbate, and aldarate metabolism were altered by AWE treatment. The significantly reversed the metabolic aberrations in AUB group by AWE facilitates to support the therapeutic effect and potential mechanisms of AWE on AUB induced by incomplete medical abortion.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

This research was funded by Health and Family Planning Commission of Chengdu‐Key disciplines of clinical pharmacy and Sichuan Medical Research Project (grant number: S18056, S19030).

Chen, T.‐T. , Zou, L. , Wang, D. , Li, W. , Yang, Y. , Liu, X.‐M. , Cao, X. , Chen, J.‐R. , Zhang, Y. , & Fu, J. (2021). Metabolomics study of Angelica sinensis (Oliv.) Diels on the abnormal uterine bleeding rats by ultra‐performance liquid chromatography–quadrupole–time‐of‐flight mass spectrometry analysis. Food Science & Nutrition, 9, 6596–6609. 10.1002/fsn3.2605

Contributor Information

Yan Zhang, Email: zhangyan@cdu.edu.cn.

Jia Fu, Email: fujia@cdu.edu.cn.

DATA AVAILABILITY STATEMENT

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

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Data Availability Statement

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

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