Characterization Of Metabolites Research Articles

An integrated approach for studying exposure, metabolism, and disposition of traditional Chinese medicine using PATBS and MDRB tools: a case study of semen Armeniacae Amarum.

Deciphering the in vivo processes of traditional Chinese medicine (TCM) is crucial for identifying new pharmacodynamic substances and new drugs. Due to the complexity and diversity of components, investigating the exposure, metabolism, and disposition remains a major challenge in TCM research. In recent years, a number of non-targeted smart mass-spectrometry (MS) techniques, such as precise-and-thorough background-subtraction (PATBS) and metabolomics, have realized the intelligent identification of in vivo components of TCM. However, the metabolites characterization still largely relies on manual identification in combination with online databases. We developed a scoring approach based on the structural similarity and minimal mass defect variations between metabolites and prototypes. The current method integrates three dimensions of mass spectral data including m/z, mass defect of MS1 and MS2, and the similarity of MS2 fragments, which was sequentially analyzed by a R-based mass dataset relevance bridging (MDRB) data post-processing technique. The MDRB technology constructed a component relationship network for TCM, significantly improving metabolite identification efficiency and facilitating the mapping of translational metabolic pathways. By combining MDRB with PATBS through this non-targeted identification technology, we developed a comprehensive strategy for identification, characterization and bridging analysis of TCM metabolite in vivo. As a proof of concept, we adopted the proposed strategy to investigate the process of exposure, metabolism, and disposition of Semen Armeniacae Amarum (CKXR) in mice. The currently proposed analytical approach is universally applicable and demonstrates its effectiveness in analyzing complex components of TCMs in vitro and in vivo. Furthermore, it enables the correlation of in vitro and in vivo data, providing insights into the metabolic transformationsamong components sharing the same parent nucleus structure. Finally, the developed MDRB platform is publicly available for ( https://github.com/933ZhangDD/MDRB ) for accelerating TCM research for the scientific community.

Stability in fecal metabolites amid a diverse gut microbiome composition: a one-month longitudinal study of variability in healthy individuals

ABSTRACT An extensive network of microbial-host interactions exists in the gut, making the gut microbiome a complex ecosystem to untangle. The microbial composition and the fecal metabolites are important readouts to investigate intricate microbiota-diet-host interplay. However, this ecosystem is dynamic, and it is of interest to understand the degree and timescale of changes occurring in the gut microbiota, during disease as well as in healthy individuals. Cross-sectional study design is often used to investigate the microbiome, but this design provides a static snapshot and cannot provide evidence on the dynamic nature of the gut microbiome. Longitudinal studies are better suited to extrapolate causation in a study or assess changes over time. This study investigates longitudinal change in the gut microbiome and fecal metabolites in 14 healthy individuals with weekly sampling over a period of one-month (four time points), to elucidate the temporal changes occurring in the gut microbiome composition and fecal metabolites. Utilizing 16S rRNA amplicon sequencing for microbiome analysis and NMR spectroscopy for fecal metabolite characterization, we assessed the stability of these two types of measurable parameters in fecal samples during the period of one month. Our results show that the gut microbiome display large variations between healthy individuals, but relatively lower within-individual variations, which makes it possible to uniquely identify individuals. The fecal metabolites showed higher stability over time compared to the microbiome and exhibited consistently smaller variations both within and between individuals. This relative higher stability of the fecal metabolites suggests a balanced, consistent output even amid individual’s differences in microbial composition and they can provide a viable complementary readout to better understand the microbiome activity.

Substantiable <i>Eruca sativa</i> biomass: from mass spectrometry to insights for biopharmacotherapy

The use of medicinal plants to treat human ailments has received more attention recently. Eruca sativa (E. sativa; jarjeer) is a very popular edible plant of the Brassicaceae family with remarkable nutritional and therapeutic properties. In this frame, bioactive metabolites derived from E. sativa biomass have captured more attention in pharmacotherapy and biomedicine. This review focuses on recent advances in high-resolution mass spectrometry (HRMS), which have made the characterization of Eruca metabolites, such as glucosinolates and their derivatives, more feasible. Moreover, bio-pharmacological impact, viz. antiulcer, anticancer, antioxidant and antimicrobial, anti-obesity and hypoglycemic activities, cardiovascular benefits, and neuroprotective effects are discussed. This review concentrates all important research findings on these topics, and stresses upon the activities of chemistry and pharmacological areas. By stressing upon additional investigations, this paper emphasizes and appeals researchers and industrials to attempt a comprehensive knowledge of the pharmacological implications of Eruca species and improve their application in modern medicine.

Comparative analysis of four hop cultivars grown in Brazil and the USA by GC-MS-based metabolomics

Why was the work done: Although the third largest beer producer in the world, Brazil currently imports the majority of its hops. A recent development is the cultivation of hops (Humulus lupulus L.) in Brazil. In addition to genetic factors, the chemical composition of hops can exhibit variations due to conditions of cultivation. Accordingly, it is of value to characterise and differentiate hop cultivars grown in Brazil with the same cultivars grown in a long established location such as the United States of America. How was the work done: Centennial, Chinook, Columbus, and Nugget cultivars grown in Brazil or in the USA were compared by metabolomic analyses of the chemical profiles using gas chromatography coupled to mass spectrometry. Principal component analysis showed sample grouping according to where the hops were grown. Partial Least Squares Discriminant Analysis allowed the characterisation of the main metabolites that discriminated hop samples from the two countries. A total of 31 metabolites were putatively identified, including monoterpenes, sesquiterpenes, oxygenated mono- and sesquiterpenes, esters, alcohols, and ketones. What are the main findings: There were clear metabolic differences between the same hop varieties grown in Brazil or the USA. The metabolites with the greatest discriminating power for Brazilian hops were trans-α-bergamotene, 2-decanone, and ι-gurjunene, while American hops presented β-copaene, humuladienone, and isopentyl isobutyrate. Notably, trans-α-bergamotene was present in Brazilian hops but absent from American hops. Why is the work important: This study sheds light on the differences in the chemical composition of hops cultivated in Brazil compared those cultivated in the USA. This knowledge may stimulate new producers and contribute to the development of hop cultivation in Brazil.

Advanced Bionic Technology Combining Online Electrochemistry-Mass Spectrometry and Offline Electrochemistry-Liquid Chromatography-Mass Spectrometry for Simulating and Characterizing Metabolic Processes of Bioactive phenolic acids in Natural Products.

The metabolism research of bioactive phenolic acids widely found in natural products is of great significance for elucidating pharmacologic mechanisms and screening lead compounds. However, it is time-consuming and vulnerable to interference to conduct the traditional metabolism approach by applying organisms or biomaterials. Herein, a bionic technology was established by combining online electrochemistry-mass spectrometry (EC-MS) with offline electrochemistry-liquid chromatography-mass spectrometry (EC-LC-MS) to investigate the oxidative transformation and metabolic processes of the active phenolic acids (including salvianolic acid A, caffeic acid, 3, 5-O-dicaffeoylquinic acid, ferulic acid, salvianic acid A, and protocatechuic acid). Phase I metabolism of the phenolic acids were simulated by applying a three-electrode controlled potential electrochemical reactor with a boron-doped diamond electrode, with glutathione mixed into the oxidative products simultaneously for obtaining the phase II metabolites. Finally, structural characterization of the simulated metabolites of the phenolic acids was achieved successfully, including hydroxylation, methylation, demethylation, decarboxylation, etc. It was revealed that the simulated metabolism process based on an electrochemical system was effective in yielding a wide variety of metabolites for these compounds, which was also compared with the metabolism results applying rat liver microsomes. Consequently, this bionic technology is expected to be a powerful tool to investigate the material basis for the efficacy of active ingredients of natural products.

Diagnosis and Biomarker Screening of Endometrial Cancer Enabled by a Versatile Exosome Metabolic Fingerprint Platform.

Exosomes have emerged as a revolutionary tool for liquid biopsy (LB), as they carry specific cargo from cells. Profiling the metabolites of exosomes is crucial for cancer diagnosis and biomarker discovery. Herein, we propose a versatile platform for exosomal metabolite assay of endometrial cancer (EC). The platform is based on a nanostructured composite material comprising gold nanoparticle-coated magnetic COF with aptamer modification (Fe3O4@COF@Au-Apt). The unique design and novel synthesis strategy of Fe3O4@COF@Au-Apt provide the material with a large specific surface area, enabling the efficient and specific isolation of exosomes. The exosomes captured Fe3O4@COF@Au-Apt can be directly used as the laser desorption/ionization mass spectrometry (LDI-MS) matrix for rapid exosomal metabolic patterns. By integrating these functionalities into a single platform, the analytical process is simplified, eliminating the need for additional elution steps and minimizing potential sample loss, resulting in large-scale exosomal metabolic fingerprints. Combining with machine learning algorithms on the metabolic patterns, accurate discrimination between endometrial patients (EGs) and benign controls (CGs) was achieved, and the area under the receiver operating characteristic curve of the blind test cohort was 0.924. Confusion matrix analysis of important metabolic fingerprint features further demonstrates the high accuracy of the proposed approach toward EC diagnosis, with an overall accuracy of 94.1%. Moreover, four metabolites, namely, hydroxychalcone, l-acetylcarnitine, elaidic acid, and glutathione, have been identified as potential biomarkers of EC. These results highlight the great value of the integrated exosome metabolic fingerprint platform in facilitating low-cost and high-throughput characterization of exosomal metabolites for cancer diagnosis and biomarker discovery.

Isolation and Characterization of Some Potential Antibacterial Metabolites from Marine Microorganisms Collected in Egypt

Isolation and Characterization of Some Potential Antibacterial Metabolites from Marine Microorganisms Collected in Egypt

Identification and Characterization of Peanut Seed Coat Secondary Metabolites Inhibiting Aspergillus flavus Growth and Reducing Aflatoxin Contamination.

The peanut seed coat acts as a physical and biochemical barrier against Aspergillus flavus infection; however, the nature of the inhibitory chemicals in the peanut seed coat in general is not known. This study identified and characterized peanut seed coat metabolites that inhibit A. flavus growth and aflatoxin contamination. Selected peanut accessions grown under well-watered and water-deficit conditions were assayed for A. flavus resistance, and seed coats were metabolically profiled using liquid chromatography mass spectrometry. Kyoto Encyclopedia of Genes and Genome phenylpropanoid pathway reference analysis resulted in the identification of several seed coat metabolic compounds, and ten selected metabolites were tested for inhibition of A. flavus growth and aflatoxin contamination. Radial growth bioassay demonstrated that 2,5-dihydroxybenzaldehyde inhibited A. flavus growth (98.7%) and reduced the aflatoxin contamination estimate from 994 to 1 μg/kg. Scanning electron micrographs showed distorted hyphae and conidiophores in cultures of 2,5-dihydroxybenzaldehyde-treated A. flavus, indicating its potential use for field application as well as seed coat metabolic engineering.

Met4DX: A Unified and Versatile Data Processing Tool for Multidimensional Untargeted Metabolomics Data.

Liquid chromatography-mass spectrometry (LC-MS) is a powerful tool in untargeted metabolomics, enabling the high-sensitivity and high-specificity characterization of metabolites. The integration of ion mobility (IM) with LC-MS, known as LC-IM-MS, enhances the analytical depth, facilitating more comprehensive metabolite profiling. However, the complexity of data generated by these technologies presents significant challenges in data processing. Addressing these challenges, we developed Met4DX, a unified and versatile software tool for processing both 3D and 4D untargeted metabolomics data. Met4DX incorporates a new MS1-oriented peak detection approach coupled with our bottom-up assembly algorithm, enabling highly sensitive and comprehensive peak detection in untargeted metabolomics data. Additionally, Met4DX employs a uniform quantification strategy to enhance the precision of peak integration across different samples. The software provides a user-friendly interface that simplifies data processing with default parameter sets, consolidating peak detection, alignment, quantification, and other procedures into a single streamlined workflow. Together, Met4DX offers a comprehensive solution for multidimensional metabolomics data processing, transforming raw data from diverse MS instruments into a final feature table containing quantification and identification results. We postulate Met4DX facilitates metabolite discovery in biological samples by deciphering the complex untargeted metabolomics data. Met4DX is freely available on the Internet (https://met4dx.zhulab.cn/).

Chemical and Biological Characterization of Metabolites from Silene viridiflora Using Mass Spectrometric and Cell-Based Assays.

A comprehensive metabolite profiling of the medicinal plant Silene viridiflora using an UHPLC-ESI-MS/MS method is described for the first time. A total of 71 compounds were identified and annotated, the most common of which were flavonoids, triterpene glycosides, and ecdysteroids. The three major compounds schaftoside, 26-hydroxyecdysone, and silviridoside can be chosen as the markers for the assessment of the quality of S. viridiflora preparations. The methanol extract and a variety of metabolites identified in S. viridiflora were screened for their cytotoxic and Wnt pathway-inhibiting activities against triple-negative breast cancer (TNBC), the deadliest form of cancer in women. 2-Deoxy-20-hydroxyecdysone with submicromolar IC50 was identified as a result. The structure-activity relationship derived from the data from the in vitro proliferation assay showed that the hydroxyl group present at position C-2 of steroid core reduces the ecdysteroids' cytotoxicity against cancer cells.

Effects of priming duration and rhizosphere bacteria metabolite concentration on the germinability of cowpea, soybean, sesame, and okra seeds

Seed priming enhances germination and growth, which are important determinants of crop yield. This study was carried out to assess the effect of priming duration and metabolite concentration on the priming of five (5) crops using the metabolites of five (5) bacterial isolates. The crop seeds were treated in the cold-extracted metabolites of the five isolates at five (5) different priming durations (1, 2, 3, 4, and 5 h) and then in five metabolite concentrations (200, 400, 600, 800, and 1000 mg/L) of the five extracted metabolites at the optimal priming duration determined in the first experiment. Characterization of the cold-extracted metabolites was also carried out using gas-chromatography-mass spectrometry (GC-MS). Results revealed that priming cowpea and soybean seeds for longer durations (< 3 h) could hinder their growth and development. Lower concentrations were observed to be optimal for cowpea and soybean, but for sesame and okra, there was no detectable pattern with metabolite concentration. The GC-MS revealed the presence of some molecules (e.g. hexadecanoic acid) that have shown plant growth promotion potential in other studies. This study showed that seeds with large endosperms, such as those of cowpea and soybean, are more prone to the harmful effects of treatment for longer durations. Further experiments should be undertaken to isolate and purify the bioactive moieties for further studies and application.

GCMS and LCMS/MS Based Analysis for the Identification and Characterization of Bioactive Metabolites of Seaweed Kappaphycus striatus as an Anti-hypertensive and Anti-hypercholesterolemic Agent

GCMS and LCMS/MS Based Analysis for the Identification and Characterization of Bioactive Metabolites of Seaweed Kappaphycus striatus as an Anti-hypertensive and Anti-hypercholesterolemic Agent

Quantitation of Usnic acid from three Usnea species, its commercial formulations Using High-Performance Thin-Layer Chromatography-Mass Spectrometry (HPTLC-MS) and their metabolite profiling using UPLC-QTof-MSE.

The genus Usnea (Parmeliaceae; lichenized Ascomycetes) is pale grayish-green fruticose lichens which grow as leafless mini-shrubs and comprise about 360 species. Most of the Usnea species are edible and is utilized in preparation of traditional foods as well as in medicines to combat wide range of ailments. The goal of this work was to quantify usnic acid in three Usnea spp. [Usnea ghattensis (UG), Usnea orientalis (UO) and Usnea undulata (UU)] using HPTLC-MS and chemical profiling of acetone extracts using UPLC-QTof-MSE resulted in the identification of sixteen compounds based on their MS/MS fragmentation patterns. Hyphenated techniques, HPTLC-MS and UPLC-QTof-MSE have been proposed to quantify usnic acid and analysis of metabolites in the crude extracts qualitatively. This method allowed tentative characterization of metabolites from Usnea spp. The quantification study showed the excellent linearity of the usnic acid at 0.25-1 µg/band with a correlation coefficient r 2>0.99, and LOD, LOQ was found to be 51.7 and 156.6 ng/band, respectively. Further, UPLC-QTof-MSE analysis of crude extract led identification of lichen substances through their exact molecular masses and MS/MS fragmentation studies. The present study summarizes HPTLC method for quantification of usnic acid in three different Usnea spp. Along with two herbal formulations containing Usnea spp. as the ingredient and developed method was validated as per the ICH guidelines and further UPLC-QTof-MSE analysis provides characterization of the sixteen different secondary metabolites based on their mass fragmentation studies. Rapid HPTLC method for quantification of usnic acid in three different Usnea spp. along with two herbal formulations and metabolite profiling using UPLC-QTof-MSE.

Role of grass endophytic fungi as a natural resource of bioactive metabolites.

Grass endophytic fungi have garnered increasing attention as a prolific source of bioactive metabolites with potential application across various fields, including pharmaceticals agriculture and industry. This review paper aims to synthesize knowledge on the diversity, isolation, and bioactivity of metabolites produced by grass endophytic fungi. Additionally, this approach aids in the conservation of rare and endangered plant species. Advanced analytical techniques such as high-performance liquid chromatography, liquid chromatograpy-mass spectrometry and gas chromatography are discussed as critical tools for metabolite identification and characterization. The review also highlights significant bioactive metabolites discovered to date, emphasizing their antimicrobial, antioxidant, and insecticidal activities and plant growth regulation properties. Besides address the challenges and future prospects in harnessing grass endophytic fungi for sustainable biotenological applications. By consolidating recent advancements and identifying agaps in the current research, this paper provides a comprehensive overview of the potential grass endophytic fungi as a valuable resource for novel bioactive compounds.

Role of Gut Microbial Metabolites in Cardiovascular Diseases-Current Insights and the Road Ahead.

Cardiovascular diseases (CVDs) are the leading cause of premature morbidity and mortality globally. The identification of novel risk factors contributing to CVD onset and progression has enabled an improved understanding of CVD pathophysiology. In addition to the conventional risk factors like high blood pressure, diabetes, obesity and smoking, the role of gut microbiome and intestinal microbe-derived metabolites in maintaining cardiovascular health has gained recent attention in the field of CVD pathophysiology. The human gastrointestinal tract caters to a highly diverse spectrum of microbes recognized as the gut microbiota, which are central to several physiologically significant cascades such as metabolism, nutrient absorption, and energy balance. The manipulation of the gut microbial subtleties potentially contributes to CVD, inflammation, neurodegeneration, obesity, and diabetic onset. The existing paradigm of studies suggests that the disruption of the gut microbial dynamics contributes towards CVD incidence. However, the exact mechanistic understanding of such a correlation from a signaling perspective remains elusive. This review has focused upon an in-depth characterization of gut microbial metabolites and their role in varied pathophysiological conditions, and highlights the potential molecular and signaling mechanisms governing the gut microbial metabolites in CVDs. In addition, it summarizes the existing courses of therapy in modulating the gut microbiome and its metabolites, limitations and scientific gaps in our current understanding, as well as future directions of studies involving the modulation of the gut microbiome and its metabolites, which can be undertaken to develop CVD-associated treatment options. Clarity in the understanding of the molecular interaction(s) and associations governing the gut microbiome and CVD shall potentially enable the development of novel druggable targets to ameliorate CVD in the years to come.

Characterization and comparison of metabolites in colostrum from yaks, buffaloes, and cows based on UPLC-QTRAP-MS metabolomics

Colostrum from yaks and buffaloes possesses substantial nutritional value, yet the complete array of metabolites within remains insufficiently elucidated. This study scrutinizes the metabolite profiles of yak, buffalo, and cow colostrum utilizing targeted metabolomics paired with ultra-performance liquid chromatography-tandem triple quadrupole linear ion trap mass spectrometry (UPLC-QTRAP-MS). The analysis detected 362 metabolites across all samples. Furthermore, 63, 77, and 46 differential metabolites were selected between yak and buffalo colostrum, yak and cow colostrum, and buffalo and cow colostrum, respectively. Yak colostrum notably contained higher concentrations of inositol, glycine, and carnitine, whereas buffalo colostrum was distinguished by a substantial presence of primary bile acids, which facilitate fat digestion. These findings offer profound insights into yak and buffalo colostrum, providing critical data to propel advancements in the dairy industry.

Phenolic profile, antioxidant capacity and oxidoreductase enzyme activity in autochthonous grape varieties from Bosnia and Herzegovina

The variability of phenolic compounds among grape varieties has an important role in selecting winemaking techniques, but the use of phenolic profiles for quality control is still fragmented and incomplete. Given the recent climate change and global warming, biochemical characterisation of secondary metabolites in autochthonous grape varieties is a very important factor for their preservation and sustainable agriculture. Two autochthonous grape varieties from the western Herzegovina region in Bosnia and Herzegovina have been selected for the research targeting at the evaluation of their phenolic profiles, antioxidant activities, and the correlation with oxidoreductase enzymes polyphenol oxidase and Class III peroxidase, in different berry tissues. The obtained results indicate a similar qualitative profile of phenolic compounds in exocarp and mesocarp in both varieties, but their concentrations and antioxidant activity vary significantly. The correlation between phenolic compounds and oxidoreductase enzyme activities in different grape berry tissues is discussed in this article.

Chemical profiling and probiotic viability assessment in Gueuze-style beer: Fermentation dynamics, metabolite and sensory characterization, and in vitro digestion resistance

Probiotic viability, metabolite concentrations, physicochemical parameters, and volatile compounds were characterized in Gueuze beers formulated with probiotic lactic acid bacteria (LAB) and yeast. Additionally, the sensory profile of the beers and the resistance of the probiotics to digestion were determined. The use of 2 International Bitterness Units resulted in high concentrations of probiotic LAB but a decline in probiotic yeast as pH decreased. Secondary fermentation led to the consumption of maltose, citric acid, and malic acid, and the production of lactic and propionic acids. Carbonation and storage at 4 °C had minimal impact on probiotic viability. The addition of probiotic LAB resulted in a distinct aroma profile with improved sensory characteristics. Our results demonstrate that sour beers produced with probiotic LAB and a probiotic yeast, and fermented using a two-step fermentation process, exhibited optimal physicochemical parameters, discriminant volatile compound profiles, promising sensory characteristics, and high probiotic concentrations after digestion.

Characterization of Neonicotinoid Metabolites by Cytochrome P450-Mediated Metabolism in Poultry.

Neonicotinoids, a neuro-effective class of insecticides, are heavily applied in agricultural activities worldwide. Poultry can be exposed to neonicotinoids by several routes, but the knowledge of neonicotinoid's metabolism in poultry and its associated interspecies differences is highly limited. Hence, this study aims to investigate the species differences in metabolite formations, as well as cytochrome P450 (CYP)-dependent metabolism of four major neonicotinoid compounds, acetamiprid, imidacloprid, clothianidin, and thiamethoxam, in poultry. In vitro biotransformation assays using hepatic microsomes of chicken, ducks, geese, quails, and rats were conducted. Metabolites of neonicotinoids were then screened by LC/Q-TOF and quantified by LC/MS/MS. The results revealed an existence of interspecies differences in the formations of N-[(6-chloro-3-pyridyl) methyl] -N-methyl acetamidine (IM-1-5) of acetamiprid and dm-clothianidin of clothianidin between chicken and other species. In addition, the greatest CYP activities in the metabolism of most neonicotinoid substrates, such as acetamiprid to dm-acetamiprid, imidacloprid to hydroxylated-imidacloprid and imidacloprid-olefin, clothianidin to dm-clothianidin, and thiamethoxam to clothianidin, were found in chicken. These results suggested that the CYPs in chicken may have a greater capacity for metabolism of neonicotinoids compared to other poultry. This study further revealed that the maximum intrinsic clearance of dn-imidacloprid and dn-clothianidin in ducks may be superintended by CYP-mediated nitro-reductions of imidacloprid and clothianidin. Further studies employing CYP recombinant enzymes may be required to elucidate the specific CYP isoforms that may be involved in neonicotinoid metabolism in avian species.

Thunbergia's Flowers Secondary Metabolites a Natural Armor Against Kidney Damage by Diclofenac.

Nephrotoxicity is a common side effect arising from exposure to drugs or toxins. The study investigates the therapeutic effects of Thunbergia alata and Thunbergia erecta flowers on diclofenac-induced renal injury. Secondary metabolite characterization by positive mode high-resolution-ESI (LC-HR-ESI-MS) was followed by assessing their renal protection against diclofenac-induced damage and molecular docking studies. Using positive LC-HR-ESI-MS, 18 compounds from T. erecta and T. alata were identified. Diclofenac administration induced significant deterioration of all parameters in the kidney in addition to renal tissue contents of several inflammatory markers. The flower extracts of T. alata and T. erecta showed a clear improvement in the treated groups compared to the diclofenac-control group. The results were confirmed by histopathological examinations followed by immunohistochemical determination of vascular endothelial growth factor (VEGF), nuclear factor erythroid 2-related factor 2 (Nrf2), and transforming growth factor beta 1 (TGF-β1) expression. Furthermore, a protein-protein network to understand the complex interplay between the target proteins and their counterparts was done in addition to a molecular docking study of the de-replicated compounds in the active sites of NF-κB, TGF-β1, and VEGFR.