Metabolomic profiling of wild rooibos (Aspalathus linearis) ecotypes and their antioxidant-derived phytopharmaceutical potential.

INTRODUCTION: Aspalathus linearis (commonly known as rooibos) is endemic to the Cape Floristic Region of South Africa and is a popular herbal drink and skin phytotherapeutic ingredient, with health benefits derived primarily from its unique phenolic content. Several, seemingly habitat-specific ecotypes from the Cederberg (Western Cape) and Northern Cape have morphological, ecological, genetic and biochemical differences. OBJECTIVES AND METHODS: Despite the commercial popularity of the cultivated variety, the uncultivated ecotypes are largely understudied. To address gaps in knowledge about the biochemical constituency, ultra-performance liquid chromatography-mass spectrometry analysis of fifteen populations was performed, enabling high-throughput metabolomic fingerprinting of 50% (v/v) methanolic extracts. Antioxidant screening of selected populations was performed via three assays and antimicrobial activity on two microbial species was assessed. The metabolomic results were corroborated with total phenolic and flavonoid screening of the extracts. RESULTS AND DISCUSSION: Site-specific chemical lineages of rooibos ecotypes were confirmed via multivariate data analyses. Important features identified via PLS-DA disclosed higher relative abundances of certain tentative metabolites (e.g., rutin, aspalathin and apiin) present in the Dobbelaarskop, Blomfontein, Welbedacht and Eselbank sites, in comparison to other locations. Several unknown novel metabolites (e.g., m/z 155.0369, 231.0513, 443.1197, 695.2883) are responsible for metabolomic separation of the populations, four of which showed higher amounts of key metabolites and were thus selected for bioactivity analysis. The Welbedacht and Eselbank site 2 populations consistently displayed higher antioxidant activities, with 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activities of 679.894 ± 3.427 µmol Trolox/g dry matter and 635.066 ± 5.140 µmol Trolox/g dry matter, respectively, in correlation with a high number of phenolic and flavonoid compounds. The contribution of the individual metabolites to the pharmacological effectiveness of rooibos remains unknown and as such, further structural elucidation and phytopharmacological testing is thus urgently needed.

Sample Preparation from Plant Tissue for Gas Chromatography-Mass Spectrometry (GC-MS)we.

Metabolites are intermediate products formed during metabolism. Metabolites play different roles, including providing energy, supporting structure, transmitting signals, catalyzing reactions, enhancing defense, and interacting with other species. Plant metabolomics research aims to detect precisely all metabolites found within tissues of plants through GC-MS. This chapter primarily focuses on extracting metabolites using chemicals such as methanol, chloroform, ribitol, MSTFA, and TMCS. The metabolic analysis method is frequently used according to the specific kind of sample or matrix being investigated and the analysis objective. Chromatography (LC, GC, and CE) with mass spectrometry and NMR spectroscopy is used in modern metabolomics to analyze metabolites from plant samples. The most frequently used method for metabolites analysis is the GC-MS. It is a powerful technique that combines gas chromatography's separation capabilities with mass spectrometry, offering detailed information, including structural identification of each metabolite. This chapter contains an easy-to-follow guide to extract plant-based metabolites. The current protocol provides all the information needed for extracting metabolites from a plant, precautions, and troubleshooting.

[Metabolomics of quality formation of different cultivars of Peucedanum praeruptorum].

This study aims to reveal the quality formation of different cultivars of Peucedanum praeruptorum based on the metabolic differences and provide a theoretical basis for the development and utilization of this medicinal herb. The non-target metabonomics analysis based on ultra-high performance liquid chromatography tandem mass spectrometry(UHPLC-MS/MS) was conducted for six cultivars(YS, H, LZ, LY, LX, and Z) of P. praeruptorum of the same origin and at the same development stage. The principal component analysis, orthogonal partial least squares discriminant analysis, and univariate statistical analysis were carried out to screen the differential metabolites of different cultivars. The potential biomarkers associated with quality formation were predicted based on the mass-to-charge ratio, Kyoto Encyclopedia of Genes and Genomes pathway enrichment, information of relevant literature, and correlation analysis. The results showed that metabolites differed significantly among the six cultivars, and 571 and 465 differential metabolites were obtained in the positive and negative ion modes, respectively. From the differential metabolites, 22 potential biomarkers related to quality formation were predicted, which involved 9 metabolic pathways, including phenylalanine, tyrosine and tryptophan biosynthesis, biosynthesis of phenylpropanoids, and biosynthesis of plant hormones. Compared with the YS cultivar, other cultivars showed decreased concentrations of psoralen, imperatorin, and luvangetin and increased concentrations of 7-hydroxycoumarine, esculetin, columbianetin, and jasmonic acid, which were involved in the biosynthesis of phenylpropanoids. The concentrations of 2-succinylbenzoate, heraclenol, and L-tyrosine involved in other metabolic pathways decreased, especially in the Z and H cultivars. Therefore, regulating the biosynthesis of phenylpropanoids is one of the key mechanisms for improving the cultivar quality of P. praeruptorum. The Z and H cultivars have better quality and metabolic processes than other cultivars and thus can be used for the screening and breeding of high-quality germplasm.

[Research on mechanism of hypolipidemic effect of Massa Medicata Fermentata based on metabolomics].

This paper aims to study the therapeutic effect of Massa Medicata Fermentata on hyperlipidemia model rats and investigate its mechanism of hypolipidemic effect with the help of non-targeted metabolomics. The mixed hyperlipidemia model rats were constructed by giving high-fat chow. After successful modeling, the rats were divided into the model group, pravastatin sodium group(4.4 mg·kg~(-1)), lipotropic group(0.1 g·kg~(-1)), high-dose group(2.4 g·kg~(-1)), medium-dose group(1.2 g·kg~(-1)), and low-dose group(0.6 g·kg~(-1)) of Massa Medicata Fermentata, and they were administered for four weeks once daily. An equal volume of ultrapure water was given to the blank group and model group. Serum lipid level and liver hematoxylin-eosin(HE) staining were used as indicators to estimate the intervention effect of Massa Medicata Fermentata on mixed hyperlipidemia, and the changes in metabolites in plasma of mixed hyperlipidemia model rats were analyzed by non-targeted metabolomics. The mechanism of the hypolipidemic effect of Massa Medicata Fermentata was analyzed through metabolite pathway enrichment. The results showed that compared with the model group, the Massa Medicata Fermentata administration group, especially the high-dose group, could significantly reduce the content of total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-c)(P<0.05 or P<0.01), and liver HE staining revealed that the number of adipocytes in the high-dose group was reduced to some extent. The potential biomarkers obtained by non-targeted metabolomics screening included glycerol 3-phosphate, sphingomyelin, sphingosine 1-phosphate, and deoxyuridine, which were mainly involved in the sphingolipid metabolism process, glycerophospholipid metabolism process, glycerol ester metabolism pathway, and pyrimidine metabolism pathway, totaling four possible metabolic pathways related to lipid metabolism. This study provides a reference for an in-depth investigation of the hypolipidemic mechanism of Massa Medicata Fermentata, which is of great significance for further promoting the clinical application of Massa Medicata Fermentata and increasing the indications.

Targeted metabolomics profiling in pregnancy associated with vitamin D deficiency.

BACKGROUND: Vitamin D deficiency is common in pregnancy, however, its effects has not been fully elucidated. Here, we conducted targeted metabolomics profiling to study the relationship. METHODS: This study enrolled 111 pregnant women, including sufficient group (n = 9), inadequate group (n = 49) and deficient group (n = 53). Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-based targeted metabonomics were used to characterize metabolite profiles associated with vitamin D deficiency in pregnancy. RESULTS: Many metabolites decreased in the inadequate and deficient group, including lipids, amino acids and others. The lipid species included fatty acyls (FA 14:3, FA 26:0; O), glycerolipids (MG 18:2), glycerophospholipids (LPG 20:5, PE-Cer 40:1; O2, PG 29:0), sterol lipids (CE 20:5, ST 28:0; O4, ST 28:1; O4). Decreased amino acids included aromatic amino acids (tryptophan, phenylalanine, tyrosine) and branched-chain amino acids (valine, isoleucine, leucine), proline, methionine, arginine, lysine, alanine, L-kynurenine,5-hydroxy-L-tryptophan, allysine. CONCLUSIONS: This targeted metabolomics profiling indicated that vitamin D supplementation can significantly affect lipids and amino acids metabolism in pregnancy.

Exploring the Hypocholesterolemic Potential of a Fucus vesiculosus Extract: Omic Insights into Molecular Mechanisms at the Intestinal Level.

High blood cholesterol levels are a major risk factor for cardiovascular diseases. A purified aqueous extract of Fucus vesiculosus, rich in phlorotannins and peptides, has been described for its potential to inhibit cholesterol biosynthesis and intestinal absorption. In this work, the effect of this extract on intestinal cells' metabolites and proteins was analysed to gain a deeper understanding of its mode of action on lipids' metabolism, particularly concerning the absorption and transport of exogenous cholesterol. Caco-2 cells, differentiated into enterocytes, were exposed to the extract, and analysed by untargeted metabolomics and proteomics. The results of the metabolomic analysis showed statistically significant differences in glutathione content of cells exposed to the extract compared to control cells, along with an increased expression of fatty acid amides in exposed cells. A proteomic analysis showed an increased expression in cells exposed to the extract compared to control cells of FAB1 and NPC1, proteins known to be involved in lipid metabolism and transport. To the extent of our knowledge, this study is the first use of untargeted metabolomics and a proteomic analysis to investigate the effects of F. vesiculosus on differentiated Caco-2 cells, offering insights into the molecular mechanism of the extract's compounds on intestinal cells.

Extensive targeted metabolomics analysis reveals the identification of major metabolites, antioxidants, and disease-resistant active pharmaceutical components in Camellia tuberculata (Camellia L.) seeds.

Sect. tuberculata plant belongs to the Camellia genus and is named for the "tuberculiform protuberance on the surface of the ovary and fruit". It is a species of great ornamental value and potential medicinal value. However, little has been reported on the metabolites of C. tuberculata seeds. Therefore, this study was conducted to investigate the metabolites of C. tuberculata seeds based on UPLC/ESI-Q TRAP-MS/MS with extensively targeted metabolomics. A total of 1611 metabolites were identified, including 107 alkaloids, 276 amino acids and derivatives, 283 flavonoids, 86 lignans and coumarins, 181 lipids, 68 nucleotides and derivatives, 101 organic acids, 190 phenolic acids, 10 quinones, 4 steroids, 17 tannins, 111 terpenoids, and 177 other metabolites. We compared the different metabolites in seeds between HKH, ZM, ZY, and LY. The 1311 identified different metabolites were classified into three categories. Sixty-three overlapping significant different metabolites were found, of which lignans and coumarins accounted for the largest proportion. The differentially accumulated metabolites were enriched in different metabolic pathways between HKH vs. LY, HKH vs. ZM, HKH vs. ZY, LY vs. ZY, ZM vs. LY and ZM vs. ZY, with the most abundant metabolic pathways being 4, 2, 4, 7, 7 and 5, respectively (p < 0.05). Moreover, among the top 20 metabolites in each subgroup comparison in terms of difference multiplicity 7, 8 and 13. ZM and ZY had the highest phenolic acid content. Ninety-six disease-resistant metabolites and 48 major traditional Chinese medicine agents were identified based on seven diseases. The results of this study will not only lead to a more comprehensive and in-depth understanding of the metabolic properties of C. tuberculata seeds, but also provide a scientific basis for the excavation and further development of its medicinal value.

Widely targeted metabolomics reveals differences in metabolites of Paeonia lactiflora cultivars.

INTRODUCTION: Paeonia lactiflora contains diverse active constituents and exhibits various pharmacological activities. However, only partial identification of biologically active substances from P. lactiflora has been achieved using low-throughput techniques. Here, the roots of P. lactiflora, namely, Fenyunu (CK), Dafugui (DFG), and Red Charm (HSML), were studied. The primary and secondary metabolites were investigated using ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESIMS/MS). METHODS: The chemical compounds and categories were detected using broadly targeted UPLC-MS/MS. Principal component analysis (PCA), orthogonal partial least-squares discriminant analysis (OPLS-DA), and hierarchical clustering analysis (HCA) were carried out for metabolites of different varieties of P. lactiflora. RESULTS: A total of 1237 compounds were detected and classified into 11 categories. HCA, PCA, and OPLS-DA of these metabolites indicated that each variety of P. lactiflora was clearly separated from the other groups. Differential accumulated metabolite analysis revealed that the three P. lactiflora varieties contained 116 differentially activated metabolites (DAMs) involved in flavonoid, flavone, and flavonol metabolism. KEGG pathway analysis revealed that, in 65 pathways, 336 differentially abundant metabolites (DMs) were enriched in the CK and DFG groups; moreover, the type and content of terpenoids were greater in the CK group than in the DFG group. The CK and HSML groups contained 457 DMs enriched in 61 pathways; the type and amount of flavonoids, terpenoids, and tannins were greater in the CK group than in the HSML group. The DFG and HSML groups contained 497 DMs enriched in 65 pathways; terpenoids and alkaloids were more abundant in the HSML variety than in the DFG variety. CONCLUSIONS: A total of 1237 compounds were detected, and the results revealed significant differences among the three P. lactiflora varieties. Among the three P. lactiflora varieties, phenolic acids and flavonoids composed the largest and most diverse category of metabolites, and their contents varied greatly. Therefore, CK is suitable for medicinal plant varieties, and DFG and HSML are suitable for ornamental plant varieties. Twelve proanthocyanidin metabolites likely determined the differences in color among the three varieties.

Rapid characterization of non-volatile phenolic compounds reveals the reliable chemical markers for authentication of traditional Chinese medicine Xiang-ru among confusing Elsholtzia species.

The aerial parts of Mosla chinensis Maxim. and Mosla chinensis cv. 'Jiangxiangru' (MCJ) are widely utilized in traditional Chinese medicine (TCM), known collectively as Xiang-ru. However, due to clinical effectiveness concerns and frequent misidentification, the original plants have increasingly been substituted by various species within the genera Elsholtzia and Mosla. The challenge in distinguishing between these genera arises from their similar morphological and metabolic profiles. To address this issue, our study introduced a rapid method for metabolic characterization, employing high-resolution mass spectrometry-based metabolomics. Through detailed biosynthetic and chemometric analyses, we pinpointed five phenolic compounds-salviaflaside, cynaroside, scutellarein-7-O-D-glucoside, rutin, and vicenin-2-among 203 identified compounds, as reliable chemical markers for distinguishing Xiang-ru from closely related Elsholtzia species. This methodology holds promise for broad application in the analysis of plant aerial parts, especially in verifying the authenticity of aromatic traditional medicinal plants. Our findings underscore the importance of non-volatile compounds as dependable chemical markers in the authentication process of aromatic traditional medicinal plants.

Differences of Typical Wuyi Rock Tea in Taste and Nonvolatiles Profile Revealed by Multisensory Analysis and LC-MS-Based Metabolomics.

Wuyi Rock tea, specifically Shuixian and Rougui, exhibits distinct sensory characteristics. In this study, we investigated the sensory and metabolite differences between Shuixian and Rougui. Quantitative description analysis revealed that Rougui exhibited higher intensity in bitter, thick, harsh, and numb tastes, while Shuixian had stronger salty and umami tastes. Nontargeted metabolomics identified 151 compounds with 66 compounds identified as key differential metabolites responsible for metabolic discrimination. Most of the catechins and flavonoids were enriched in Rougui tea, while epigallocatechin-3,3'-di-O-gallate, epigallocatechin-3,5-di-O-gallate, gallocatechin-3,5-di-O-gallate, isovitexin, and theaflavanoside I were enriched in Shuixian tea. Catechins, kaempferol, quercetin, and myricetin derivatives were positively correlated with bitter taste and numb sensation. Sour taste was positively correlated to organic acids. Amino acids potentially contributed to salty and umami tastes. These results provide further insights into the taste characteristics and the relationship between taste attributes and specific metabolites in Wuyi Rock tea.

Metabolomics reveals the importance of metabolites in Mussaenda pubescens for antioxidant properties and quality traits.

Mussaenda pubescens (Mp) is a valuable medicinal plant that has traditionally been used for medicinal purposes or as a tea substitute. However, there are few studies on the comprehensive and dynamic evaluation of Mp metabolites. This study used an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach and biochemical analysis to investigate substance changes in leaves at three different stages and elucidate the relationship between metabolites and antioxidant capacity. The findings showed that Mp leaves contained 957 metabolites, the majority of which were phenolic acids, lipids, and terpenoids. The metabolite profiling of Mp leaves was significantly influenced by their growth and development at different stages. A total of 317 differentially accumulated metabolites (DAMs) were screened, including 150 primary metabolites and 167 secondary metabolites, with 202 DAMs found in bud leaf vs. tender leaf, 54 DAMs in tender leaf vs. mature leaf, and 254 DAMs in bud leaf vs. mature leaf. Total phenolics, flavonoids, and anthocyanin concentrations decreased as Mp leaves grew and developed, whereas terpenoids increased significantly. The secondary metabolites also demonstrated a positive correlation with antioxidant activity. Phenolics, flavonoids, terpenoids, and anthocyanins were the primary factors influencing the antioxidant activity of leaves. These findings provide new insights into the metabolite formation mechanism, as well as the development and utilization of Mp tea.

[Non-targeted metabolomics of spleen and liver metabolism in mice treated with Pruni Semen processed with different methods].

Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was employed to investigate the impacts of Pruni Semen processed with different methods(raw and fried) on the liver and spleen metabolism in mice. A total of 24 male mice were randomly assigned to three groups: raw Pruni Semen group, fried Pruni Semen group, and control(deionized water) group. Mice in the three groups were orally administrated with 0.01 g·mL~(-1) Pruni Semen decoction or deionized water for one week. After that, the liver and spleen tissues were collected, and liquid chromatography-mass spectrometry(LC-MS)-based metabolomic analysis was carried out to investigate the impact of Pruni Semen on the liver and spleen metabolism in mice. Compared with thte control group, the raw Pruni Semen group showed up-regulation of 11 metabolites and down-regulation of 57 metabolites in the spleen(P<0.05), as well as up-regulation of 15 metabolites and down-regulation of 58 metabolites in the liver(P<0.05). The fried Pruni Semen group showed up-regulation of 31 metabolites and down-regulation of 10 metabolites in the spleen(P<0.05), along with up-regulation of 26 metabolites and down-regulation of 61 metabolites in the liver(P<0.05). The differential metabolites identified in the raw Pruni Semen group were primarily associated with alanine, aspartate, and glutamate metabolism, purine metabolism, amino sugar and nucleotide sugar metabolism, and D-glutamine and D-glutamate metabolism. The differential metabolites identified in the fried Pruni Semen group predominantly involved riboflavin metabolism, amino sugar and nucleotide sugar metabolism, purine metabolism, alanine, aspartate, and glutamate metabolism, D-glutamine and D-glutamate metabolism, and glutathione metabolism. The findings suggest that both raw and fried Pruni Semen have the potential to modulate the metabolism of the liver and spleen in mice by influencing the glutamine and glutamate metabolism.

LC-MS based metabonomics study on protective mechanism of ESWW in cerebral ischemia via CYTC/Apaf-1/NDRG4 pathway.

BACKGROUND: Ershiwuwei Zhenzhu pills was originally recorded in the Tibetan medical book Si Bu Yi Dian in the 8th century AD and is now included in the Pharmacopoeia of the People's Republic of China (2020). The pills can calm the nerves and open the mind as well as treat cerebral ischemia reperfusion injury, stroke, hemiplegia. However, its quality standards have not yet been established, and the therapeutic effect on cerebral ischemia by regulating the mitochondrial apoptosis pathway has not been elucidated. STUDY DESIGN AND METHODS: LC-MS was used to establish quality standards for Ershiwuwei Zhenzhu pills. Metabonomics, molecular docking, neuroethology, cerebral infarction ratio, pathological detection of diencephalon, cortex, and hippocampus, and molecular biology techniques were used to reveal the mechanism of the pills in regulating the mitochondrial apoptosis pathway to treat cerebral ischemia. RESULTS: The contents of 20 chemical components in Ershiwuwei Zhenzhu pills from 12 batches and 8 manufacturers was determined for the first time. Eleven differential metabolites and three metabolic pathways, namely, fructose and mannose metabolism, glycerophospholipid metabolism, and purine metabolism, were identified by metabonomics. The pills improved the neuroethology abnormalities of MCAO rats and the pathological damage in the diencephalon and decreased the ratio of cerebral infarction. It also significantly reduced the mRNA expression of AIF, Apaf-1, cleared caspase8, CytC, and P53 mRNA in the brain tissue and the protein expression of Apaf-1 and CYTC and increased the protein expression of NDRG4. CONCLUSION: In vitro quantitative analysis of the in vitro chemical components of Ershiwuwei Zhenzhu pills has laid the foundation for improving its quality control. The potential mechanism of the pills in treating cerebral ischemia may be related to the Apaf-1/CYTC/NDRG4 apoptosis pathway. This work provides guidance for clinical drug use for patients.

Integrated metabolomics and network pharmacology study on the mechanism of herbal pair of danggui-kushen for treating ischemia heart disease.

DangGui-KuShen (DK) is a well-known classic traditional Chinese medicine recipe that improves blood circulation, eliminates moisture, and detoxifies, and is frequently used in the treatment of cardiovascular problems. Some protective effects of DK on cardiovascular disease have previously been identified, but its precise mechanism remains unknown. The goal of this study is to combine metabolomics and network pharmacology to investigate DK's protective mechanism in Ischemic Heart Disease(IHD) rat models. A combination of metabolomics and network pharmacology based on UPLC-Q-TOF/MS technology was used in this study to verify the effect of DK on IHD through enzyme-linked immunosorbent assay, HE staining, and electrocardiogram, and it was determined that DK improves the synergistic mechanism of IHD. In total, 22 serum differential metabolites and 26 urine differential metabolites were discovered, with the majority of them involved in phenylalanine, tyrosine, and tryptophan biosynthesis, glycine, serine, and threonine metabolism, arginine and proline metabolism, aminoacyl-tRNA biosynthesis, purine metabolism, and other metabolic pathways. Furthermore, using network pharmacology, a composite target pathway network of DangGui and KuShen for treating IHD was created, which is primarily associated to the tumor necrosis factor (TNF) signaling pathway, P53 signaling, and HIF-1 signaling pathways. The combined research indicated that the NF-B signaling pathway and the HIF-1 signaling pathway are critical in DK treatment of IHD. This study clearly confirms and expands on current knowledge of the synergistic effects of DG and KS in IHD.

Transcriptomic and metabolomic profiling provide insight into the role of sugars and hormones in leaf senescence of Pinellia ternata.

KEY MESSAGE: The interaction network and pathway map uncover the potential crosstalk between sugar and hormone metabolisms as a possible reason for leaf senescence in P. ternata. Pinellia ternata, an environmentally sensitive medicinal plant, undergoes leaf senescence twice a year, affecting its development and yield. Understanding the potential mechanism that delays leaf senescence could theoretically decrease yield losses. In this study, a typical senescent population model was constructed, and an integrated analysis of transcriptomic and metabolomic profiles of P. ternata was conducted using two early leaf senescence populations and two stay-green populations. The result showed that two key gene modules were associated with leaf senescence which were mainly enriched in sugar and hormone signaling pathways, respectively. A network constructed by unigenes and metabolisms related to the obtained two pathways revealed that several compounds such as D-arabitol and 2MeScZR have a higher significance ranking. In addition, a total of 130 hub genes in this network were categorized into 3 classes based on connectivity. Among them, 34 hub genes were further analyzed through a pathway map, the potential crosstalk between sugar and hormone metabolisms might be an underlying reason of leaf senescence in P. ternata. These findings address the knowledge gap regarding leaf senescence in P. ternata, providing candidate germplasms for molecular breeding and laying theoretical basis for the realization of finely regulated cultivation in future.

Mechanism and functional substances of Saiga antelope horn in treating hypertension with liver-yang hyperactivity syndrome explored using network pharmacology and metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Saiga antelope horn (SAH) is a traditional Chinese medicine for treating hypertension with liver-yang hyperactivity syndrome (Gan-Yang-Shang-Kang, GYSK), that has a long history of clinical application and precise efficacy, but its mechanism and functional substances are still unknown. Based on the demand for alternative research on the rare and endangered SAH, the group designed and carried out the following studies. AIM OF THE STUDY: The purpose of this research was to demonstrate the functional substances and mechanisms of SAH in the treatment of GYSK hypertension. MATERIALS AND METHODS: The GYSK-SHR model was constructed by administering a decoction of aconite to spontaneously hypertensive rats (SHRs). Blood pressure (BP), behavioural tests related to GYSK, and pathological changes in the kidneys, heart and aorta were measured to investigate the effects of SAH on GYSK-SHRs. Proteomic analysis was used to identify the keratins and peptides of SAH. Moreover, network pharmacology and plasma metabolomics studies were carried out to reveal the mechanisms by which functional peptides in SAH regulate GYSK-hypertension. RESULTS: SAH has a significant antihypertensive effect on GYSK hypertensive animals. It has also been proven to be effective in protecting the function and structural integrity of the kidneys, heart and aorta. Moreover, SAH improved the abnormalities of 31 plasma biomarkers in rats. By constructing a "biomarker-target-peptide" network, 10 functional peptides and two key targets were screened for antihypertensive effects of SAH. The results indicated that SAH may exert a therapeutic effect by re-establishing the imbalance of renin-angiotensin (RAS) system. CONCLUSIONS: Functional peptides from keratin contained in SAH are the main material basis for the treatment of GYSK-hypertension and exhibited the protective effect on the GYSK-SHR model through the RAS system.

Multi-omics and chemical profiling approaches to understand the material foundation and pharmacological mechanism of sophorae tonkinensis radix et rhizome-induced liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Sophorae tonkinensis Radix et Rhizoma (STR) is an extensively applied traditional Chinese medicine (TCM) in southwest China. However, its clinical application is relatively limited due to its hepatotoxicity effects. AIM OF THE STUDY: To understand the material foundation and liver injury mechanism of STR. MATERIALS AND METHODS: Chemical compositions in STR and its prototypes in mice were profiled by ultra-performance liquid chromatography coupled quadrupole-time of flight mass spectrometry (UPLC-Q/TOF MS). STR-induced liver injury (SILI) was comprehensively evaluated by STR-treated mice mode. The histopathologic and biochemical analyses were performed to evaluate liver injury levels. Subsequently, network pharmacology and multi-omics were used to analyze the potential mechanism of SILI in vivo. And the target genes were further verified by Western blot. RESULTS: A total of 152 compounds were identified or tentatively characterized in STR, including 29 alkaloids, 21 organic acids, 75 flavonoids, 1 quinone, and 26 other types. Among them, 19 components were presented in STR-medicated serum. The histopathologic and biochemical analysis revealed that hepatic injury occurred after 4 weeks of intragastric administration of STR. Network pharmacology analysis revealed that IL6, TNF, STAT3, etc. were the main core targets, and the bile secretion might play a key role in SILI. The metabolic pathways such as taurine and hypotaurine metabolism, purine metabolism, and vitamin B6 metabolism were identified in the STR exposed groups. Among them, taurine, hypotaurine, hypoxanthine, pyridoxal, and 4-pyridoxate were selected based on their high impact value and potential biological function in the process of liver injury post STR treatment. CONCLUSIONS: The mechanism and material foundation of SILI were revealed and profiled by a multi-omics strategy combined with network pharmacology and chemical profiling. Meanwhile, new insights were taken into understand the pathological mechanism of SILI.

Understanding PFAS toxicity through cell culture metabolomics: Current applications and future perspectives.

Per- and polyfluoroalkyl substances (PFAS), ubiquitous environmental contaminants, pose significant challenges to ecosystems and human health. While cell cultures have emerged as new approach methodologies (NAMs) in ecotoxicity research, metabolomics is an emerging technique used to characterize the small-molecule metabolites present in cells and to understand their role in various biological processes. Integration of metabolomics with cell cultures, known as cell culture metabolomics, provides a novel and robust tool to unravel the complex molecular responses induced by PFAS exposure. In vitro testing also reduces reliance on animal testing, aligning with ethical and regulatory imperatives. The current review summarizes key findings from recent studies utilizing cell culture metabolomics to investigate PFAS toxicity, highlighting alterations in metabolic pathways, biomarker identification, and the potential linkages between metabolic perturbations. Additionally, the paper discusses different types of cell cultures and metabolomics methods used for studies of environmental contaminants and particularly PFAS. Future perspectives on the combination of metabolomics with other advanced technologies, such as single-cell metabolomics (SCM), imaging mass spectrometry (IMS), extracellular flux analysis (EFA), and multi-omics are also explored, which offers a holistic understanding of environmental contaminants. The synthesis of current knowledge and identification of research gaps provide a foundation for future investigations that aim to elucidate the complexities of PFAS-induced cellular responses and contribute to the development of effective strategies for mitigating their adverse effects on human health.

Comprehensive Metabolomic Analysis of Human Heart Tissue Enabled by Parallel Metabolite Extraction and High-Resolution Mass Spectrometry.

The heart contracts incessantly and requires a constant supply of energy, utilizing numerous metabolic substrates, such as fatty acids, carbohydrates, lipids, and amino acids, to supply its high energy demands. Therefore, a comprehensive analysis of various metabolites is urgently needed for understanding cardiac metabolism; however, complete metabolome analyses remain challenging due to the broad range of metabolite polarities, which makes extraction and detection difficult. Herein, we implemented parallel metabolite extractions and high-resolution mass spectrometry (MS)-based methods to obtain a comprehensive analysis of the human heart metabolome. To capture the diverse range of metabolite polarities, we first performed six parallel liquid-liquid extractions (three monophasic, two biphasic, and one triphasic) of healthy human donor heart tissue. Next, we utilized two complementary MS platforms for metabolite detection: direct-infusion ultrahigh-resolution Fourier-transform ion cyclotron resonance (DI-FTICR) and high-resolution liquid chromatography quadrupole time-of-flight tandem MS (LC-Q-TOF-MS/MS). Using DI-FTICR MS, 9644 metabolic features were detected where 7156 were assigned a molecular formula and 1107 were annotated by accurate mass assignment. Using LC-Q-TOF-MS/MS, 21,428 metabolic features were detected where 285 metabolites were identified based on fragmentation matching against publicly available libraries. Collectively, 1340 heart metabolites were identified in this study, which span a wide range of polarities including polar (benzenoids, carbohydrates, and nucleosides) as well as nonpolar (phosphatidylcholines, acylcarnitines, and fatty acids) compounds. The results from this study will provide critical knowledge regarding the selection of appropriate extraction and MS detection methods for the analysis of the diverse classes of human heart metabolites.