Effect of Wuzi Yanzong prescription on oligoasthenozoospermia rats based on UPLC-Q-TOF-MS metabolomics

Abstract Context Wuzi Yanzong Prescription (WYP) has long been used to treat male infertility, with convincing clinical evidence. However, its mechanism of action is not clear. Objective WYP chemical components were quantified by HPLC, and the effect on oligoasthenozoospermia rats was explored based on UPLC-Q-TOF-MS metabonomics technology. Materials and methods The solution was extracted by alcohol and water; the content of eight components in the extracting solution was determined by HPLC. Twenty-four Sprague-Dawley rats were randomly divided into control (CG), model (MG) and administration (PG) groups. Oligoasthenozoospermia was induced for 30 days in all, but the CG with daily oral doses of 20 mg/kg Tripterysium glycosides (TG). PG was given daily oral doses of WYP solution (1.08 g/kg), CG and MG received the same volume of distilled water, all administered for 30 days. After the last administration, the serum samples were collected and detected by UPLC-Q-TOF-MS. Results An HPLC method for simultaneous determination of chlorogenic acid (0.081%), ellagic acid (0.021%), hyperoside (0.032%), verbascoside (0.011%), isoquercitrin (0.041%), astragalin (0.026%), kaempferol (0.009%) and schisandrin (0.014%) was established. Moreover, the 74 potential biomarkers and eight metabolic pathways related to oligoasthenozoospermia were identified by multivariate analysis and metabolite profiling. WYP can regulate 36 markers, mainly involving amino acid metabolism, lipid metabolism and nucleotide metabolism. Discussion and conclusions This is a simple and accurate method for quality control of WYP. It further enriches the potential mechanism of WYP in the treatment of oligoasthenozoospermia. It can provide a theoretical basis for the rational application of WYP.


Introduction
Infertility and subfertility affect a significant proportion of humanity. According to the World Health Organisation (WHO) definition, infertility is a disease of the male or female reproductive system defined by the failure to achieve a pregnancy after 12 months or more of regular unprotected sexual intercourse. Population-based studies have reported that about 15% of couples may suffer from infertility, thus representing a considerable issue for the global health community. In this context, approximately 40%-50% of infertile couples are unable to conceive as a consequence of male reproductive impairment (Capogrosso et al. 2021;Pillai and McEleny 2021). In the male reproductive system, infertility is most commonly caused by problems in the ejection of semen, absence or low levels of sperm, or abnormal shape (morphology) and movement (motility) of the sperm. Oligoasthenospermia is caused by a variety of diseases or factors and usually manifests itself as 'spermlessness', 'heirlessness', 'sterility', 'infertility', 'cold sperm', 'clear sperm', 'low sperm', etc. (Fuxing et al. 2020). It is the most common type of semen abnormalities in male infertility patients (Wang et al. 2021).
At present, the treatment of male infertility by western medicine mainly includes the treatment of primary diseases, semen drugs, assisted reproductive technology, etc., but its therapeutic effects are limited and cannot fundamentally solve the problem of male infertility. The theory of traditional Chinese medicines (TCM) believes that male infertility with abnormal semen and few weak sperm is often caused by insufficient kidney essence, and should take the method of nourishing the kidney and improving the essence. A classical TCM formula for this type of infertility, Wuzi Yanzong prescription (WYP), was first recorded in the 'she sheng zong miao fang' text of the Ming Dynasty and is a mixture of Lycii fructus (the dried ripe fruit Lycium barbarum L.), stir-fried cuscutae semen (the dried ripe seed of Cuscuta australis R.Br. or Cuscuta chinensis Lam.), rubi fructus (the dried fruit of Rubus chingii Hu), saltwater stir-fried plantaginis semen (the dried ripe seed of Plantago asiatica L. or Plantago depressa Willd.) and steamed schisandrae chinensis fructus [the dried ripe fruit of Schisandra chinensis (Turcz.) Baill.]. It nourishes the kidney and strengthens the essence and therefore has been used to treat oligoasthenozoospermia secondary to kidney essence insufficiency as 'The Number One Herbal Formula for Assisting Fertility' . Modern studies have shown that WYP has a good protective effect on reproductive system (Sheng et al. 2018;Hu et al. 2019), immune system (Zhuo et al. 2019) and nervous system (Ruixue et al. 2019).
In the early stage, we have carried out research on WYP's chemical composition, pharmacodynamics and other aspects. The results showed that WYP could restore testicular structure and significantly increase the serum levels of GnRH, LH, E2, T and decrease FSH, TGf-b1, Smad2 and Sma4 expression levels . The main components of WYP include polysaccharide, fatty acids, flavonoids, phenylpropanoids, organic acids, alkaloids, terpenoids, etc. But the research is relatively single and scattered, which cannot reflect the holistic view of WYP in the treatment of diseases. Therefore, this study intends to explore the intervention effect and regulatory mechanism of WYP on oligoasthenozoospermia rats based on ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) metabonomics to more comprehensively elaborate the metabolic pathway of WYP in the treatment of oligoasthenozoospermia, and provide reference for the clinical treatment of male reproductive system diseases.

Preparation of WYPextracting solution
Stir-fried cuscutae semen, saltwater stir-fried plantaginis semen and steamed schisandrae chinensis fructus were prepared according to Pharmacopoeia of the People's Republic of China 2020 Vision (ChP 2020). Lycii fructus, stir-fried cuscutae semen, rubi fructus, steamed schisandrae chinensis fructus and saltwater stirfried plantaginis semen were combined in the proportion of 8:8:4:2:1 to form WYP (200 g), as outlined in the ChP 2020. WYP extracting solution was obtained by reflux exaction, adding six times 95%, 70% and 50% ethanol for 1.5 h each time. The dregs were refluxed successively adding six times water and four times water for 1.5 h each time. The filtrates were mixed five times. The decoction was concentrated to 463 mL by vacuum concentration.

Animals
Twenty-four SD rats (male, 220 ± 20 g) were provided by Chengdu Dashuo Experimental Animal Co., Ltd. (Chengdu, China). All animals were housed under controlled temperature (25 ± 2 C) and a 12-h light/dark cycle and fed standard diet and water. The animal experiments were reviewed by the Committee of Scientific Research and the Committee of Animal Care of the Chengdu University of TCM. All the animals were allowed to acclimatize in metabolism cages for 1 week prior to treatment.

Establishment of oligoasthenozoospermia model and treatment
The rats were randomly divided into three groups, including control group (CG, n ¼ 8), model group (MG, n ¼ 8) and administration group (PG, n ¼ 8). Oligoasthenozoospermia was induced for 30 days in all but the CG with daily oral doses of 20 mg/kg TG in the morning . Meanwhile, PG was given daily oral doses of 1.08 g/kg extracting solution of WYP, while the CG rats and model rats received the same volume of distilled water. On the 31st day, blood samples were collected from the abdominal aorta of all the rats after 45 min of intragastric administration. Prior to this, all of the rats were prohibited any food for 12 h before the last administration, but were allowed free access to water. The blood samples were left at room temperature for 1 h, and were centrifuged at 3500 rpm for 10 min at 4 C. Then the supernatant was transferred into frozen pipes and stored at À80 C until analysis.

Sample preparation
Take about 1 mL of WYP extracting solution (to a 10 mL volumetric flask), adding a moderate amount of methanol, dilute to 10 mL. The material was filtered (0.45 lm pore size) before HPLC injection.

Metabolomic study
Metabolite extraction A blood sample (100 lL) was transferred to an Eppendorf (EP) tube, and 400 lL extract solution (acetonitrile: methanol ¼ 1: 1) (V/V) containing isotopically-labelled internal standard mixture was added. After 30-s vortex, the samples were sonicated for 5 min in ice-water bath. Then the samples were incubated at À40 C for 1 h and centrifuged at 12,000 rpm for 15 min at 4 C. Supernatant (400 lL) was transferred to a fresh tube and dried in a vacuum concentrator at 37 C. Then, the dried samples were reconstituted in 200 lL of 50% acetonitrile by sonication on ice for 10 min. The constitution was then centrifuged at 13,000 rpm for 15 min at 4 C, and 75 lL of supernatant was transferred to a fresh glass vial for LC/MS analysis. The quality control (QC) sample was prepared by mixing an equal aliquot of the supernatants from all of the samples.
The Triple TOF 6600 mass spectrometry (AB Sciex) was used for its ability to acquire MS/MS spectra on an informationdependent basis (IDA) during an LC/MS experiment. In this mode, the acquisition software (Analyst TF 1.7, AB Sciex) continuously evaluates the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on preselected criteria. In each cycle, the most intensive 12 precursor ions with intensity above 100 were chosen for MS/MS at collision energy (CE) of 30 eV. The cycle time was 0.56 s. ESI source conditions were set as following: Gas 1 as 60 psi, Gas 2 as 60 psi, Curtain Gas as 35 psi, Source Temperature as 600 C, Declustering potential as 60 V, Ion Spray Voltage Floating (ISVF) as 5000 V or À4000 V in positive or negative modes, respectively.
Data preprocessing and analysis MS raw data (.wiff) files were converted to the mzXML format by ProteoWizard and processed by R package XCMS (version 3.2). The process includes peak deconvolution, alignment and integration. Minfrac and cut off are set as 0.5 and 0.3, respectively. The resultant data matrices were imported to SIMCA-P (V15.0.2) for the principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The PCA was used to evaluate the MS data in a first overview of the cluster of groups. The following analysis with OPLS-DA scores was performed to obtain the VIP scores and dataset of the relative intensity of metabolites to identify biomarkers. In-house MS2 database (Biotree biomedical technology co., LTD) was applied for metabolite identification. The combination approaches of Human Metabolome Database (http://www. hmdb.ca) and mzcloud (https://www.mzcloud.org) were applied to analyse biomarkers and relate biological information. The pathway analysis and inner relation among them were undertaken with MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/) and KEGG database (http://www.kegg.jp/). The differences in metabolites among groups were compared by Student's t-test, and p < 0.05 was considered statistically significant.

Identification and determination of components from WYP extracting solution
The chromatograms of mixed standards and samples are shown in Figure 1. The chromatographic peaks are well separated, the retention time is accurate and the determination results are reliable. By comparing the retention time in HPLC analysis with an accepted standard, nine components were identified, including chlorogenic acid, kaempferol, hyperoside, verbascoside, schisandrin, isoquercitrin, ellagic acid, quercetin and astragalin. The contents of 8 components were determined. The results are shown in Table 1.

Results of the serum metabolomics
Total ion flow chromatogram of rat serum UPLC-Q-TOF-MS was used to analyse the serum of CG, MG and PG. The total ion flow chromatogram is shown in Figure 2. The endogenous markers obtained excellent separation within 12 min. Further analysis of the total ion flow chromatograms under the positive and negative ion modes showed that the total ion flow chromatograms of the samples in each group were basically similar, but there were some differences in the peak type and peak area of each group, indicating that some metabolites in rats were changed. The reason for this difference may be that the endogenous metabolites and their metabolic pathways changed in rats before and after WYP treatment.

Multivariate analysis and metabolite profiling
As shown in Figure 3, the PCA score plot was introduced to distinguish and evaluate the status of each group. In the positive and negative mode, the distribution of QC samples is concentrated, showing good reproducibility and reliability. The data of MG and other groups were well distinguished, and the distance between the CG and MG was far, indicating that the model was successfully established. The CG had some discrete data, which may be caused by individual differences and injection time differences of rats. OPLS-DA is a noise reduction method based on partial least squares method, which can remove some variations in the independent variables that were not related to the dependent variables, reduce the complexity of model, enhance the model forecast capability and better reveal the differences between groups (Zhou et al. 2016). The OPLS-DA model was used to further differentiate the differences among the groups. As shown in Figure 3, The OPLS-DA score plots (R2X (cum) ¼ 0.469, R2Y (cum) ¼ 0.970, Q2 (cum) ¼ 0.592 in positive mode and R2X (cum) ¼ 0.357, R2Y (cum) ¼ 0.946, Q2 (cum) ¼ 0.535 in negative mode) showed the prediction ability of the model is good, and the metabolites in each group were completely separated either on the positive or negative mode. The samples in the PG were more inclined to the CG, indicating that WYP had a callback effect on the abnormal endogenous metabolites.
To gain a better discrimination between the CG and MG, the OPLS-DA approaches were applied to differentiate the metabolic profile and find the potential biomarkers. The OPLS-DA score plots (R2X (cum) ¼ 0.424, R2Y (cum) ¼ 0.997, Q2(cum) ¼ 0.824 in positive mode and R2X (cum) ¼ 0.285, R2Y (cum) ¼ 0.982, Q2(cum) ¼ 0.724 in negative mode) showed an overall goodness of fit between the MG and CG (Figure 4). The results of 200 permutation tests showed that the intercept of Q2 regression line is less than 0, which indicates that the model has not been fitted, and the OPLS-DA model is reliable and stable. The metabolites were identified under the reference standard of same retention times and same molecular weights, while p-values less than 0.05. Based on the protocols described above and the comparison of the metabolites between the CG and MG, 74 differential biomarkers were identified, which could be considered as the endogenous biomarkers of oligoasthenozoospermia. Fortyfour endogenous metabolites were regulated significantly by WYP (p < 0.05). The p-values were derived using a one-way ANOVA. The results are shown in Table 2.

Metabolic pathway analysis
To explore the potential metabolic pathways of oligoasthenozoospermia, 68 endogenous biomarkers were imported into Metaboanalyst 5.0. The pathway impact greater than 0.1 was used as the standard to screen the main metabolic pathway. As shown in Table 3 and Figure 5, 8, corresponding pathways, which could be positively related to oligoasthenozoospermia were constructed, including the phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, arachidonic acid metabolism, glycerophospholipid metabolism, pyrimidine metabolism, and tyrosine metabolism.

Discussion
As a unique theory of TCM, syndrome differentiation treatment is the soul of the basic theory of TCM and plays an important role in the diagnosis of TCM. To study the mechanism of WYP, we must first establish relevant animal pathological models. The previous research showed that the establishment of    To ensure the stability of the main chemical components in WYP extracts solution, we established an analytical method to identify the eight components, the results showed that the eight components showed good linearity in their respective ranges. Furthermore, through the analysis of serum metabolomics, 74 potential biomarkers and eight metabolic pathways were identified to be related to oligoasthenozoospermia. It is mainly related to amino acid metabolism, lipid metabolism and nucleotide metabolism. This is consistent with the literature report that WYP can regulate the concentration of amino acids, lipids and other metabolites of nonobstructive oligoasthenospermia, normalize the metabolic phenotype and regulate metabolic disorders (Zou et al. 2019).There are many TCM syndrome types. The model of oligoasthenospermia prepared by TG in this paper belongs to the syndrome of deficiency of kidney essence. Compared with the model of orchiectomy, it has a wider scope of application and more representative significance, which enriches the research on the mechanism of WYP. According to the enriched metabolic pathway, the most significant metabolic feature of oligoasthenozoospermia is the change of amino acid metabolism. A variety of amino acid metabolic pathways, including phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, tyrosine metabolism, alanine, aspartate and glutamate metabolism, arginine and proline metabolism, suggest that amino acids play an important role in spermatogenesis, development and maturation. Free amino acids participate in the cellular metabolism of motile sperm and are the source of energy supplement, which can promote its activity and reduce the harmful effects of toxins. Essential amino acids are important raw materials for the synthesis of nucleic acids. Supplementing essential amino acids will improve the body's ability to synthesize nucleic acids and promote spermatogenesis (Han et al. 2017).
In addition to amino acid metabolism, lipid metabolism and nucleoside metabolism also play a very important role in oligoasthenozoospermia, such as arachidonic acid metabolism, glycerophospholipid metabolism and pyrimidine metabolism. Modern research shows that lipid is an important part of sperm membrane, which participates in the changes of sperm structure in the process of spermatogenesis, and lipid metabolism provides energy for sperm (Hua et al. 2020). As the important components of various biofilms, fatty acids and cholesterol deletion will cause sperm damage (Huiqing et al. 2017). Other studies have shown that abnormal arachidonic acid metabolism can reduce sperm motility by activating p38 MAPK pathway through lipoxygenase, cytochrome P450 and cyclooxygenase pathways (Yu et al. 2019). This study found that WYP can improve metabolic disorder by regulating 36 biomarkers. It is suggested that WYP activity depends on multiple chemical components and effectively acts on multiple targets reflecting different potential pathways. In future research, the association of components, targets and efficacy can be realized through the research on serum pharmacochemistry and network pharmacology of WYP, combined with data analysis technologies to provide more clear and sufficient evidence for the mechanism of WYP, and to provide a basis for clinical application of WYP.

Conclusions
In this study, a method for the determination of eight components in WYP extracting solution was established. The method is simple, stable and feasible, and can be used for the quality control of WYP. WYP mainly treats oligoasthenozoospermia by regulating amino acid metabolism, lipid metabolism and nucleotide metabolism. It further enriches the potential mechanism of WYP  in the treatment of oligoasthenozoospermia. It can provide a theoretical basis for the rational application of WYP in clinic.