Untargeted Metabolomics Based on Ultra-High-Performance Liquid Chromatography Coupled with Quadrupole Orbitrap High-Resolution Mass Spectrometry for Differential Metabolite Analysis of Pinelliae Rhizoma and Its Adulterants.

The present study investigates the chemical composition variances among Pinelliae Rhizoma, a widely used Chinese herbal medicine, and its common adulterants including Typhonium flagelliforme, Arisaema erubescens, and Pinellia pedatisecta. Utilizing the non-targeted metabolomics technique of employing UHPLC-Q-Orbitrap HRMS, this research aims to comprehensively delineate the metabolic profiles of Pinelliae Rhizoma and its adulterants. Multivariate statistical methods including PCA and OPLS-DA are employed for the identification of differential metabolites. Volcano plot analysis is utilized to discern upregulated and downregulated compounds. KEGG pathway analysis is conducted to elucidate the differences in metabolic pathways associated with these compounds, and significant pathway enrichment analysis is performed. A total of 769 compounds are identified through metabolomics analysis, with alkaloids being predominant, followed by lipids and lipid molecules. Significant differential metabolites were screened out based on VIP > 1 and p-value < 0.05 criteria, followed by KEGG enrichment analysis of these differential metabolites. Differential metabolites between Pinelliae Rhizoma and Typhonium flagelliforme, as well as between Pinelliae Rhizoma and Pinellia pedatisecta, are significantly enriched in the biosynthesis of amino acids and protein digestion and absorption pathways. Differential metabolites between Pinelliae Rhizoma and Arisaema erubescens are mainly enriched in tyrosine metabolism and phenylalanine metabolism pathways. These findings aim to provide valuable data support and theoretical references for further research on the pharmacological substances, resource development and utilization, and quality control of Pinelliae Rhizoma.

[Existence forms of Tiantian Capsules and its raw material Aloe in rats].

This study explored the existence forms(original constituents and metabolites) of Tiantian Capsules, Aloe, and Tiantian Capsules without Aloe in rats for the first time, aiming to clarify the contribution of Aloe to the existence form of Tiantian Capsules. Rats were administrated with corresponding drugs by gavage once a day for seven consecutive days. All urine and feces samples were collected during the seven days of administration, and blood samples were collected 0.5, 1, and 1.5 h after the last administration. UHPLC-Q-TOF-MS was employed to detect and identify the original constituents and metabolites in the samples. A total of 34, 28, and 2 original constituents and 64, 94, and 0 metabolites were identified in the samples of rats administrated with Aloe, Tiantian Capsules, and Tiantian Capsules without Aloe, respectively. The main metabolic reactions were methylation, hydrogenation, hydroxylation, dehydroxylation, glucuronidation, and sulfation. This study clarified for the first time the existence forms and partial metabolic pathways of Aloe, Tiantian Capsules, and Tiantian Capsules without Aloe in rats, laying a foundation for revealing their effective forms. The findings are of great significance to the research on the functioning mechanism and quality control of Aloe and Tiantian Capsules.

Epiberberine inhibits bone metastatic breast cancer-induced osteolysis.

ETHNOPHARMACOLOGICAL RELEVANCE: The anti-tumor related diseases of Coptidis Rhizoma (Huanglian) were correlated with its traditional use of removing damp-heat, clearing internal fire, and counteracting toxicity. In the recent years, Coptidis Rhizoma and its components have drawn extensive attention toward their anti-tumor related diseases. Besides, Coptidis Rhizoma is traditionally used as an anti-inflammatory herb. Epiberberine (EPI) is a significant alkaloid isolated from Coptidis Rhizoma, and exhibits multiple pharmacological activities including anti-inflammatory. However, the effect of epiberberine on breast cancer and the inflammatory factors of metastatic breast cancer-induced osteolysis has not been demonstrated clearly. AIM OF THE STUDY: Bone metastatic breast cancer can lead to osteolysis via inflammatory factors-induced osteoclast differentiation and function. In this study, we try to analyze the effect of epiberberine on breast cancer and the inflammatory factors of metastatic breast cancer-induced osteolysis. METHODS: To evaluate whether epiberberine could suppress bone metastatic breast cancer-induced osteolytic damage, healthy female Balb/c mice were intratibially injected with murine triple-negative breast cancer 4T1 cells. Then, we examined the inhibitory effect and underlying mechanism of epiberberine on breast cancer-induced osteoclastogenesis in vitro. Xenograft assay was used to study the effect of epiberberine on breast cancer cells in vivo. Moreover, we also studied the inhibitory effects and underlying mechanisms of epiberberine on RANKL-induced osteoclast differentiation and function in vitro. RESULTS: The results show that epiberberine displayed potential therapeutic effects on breast cancer-induced osteolytic damage. Besides, our results show that epiberberine inhibited breast cancer cells-induced osteoclast differentiation and function by inhibiting secreted inflammatory cytokines such as IL-8. Importantly, we found that epiberberine directly inhibited RANKL-induced differentiation and function of osteoclast without cytotoxicity. Mechanistically, epiberberine inhibited RANKL-induced osteoclastogensis via Akt/c-Fos signaling pathway. Furthermore, epiberberine combined with docetaxel effectively protected against bone loss induced by metastatic breast cancer cells. CONCLUSIONS: Our findings suggested that epiberberine may be a promising natural compound for treating bone metastatic breast cancer-induced osteolytic damage by inhibiting IL-8 and is worthy of further exploration in preclinical and clinical trials.

Yinhuang buccal tablet alters airway microbiota composition and metabolite profile in healthy humans.

ETHNOPHARMACOLOGICAL RELEVANCE: Perturbations in airway microbiota composition and disruption of microbe-metabolite interactions have been observed in respiratory infectious diseases (RIDs). The Yinhuang (YH) buccal tablet, as an ancient Chinese medicinal formula, has been traditionally employed for the management of upper RIDs. However, there is a lack of evidence for the effects of YH buccal tablets on upper respiratory tract microbiota and circulating metabolites. AIM OF THE STUDY: The aim of this study was to analyze the changes in respiratory microbiota composition and circulating metabolite profile after YH buccal tablets administration. MATERIALS AND METHODS: Throat swab samples and serum samples were collected from 60 healthy subjects for high-throughput 16S ribosomal RNA gene (16S rRNA) sequencing and non-targeted Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. RESULTS: Airway microbial composition changed significantly after YH administration. The abundance of Actinomyces and Prevotella_7 increased, while the abundance of potentially pathogenic Pseudomonas and Corynebacterium decreased. A total of 168 significant HMDB taxonomic metabolites were identified in serum samples, of which lipid metabolites accounted for the largest proportion. Correlation analysis showed that circulatory metabolites were significantly correlated with changes in airway microbiota composition. CONCLUSIONS: YH buccal tablets can inhibit opportunistic pathogens, increase beneficial microorganisms in the upper respiratory tract, and regulate the body's metabolic pathways. These findings provide insights into the mechanism of action of YH buccal tablets in the treatment and prevention of respiratory diseases.

Elevated KDM4D Expression in Pterygium: Impact and Potential Inhibition by Lycium Barbarum Polysaccharide.

Purpose: This study aimed to explore the effects of elevated KDM4D expression and potential therapeutic effects of Lycium barbarum polysaccharide (LBP) on pterygium. Methods: The expression levels of KDM4D in the primary pterygium (n = 29) and normal conjunctiva (n = 14) were detected by immunohistochemistry. The effects of KDM4D on pterygium fibroblasts were detected by the CCK-8 assay, liquid chromatography-mass spectrometry assay, flow cytometry, and scratch wound healing assay. The relative expression of KDM4D in pterygium fibroblasts stimulated by interleukin (IL)-1ß, IL-6, IL-8, and LBP was detected by quantitative real-time PCR and Western blot. The effects of LBP on pterygium fibroblasts were detected using flow cytometry and scratch wound healing assays. Results: The expression level of KDM4D in pterygium was higher than that in normal conjunctiva. KDM4D increased the cell viability of pterygium fibroblasts. The differentially expressed genes identified in the LM-MS assay enriched in "actin filament organization" and "apoptosis." KDM4D promoted migration and inhibited apoptosis of pterygium fibroblasts in vitro. Inflammatory cytokines, including IL-1ß, IL-6, and IL-8, enhanced the expression of KDM4D in pterygium fibroblasts. LBP inhibited the expression of KDM4D in pterygium fibroblasts and decreased their cell viability. Moreover, LBP attenuated the KDM4D effects on migration and apoptosis of pterygium fibroblasts. Conclusions: Elevated KDM4D expression is a risk factor for pterygium formation. LBP inhibits the expression of KDM4D in pterygium fibroblasts and may be a potential drug for delaying pterygium development.

Effective substances and molecular mechanisms guided by network pharmacology: An example study of Scrophulariae Radix treatment of hyperthyroidism and thyroid hormone-induced liver and kidney injuries.

ETHNOPHARMACOLOGICAL RELEVANCE: Scrophulariae Radix (Xuanshen [XS]) has been used for several years to treat hyperthyroidism. However, its effective substances and pharmacological mechanisms in the treatment of hyperthyroidism and thyroid hormone-induced liver and kidney injuries have not yet been elucidated. AIM OF THE STUDY: This study aimed to explore the pharmacological material basis and potential mechanism of XS therapy for hyperthyroidism and thyroid hormone-induced liver and kidney injuries based on network pharmacology prediction and experimental validation. MATERIALS AND METHODS: Based on 31 in vivo XS compounds identified using ultra-performance liquid chromatography tandem quadruple exactive orbitrap high-resolution accurate-mass spectrometry (UPLC-QE-HRMS), a network pharmacology approach was used for mechanism prediction. Systematic networks were constructed to identify the potential molecular targets, biological processes (BP), and signaling pathways. A component-target-pathway network was established. Mice were administered levothyroxine sodium through gavage for 30 d and then treated with different doses of XS extract with or without propylthiouracil (PTU) for 30 d. Blood, liver, and kidney samples were analyzed using an enzyme-linked immunosorbent assay (ELISA) and western blotting. RESULTS: A total of 31 prototypes, 60 Phase I metabolites, and 23 Phase II metabolites were tentatively identified in the plasma of rats following the oral administration of XS extract. Ninety-six potential common targets between the 31 in vivo compounds and the diseases were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that Bcl-2, BAD, JNK, p38, and ERK1/2 were the top targets. XS extract with or without PTU had the following effects: inhibition of T3/T4/fT3/fT4 caused by levothyroxine; increase of TSH levels in serum; restoration of thyroid structure; improvement of liver and kidney structure and function by elevating the activities of anti-oxidant enzymes catalase (CAT),superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px); activation anti-apoptotic proteins Bcl-2; inhibition the apoptotic protein p-BAD; downregulation inflammation-related proteins p-ERK1/2, p-JNK, and p-p38; and inhibition of the aggregation of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6, as well as immune cells in the liver. CONCLUSION: XS can be used to treat hyperthyroidism and liver and kidney injuries caused by thyroid hormones through its anti-oxidant, anti-inflammatory, and anti-apoptotic properties. In addition, serum pharmacochemical analysis revealed that five active compounds, namely 4-methylcatechol, sugiol, eugenol, acetovanillone, and oleic acid, have diverse metabolic pathways in vivo and exhibit potential as effective therapeutic agents.

A study integrated metabolomics and network pharmacology to investigate the effects of Shicao in alleviating acute liver injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Shicao is the aerial part of Achillea alpina L., a common herb found mainly in Europe, Asia, and North America. Traditional Chinese medicine has a history of thousands of years and is widely used to treat various diseases. AIM OF STUDY: To explore the hepatoprotective effects of Shicao on CCl4-induced acute liver injury. METHODS: A rat model of acute liver injury was established and liver function indices were assessed to evaluate the protective effect of Shicao on the liver. Untargeted metabolomics of the serum and liver tissues was conducted using UPLC-Q-TOF/MS to identify differential metabolites related to acute liver injury. A network of metabolite-reaction-enzyme-gene constituents was constructed using network pharmacology. Hub targets and key components of the effect of Shicao on acute liver injury were screened from the network. RESULTS: Compared to the model group, Shicao improved the degree of liver damage through the assessment of the liver index, ALT and AST levels, and hepatic pathology slices, demonstrating its hepatoprotective effect against acute liver injury in rats. 10 and 38 differential metabolites involved in acute liver injury were identified in serum and liver tissues, respectively. Most of these were regulated or restored following treatment with Shicao, which mainly consisted of bile acids, lipids, and nucleotides such as taurocholic acid, LysoPC (17:0), and adenosine diphosphate ribose. Through the network of metabolite-reaction-enzyme-gene-constituents, 10 key components and 5 hub genes, along with 7 crucial differential metabolites, were mainly involved in glycerophospholipid metabolism, purine metabolism, biosynthesis of unsaturated fatty acids, and primary bile acid biosynthesis, which may play important roles in the prevention of acute liver injury by Shicao. CONCLUSION: This study revealed that Shicao had protective effects against CCl4-induced liver injury in rats. It was speculated that the ingredients of Shicao might be closely related to the hub targets, thereby regulating the levels of key metabolites, affecting inflammatory response and oxidative stress and attenuate the liver injury consequently. This study provides a basis for further investigation of its therapeutic potential and the mechanism of action.

Effect and pharmacological mechanism of Salvia miltiorrhiza and its characteristic extracts on diabetic nephropathy.

ETHNOPHARMACOLOGICAL RELEVANCE: Diabetic nephropathy (DN) is a severe diabetic microvascular complication with an increasing prevalence rate and lack of effective treatment. Traditional Chinese medicine has been proven to have favorable efficacy on DN, especially Salvia miltiorrhiza Bunge (SM), one of the most critical and conventional herbs in the treatment. Over the past decades, studies have demonstrated that SM is a potential treatment for DN, and the exploration of the underlying mechanism has also received much attention. AIM OF THIS REVIEW: This review aims to systematically study the efficacy and pharmacological mechanism of SM in the treatment of DN to understand its therapeutic potential more comprehensively. MATERIALS AND METHODS: Relevant information was sourced from Google Scholar, PubMed, Web of Science, and CNKI databases. RESULTS: Several clinical trials and systematic reviews have indicated that SM has definite benefits on the kidneys of diabetic patients. And many laboratory studies have further revealed that SM and its characteristic extracts, mainly including salvianolic acids and tanshinones, can exhibit pharmacological activity against DN by the regulation of metabolism, renal hemodynamic, oxidative stress, inflammation, fibrosis, autophagy, et cetera, and several involved signaling pathways, thereby preventing various renal cells from abnormal changes in DN, including endothelial cells, podocytes, epithelial cells, and mesangial cells. CONCLUSION: As a potential drug for the treatment of DN, SM has multi-component, multi-target, and multi-pathway pharmacological effects. This work will not only verify the satisfactory curative effect of SM in the treatment of DN but also provide helpful insights for the development of new anti-DN drugs and the application of traditional Chinese medicine.

Unlocking the hidden potential: Enhancing the utilization of stems and leaves through metabolite analysis and toxicity assessment of various parts of Aconitum carmichaelii.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconitum carmichaelii is widely used in traditional Chinese medicine clinics as a bulk medicinal material. It has been used in China for more than two thousand years. Nevertheless, the stems and leaves of this plant are usually discarded as non-medicinal parts, even though they have a large biomass and exhibit therapeutic properties. Thus, it is crucial to investigate metabolites of different parts of Aconitum carmichaelii and explore the relationship between metabolites and toxicity to unleash the utilization potential of the stems and leaves. AIM OF THE STUDY: Using plant metabolomics, we aim to correlate different metabolites in various parts of Aconitum carmichaelii with toxicity, thereby screening for toxicity markers. This endeavor seeks to offer valuable insights for the development of Aconitum carmichaelii stem and leaf-based applications. MATERIALS AND METHODS: UHPLC-Q-Orbitrap MS/MS-based plant metabolomics was employed to analyze metabolites of the different parts of Aconitum carmichaelii. The cardiotoxicity and hepatotoxicity of the extracts from different parts of Aconitum carmichaelii were also investigated using zebrafish as animal model. Toxicity markers were subsequently identified by correlating toxicity with metabolites. RESULTS: A total of 113 alkaloids were identified from the extracts of various parts of Aconitum carmichaelii, with 64 different metabolites in stems and leaves compared to daughter root (Fuzi), and 21 different metabolites in stems and leaves compared to mother root (Wutou). The content of aporphine alkaloids in the stems and leaves of Aconitum carmichaelii is higher than that in the medicinal parts, while the content of the diester-diterpenoid alkaloids is lower. Additionally, the medicinal parts of Aconitum carmichaelii exhibited cardiotoxicity and hepatotoxicity, while the stems and leaves have no obvious toxicity. Finally, through correlation analysis and animal experimental verification, mesaconitine, deoxyaconitine, and hypaconitine were used as toxicity markers. CONCLUSION: Given the low toxicity of the stems and leaves and the potential efficacy of aporphine alkaloids, the stems and leaves of Aconitum carmichaelii hold promise as a valuable medicinal resource warranting further development.

Study on Fu-Fang-Jin-Qian-Cao Inhibiting Autophagy in Calcium Oxalate-induced Renal Injury by UHPLC/Q-TOF-MS-based Metabonomics and Network Pharmacology Approaches.

INTRODUCTION: Fu-Fang-Jin-Qian-Cao is a Chinese herbal preparation used to treat urinary calculi. Fu-Fang-Jin-Qian-Cao can protect renal tubular epithelial cells from calcium oxalateinduced renal injury by inhibiting ROS-mediated autopathy. The mechanism still needs further exploration. Metabonomics is a new subject; the combination of metabolomics and network pharmacology can find pathways for drugs to act on targets more efficiently. METHODS: Comprehensive metabolomics and network pharmacology to study the mechanism of Fu-Fang-Jin-Qian-Cao inhibiting autophagy in calcium oxalate-induced renal injury. Based on UHPLC-Q-TOF-MS, combined with biochemical analysis, a mice model of Calcium oxalateinduced renal injury was established to study the therapeutic effect of Fu-Fang-Jin-Qian-Cao. Based on the network pharmacology, the target signaling pathway and the protective effect of Fu- Fang-Jin-Qian-Cao on Calcium oxalate-induced renal injury by inhibiting autophagy were explored. Autophagy-related proteins LC3-II, BECN1, ATG5, and ATG7 were studied by immunohistochemistry. RESULTS: Combining network pharmacology and metabolomics, 50 differential metabolites and 2482 targets related to these metabolites were found. Subsequently, the targets enriched in PI3KAkt, MAPK and Ras signaling pathways. LC3-II, BECN1, ATG5 and ATG7 were up-regulated in Calcium oxalate-induced renal injury. All of them could be reversed after the Fu-Fang-Jin-Qian- Cao treatment. CONCLUSIONS: Fu-Fang-Jin-Qian-Cao can reverse ROS-induced activation of the MAPK signaling pathway and inhibition of the PI3K-Akt signaling pathway, thereby reducing autophagy damage of renal tubular epithelial cells in Calcium oxalate-induced renal injury.

Network pharmacology integrated with molecular docking reveals the anticancer mechanism of Jasminum sambac Linn. essential oil against human breast cancer and experimental validation by in vitro and in vivo studies.

Jasminum sambac L. (J. sambac) belongs to the family Oleaceae and it is an ornamental subtropical evergreen shrub used in traditional treatments of certain ailments and diseases. This study aimed at devising an integrated strategy attempts to evaluate the bioactive components in the J. sambac essential oil (JEO) against human breast cancer. JEO extracted by distillation process and analyzed by GC-MS was subjected to screening of therapeutic components in their allegiance to the drug-likeness index. The utility and efficacy of its molecular mechanism relating to anticancer potential were probed with network pharmacology analysis. Gene ontology, pathway enrichment, and compound-target-pathway network by Cytoscape helped to harp on hub targets and pathways involved in curative action. Drawing from the network data, molecular docking analysis of selected compounds on breast cancer targets was approached. The anti-proliferative study was carried out in MCF-7 and MDA-MB-231 to evaluate the cytotoxicity of JEO. Finally, in vivo anticancer activity was verified using rat models. The results showed MDA-MB-231 cell growth was highly inhibited than the MCF-7 cell line. Alongside this in vitro trial, in situ effectiveness of JEO was evaluated using female Sprague-Dawley rat animal models. In vivo experiments and histopathological analysis showed convincing results in DMBA tumor-induced rats. The larger aim of this study is to identify the potential ingredients of the JEO in cancer apoptosis by integrating network pharmacology and experimental validation achieved to certain extent confers credence to the concept of hiring J. sambac as floral therapy in dealing with the disastrous disease.

Exploring the mechanism of Bushen Huoxue prescription in the treatment of early diabetic retinal edema from the perspective of inner blood-retinal barrier injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Diabetic retinopathy (DR) is one of the most severe complications of diabetes mellitus, diabetes belongs to the category of "emaciation-thirst disease" in traditional Chinese medicine (TCM). Bushen Huoxue Prescription (BHP) is composed of traditional Chinese materia medica, which has therapeutic effects on DR and early diabetic retinal edema (EDRE). However, the therapeutic mechanism is unclear. AIM OF THE STUDY: Exploring the mechanism of BHP against EDRE. METHODS: Feeding Sprague Dawley (SD) rats a high-fat, high-sugar diet as well as providing intraperitoneal injections of streptozotocin (STZ) to promote inner blood-retinal barrier (iBRB) damage that can trigger EDRE, evaluating the therapeutic effect of BHP by the level of expressiveness of TJ proteins (ZO-1,Occludin) of the iBRB and the leakage of rhodamine B isothiocyanate (RITC) in the retina. The combination of network pharmacology and metabolomics was employed to study the mechanism of BHP in preventing of EDRE, then four proteins which were closely to the damage of iBRB were chosen for the validation by employing Western Blot (WB). RESULTS: Research of network pharmacology had shown that BHP had efficacy against EDRE by regulating targets such as AKT1, ALB, TNF, PPARG, etc, its potential pathways mainly involving signaling pathways such as HIF-1. In untargeted metabolomics analysis of serum, 15 differential metabolites were identified, with the metabolic pathways focusing on ketone body metabolism and synthesis, sphingolipid metabolism and phenylalanine metabolism. The conclusions of metabolomics and network pharmacology revealed that BHP can treat EDRE by alleviating hypoxia and oxidative stress and exerting protection of the iBRB. Finally, BHP's protection behavior of the iBRB was validated by WB experiments. CONCLUSION: Through integrating pharmacodynamics, network pharmacology and metabolomics, BHP was discovered to have a crucial function in EDRE therapy by preserving the integrity of iBRB. This comprehensive strategy also provided a reasonable way to reveal the multi-components, multi-targets, multi-pathways mechanism of TCM.

Effects of slit dual-frequency ultrasound-assisted pulping on the structure, functional properties and antioxidant activity of Lycium barbarum proteins and in situ real-time monitoring process.

To improve the protein dissolution rate and the quality of fresh Lycium barbarum pulp (LBP), we optimized the slit dual-frequency ultrasound-assisted pulping process, explored the dissolution kinetics of Lycium barbarum protein (LBPr), and established a near-infrared spectroscopy in situ real-time monitoring model for LBPr dissolution through spectral information analysis and chemometric methods. The results showed that under optimal conditions (dual-frequency 28-33 kHz, 300 W, 31 min, 40 °C, interval ratio 5:2 s/s), ultrasonic treatment not only significantly increased LBPr dissolution rate (increased by 71.48 %, p < 0.05), improved other nutrient contents and color, but also reduced the protein particle size, changed the amino acid composition ratio and protein structure, and increased the surface hydrophobicity, zeta potential, and free sulfhydryl content of protein, as well as the antioxidant activity of LBPr. In addition, ultrasonication significantly improved the functional properties of the protein, including thermal stability, foaming, emulsification and oil absorption capacity. Furthermore, the real-time monitoring model of the dissolution process was able to quantitatively predict the dissolution rate of LBPr with good calibration and prediction performance (Rc = 0.9835, RMSECV = 2.174, Rp = 0.9841, RMSEP = 1.206). These findings indicated that dual-frequency ultrasound has great potential to improve the quality of LBP and may provide a theoretical basis for the establishment of an intelligent control system in the industrialized production of LBP and the functional development of LBPr.

Identification of novel flavin-dependent monooxygenase from Strobilanthes Cusia reveals molecular basis of indoles' biosynthetic logic.

BACKGROUND: Strobilanthes cusia (Nees) Kuntze is a traditional medical plant distributed widely in south China. The indole compounds that originated from the plant are responsible for its pharmacological activities. However, the reason why indole ingredients are accumulated in this herb and how it is biosynthesized has remained largely unknown. RESULTS: In this study, metabolic and transcriptional profiling measurement experiments of different S. cusia organs were carried out to understand the underlying molecular basis of indoles' biosynthetic logic. A metabolic investigation demonstrated that the indoles are primarily accumulated mainly in aerial parts, particularly in leaves. RNA-seq was employed to reveal the organ specific accumulation of indoles in different S. cusia organs. Meanwhile, a flavin-dependent monooxygenase gene (ScFMO1) was found in S. cusia, and it has capacity to produce indoxyl from indole by the fermentation assay. Finally, we assessed the outcomes of transient expression experiment in tobacco and confirmed that ScFMO1 localizes in cytoplasm. CONCLUSIONS: Our results suggest that ScFMO1 plays a key role in biosynthesis of indoles (Indigo, indirubin, indican, etc.), it will be useful for illuminating the molecular basis of the medicinal indoles' biosynthesis and developing strategies for improving their yields.

Simultaneous determination and pharmacokinetics study of three triterpenoid saponins in rat plasma by ultra-high-performance liquid chromatography tandem mass-spectrometry after oral administration of Astragalus Membranaceus leaf extract.

A selective and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of three triterpenoid saponins isolated from Astragalus membranaceus leaf extract. In this article, a method for simultaneous determination of Huangqiyenin A, Huangqiyenin E, and Huangqiyenin K was established for the first time. The method was successfully applied to the pharmacokinetic study of Astragalus membranaceus leaf extract after oral administration. Liquid-liquid extraction was applied to plasma sample preparation. Multiple reaction monitoring mode with an electrospray ion source in positive electrospray ionization was chosen to quantify the analytes. Chromatographic separation was performed on a Waters HSS T3 column, using gradient elution with a mobile phase composed of acetonitrile and 5 mM ammonium acetate/water. The pharmacokinetic results showed that all three compounds had the characteristics of rapid absorption-slow metabolism trend. The time of maximum plasma concentration of Huangqiyenin A is higher than Huangqiyenin E and Huangqiyenin K. And the maximum plasma concentration of Huangqiyenin A is higher as well. The pharmacokinetic results revealed the pharmacokinetic characteristics of the three analytes in rat plasma, which could provide a helpful reference for the further study of Astragalus membranaceus leaf extract.

Advances in the pharmacological study of Chinese herbal medicine to alleviate diabetic nephropathy by improving mitochondrial oxidative stress.

Diabetic nephropathy (DN) is one of the serious complications of diabetes mellitus, primarily arising from type 2 diabetes (T2DM), and can progress to chronic kidney disease (CKD) and end stage renal disease (ESRD). The pathogenesis of DN involves various factors such as hemodynamic changes, oxidative stress, inflammatory response, and lipid metabolism disorders. Increasing attention is being given to DN caused by oxidative stress in the mitochondrial pathway, prompting researchers to explore drugs that can regulate these target pathways. Chinese herbal medicine, known for its accessibility, rich historical usage, and remarkable efficacy, has shown promise in ameliorating renal injury caused by DN by modulating oxidative stress in the mitochondrial pathway. This review aims to provide a reference for the prevention and treatment of DN. Firstly, we outline the mechanisms by which mitochondrial dysfunction impairs DN, focusing on outlining the damage to mitochondria by oxidative stress. Subsequently, we describe the process by which formulas, herbs and monomeric compounds protect the kidney by ameliorating oxidative stress in the mitochondrial pathway. Finally, the rich variety of Chinese herbal medicine, combined with modern extraction techniques, has great potential, and as we gradually understand the pathogenesis of DN and research techniques are constantly updated, there will be more and more promising therapeutic targets and herbal drug candidates. This paper aims to provide a reference for the prevention and treatment of DN.

Astragulus embranaceus (Fisch.) Bge-Dioscorea opposita Thunb herb pair ameliorates sarcopenia in senile type 2 diabetes mellitus through Rab5a/mTOR-mediated mitochondrial dysfunction.

ETHNOPHARMACOLOGICAL RELEVANCE: The combination of Astragulus embranaceus (Fisch.) Bge (Huangqi) and Dioscorea opposita Thunb (Shanyao) are one of the most widely accepted herb pairs in traditional Chinese medicine prescriptions for treating sarcopenia. However, the mechanisms underlying the combination of these herbs for anti-sarcopenia treatment are not yet fully understood. AIM OF THE STUDY: To investigate the potential effect of the Astragulus embranaceus (Fisch.) Bge and Dioscorea opposita Thunb herb pair (Ast-Dio) on sarcopenia in mice that have been induced with senile type 2 diabetes mellitus, as well as to explore the underlying mechanisms related to the Rab5a/mTOR signaling pathway and mitochondrial quality control. MATERIALS AND METHODS: Network pharmacology was utilized to identify the main active ingredients of Ast-Dio and potential therapeutic targets for sarcopenia. Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted to explore the underlying mechanisms of Ast-Dio in treating sarcopenia. The high-performance liquid chromatography method coupled with triple-quadrupole tandem mass spectrometry was developed to quantify the major constituents of Ast-Dio. Male C57/BL6 mice, aged 12 months, induced with type 2 diabetes mellitus via streptozotocin were divided into three groups for 8 weeks: the model group, Ast-Dio treatment group (7.8 g/kg), and metformin treatment group (100 mg/kg). Normal control groups included mice aged 3 and 12 months, respectively. The study monitored changes in fasting blood glucose levels, grip strength, and body weight during 8 weeks of intragastric administration. Liver and kidney function in mice was evaluated by measuring the levels of serum creatinine, alanine transaminase, and aspartate transaminase. Skeletal muscle mass condition was evaluated by muscle weight, and hematoxylin and eosin staining. Protein and mRNA expressions related to muscle atrophy, mitochondrial quality control, and the Rab5a/mTOR signaling pathway were detected using immunofluorescence staining, immunohistochemical staining, Western blotting, and quantitative real-time polymerase chain reaction. In addition, transmission electron microscopy was employed to investigate the condition of mitochondria in the groups. RESULTS: Through the prediction analysis of network pharmacology, we identified mTOR as one of the primary targets for Ast-Dio therapy of sarcopenia. Gene Ontology functional enrichment analysis revealed that mitochondrial control quality is crucial in the treatment of sarcopenia with Ast-Dio. Our findings showed that senile type 2 diabetes mellitus induced muscle mass loss and a reduction in grip strength, both of which were dramatically restored by Ast-Dio treatment. Notably, Ast-Dio increased Myogenin expression while decreasing Atrogin-1 and MuRF-1 expression. Additionally, Ast-Dio activated Rab5a/mTOR and its downstream effector AMPK. Moreover, Ast-Dio modulated mitochondrial quality control by decreasing Mitofusin-2 expression while increasing the expression of TFAM, PGC-1α, and MFF. CONCLUSIONS: Our results suggest that Ast-Dio treatment may alleviate sarcopenia in mice with senile type 2 diabetes mellitus through its effects on the Rab5a/mTOR pathway and mitochondrial quality control.

An integrated analytical strategy to decipher the metabolic profile of alkaloids in Compound Kushen injection based on UHPLC-ESI-QTOF/MSE.

Compound Kushen injection (CKI) is a kind of sterilised water-soluble traditional Chinese medicine preparation that has been used for the clinical treatment of a variety of cancers (hepatocellular carcinoma, lung cancer, etc.) for 19 years. However, to date, the metabolism-related study on CKI in vivo has not been conducted.An integrated analytical strategy was established to investigate the metabolic profile of alkaloids of CKI in rat plasma, urine, and faeces based on ultra-high performance liquid chromatography-electrospray quadrupole time-of-flight mass spectrometry in MSE mode (UHPLC-ESI-QTOF/MSE).Nineteen prototype alkaloids (including 12 matrine-type alkaloids, 2 cytisine-type alkaloids, 3 lupinine-type alkaloids, and 2 aloperine-type alkaloids) of CKI were identified in vivo. Furthermore, 71 metabolites of alkaloids (including 11 of lupanine-related metabolites, 14 of sophoridine-related metabolites, 14 of lamprolobine-related metabolites, and 32 of baptifoline-related metabolites) were tentatively characterised. Metabolic pathways involved in the metabolism of phase I (include oxidation, reduction, hydrolysis, and desaturation), phase II (mainly include glucuronidation, acetylcysteine or cysteine conjugation, methylation, acetylation, and sulphation), and associated combination reactions.The integrated analytical strategy was successfully used to characterise the prototype alkaloids and their metabolites of CKI in vivo, and the results laying a foundation for further study its pharmacodynamic substances.

The Chinese herbal medicine Fufang Zhenzhu Tiaozhi ameliorates diabetic cardiomyopathy by regulating cardiac abnormal lipid metabolism and mitochondrial dynamics in diabetic mice.

Diabetic cardiomyopathy (DCM) is an important complication leading to the death of patients with diabetes, but there is no effective strategy for clinical treatments. Fufang Zhenzhu Tiaozhi (FTZ) is a patent medicine that is a traditional Chinese medicine compound preparation with comprehensive effects for the prevention and treatment of glycolipid metabolic diseases under the guidance of "modulating liver, starting pivot and cleaning turbidity". FTZ was proposed by Professor Guo Jiao and is used for the clinical treatment of hyperlipidemia. This study was designed to explore the regulatory mechanisms of FTZ on heart lipid metabolism dysfunction and mitochondrial dynamics disorder in mice with DCM, and it provides a theoretical basis for the myocardial protective effect of FTZ in diabetes. In this study, we demonstrated that FTZ protected heart function in DCM mice and downregulated the overexpression of free fatty acids (FFAs) uptake-related proteins cluster of differentiation 36 (CD36), fatty acid binding protein 3 (FABP3) and carnitine palmitoyl transferase 1 (CPT1). Moreover, FTZ treatment showed a regulatory effect on mitochondrial dynamics by inhibiting mitochondrial fission and promoting mitochondrial fusion. We also identified in vitro that FTZ could restore lipid metabolism-related proteins, mitochondrial dynamics-related proteins and mitochondrial energy metabolism in PA-treated cardiomyocytes. Our study indicated that FTZ improves the cardiac function of diabetic mice by attenuating the increase in fasting blood glucose levels, inhibiting the decrease in body weight, alleviating disordered lipid metabolism, and restoring mitochondrial dynamics and myocardial apoptosis in diabetic mouse hearts.

Effects of Banxia Xiexin Decoction on apoptosis of interstitial cells of Cajal via regulation of MiR-451-5p: An in vivo and in vitro study.

ETHNOPHARMACOLOGICAL RELEVANCE: Banxia Xiexin Decoction (BXD) is a traditional Chinese medical formula applied to gastrointestinal (GI) motility disorders. Previous studies showed that miR-451-5p was down-regulated in rats with GI motility disorders induced by gastric electrical dysrhythmia. Interstitial cells of Cajal (ICCs) are pacemakers for GI motility, while loss of ICCs is responsible for GI motility disturbance. Thus, the underlying interaction mechanisms for BXD regulating ICCs apoptosis via miR-451-5p remain to be explored. AIM OF THE STUDY: In this work, the main objectives were to examine the efficacy of BXD on ICCs via miR-451-5p both in GI motility disorders rats model and in vitro, as well as the potential contributions of SCF/c-kit signaling. MATERIALS AND METHODS: Rats with gastric electrical dysrhythmia were established in male SD rats by using a single-day diet and a double fasting method (drinking diluted hydrochloric acid water during the period) for 4 weeks. The gastric slow wave (GSW) recording, RT-qPCR, and western blot were performed to examine the effects of BXD on ICCs apoptosis in rats with GED and miR-451-5p expression. In vitro assays included CCK-8, flow cytometry analysis, RT-qPCR, and western blot were applied to investigate the potential molecular mechanism of BXD on ICCs apoptosis via miR-451-5p. RESULTS: BXD promoted gastric motility, reduced ICCs apoptosis, and elevated miR-451-5p in GED rats. In addition, miR-451-5p was significantly up-regulated in ICCs after BXD treatment compared with that in ICCs with miR-451-5p inhibitor transfection. Meanwhile, high miR-451-5p expression with either BXD treatment or miRNA mimics enhanced ICCs proliferation and inhibit apoptosis. Moreover, overexpression of miR-451-5p can reverse G0/G1 arrest in ICCs by BXD treatment. Further, SCF and c-kit protein levels were detected to demonstrate that modulation of miR-451-5p by BXD treatment was involved in this signaling. CONCLUSIONS: Through this study, we demonstrated that BXD could promote ICCs proliferation and inhibit apoptosis via miR-451-5p and may involve the modulations of SCF/c-kit signaling, thus suggesting a new therapy basis for GI motility dysfunction from the perspective of modulation of ICCs apoptosis by targeting miR-451-5p.