1 Introduction

WZYZW is a traditional TCM formula for tonifying the kidney and essence. It is a combination of cuscutae semen (Cuscuta australis R.Br. or Cuscuta chinensis Lam., chinese name Tusizi), Lycii fructus (Lycium barbarum L., chinese name Gouqizi), plantaginis semen (Plantago asiatica L. or Plantago depressa Willd., chinese name Cheqianzi), rubi fructus (Rubus chingii Hu,chinese name Fupengzi), and schisandrae chinensis fructus [Schisandra chinensis (Turcz.) Baill., chinese name Wuweizi]. Because of its effects of tonifying the essence, inducing kidney qi and reproduction, WZYZW has been respected by generations of doctors, being called “The Number One Herbal Formula for Assisting Fertility” (Chinese Pharmacopoeia Commission, 2020). In this formula, Lycii fructus warms the kidney and enhances the essence, and rubi fructus nourishes the kidney and fills the essence, the two drugs supplement yin and yang together, and are the monarch; schisandrae chinensis fructus invigorates qi、strengthens yin and astringent essence, cuscutae semen warms the kidneys without drying, solidifying the essence but not coagulation, and are the minister; plantaginis semen is both astringent, tonic but not stagnant, used as an assistant medicine (Li et al., 2019), Clinically, it has an extensive range of applications in reproductive system and nervous system diseases.

WZYZW is composed of multiple herbs, the compatibility and composition of medicinal flavors are more complex, and the quality of decoction pieces is also uneven. At present, there are few researches on the integrated quality control of WZYZW. As a consequence of that, it is of great significance to develop a method that can comprehensively explain the characteristics of WZYZW compounds. Based on this background, Academician Changxiao Liu proposed a new thinking of about Q-markers in TCM and clarified that the five principles of Q-marker are uniqueness, validity, transmission and traceability, measurability, and matching environment. (Liu et al., 2016) The fingerprint of TCM can comprehensively and quantitatively analyze the chemical information contained in TCM, which is an important means of quality control of it. At the same time, it also becomes an important means in the research of substance standard of single-flavor drugs and compound formulas of TCM (Wei et al., 2018). The complexity of the TCM components determines that it is challenging to express the quality by single ingredient or index evaluation, so the model of multi-component quality control is proposed. QAMS is a multi-index quality evaluation model that is suitable for difficult preparation, high cost or unstable reference product by selecting a stable and easily prepared component as the internal reference within the corresponding linear range through the internal functional relationship between components in the sample to be tested (Wang et al., 2006). The two methods have been widely used in the quality control research of TCM herbs, pieces, and compounds. Based on the theory of systems biology, network pharmacology explores the correlation between drugs and diseases from a holistic perspective by establishing prediction models of the relationship between drugs and targets, and between diseases, and therapeutic targets (Wang et al., 2021). It examines the potential functional ingredients and targets of Chinese medicine compounds at a systematic level, and also theoretically elucidates the effectiveness of herbal medicines. At present, the studies of WYZW include fingerprint similarity evaluation, network pharmacology screening of potential targets and pathways, and QAMS (Liao et al., 2021a, 2021b, Wang et al., 2022a, 2022b, Tong et al., 2023), but few of them analyze Q-markers in combination with fingerprint, network pharmacology and QAMS. Therefore, based on Q-Marker theory, this study established an analysis method combining fingerprint, network pharmacology and QAMS, analyzed its components from the substance component level, predicted its potential targets and pathways from the molecular level, and finally predicted the Q-marker of WZYZW. The purpose of this study was to provide ideas for the new research model of WZYZW, to provide a basis for the quality control of WZYZW, and to provide a reference for the study of the mechanism of WZYZW.

2 Materials and methods

3 Results

3.1

3.1 Establishment fingerprint of WZYZW

3.1.1

3.1.1 Method validation

3.1.1.1

3.1.1.1 Precision

Take WZYZW (S5) test solution, according to “1.2”, inject 6 times continuously, and record the chromatogram. The similarity was greater than 0.90, the results showed that the instrument has good accuracy.

3.1.1.2

3.1.1.2 Repeatability test

Six test samples of WZYZW (S5) were prepared in parallel according to the method in “1.4”, injected according to the chromatographic conditions in “1.2” and the chromatograms were recorded. The similarity was greater than 0.99 and the results exhibited that the method was reproducible.

3.1.1.3

3.1.1.3 Stability test

The test solution of WZYZW (S5) was injected into the HPLC at 0, 2, 4, 8, 10, and 24 h according to the conditions in “1.2”, recording the chromatogram. The similarity was greater than 0.93, and the results showed that the test solution had a good stability in 24 h.

3.1.2

3.1.2 Fingerprint mapping and similarity valuation

Take ten lots of WZYZW samples to prepare test solutions according to “1.4”, inject samples according to the chromatographic conditions under “1.2”, and the data with AIA style was imported into the “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine”. The S1 sample was set as a reference spectrum (median method with a time width of 0.1) (Fig. 1). A total of 28 common peaks were calibrated, and we identified 12 characteristic peaks by comparing them with the reference standards (Fig. 2), of which peak 1 was gallic acid, peak 3 was geniposidic acid, peak 8 was chlorogenic acid, peak 16 was ellagic acid, peak 17 was rutin, peak 18 was hyperoside, peak 19 was verbascoside, peak 20 was astragalin, peak 23 was kaempferol, peak 25 was Schisandrol A, peak 26 was Schisandrol B, and peak 28 was schisandrin B. The structures of the 12 components are shown in Fig. 3. In Table 1, we exhibited the SA information, in which the similarity of S2, S5, S6, and S8 is less than 0.9, indicating that there is a difference in quality between different batches of WZYZW, and the results of cluster analysis of each batch are shown in Fig. 4.

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Fig. 1

HPLC fingerprints for ten lots (S1 ∼ S10) of WZYZW, and batch R is the simulated chromatogram.

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Fig. 2

HPLC chromatogram of WZYZW test solution (1) and mixed standard solution (2). Note. 1, gallic acid. 3, geniposidic acid. 8, chlorogenic acid. 16, ellagic acid. 17, rutin. 18, hyperoside. 19, verbascoside. 20, astragalin. 23, kaempferol. 25, schisandrol A. 26, schisandrol B. 28, schisandrin B.

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Fig. 3

Chemical structures of gallic acid (a), geniposidic acid (b), chlorogenic acid (c), ellagic acid (d), rutin (e), hyperoside (f), verbascoside (g), astragalin (h), kaempferol (i), schisandrol A (j), schisandrol B (k), schisandrin B (l).

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Table 1 SA results for 10 batches of WZYZW samples.

No.	S1	S2	S3	S4	S5	S6	S7	S8	S9	S10	R
S1	1.000	0.965	0.896	0.963	0.983	0.991	0.979	0.983	0.983	0.970	0.986
S2	0.965	1.000	0.936	0.983	0.984	0.964	0.966	0.962	0.984	0.974	0.986
S3	0.896	0.936	1.000	0.971	0.889	0.876	0.952	0.892	0.920	0.961	0.946
S4	0.963	0.983	0.971	1.000	0.964	0.958	0.985	0.962	0.981	0.990	0.991
S5	0.983	0.984	0.889	0.964	1.000	0.982	0.965	0.981	0.989	0.964	0.984
S6	0.991	0.964	0.876	0.958	0.982	1.000	0.973	0.990	0.984	0.967	0.982
S7	0.979	0.966	0.952	0.985	0.965	0.973	1.000	0.978	0.981	0.995	0.993
S8	0.983	0.962	0.892	0.962	0.981	0.990	0.978	1.000	0.991	0.980	0.986
S9	0.983	0.984	0.920	0.981	0.989	0.984	0.981	0.991	1.000	0.986	0.995
S10	0.970	0.974	0.961	0.990	0.964	0.967	0.995	0.980	0.986	1.000	0.995
R	0.986	0.986	0.946	0.991	0.984	0.982	0.993	0.986	0.995	0.995	1.000

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Fig. 4

Clustering analysis of 10 batches of WZYZW.

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3.2

3.2 Network pharmacology predictive analysis

3.2.1

3.2.1 Prediction of compound targets

The SMILES of 12 compounds of gallic acid, geniposidic acid, hyperoside, rutin, chlorogenic acid, astragalin, ellagic acid, verbascoside, kaempferol, schisandrol A, schisandrol B, and schizandrin B were searched in the Pub Chem database, and SMILES was imported into the Swiss Target Prediction database for predictive analysis of the corresponding targets. Removal of repeated targets, we yielded 276 targets in all associated with 12 compounds.

3.2.2

3.2.2 PPI network construction

The 268 target proteins obtained were imported into the STRING database, set species selection as homo sapiens, and the highest confidence protein interaction parameter score value installed was >0.9, other parameter settings remain unchanged, remove a single node in the network, and obtain a PPI network diagram, see Fig. 5-a. The topological characterization of PPI is shown in Fig. 5-b, where the color was darker and the shape was larger, indicating that the target was more important. Points with a degree, betweenness, and closeness greater than the median and degree ≥35 were selected as core targets. 41 key targets in total were obtained after screening. The top 20 were AKT1, TNF, EGFR, SRC, HSP90AA1, STAT3, JUN, MTOR, ESR1, ERBB2, PTGS2, MMP9, PIK3CA, BCL2L1, MDM2, KDR, IL2, PIK3R1, JAK2 and AR (Table 2).

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Fig. 5

PPI network.

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Table 2 Key node information in the PPI network.

Target	Name	Degree	BetweennessCentrality
AKT1	Serine/threonine-protein kinase AKT	126	0.1412
TNF	TNF-alpha	118	0.1102
EGFR	Epidermal growth factor receptor erbB1	100	0.0490
SRC	Epidermal growth factor receptor erbB1	99	0.0709
HSP90AA1	Heat shock protein HSP 90-alpha	95	0.0524
STAT3	Signal transducer and activator of transcription 3	86	0.0222
JUN	Proto-oncogene c-JUN	85	0.0238
MTOR	Serine/threonine-protein kinase mTOR	84	0.0359
ESR1	Estrogen receptor alpha	78	0.0435
ERBB2	Receptor protein-tyrosine kinase erbB-2	74	0.0179
PTGS2	Cyclooxygenase-2	71	0.0322
MMP9	Matrix metalloproteinase 9	67	0.0132
PIK3CA	PI3-kinase p110-alpha subunit	64	0.0093
BCL2L1	Apoptosis regulator Bcl-X	61	0.0111
MDM2	p53-binding protein Mdm-2	57	0.0119
KDR	Vascular endothelial growth factor receptor 2	55	0.0060
IL2	Interleukin-2	55	0.0090
PIK3R1	PI3-kinase p85-alpha subunit	54	0.0070
JAK2	Tyrosine-protein kinase JAK2	53	0.0057
AR	Androgen Receptor	52	0.0267

3.2.3

3.2.3 GO enrichment and KEGG pathway analysis

GO (Gene Ontology) functional and KEGG pathway enrichment analysis of 41 potential core target were performed using the DAVID 2021 database. Both analysis had statistical significance (P < 0.05).

After GO enrichment analysis, some significant items (20 biological processes (BP), 15 molecular functions (MF), and 15 cellular components (CC)) were selected for display, as shown in Fig. 6. The results of GO analysis showed that BP was significantly gathered in protein phosphorylation、signal transduction, intracellular, signal transduction, positive regulation of gene expression and negative regulation of the apoptotic process. MF is mainly involved in the regulation of protein, adenosine triphosphate (ATP), the activity of protein kinase, transmembrane receptor protein tyrosine kinase, protein kinase, etc. CC is mainly enriched in cell fluid, cytoplasm, plasma membrane, etc.

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Fig. 6

GO enrichment analysis.

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140 pathways in total were gained by the KEGG enrichment study, and the pathway with a related gene number greater than 20 was chosen for display, as shown in Fig. 7, the enriched pathways mainly involved pathways in cancer, PI3K-Akt signaling pathway, proteoglycans in cancer, Endocrine resistance, EGFR tyrosine kinase inhibitor resistance, prostate cancer, Fluid shear stress and atherosclerosis, which indicated that these 41 core targets may be mainly regulated by regulating these pathways to achieve the purpose of intervening in disease.

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Fig. 7

KEGG enrichment analysis.

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3.2.4

3.2.4 “Drug-components-targets-pathways” network construction

The “drug-components-targets-pathways” network was constructed using Cytoscape 3.7.2 software based on the screened 12 components, 41 targets, and 20 pathways, and the results are shown in Fig. 8. The figure showed that WZYZW achieves its therapeutic effect through the synergistic effect of various components, various targets, and various pathways, and there was a close connection between ingredients, targets, and pathways; Among the 41 core targets, the connectivity of PIK3CA, AKT1, PIK3CB, PIK3CD, PIK3R1, EGFR, SRC, GSK3B, MAP2K1, PTK2, and IGF1R was higher than other targets, which may be the key target for WZYZW to exert efficacy. Among the 20 pathways, there is little difference in connectivity and all are likely to be key signaling pathways for WZYZW.

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Fig. 8

Network of “drug-components-targets-pathways”. Blue represents pathways; Red represents targets; Yellow represents components; Green represents WZYZW. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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3.2.5

3.2.5 Integrated analysis

As a classic prescription for tonifying the kidney and invigorating essence, WZYZW is comprehensively used in clinical applications with significant efficacy in the treatment of reproductive, tumor, urinary, nerve, and other diseases (Duan et al., 2015, Fu et al., 2017). GO enrichment analysis showed that WZYZW had a wide range of regulatory effects on cell signal transduction, positive regulation of gene expression, negative regulation of the apoptosis process, the activity of protein kinase and transmembrane receptor protein tyrosine kinase. In the KEGG enrichment analysis, WZYZW mainly regulates the activities of PI3K-Akt signaling and cancer-related receptor activity.

Schisandrol A is one of the main component of WZYZW, which can exert neuroprotective effects via stimulating the PI3K/Akt pathway, inhibiting the IKK/nuclear factor κB inhibitory protein pathway, reducing neuronal inflammation and oxidative stress response (Yan et al., 2019), and has protective effects on depression and cerebral ischemia (Ru et al., 2022); Ellagic acid could protective against cerebral ischaemia and kidney injury (Chen et al., 2021a, 2021b, 2021c Sun et al., 2022); Kaempferol can regulate the phosphorylation level of Akt protein in brain tissue to exert brain protective effects (Xu et al., 2022a, 2022b), can also restrain the multiplication of human colorectal cancer cells and induce their apoptosis, and downregulate the PI3K/Akt signaling pathway to induce autocannibalism in cancer cells (Gu et al., 2020); Researches have diaplayed that the pharmacological effects of hyperoside are manifested in the protection, anti-tumor, antidepressant and other aspects of kidney and liver (Chen et al., 2019, Liang et al., 2020, Zhu et al., 2022), AKT1 can be activated by extracellular signaling through PI3K-dependent mechanisms, is the core factor in the PI3K/AKT signaling pathway, and is related to the expression of cancer (Herberts et al., 2020, Li et al., 2022), and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene is closely related to the clinicopathological characteristics of breast cancer (Lin and Chen, 2022). The PI3K-AKT signaling pathway has also been associated with the treatment of depression, moreover the studies have shown that depression affects reproductive function, so WZYZW may modulate this signaling pathway to treat infertility (Huang et al., 2020, Zhu et al., 2021).

Bladder cancer is a common disease of the urinary system, studies have found that the process of bladder cancer will induce the expression of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB), p-AKT proteins, while PI3K/AKT signaling pathway is further activated, the connection of growth factors to their receptor tyrosine kinase (RTK) or G protein-coupled receptor (GPCR) stimulates the production of phosphatidylinositide 3-kinases (PI3K) isoform phosphatidyl-inositol, 3, 4, 5 triphosphate (PIP3), PIP3 as a second messenger, in turn, helps activate Akt, once activated, Akt can participate in a series of processes such as apoptosis and protein synthesis through phosphorylation pathways (Yang et al., 2021), and the application of WZYZW in urology may be related to inhibiting the activation of this signaling pathway. Epidermal growth factor receptor (EGFR) is a tyrosine kinase, the mutations in the EGFR gene and overexpression of proteins can cause cancer, and WZYZW's treatment for cancer may be to inhibit the activity of this enzyme, inhibit its gene mutation, and overexpression (Floc'h et al., 2020). WZYZW modulates the PI3K/Akt signaling pathway, phosphorylates Akt, activates the downstream transcription factor Nrf2, and thus playing a preventive role in the prevention and treatment of neural tube malformations through antioxidant stress and anti-apoptosis pathway (Chen et al., 2022a, 2022b), the above research shows that the results of network pharmacology prediction are close to the existing relevant literature, indicating that the research method has certain accuracy and reliability, and is also in line with the concept of the integrity of TCM. It provides a reference for further elaboration on the material basis of WZYZW. Multiple key components in WZYZW can act on multiple core targets, thereby intervening in multiple pathways and playing a role in treating or intervening in diseases. The 12 components identified in WZYZW have certain pharmacological effects, and the targets and pathways predicted by these components are relevant to the diseases treated by WZYZW. Combined with the essential request of Q-Markers, it can be preliminarily predicted that the 12 components are potential Q-markers for WZYZW. In order to further verify the rationality of this prediction, we will use QAMS to determine the content of these components in multiple batches of WZYZW.

4 Discussion

The quality of TCM is the lifeline of it and related industries, which has a bearing on clinical efficacy and the quality development of the TCM industry. The establishment of Q-Markers for TCM can promote quality control research and strengthen the quality evaluation system for further improvement.

In this research, the selection of the extraction process, mobile phase composition, flow rate, column temperature, detection wavelength, and chromatographic conditions of the test solution was based on the “2020” version of Chinese Pharmacopoeia (Ch.P.) (Chinese Pharmacopoeia Commission, 2020), combined with the preliminary research (Liao et al., 2021a, 2021b), and finally determined that 0.4 % phosphate-acetonitrile was used as the mobile phase, the volume flow was set to 1.0 mL/min, set the column temperature as 40℃, and set the wavelength as 245 nm so that the reference and test products had a suitable degree of resolution under such conditions. Therefore, based on these conditions, 10 batches of WZYZW fingerprints were established, and 12 components were identified from 28 common peaks.

In order to further search for the potential targets and pathways of these components, we used network pharmacology to analyze it from the perspective of effectiveness. The results showed that the 12 active components acted on cancer pathways, PI3K-Akt signaling pathways, prostate cancer, and other key targets by regulating PIK3CA, AKT1, PIK3CB, PIK3R1, EGFR, SRC, GSK3B, MAP2K1, PTK2, IGF1R to play the therapeutic role. Numerous investigations have shown that the 12 components have significant pharmacological effects on neurological diseases, liver damage, kidney damage, anticancer, anti-tumor, anti-testicular inflammation, etc. by multiple targets and pathways. As a result, we used the QAMS approach to assess the quality of WZYZW based on the 12 components.

Modern pharmacological research have revealed that these 12 components have considerable effects, and the interaction of these pharmacological effects is associated with the therapy of WZYZW. Geniposidic acid can increase the expression of growth-associated protein 43(GAP43) through activation PI3K/AKT pathway. Ultimately, it improved nerve damage in mice with Alzheimer's disease (AD), thereby alleviating AD (Chen et al., 2022a, 2022b); as a novel NLRP3-specific covalent inhibitor, geniposidic acid protects against liver injury due to excessive accumulation of bile acids by inhibiting its expression, thereby reducing bile acid-induced inflammation, and by participating in the regulation of bile transporters such as Bsep and Mrp2, thereby protecting against liver injury (Chen et al., 2016, Song et al., 2022), at the same time, geniposidic acid is also used in ischemic brain injury. Schisandrin B has shown strong pharmacological activity in anti-inflammatory (Chen et al., 2021a, 2021b, 2021c, Zhang et al., 2021), anti-tumor (Li et al., 2021a, 2021b), hepatoprotective (Wang et al., 2020, Chen et al., 2021a, 2021b, 2021c, Li et al., 2021a, 2021b, Yan et al., 2022), infertility (Zou et al., 2021) and other applications. Gallic acid can inhibit the production of colon cancer cells by regulating the formation of ferroptosis to play an anti-cancer effect (Hong et al., 2021), and other studies have shown that it exerts anti-tumor aversion by inhibiting the reproduction of cancer cells to make it apoptosis (Priyadarshi et al., 2021). A certain dose of gallic acid can also reduce the kidney tissue damage caused by diclofenac (DIC), Gallic acid treatment can significantly improve DIC-induced oxidative stress abnormalities and serum biochemical parameters (Moradi et al., 2021). AD is a persistent neurodegenerative disease manifested by neuroinflammation, mitochondrial dysfunction, increased oxidative stress, weakened antioxidant defenses, and elevated acetylcholinesterase activity. Other manifestation was amyloidosis in the brain, which was found that given gallic acid treatment to mice with the characteristics of the disease, amyloidosis β in the cerebral cortex and cerebral vessels was alleviated, and the mechanism was related to direct inhibition of β-site APP-cleaving enzyme 1 (BACE1) activity (Mori et al., 2020). As a dimer of gallic acid, ellagic acid protects neurons through antioxidant action, iron chelation, and mitochondrial protection (Javaid et al., 2021). Chlorogenic acid plays a pharmacological role in anti-inflammatory, neuroprotective, anti-cancer, etc. (Naveed et al., 2018, Munteanu and Apetrei, 2021). Hyperoside is a natural flavonoid with antitumor, anticancer, antidepressant, and other effects (Wang et al., 2022a, 2022b, Xu et al., 2022a, 2022b), which can inhibit the proliferation of hepatocellular carcinoma (HCC) by inhibiting the PI3K/AKT signaling pathway. The mechanism is that hyperoside can reduce the expression of bone morphogenetic protein 7 (BMP-7), thereby inducing cell cycle arrest in HepG2 cells (Wei et al., 2021). Verbascoside have pharmacological effects such as anticancer, neuropathy, and anti-testicular injury (Alipieva et al., 2014, Han et al., 2021, Wu et al., 2021). It is possible to improve sperm levels by resisting testicular damage, thus treating male infertility, which is consistent with the clinical application of WZYZW. Therefore, it is necessary to carry out a quality evaluation to provide a reference for the construction of the WZYZW quality evaluation system.

In summary, based on fingerprint and network pharmacology, this study preliminarily predicted that gallic acid, geniposidic acid, rutin, hyperoside, chlorogenic acid, ellagic acid, verbascoside, astragalin, kaempferol, schisandrol A, schisandrol B, and schisandrin B were the potential Q-markers of WZYZW, and conducted quality studies on 12 components in multiple batches of WZYZW, to provide a foundation and reference for further studies on quality control as well as theoretical support for in-depth studies on the mechanism of WZYZW. However, the current research on WZYZW is still insufficient. For example, the basis of some chemical substances related to traditional efficacy is not clear, and it still stays in the stage of crude extract researching, the research on pharmacological effects and mechanisms is not deep enough, and the quality control research is not systematic enough, so the development and utilization of WZYZW is limited to a certain extent. Therefore, further research on WZYZW can be conducted with the help of cutting-edge technologies such as metabolomics and mass spectrometry imaging according to the overall model of Q-Marker research.

5 Conclusion

In this study, a fingerprint of 10 batches of WZYZW was established. By comparing with the standards, 12 components were identified from 28 common peaks, which are gallic acid, geniposidic acid, rutin, hyperoside, chlorogenic acid, astragalin, ellagic acid, verbascoside, kaempferol, schisandrol A, schisandrol B, and schisandrin B. Then, based on network pharmacology, the “drug-components-targets-pathways” network was constructed, its potential targets and pathways were analyzed. It was found that WZYZW can exert therapeutic effects in a multi-component, multi-target, and multi-pathway. Finally, a QAMS method capable of simultaneously determining the content of 12 components in WZYZW was established and their quality was analyzed. In conclusion, the Q-marker of WZYZW was preliminarily predicted from three fingerprint, network pharmacology and QAMS as well as could lay the foundation of quality control research of WZYZW. However, the effective constituents, active targets and signaling pathways obtained in research need to be confirmed and validated by animal experiment in further studies.

CRediT authorship contribution statement

Bao-Hua Dong: Writing – original draft, Writing – review & editing. Jie Wu: Software, Visualization. Ying Peng: Supervision. Yun-Xiu Jiang: Supervision. Ma-Yi-Jie Cao: Supervision. Yu Huang: Investigation, Supervision. Chang-Jiang Hu: Funding acquisition, Supervision. Ling-ying Yu: Supervision. Zhi-Min Chen: Conceptualization, Project administration, Supervision, Funding acquisition.