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Zhi-Ke-Bao pills (ZKB), a traditional Chinese medicine preparation composed of 13 herbs, is generally used to treat cough caused by external wind cold, phlegm, etc in clinical applications, and it plays a core role in relieving cough caused by COVID-19 and influenza in China. Till now, the understanding of its chemical constituents was dramatically limited due to its chemical complexity, restricting its clinical application or development. In this work, a developed ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) method, a targeted and non-targeted strategy and network pharmacology were used to comprehensively characterize the chemical compositions in ZKB and predict its mechanism against cough. A total of 164 compounds (148 targeted compounds and 16 non-targeted ones) were identified or tentatively characterized in ZKB, including 65 flavonoids, 25 alkaloids, 19 organic acids, 41 saponins, 9 coumarins, 2 phenylpropanoids, 2 anthraquinones, and 1 other types. Among them, 37 compounds were unambiguously identified by comparison to reference standards. Meanwhile, the fragmentation behaviors of five main chemical structure types were also summarized. 309 targets and two core signaling pathways of ZKB against cough were predicted by network pharmacology, including MAPK and PI3K-Akt signaling pathways. It was the first time to characterize the chemical compounds of ZKB and reveal its potential mechanism against cough, providing the material basis for further quality control or pharmacodynamic evaluation of ZKB., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Feng-Xiang Zhang reports financial support was provided by Teyi Pharmaceutical Group Co., Ltd. Qing-Min Zhang reports a relationship with Teyi Pharmaceutical Group Co., Ltd that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Rationale: Isopsoralen (ISO), a quality control marker (Q-marker) in Psoraleae Fructus, is proven to present an obvious anti-osteoporosis effect. Until now, the metabolism and anti-osteoporosis mechanisms of ISO have not been fully elucidated, greatly restricting its drug development., Methods: The metabolites of ISO in rats were profiled by using ultrahigh-performance liquid chromatography coupled with time-of-flight mass spectrometry. The potential anti-osteoporosis mechanism of ISO in vivo was predicted by using network pharmacology., Results: A total of 15 metabolites were characterized in rats after ingestion of ISO (20 mg/kg/day, by gavage), including 2 in plasma, 12 in urine, 6 in feces, 1 in heart, 3 in liver, 1 in spleen, 1 in lung, 3 in kidney, and 2 in brain. The pharmacology network results showed that ISO and its metabolites could regulate AKT1, SRC, NFKB1, EGFR, MAPK3, etc., involved in the prolactin signaling pathway, ErbB signaling pathway, thyroid hormone pathway, and PI3K-Akt signaling pathway., Conclusions: This is the first time for revealing the in vivo metabolism features and potential anti-osteoporosis mechanism of ISO by metabolite profiling and network pharmacology, providing data for further verification of pharmacological mechanism., (© 2024 John Wiley & Sons Ltd.)

Rationale: Eucommia cortex is the core herb in traditional Chinese medicine preparations for the treatment of osteoporosis. Pinoresinol diglucoside (PDG), the quality control marker and the key pharmacodynamic component in Eucommia cortex, has attracted global attention because of its definite effects on osteoporosis. However, the in vivo metabolic characteristics of PDG and its anti-osteoporotic mechanism are still unclear, restricting its development and application., Methods: Ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used to analyze the metabolic characteristics of PDG in rats, and its anti-osteoporosis targets and mechanism were predicted using network pharmacology., Results: A total of 51 metabolites were identified or tentatively characterized in rats after oral administration of PDG (10 mg/kg/day), including 9 in plasma, 28 in urine, 13 in feces, 10 in liver, 4 in heart, 3 in spleen, 11 in kidneys, and 5 in lungs. Furan-ring opening, dimethoxylation, glucuronidation, and sulfation were the main metabolic characteristics of PDG in vivo. The potential mechanism of PDG against osteoporosis was predicted using network pharmacology. PDG and its metabolites could regulate BCL2, MARK3, ALB, and IL6, involving PI3K-Akt signaling pathway, estrogen signaling pathway, and so on., Conclusions: This study was the first to demonstrate the metabolic characteristics of PDG in vivo and its potential anti-osteoporosis mechanism, providing the data for further pharmacological validation of PDG in the treatment of osteoporosis., (© 2024 John Wiley & Sons Ltd.)

Background: Quality control exerted great importance on the clinical application of drugs for ensuring effectiveness and safety. Due to chemical complexity, diversity among different producing areas and harvest seasons, as well as unintentionally mixed with non-medicinal parts, the current quality standards of traditional Chinese medicine (TCM) still faced challenges in evaluating the overall chemical consistency.Purpose: We aimed to develop a new strategy to discover potential quality marker (Q-marker) of TCM by integrating plant metabolomics and network pharmacology, using Periplocae Cortex (GP, the dried root bark of Periploca sepium Bge.) as an example.Methods: First, plant metabolomics analysis was performed by UPLC/Q-TOF MS in 89 batches of samples to discover chemical markers to distinguish medicinal parts (GP) and non-medicinal parts (the dried stem bark of Periploca sepium Bge. (JP)), harvest seasons and producing region of Periplocae Cortex. Second, network pharmacology was applied to explore the initial linkages among chemical constituents, targets and diseases. Last, potential Q-marker were selected by integrating analysis of plant metabolomics and network pharmacology, and the quantification method of Q-marker was developed by using UPLC-TQ-MS.Results: The chemical profiling of GP and JP was investigated. Fifteen distinguishing features were designated as core chemical markers to distinguish GP and JP. Besides, the content of 4-methoxybenzaldehyde-2-O-β-d-xylopyranosyl-(1→6)-β-d-glucopyranoside could be used to identify Periplocae Cortex harvested in spring-autumn or summer. Meanwhile, a total of 15 components targeted rheumatoid arthritis were screened out based on network pharmacology. Taking absorbed constituents into consideration, 23 constituents were selected as potential Q-marker. A simultaneous quantification method (together with 11 semi-quantitative analysis) was developed and applied to the analysis of 20 batches of commercial Periplocae Cortex on the market. The PLS-DA model was successfully developed to distinguish GP and JP samples. In addition, the artificially mixed GP sample, which contained no less than 10% of the adulterant (JP), could also be correctly identified.Conclusion: Our results indicated that 9 ingredients could be considered as Q-marker of Periplocae Cortex. This study has also demonstrated that the plant metabolomics and network pharmacology could be used as an effective approach for discovering Q-marker of TCM to fulfill the evaluation of overall chemical consistency among samples from different producing areas, harvest seasons, and even those commercial crude drugs, which might be mixed with a small amount of non-medicinal parts. [ABSTRACT FROM AUTHOR]

Shuang-Huang-Lian preparation has captured wide attention since its clinical applications for the successful treatment of upper respiratory tract infection. However, its functional basis under actual therapeutic dose in vivo was still unrevealed. This study aimed to reveal the anti-flu substances and mechanism of Shuang-Huang-Lian water extract (SHL) on H1N1 infected mouse model by a strategy based on serum pharmaco-chemistry under actual therapeutic dose and network pharmacology. H1N1 infected mouse model was employed for evaluation of the anti-flu effects of SHL. A simultaneous quantification method was developed by UPLC-TQ-XS MS coupled switch-ions mode and applied to characterize the pharmacokinetics of the multiple components of SHL under actual therapeutic dose. The potential active ingredients were screened out based on their pharmacokinetic parameters. And then, a compound mixture of these active candidates was re-evaluated for the anti-flu activity on H1N1 infected mouse model. Furthermore, the anti-flu mechanism of SHL was also predicted by network pharmacology coupled with the experimental result. SHL significantly increased the survival rate and prolonged survival days on H1N1 infected mice at a dosage of 20 g crude drug/kg/day by reversing the increased lung index, down-regulating the inflammatory cytokines (TNF-α, IL-1β, IL-6) and inhibiting the release of IFN-β in bronchoalveolar lavage fluids (BALF). Concomitantly, the pharmacokinetic parameters of fourteen quantified and twenty-one semi-quantified constituents of SHL were characterized. And then, five compounds (baicalin, sweroside, chlorogenic acid, forsythoside A and phillyrin), which displayed satisfactory pharmacokinetic features, were considered as potential active ingredients. Thus, a mixture of these five ingredients was administered to H1N1-infected mice at a dose of 4.24 mg/kg/day. As a result, the therapeutical effects of the mixture were similar to SHL in terms of survival rate, lung index and the release of cytokines (TNF-α, IL-1β and IL-6) in BALF. Moreover, network pharmacology analysis indicated that the TNF-signal pathways might play a role in the anti-flu mechanism of SHL. A mixture of five compounds (baicalin, sweroside, chlorogenic acid, forsythoside A and phillyrin) were the anti-flu substances of SHL. The strategy based on serum pharmaco-chemistry under actual therapeutic dose provided a new sight on exploring in vivo effective substances of TCM. Image 1 • A new strategy based on serum pharmaco-chemistry and network pharmacology was proposed and applied to explore anti-flu substances of SHL. • SHL showed significant anti-flu effects on H1N1 infected mice at a therapeutic dose (i.g. 20 g crude drug/kg/day). • Anti-flu effect of a mixture (baicalin, sweroside, chlorogenic acid, forsythoside A and phillyrin) was similar to SHL. • Network pharmacology analysis indicated that the TNF-signal pathways might play a role in the anti-flu mechanism of SHL. • The new strategy proposed in this paper provided a new sight on exploring in vivo effective substances of TCM. [ABSTRACT FROM AUTHOR]

Morusin, a prenylated flavone isolated from Morus species, was treated as a potential anti-tumor drug since it inhibited effects on numerous types of human cancer cells. In some sense, drugs-related metabolites always contribute to pharmacological changes, inducing therapy improvement, reduced efficacy, or side-effects. Thus, the characterization of metabolites and the potential functions exerted great importance in clinical applications. Till now, the metabolism feature of morusin was still unclear, and its pharmacological changes were still unconducted, too. In this work, an integrated strategy based on metabolites profiling and network pharmacological was applied to characterize the metabolism feature and reveal pharmacological changes of morusin in vivo. As a result, a total of 31 metabolites (19 in plasma, 8 in urine, 30 in feces, 6 in heart, 17 in liver, 4 in spleen, 6 in lung, 6 in kidney, and 3 in brain) were screened out in rats, and 11 of them were characterized for the first time. Among them, metabolites M6 , M18 , M19 , M20 , and M28 , were the main metabolites. Phase I metabolic reactions of hydroxylation, dehydrogenation (cyclization), isomerization and phase II reactions of glucuronidation occurred, and glucuronidation and hydroxylation were the two main metabolic reactions. Moreover, the pharmacological difference between morusin and five main metabolites was predicted by an network pharmacological method, and these 6 candidates targeted 177 targets. Meanwhile, in addition to common pathways (PI3K-Akt signaling pathway, proteoglycans in cancer, hepatitis B, cAMP signaling pathway, and viral carcinogenesis) of morusin in cancer, six metabolites' targets were involved in prostate cancer, chemokine signaling pathway, ras signaling pathway and neuroactive ligand-receptor interaction, indicating that these functional changes might result in novel pharmacological mechanism and new indications. Our work provided the metabolism feature and functional modifications of morusin in vivo for the first time, and meaningful information for further pharmacological validations or potential indications in clinic were supplied. [ABSTRACT FROM AUTHOR]

• The chemical components of Lonicerae japonicae flos was systematically profiled by UHPLC/Q-TOF MS, especially for N-contained iridoid glycosides. • Ten potential novel N-contained iridoid glycosides were found in Lonicerae japonicae flos. • A strategy based on in vivo substances' profiling and network pharmacology was proposed and successfully applied. Lonicerae japonicae flos. (LJF) was widely used as a drug to treat upper respiratory tract infection or a tea to clear heat in Asian countries for thousands of years. Despite of its curative effects confirmed by modern pharmacological methods, its functional materials and mechanism against influenza were still unclear and needed further investigation. In this study, an integrated strategy based on in vivo substances profiling and network pharmacology was proposed and applied to screen out the potential anti-influenza substances and mechanism of LJF. An UHPLC/Q-TOF MS method was utilized to profile the chemical components in LJF and their metabolites in rats. The targets of absorbed prototypes were predicted by Swiss Target Prediction, and they were further analyzed by String and Kyoto Encyclopedia of Genes and Genomes (KEGG). As a result, a total of 126 chemical components mainly featuring three chemical structure types were characterized, including 70 iridoid glycosides, 17 caffeoylquinic acids, 24 flavonoids, and 15 other types compounds. Among them, ten N-contained iridoid glycosides were characterized as potential novel compounds. Moreover, 141 xenobiotics (74 prototypes and 67 metabolites) were clearly screened out in rat plasma and urine after ingestion of LJF. Phase II reactions (sulfation, glucuronidation, methylation) and phase I reactions (dehydroxylation, hydrogenation, hydrolysis, N-heterocyclization) were the main metabolic reactions of LJF in rats. Further, a total of 338 targets were predicted and TNF, PTGS2 and EGFR were the three main targets involved in the pathology of influenza. In addition to normal NF-κB pathway, T cell signal pathway and mTOR signal pathway were the other patterns for LJF to achieve its anti-flu effects. Our work provided the meaningful data for further pharmacological validation of LJF against influenza, and a new strategy was also proposed for minimizing the process to reveal the mechanism and functional basis of TCMs. [ABSTRACT FROM AUTHOR]

• An integrated strategy based on metabolites profiling and network pharmacology was proposed. • A total of 53 arctiin-related metabolites were characterized in rats. • One metabolic pattern and 38 metabolites of arctiin were reported for the first time. • The potential effects of arctiin against hepatitis B or viral carcinogenesis were revealed. Traditional Chinese medicine was widely used in China since its definite effects and therapy. The components of TCM were absorbed into the circle system as the format of prototypes or metabolites, which contributed to the therapy or side effects. Declaring the functional changes in this process was of great importance to the clinical applications. In this work, an integrated strategy based on metabolites' profiling and network pharmacology was proposed for exploring the pharmacological changes of compounds in vivo. Arctiin, the main component in Fructus Arctii with various kinds of bioactivities, was used as an example. An ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry and metabolynx™ software was applied to characterize the metabolites of arctiin in rats at a dosage of 100 mg/kg; network pharmacology was applied to characterize the functional changes. As a result, fifty-three metabolites (32 in plasma, 40 in urine, 19 in bile, 20 in feces, 1 in brain, 12 in liver and 4 in lungs) were screened out and characterized, and 3 of them were unambitiously identified by comparison with standard substances. Among them, 38 metabolites were reported for the first time. It was found the major metabolic pathways of arctiin in rats were demethylation, lactone-opening and phase II conjugations with sulfate and glucuronide. It also confirmed that M14, M15, M18, M23, M22, M43 and M45 were the major circulating forms of arctiin in rats following oral administration. In addition to the above metabolic reactions, phase I reactions of hydrolysis, demethylation, dehydroxylation were also observed, and dehydrogenation were first revealed metabolic patterns of arctiin in rats. Meanwhile, in addition to the main targets of arctiin (MTOR, EGFR and MAPK14), its metabolites targeted additional 392 targets with additional functions of anti-hepatitis B or viral carcinogenesis (SRC, CAPS3, PIK3CA, CDK4, ESR1, MMP9 and ERBB2). The above results provided very important information for understanding the metabolism and functional changes of arctiin in vivo, and supporting data for further pharmacological evaluation. Our work also provided a newsight for elucidation of functional changes of TCMs in vivo. [ABSTRACT FROM AUTHOR]