Comparative Metabolomics of Reproductive Organs in the Genus Aesculus (Sapindaceae) Reveals That Immature Fruits Are a Key Organ of Procyanidin Accumulation and Bioactivity

Fruit from A. hippocastanum L. are used commercially for chronic venous insufficiency (CVI). The isomeric mixture of pentacyclic triterpenoid saponins (β-aescin) exert anti-inflammatory effects. Hence, research has focused on β-aescin, yet the diversity, accumulation, and bioactivity of organ-specific secondary metabolites represent missed pharmacological opportunities. To this end, we applied an untargeted metabolomics approach by liquid chromatography—tandem mass spectrometry (LC–MS/MS) to the chemical profiles of flowers, immature fruits, and pedicels from 40 specimens across 18 species of Aesculus. Principal component analysis (PCA), orthogonal partial least squares (OPLS-DA), and molecular networking revealed stronger chemical differences between plant organs, than between species. Flowers are rich in glycosylated flavonoids, pedicels in organic acids and flavonoid aglycones, and immature fruits in monomeric flavan-3-ols and procyanidins. Although a high diversity of flavonoids and procyanidins was observed, the relative amounts differed by plant organ. Fruit extracts demonstrated the strongest antifungal (Saccharomyces cerevisiae) and antioxidant activity, likely from the procyanidins. Overall, secondary metabolite profiles are organ-specific, and fruits accumulate antifungal and antioxidant compounds. Due to the chemical similarity between species, similar effects may be achieved between species. This creates incentives for further exploration of the entire genus, in bioprospecting for potential therapeutic leads.

Study on Active Components of Cuscuta chinensis Promoting Neural Stem Cell Proliferation: Bioassay-Guided Fractionation

Neural stem cells (NSCs) exist in the central nervous system of adult animals and capable of self-replication. NSCs have two basic functions, namely the proliferation ability and the potential for multi-directional differentiation. In this study, based on the bioassay-guided fractionation, we aim to screen active components in Cuscuta chinensis to promote the proliferation of NSCs. CCK-8 assays were used as an active detection method to track the active components. On the basis of isolating active fraction and monomer compounds, the structures of these were identified by LC-MS and (1H, 13C) NMR. Moreover, active components were verified by pharmacodynamics and network pharmacology. The system solvent extraction method combined with the traditional isolation method were used to ensure that the fraction TSZE-EA-G6 of Cuscuta chinensis exhibited the highest activity. Seven chemical components were identified from the TSZE-EA-G6 fraction by UPLC-QE-Orbitrap-MS technology, which were 4-O-p-coumarinic acid, chlorogenic acid, 5-O-p-coumarinic acid, hyperoside, astragalin, isochlorogenic acid C, and quercetin-3-O-galactose-7-O-glucoside. Using different chromatographic techniques, five compounds were isolated in TSZE-EA-G6 and identified as kaempferol, kaempferol-3-O-glucoside (astragalin), quercetin-3-O-galactoside (hyperoside), chlorogenic acid, and sucrose. The activity study of these five compounds showed that the proliferation rate of kaempferol had the highest effects; at a certain concentration (25 μg/mL, 3.12 μg/mL), the proliferation rate could reach 87.44% and 59.59%, respectively. Furthermore, research results using network pharmacology techniques verified that kaempferol had an activity of promoting NSCs proliferation and the activity of flavonoid aglycones might be greater than that of flavonoid glycosides. In conclusion, this research shows that kaempferol is the active component in Cuscuta chinensis to promote the proliferation of NSCs.

Naturally Available Flavonoid Aglycones as Potential Antiviral Drug Candidates against SARS-CoV-2

Flavonoids are important secondary plant metabolites that have been studied for a long time for their therapeutic potential in inflammatory diseases because of their cytokine-modulatory effects. Five flavonoid aglycones were isolated and identified from the hydrolyzed aqueous methanol extracts of Anastatica hierochuntica L., Citrus reticulata Blanco, and Kickxia aegyptiaca (L.) Nabelek. They were identified as taxifolin (1), pectolinarigenin (2), tangeretin (3), gardenin B (4), and hispidulin (5). These structures were elucidated based on chromatographic and spectral analysis. In this study, molecular docking studies were carried out for the isolated and identified compounds against SARS-CoV-2 main protease (Mpro) compared to the co-crystallized inhibitor of SARS-CoV-2 Mpro (α-ketoamide inhibitor (KI), IC50 = 66.72 µg/mL) as a reference standard. Moreover, in vitro screening against SARS-CoV-2 was evaluated. Compounds 2 and 3 showed the highest virus inhibition with IC50 12.4 and 2.5 µg/mL, respectively. Our findings recommend further advanced in vitro and in vivo studies of the examined isolated flavonoids, especially pectolinarigenin (2), tangeretin (3), and gardenin B (4), either alone or in combination with each other to identify a promising lead to target SARS-CoV-2 effectively. This is the first report of the activity of these compounds against SARS-CoV-2.

Molecular Spectroscopic (FTIR and UV-Vis) and Hyphenated Chromatographic (UHPLC-qTOF-MS) Analysis and In Vitro Bioactivities of the Momordica balsamina Leaf Extract

Momordica balsamina (M. balsamina) is a medicinal herb comprising health-promoting secondary metabolites. This study was aimed to profile bioactive compounds in the methanolic extract of M. balsamina leaves using molecular spectroscopic (UV-Vis and FTIR) and hyphenated chromatographic (UHPLC-qTOF-MS) techniques and evaluate the biological (in vitro anti-inflammatory and cytotoxicity) activities of the extract. The preliminary phytochemical screening tests revealed the presence of cardiac glycosides, flavonoids, saponins, tannins, and terpenoids. The UV-Vis profile revealed various absorption bands ranging from 200 to 750 nm, indicating the presence of flavonoids, phenolic compounds, tannins, terpenoids, carotenoids, chlorophyll, and alkaloids. FTIR spectra confirmed the presence of alkaloids, flavonoids, terpenes, anthraquinones, and phenolic compounds. A high-resolution and accurate mass spectrometer (LC-QTOF-MS model LC-MS-9030 instrument) was used, and the results confirmed the presence of flavonoid aglycones, such as quercetin, isorhamnetin, and kaempferol, as well as pseudolaroside A and dicaffeoylquinic and feruloyl isocitric acids. To the best of our knowledge, this is the first report of pseudolaroside A dimer and feruloyl isocitric acid in M. balsamina leaves. In vitro cytotoxicity assay showed that the extract was nontoxic against human colorectal adenocarcinoma (HT29 and Caco2), Vero, and RAW 264.7 cells. However, the extract showed anti-inflammatory activity on RAW 264.7 cells. The study confirmed that M. balsamina leaves contain nontoxic secondary metabolites that may play a pivotal role in human health as anti-inflammatory agents.

The Impact of Citrus-Tea Cofermentation Process on Chemical Composition and Contents of Pu-Erh Tea: An Integrated Metabolomics Study

Ganpu tea, an emerging pu-erh compound tea, which is cofermented with the peel of Citrus reticulata “Chachi,” has been widely favored by Chinese consumers due to its potential health effects and distinct flavor and taste. So far, the influence of this cofermentation procedure on the chemical profile of pu-erh tea has barely been addressed yet. In this work, an ultra-high-performance liquid chromatography-Q Exactive Orbitrap mass spectrometry (UHPLC-QE Orbitrap MS)-based qualitative and quantitative method combined with multivariate analysis was conducted to comprehensively investigate the chemical changes in pu-erh tea after cofermented with Citrus peel. A total of 171 compounds were identified based on a three-level strategy, among which seven phenolic acids, 11 flavan-3-ols, and 27 flavonoids and flavonoid glycosides were identified from pu-erh tea for the first time. Eighty-nine main constituents were selected for further quantitative analysis using a validated method. Both the principal component analysis (PCA) of untargeted metabolomics and orthogonal partial least squares discriminant analysis (OPLS-DA) models of targeted components revealed the significant chemical profile disparity between the raw pu-erh tea and Ganpu tea. It showed that Citrus tea cofermentation process significantly decreased the total contents of phenolic acids, flavan-3-ols, and flavonoid aglycones, while most of the quercetin glycosides and myricetin glycosides as well as the vitexin were significantly increased. In addition, hesperidin, a flavonoid glycoside only existed in Citrus, was first found in pu-erh tea after cofermented with Citrus. This study clearly profiled the chemical composition and content changes of pu-erh tea after cofermented with Citrus peel, which revealed that Citrus tea cofermentation process further accelerated the fermentation of pu-erh tea and improved the unique flavor of tea.

Sustainable Extraction Techniques for Obtaining Antioxidant and Anti-Inflammatory Compounds from the Lamiaceae and Asteraceae Species

Melissa officinalis L. and Origanum majorana L., within Lamiaceae family, and Calendula officinalis L. and Achillea millefolium L., within the Asteraceae, have been considered a good source of bioactive ingredients with health benefits. In this study, the supercritical fluid extraction (SFE) using pure CO2, and the ultrasound assisted extraction (UAE) were proposed as green techniques to obtain plant-based extracts with potential antioxidant and anti-inflammatory activities. Higher values of total phenolic content and antioxidant activity were achieved in UAE ethanol:water (50:50, v/v) extracts. Meanwhile, UAE pure ethanol extracts showed greater anti-inflammatory activity. RP-HPLC-PAD-ESI-QTOF-MS/MS analysis showed a vast number of phenolic compounds in the extracts, including unreported ones. O. majorana ethanol:water extract presented the highest content of phenolics and antioxidant activity; among its composition, both rosmarinic acid and luteolin glucoside derivatives were abundant. The pure ethanol extract of A. millefolium resulted in an important content of caffeoylquinic acid derivatives, luteolin-7-O-glucoside and flavonoid aglycones, which could be related to the remarkable inhibition of TNF-α, IL-1β and IL-6 cytokines. Besides, borneol and camphor, found in the volatile fraction of A. millefolium, could contributed to this latter activity. Thus, this study points out that O. majorana and A. millefolium are considered a promising source of bioactive ingredients with potential use in health promotion.

Characterization of the CYP3A4 Enzyme Inhibition Potential of Selected Flavonoids

Acacetin, apigenin, chrysin, and pinocembrin are flavonoid aglycones found in foods such as parsley, honey, celery, and chamomile tea. Flavonoids can act as substrates and inhibitors of the CYP3A4 enzyme, a heme containing enzyme responsible for the metabolism of one third of drugs on the market. The aim of this study was to investigate the inhibitory effect of selected flavonoids on the CYP3A4 enzyme, the kinetics of inhibition, the possible covalent binding of the inhibitor to the enzyme, and whether flavonoids can act as pseudo-irreversible inhibitors. For the determination of inhibition kinetics, nifedipine oxidation was used as a marker reaction. A hemochromopyridine test was used to assess the possible covalent binding to the heme, and incubation with dialysis was used in order to assess the reversibility of the inhibition. All the tested flavonoids inhibited the CYP3A4 enzyme activity. Chrysin was the most potent inhibitor: IC50 = 2.5 ± 0.6 µM, Ki = 2.4 ± 1.0 µM, kinact = 0.07 ± 0.01 min−1, kinact/Ki = 0.03 min−1 µM−1. Chrysin caused the highest reduction of heme (94.5 ± 0.5% residual concentration). None of the tested flavonoids showed pseudo-irreversible inhibition. Although the inactivation of the CYP3A4 enzyme is caused by interaction with heme, inhibitor-heme adducts could not be trapped. These results indicate that flavonoids have the potential to inhibit the CYP3A4 enzyme and interact with other drugs and medications. However, possible food–drug interactions have to be assessed clinically.

Biotransformation of Flavonoids by Newly Isolated and Characterized Lactobacillus pentosus NGI01 Strain from Kimchi

Lactic acid bacteria (LAB) are generally recognized as safe (GRAS) microorganisms. This study aimed to identify novel LAB strains that can transform flavonoids into aglycones to improve bioavailability. We isolated 34 LAB strains from kimchi. The biotransformation activity of these 34 LAB strains was investigated based on α-L-rhamnosidase and β-D-glucosidase activities. Among them, 10 LAB strains with high activities were identified by 16S rRNA sequencing analysis. All tested LAB strains converted hesperidin to hesperetin (12.5–30.3%). Of these, only the Lactobacillus pentosus NGI01 strain produced quercetin from rutin (3.9%). The optimal biotransformation conditions for the L. pentosus NGI01 producing hesperetin and quercetin were investigated. The highest final product concentrations of hesperetin and quercetin were 207 and 78 μM, respectively. Thus, the L. pentosus NGI01 strain can be a biocatalyst for producing flavonoid aglycones in the chemical and food industries.

Network pharmacology and chemoproteomics unveil the mechanism of Total Flavonoid Aglycones Extract inhibiting EMT in A549 cell line

Non–small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality. The epithelial–mesenchymal transition (EMT) process promotes cancer cell migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, combining network pharmacology and stable isotope dimethyl–labeled proteomics technology, we found the inhibitory effect of TFAE on the EMT process of A549 cells, and established the relationship between TFAE, PI3K/Akt signaling pathways, TWIST1, and glycolysis pathway. LY294002 was incubated with A549 cells to inhibit PI3K/Akt signaling pathway, and small interfering RNA (siRNA) method was applied to knock down the TWIST1 gene in A549 cell. Compared with normal cells, LY294002–treated/ TFAE–treated/ TWIST1–silenced / TFAE and siTWIST1–treated cells showed poor migration and invasion capabilities, increased expression of epithelial markers and decreased expression of mesenchymal markers. All the results suggested that TFAE inhibits PI3K/Akt pathway and TWIST1 and then inhibits glycolysis pathway, thereby inhibiting the EMT process of A549 cells.

FLAVONOIDS OF THE EAST ASIAN SPECIES SERRATULA MANSHURICA KITAG.

One of the poorly studied and widespread plants in the Russian Far East is the Manchurian saw-wort (Serratula manshurica Kitag.). It is known that this plant is rich in phytoecdysteroids, which have high biological activity (anabolic, adaptogenic, cardiotropic, immunomodulatory effects). Two biologically active compounds of the flavonoid class were isolated from the aerial part of Serratula manshurica Kitag. Growing in the Primorsky Kray of the Far East of Russia by liquid extraction and recrystallization methods: quercetin-4'-O-β-D-glucuronopyranoside and apigenin-7-O-β-D-glucuronopyranoside. The structure of the isolated compounds was proved by UV-, NMR-spectroscopy and mass spectrometry with electrospray ionization. The quantitative content of flavonoids in the aerial part of the Serratula manshurica plant in different phenological phases of plant development (luteolin-4'-O-β-D-glucuronopyranoside, quercetin-4'-O-β-D-glucuronopyranoside, apigenin-7-O-β-D-glucuronopyranoside, 3-methylquercetin-4'-O-β-D-glucuronopyranoside, quercetin, apigenin, 3-methylquercetin, luteolin, isokaempferide) was determined by the method of reversed phase liquid chromatography (RP HPLC). It was shown that the maximum content of flavonoid aglycones in plants was observed during the beginning of the growing season and in the budding phase, and their glycosides - during the flowering phase. Thus, to obtain plant material with the maximum content of flavonoid aglycones (quercetin, 3-methylquercetin, luteolin) the collection of vegetative mass should be carried out during the beginning of the growing season, and their glycosides - during the flowering period. Serratula manshurica Kitag., which grows abundantly on the territory of Primorsky Krai, can be considered a constantly renewable and promising source for obtaining flavonoids.