Bioactivity-guided Fractionation Research Articles

From Sea to Science: Coral Aquaculture for Sustainable Anticancer Drug Development.

Marine natural products offer immense potential for drug development, but the limited supply of marine organisms poses a significant challenge. Establishing aquaculture presents a sustainable solution for this challenge by facilitating the mass production of active ingredients while reducing our reliance on wild populations and harm to local environments. To fully utilize aquaculture as a source of biologically active products, a cell-free system was established to target molecular components with protein-modulating activity, including topoisomerase II, HDAC, and tubulin polymerization, using extracts from aquaculture corals. Subsequent in vitro studies were performed, including MTT assays, flow cytometry, confocal microscopy, and Western blotting, along with in vivo xenograft models, to verify the efficacy of the active extracts and further elucidate their cytotoxic mechanisms. Regulatory proteins were clarified using NGS and gene modification techniques. Molecular docking and SwissADME assays were performed to evaluate the drug-likeness and pharmacokinetic and medicinal chemistry-related properties of the small molecules. The extract from Lobophytum crassum (LCE) demonstrated potent broad-spectrum activity, exhibiting significant inhibition of tubulin polymerization, and showed low IC50 values against prostate cancer cells. Flow cytometry and Western blotting assays revealed that LCE induced apoptosis, as evidenced by the increased expression of apoptotic protein-cleaved caspase-3 and the populations of early and late apoptotic cells. In the xenograft tumor experiments, LCE significantly suppressed tumor growth and reduced the tumor volume (PC3: 43.9%; Du145: 49.2%) and weight (PC3: 48.8%; Du145: 7.8%). Additionally, LCE inhibited prostate cancer cell migration, and invasion upregulated the epithelial marker E-cadherin and suppressed EMT-related proteins. Furthermore, LCE effectively attenuated TGF-β-induced EMT in PC3 and Du145 cells. Bioactivity-guided fractionation and SwissADME validation confirmed that LCE's main component, 13-acetoxysarcocrassolide (13-AC), holds greater potential for the development of anticancer drugs.

Screening of peptidyl arginine deiminase 4 inhibitors in traditional herbal medicines

Peptidyl arginine deiminase 4 (PAD4) is a promising target for the treatment of metabolic diseases associated with autoimmune and central nervous system disease. By now there are limited numbers of PAD4 inhibitors, and no one is ready for clinical use. This study aims to find efficient and specific PAD4 inhibitors from traditional herbal medicines and to investigate their inhibitory mechanisms. The inhibitory effects of forty-eight extracts from sixteen traditional herbal medicines which are widely used in traditional herbal medicines were investigated. Salvia miltiorrhiza was found to have the most potent PAD4 inhibitory activity. After that, a practical bioactivity-guided fractionation coupling with a chemical profiling strategy was used to identify the fractions from Salvia miltiorrhiza with strong PAD4 inhibition activity, and the major constituents in these bioactive fractions were characterized by LC-MS/MS. Seven compounds were found to have inhibition on PAD4 with IC50 values ranging from 33.52 μM to 667 μM, in which salvianolic acid A showed the most potent inhibitory activity, with an IC50 value of 33.52 μM. Inhibition kinetic analyses indicated that salvianolic acid A effectively inhibited PAD4 in a mixed inhibitory manner, and computer simulation analyses demonstrated that salvianolic acid A binds to PAD4 mainly using hydrogen bonding. Overall, our results suggest that salvianolic acid A from Salvia miltiorrhiza is a potent inhibitor of PAD4, and that salvianolic acid A can be used as a promising lead compound for the development of more potent PAD4 inhibitors.

Investigation of the uterine modulating activities and mechanisms of Brachystegia eurycoma Harms (Leguminosae) methanol stem bark extract and fractions

Dysfunctional labour (DL) is a major cause of maternal and neonatal deaths worldwide. The only clinically available drug is oxytocin which is about 50% effective. Therefore, the development of new effective drugs for DL is a necessity. The medicinal plant Brachystegia eurycoma Harms (synonym B. spiceaformis) is used for labour management in ethnomedicine. In the search for new lead compounds for DL therapy, the stem bark of B. eurycoma was subjected to bioactivity-guided fractionation procedures to elucidate its uterine-modulating activities and active compounds. B. eurycoma stem bark extract (BE) was fractionated via vacuum liquid chromatographic techniques. The fractionation process was guided by ex vivo uterine contractility assays on the mouse uterus, performed using spontaneous, oxytocin, and high KCl-induced contractility assays. Furthermore, the characterization of the chemical constituents was performed using nuclear magnetic resonance (NMR) and mass spectrometry (MS). BE produced dual effects on uterine contractility which was dependent on the reconstitution period. The freshly prepared crude extract (BEF) inhibited uterine contractility (IC50 amplitude = 0.8 ± 0.1 mg/ml; IC50 frequency = 1.4 ± 0.2 mg/ml). However, contractility was augmented when the reconstituted extract was stored for 24 h and beyond (BES) (EC50 amplitude = 0.2 ± 1.2 mg/ml; EC50 frequency = 0.91 ± 1.2 mg/ml). In addition, the compounds caffeine, genistein, and prunetin were identified from the potent BE fractions via the different spectroscopic and spectrometric tools. This study demonstrated a storage-dependent dual effect of BE where BEF induced inhibition and BES augmented uterine contractility. Findings from this study also suggest that caffeine and phytoestrogens contributed to the uterine-modulating activities of BE. Thus, further investigation of these compounds in combination and as separate lead compounds is required for DL therapeutic management.

Antimicrobial Activity of Fungal Endophytes Associated with Peperomia argyreia (Piperaceae)

The endophytic fungal biodiversity of unique plants like Peperomia argyreia (Miq.) É. Morren (Piperaceae) has antimicrobial properties and can be employed for infection treatment. Fungal isolates were obtained from appropriately treated plant tissues cultured in solid media, characterized by morphology, and identified by molecular biology using ITS and NL primers. The antimicrobial properties of fungal extracts were analyzed by combining microdilution and bioautographic assays complemented with metabolic profiling by automated thin-layer chromatography and 1H NMR techniques. Thirty-one filamentous fungi were isolated and characterized by ITS and/or D1/D2 region amplification of rDNA, identified as Thermothielavioides, Trichoderma, Cyphellophora, Cladosporium, Arcopilus, Plectosphaerella; Chaetomium, Sporothrix, Alboefibula, and Penicillium. Thermothielavioides spp. inhibited Staphylococcus aureus ATCC 25923; moreover, Penicillium westlingii P4 showed inhibitory activity on Ascochyta rabiei AR2. The bioactivity-guided fractionation of the EtOAc extract (MIC = 62.5 μg/mL) of P. westlingii P4 allowed the purification of citrinin as the main inhibitory compound (MIC = 62.5 μg/mL). Peperomia argyreia harbors a rich and diverse endophytic community able to produce bioactive molecules. Citrinin, with a minor influence of volatile compounds biosynthesized by P. westlingii P4, was responsible for the inhibition of A. rabiei AR2.

Anti-IL-17 bioactivity-guided fractionation of a pine bio-oil: Chemical characterization and impact on HaCaT human keratinocytes gene expression

Psoriasis is a dermatological inflammatory disease treated for centuries with pyrolytic pine bio-oils (pine tars), although their chemical composition and mechanism of action are poorly investigated. This study focuses on the fractionation of a standardized pine pyrolysis bio-oil, its chemical characterization (GC–MS, UV fluorescence, ESI-MS, and NMR), and the analysis of the anti-IL17 activity of fractions on HaCaT keratinocytes. Furthermore, the impact of the fraction showing the highest anti-IL-17 activity on keratinocyte gene expression was determined. Pine bio-oil comprises molecules derived from cellulose, hemicellulose, and lignin thermochemical degradation reactions, such as hydroxyacetaldehyde, levoglucosan, acetic acid, and phenols. The aqueous fraction exhibited the most consistent anti-IL-17 activity. The functional analysis suggested that this fraction affected the expression of 29 genes associated with the negative regulation of the mitogen-activated protein kinase (MAPK) activity, a pathway known for its crucial role in the inflammatory immune response.

Metabolite profiling and bioactivity guided fractionation of Lactobacillaceae and rice bran postbiotics for antimicrobial-resistant Salmonella Typhimurium growth suppression.

Probiotic-fermented supplements (postbiotics) are becoming increasingly explored for their activity against antibiotic-resistant enteropathogens. Prebiotics are often incorporated into postbiotics to enhance their efficacy, but due to strain differences in probiotic activity, postbiotic antimicrobial effects are poorly understood. To improve postbiotic antimicrobial efficacy, we investigated and compared metabolite profiles of postbiotics prepared with three lactic acid bacteria strains (L. fermentum, L. paracasei, and L. rhamnosus) cultured with and without rice bran, a globally abundant, rich source of prebiotics. At their minimum inhibitory dose, L. fermentum and L. paracasei postbiotics + rice bran suppressed S. Typhimurium growth 42-55% more versus their respective probiotic-alone postbiotics. The global, non-targeted metabolome of these postbiotics identified 109 metabolites increased in L. fermentum and L. paracasei rice bran postbiotics, including 49 amino acids, 20 lipids, and 12 phytochemicals metabolites. To identify key metabolite contributors to postbiotic antimicrobial activity, bioactivity-guided fractionation was applied to L. fermentum and L. paracasei rice bran-fermented postbiotics. Fractionation resulted in four L. fermentum and seven L. paracasei fractions capable of suppressing S. Typhimurium growth more effectively versus the negative control. These fractions were enriched in 15 metabolites that were significantly increased in the global metabolome of postbiotics prepared with rice bran versus postbiotic alone. These metabolites included imidazole propionate (enriched in L. fermentum + rice bran, 1.61-fold increase; L. paracasei + rice bran 1.28-fold increase), dihydroferulate (L. fermentum + rice bran, 5.18-fold increase), and linoleate (L. fermentum + rice bran, 1.82-fold increase; L. paracasei + rice bran, 3.19-fold increase), suggesting that they may be key metabolite drivers of S. Typhimurium growth suppression. Here, we show distinct mechanisms by which postbiotics prepared with lactic acid bacteria and rice bran produce metabolites with antimicrobial activity capable of suppressing S. Typhimurium growth. Probiotic strain differences contributing to postbiotic antimicrobial activity attract attention as adjunctive treatments against pathogens.

Antiprotozoal Activity of Plants Used in the Management of Sleeping Sickness in Angola and Bioactivity-Guided Fractionation of Brasenia schreberi J.F.Gmel and Nymphaea lotus L. Active against T. b. rhodesiense.

Folk medicine is widely used in Angola, even for human African trypanosomiasis (sleeping sickness) in spite of the fact that the reference treatment is available for free. Aiming to validate herbal remedies in use, we selected nine medicinal plants and assessed their antitrypanosomal activity. A total of 122 extracts were prepared using different plant parts and solvents. A total of 15 extracts from seven different plants exhibited in vitro activity (>70% at 20 µg/mL) against Trypanosoma brucei rhodesiense bloodstream forms. The dichloromethane extract of Nymphaea lotus (leaves and leaflets) and the ethanolic extract of Brasenia schreberi (leaves) had IC50 values ≤ 10 µg/mL. These two aquatic plants are of particular interest. They are being co-applied in the form of a decoction of leaves because they are considered by local healers as male and female of the same species, the ethnotaxon "longa dia simbi". Bioassay-guided fractionation led to the identification of eight active molecules: gallic acid (IC50 0.5 µg/mL), methyl gallate (IC50 1.1 µg/mL), 2,3,4,6-tetragalloyl-glucopyranoside, ethyl gallate (IC50 0.5 µg/mL), 1,2,3,4,6-pentagalloyl-β-glucopyranoside (IC50 20 µg/mL), gossypetin-7-O-β-glucopyranoside (IC50 5.5 µg/mL), and hypolaetin-7-O-glucoside (IC50 5.7 µg/mL) in B. schreberi, and 5-[(8Z,11Z,14Z)-heptadeca-8,11,14-trienyl] resorcinol (IC50 5.3 µg/mL) not described to date in N. lotus. Five of these active constituents were detected in the traditional preparation. This work provides the first evidence for the ethnomedicinal use of these plants in the management of sleeping sickness in Angola.

An Update on Pharmacological and Phytochemical Aspects of Costus pictus D. Don - A Promising Anti-diabetic Plant.

The genus Costus is the largest genus in the family Costaceae and encompasses about 150 known species. Among these, Costus pictus D. Don (Synonym: Costus mexicanus) is a traditional medicinal herb used to treat diabetes and other ailments. Currently, available treatment options in modern medicine have several adverse effects. Herbal medicines are gaining importance as they are cost-effective and display improved therapeutic effects with fewer side effects. Scientists have been seeking therapeutic compounds in plants, and various in vitro and in vivo studies report Costus pictus D. Don as a potential source in treating various diseases. Phytochemicals with various pharmacological properties of Costus pictus D. Don, viz. anti-cancer, anti-oxidant, diuretic, analgesic, and anti-microbial have been worked out and reported in the literature. The aim of the review is to categorize and summarize the available information on phytochemicals and pharmacological properties of Costus pictus D. Don and suggest outlooks for future research. This review combined scientific data regarding the use of Costus pictus D. Don plant for the management of diabetes and other ailments. A systematic search was performed on Costus pictus plant with anti-diabetic, anti-cancer, anti-microbial, anti-oxidant, and other pharmacological properties using several search engines such as Google Scholar, PubMed, Science Direct, Sci-Finder, other online journals and books for detailed analysis. Research data compilation and critical review of the information would be beneficial for further exploration of its pharmacological and phytochemical aspects and, consequently, new drug development. Bioactivity-guided fractionation, isolation, and purification of new chemical entities from the plant as well as pharmacological evaluation of the same will lead to the search for safe and effective novel drugs for better healthcare. This review critically summarizes the reports on natural compounds, and different extract of Costus pictus D. Don with their potent anti-diabetic activity along with other pharmacological activity. Since this review has been presented in a very interactive manner showing the geographical region of availability, parts of plant used, mechanism of action and phytoconstituents in different extracts of Costus pictus responsible for particular action, it will be of great importance to the interested readers to focus on the development of the new drug leads for the treatment of diseases.

A study of the protease inhibitory activity component of SARS-CoV-2 3CL in Bletilla striata

The protease Mpro (referred to as 3CLpro or 3CL protease) is a cysteine protease that is highly conserved in coronavirus and is indispensable for viral replication. Because there is no homologous protein for MPro in the human body, SARS-CoV-2 3CL protease is an ideal target against coronavirus. Bletilla striata (Reich. Bf.) is a well-known form of Traditional Chinese Medicine and can exert many pharmacological effects, including hemostasis, anti-microbial and anti-virus activities. Our preliminary screening showed that the n-butanol component of a methanol extract of B. striata exhibited potent inhibitory activity against SARS-CoV-2 3CL protease (58.82% at 200 μg/mL). In this study, we biologically evaluated ten isolated chemical compounds from B. striata and investigated the inhibitory activities of its constituents on SARS-CoV-2 3CL protease. Following bioactivity-guided fractionation, four bibenzyls (1, 4, 6 and 7), three phenols (5, 8 and 9), two anthraquinones (2 and 3) and one glucosyloxybenzyl 2-isobutylmalate (10) were isolated and evaluated for their ability to inhibit SARS-CoV-2 3CL protease by fluorescence resonance energy transfer (FRET) analysis. The binding mode between compounds and enzymes was investigated by molecular docking and Saturation Transfer Differences - nuclear magnetic resonance (STD-NMR). Moreover, Pleiobibenzynin B (7), Blestritin B, Gymconopin D (4), Physcion, 3′-O-methyl dioscin III (6), Gastrodin (8) and caffeic acid (5) all exhibited inhibitory activity against 3CL protease in vitro. The four bibenzyls (1, 4, 6 and 7) exhibited good inhibitory activity against SARS-CoV-2 3CL protease (7.37–39.86 μM). These findings identify potential new inhibitors against SARS-CoV-2 3CL protease, which offers promising lead compounds for the development of novel anti-SARS-CoV-2 drugs.

Abstract 5766: Fungal genomes in drug discovery: Application of CETSA to study the mechanism of action of brefeldin A

Abstract Fungal genomes encode a vast collection of chemical diversity that has evolved over millions of years to engage specific targets and modulate diverse biological processes. These Genetically Encoded small Molecules (GEMs) constitute a largely untapped source of potential therapeutics. Although fungal GEMs such as cyclosporine, lovastatin, and penicillin have revolutionized modern medicine, these GEMs were discovered serendipitously through bioactivity-guided fractionation of fungal extracts. LifeMine is redefining GEM discovery through the deployment of proprietary genomic search algorithms that enable GEM target prediction a priori. Our strategy facilitates focused search-and-retrieval campaigns to rationally discover molecules selected by Nature to modulate the desired target protein. These GEM drug leads often exhibit unique structural and mechanistic features, necessitating comprehensive deconvolution of the human target profile and mechanism of action during the development process from GEM to clinical candidate. As a proof-of-concept study, we leveraged CETSA Explore to study the biological mechanism of the fungal polyketide lactone Brefeldin A (BFA) in human THP-1 cells. This GEM, originally isolated from the filamentous fungus Penicillium brefeldianis, inhibits protein trafficking between the endoplasmic reticulum and the Golgi complex, eliciting antiviral, antifungal, and anticancer activity. Our CETSA profiling study confirmed the Golgi-associated guanine nucleotide exchange factor GBF1 as the primary target of BFA, validating the established mechanism of this GEM as an inhibitor of GBF1-mediated activation of the small GTPase ARF1. These results demonstrate the utility of integrating the LifeMine GEM discovery engine with downstream proteomic profiling technologies to facilitate the mechanistic characterization of fungal GEMs en route to novel therapeutics. Citation Format: Tomas Friman, Benjamin M. Fontaine, Alexey Chernobrovkin, Daniel Martinez Molina, Victoria Brehmer, Stina Lundgren, Paola M. Castaldi, Gregory L. Verdine. Fungal genomes in drug discovery: Application of CETSA to study the mechanism of action of brefeldin A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5766.

Anti-inflammatory activities of some Anthemis species used in the treatment of inflammation-related diseases, GC/MS and LC-MS/MS analysis with bioactivity-guided fractionation

Recent studies have focused on Anthemis sp. because of their rich chemical composition and application in the treatment of diseases associated with inflammation. The purpose of this study was to determine the active chemicals responsible for the anti-inflammatory properties of Anthemis cotula, A. tomentosa, A. austriaca, and A. tinctoria var. tinctoria. Therefore, the 5-Lipoxygenase (5-LOX) was used to assess the anti-inflammatory properties of methanol extracts that were extracted from the aerial parts of the mentioned species, with an IC50 value of 23.88 μg/mL, the methanol extract of A. tinctoria var. tinctoria demonstrated anti-inflammatory effects close to the standard (Indomethacin, IC50: 18.05 μg/mL). Compared to the other extracts, the methanol extract of A. tinctoria var. tinctoria demonstrated significantly greater anti-inflammatory activity. A. tinctoria var. tinctoria and A. tomentosa methanol extracts, which were the most active in comparison to other extracts, were fractionated using the bioactivity-guided fractionation method. Their anti-inflammatory properties were evaluated to determine the compounds responsible for the activity. With an IC50 value of 3.44 μg/mL, the ethyl acetate fraction of A. tinctoria var. tinctoria (ATiEA) was found to be the most active of all the fractions. After ATiEA was further fractionated using column chromatography, it was found that ATiEA-II, one of its sub-fractions, was more effective than other sub-fractions against the 5-LOX, with an IC50 value of 2.93 μg/mL. Furthermore, the sub-fractions' total phenol and flavonoid contents were examined to confirm their anti-inflammatory properties. The sub-fraction ATiEA-II exhibited higher total flavonoid content (743,86 μg QE/g dry weight) and total phenol content (4659.36 μg GA/g dry weight) than other sub-fractions, which was in line with its anti-inflammatory activity. GC/MS and LC-MS/MS were used to clarify the effective compounds of the sub-fraction ATiEA-II. Experiments on the anti-inflammatory properties of different Anthemis sp. have demonstrated the good anti-inflammatory potential of this species. Specifically, ATiEA-II, a sub-fraction of A. tinctoria var. tinctoria, may contain a novel anti-inflammatory drug molecule.

Asperustins A-J: Austocystins with Immunosuppressive and Cytotoxic Activities from Aspergillus ustus NRRL 5856.

Ten new (1-10) and nine known (11-19) austocystins, along with four known anthraquinones (20-23), were isolated from the culture of Aspergillus ustus NRRL 5856 by bioactivity-guided fractionation. The structures of the new compounds were elucidated by spectroscopic data analysis, X-ray crystallographic study, the modified Mosher's method, [Rh2(OCOCF3)4]-induced ECD spectral analysis, and comparison of the experimental ECD spectra with those of the similar analogues. Compounds 1-8 represent the first examples of austocystins with a C-4' oxygenated substitution. The absolute configuration of 1″-hydroxy austocystin D (11) was determined by single-crystal X-ray diffraction and consideration of its biosynthetic origin. Compounds 5, 9, and 11 exhibited significant inhibitory effects against the proliferation of ConA-induced T cells with IC50 values of 1.1, 1.0, and 0.93 μM, respectively. Furthermore, these compounds suppressed the expression of IL-6 in a dose-dependent manner. Compounds 10-12 and 14 showed pronounced cytotoxicities against MCF-7 with IC50 values of 3.9, 1.3, 0.46, and 2.3 μM, respectively.

Phytotoxic phenols from the needles of Cedrus deodara

During the course of screening for anti-seed germination phytochemicals, the methanol fraction of the Cedrus deodara fresh needles showed potent activity. Bioactivity-guided fractionation led to the isolation of thirty-eight phenolic compounds. Four ones were identified as previously undescribed including (7S,8S)-3-methoxy-9′-acetoxy-3′,7-epoxy-8,4′-oxyneoligna-4,9-diol (7), (7S,8R)-dihydro-3′-hydroxy-8-acetoxymethyl-7-(4-hydroxy-3-methoxy-phenyl)-1′-benzofuranpropanol (10), (8S)-4,9,9′-trihydroxy-3,3′-dimethoxy-8,4′-oxyneolignan (11) and (7S,8S)-4,7,9′-trihydroxy-3,3′-dimethoxy-9-acetoxy-8,4′-oxyneolignan (16), respectively. The potential phytotoxic effects of these compounds on the seed germination and root elongation of Arabidopsis thaliana were evaluated by the filter paper assay developed in our laboratory. Bioassay results indicated that caffeic acid (36) displayed most significant inhibitory activities against the seed germination and root elongation of A. thaliana, stronger than those of the commercial herbicides acetochlor and glyphosate at the same concentration of 200 μg/mL. Ditetrahydrofuran lignan (1), dihydrochalcone (25), and eight simple phenols (28, 29, 31, 33–35, 37 and 38) completely inhibited the seed germination of A. thaliana at the concentration of 400 μg/mL, which were as active as acetochlor. Dihydroflavone (21) and the simple phenols 32–34 displayed stronger inhibitory effects on the root elongation of A. thaliana than that of glyphosate. The inhibitory effects of these active compounds on the seed germination and root elongation of Amaranthus tricolor and Lactuca sativa were evaluated as well. The phytotoxic activity of 11, 16, 22, 25, 31, 34, 37 and 38 were detected for the first time. In addition, the structure-activity relationships of the same class of these phytochemicals were discussed.

Identification of acetogenin compound, sabadelin from soursop seeds, Annona muricata (Annonaceae)

Isolation and identification of one chemical compound from the seeds of soursop, Annona muricata (Annonaceae) based on bioactivity-guided fractionation by the Brine Shrimp Lethality Assay (BSLA) have been done. The powder of soursop seed was extracted successively with n-hexane, chloroform, and methanol then the methanol extract was partitioned with ethylacetate water. Isolation and purification of ethylacetate extracts were carried out by column chromatography [(SiO2; i. n-hexane-chloroform = 15:1 ~ 1:1 gave one pure isolatedcompound. Based on Ultra Violet (UV), Infra-Red (IR), Nuclear Magnetic Resonance (NMR), and mass spectra (LC-MS/MS), the isolated Fr-4 was an acetogenin compound, sabadelin which had a weak cytotoxicity LC50 of 487 ppm.

Anticancer activity of Bacopa monnieri through apoptosis induction and mitophagy-dependent NLRP3 inflammasome inhibition in oral squamous cell carcinoma

BackgroundBacopa monnieri (BM) is traditionally used in human diseases for its antioxidant, anti-inflammatory and neuroprotective effects. However, its anticancer potential has been poorly understood. AimThe aim of this study was to explore the detailed anticancer mechanism of BM against oral cancer and to identify the bioactive BM fraction for possible cancer therapeutics. ResultsWe performed bioactivity-guided fractionation and identified that the aqueous fraction of the ethanolic extract of BM (BM-AF) had a potent anticancer potential in both in vitro and in vivo oral cancer models. BM-AF inhibited cell viability, colony formation, cell migration and induced apoptotic cell death in Cal33 and FaDu cells. BM-AF at low doses promoted mitophagy and BM-AF mediated mitophagy was PARKIN dependent. In addition, BM-AF inhibited arecoline induced reactive oxygen species production in Cal33 cells. Moreover, BM-AF supressed arecoline-induced NLR family pyrin domain containing 3 (NLRP3) inflammasome activation through mitophagy in Cal33 cells. The in vivo antitumor effect of BM-AF was further validated in C57BL/6J mice through a 4-nitroquinolin-1-oxide and arecoline-induced oral cancer model. The tumor incidence was significantly reduced in the BM-AF treated group. Further, data obtained from western blot and immunohistochemistry analysis showed increased expression of apoptotic markers and decreased expression of inflammasome markers in the tongue tissue obtained from BM-AF treated mice in comparison with the non-treated tumor bearing mice. ConclusionIn conclusion, BM-AF exhibited potent anticancer activity through apoptosis induction and mitophagy-dependent inhibition of NLRP3 inflammasome activation in both in vitro and in vivo oral cancer models. Moreover, we have investigated apoptosis and mitophagy-inducing compounds from this plant extract having anticancer activity against oral cancer cells.

Targeted isolation of natural analogs of anti-mycobacterial hit compounds based on the metabolite profiling of a large collection of plant extracts

Antibiotics resistance is a clear threat to the future of current tuberculosis treatments like rifampicin, prompting the need for new treatment options in this field. While plants can offer a plethora of chemical diversity in their constitutive natural products to tackle this issue, finding potentially bioactive compounds in them has not always proven to be that simple. Classical bioactivity-guided fractionation approaches are still trendy, but they bear significant shortfalls, like their time-consuming nature as well as the ever-increasing risk of isolating known bioactive compounds. In this regard, we have developed an alternative method to the latter approach that allows for natural derivatives of a known bioactive scaffold to be efficiently targeted and isolated within a large library of plant extracts. Hence our approach allows for the anticipation of bioactive structure independently of preliminary bioassays. By relying on the chemical diversity of a set of 1,600 plant extracts analyzed by HRMS/MS, we were able to isolate and characterize several minor derivatives of a previously reported bioactive aza-anthraquinone compound from Cananga brandisiana, selected within the plant set. Assessment of bioactivity on these derivatives (especially onychine, with an IC50 value of 39 µM in infection) confirmed their expected activity on Mycobacterium marinum in our anti-infective assay. This proof-of-concept study has established an original path towards bioactive compounds isolation, with the advantage of potentially highlighting minor bioactive compounds, whose activity may not even be detectable at the extract level.

Bacillus subtilis YZ-1 surfactins are involved in effective toxicity against agricultural pests.

Insect pests negatively affect crop quality and yield. The excessive use of chemical pesticides has serious impacts on the environment and food safety. Therefore, development of effective management strategies in the form of bio-agents have important agricultural applications. Tenebrio molitor, a storage pest, causes losses of grains, medicinal materials, and various agricultural and related products in the warehouse. Bacillus subtilis YZ-1 isolated from naturally deceased Pieris rapae has been found to exhibit significant toxicity against T. molitor. Treatment with B. subtilis YZ-1 fermentation broth resulted in a 90-95% mortality rate of T. molitor within 36 h post-treatment, indicating some active substances may have insecticidal activity in the bacterial supernatant. A bioactivity-guided fractionation method was used to isolate the insecticidal compounds from YZ-1, which led to the identification of surfactins. Additionally, a surfactin deletion mutant YZ-1△srfAA was constructed and the surfactin production by the mutant YZ-1△srfAA was verified through liquid chromatography-mass spectrometry (LC-MS). Further, YZ-1△srfAA exhibited loss of insecticidal activity against T. molitor, Plutella xylostella and Achelura yunnanensis. The insecticidal activity and surfactins contents of several strains of Bacillus sp. were also tested and correlation was found between varying surfactins yield and insecticidal activity exhibited by different strains. Conclusively, our results suggest that B. subtilis YZ-1 may provide a novel approach for plant protection against agricultural pests. © 2023 Society of Chemical Industry.

Cytotoxic triterpenoid saponins from the root of Olax subscorpioidea Oliv. (Olacaceae)

Bioactivity-guided phytochemical fractionation of the methanol extract of Olax subscorpioidea root has led to the isolation of six triterpenes. Three of these compounds are previously undescribed triterpenoid saponins: oleanolic acid 3-O-[α-L-rhamnopyranosyl-(1→3)-β-D-glucopyranosyl-(1 → 2)-6-O-methyl-β-D-glucuronopyranoside]-28-O-β-D-glucopyranosyl ester (2), oleanolic acid 3-O-[β-D-glucopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 3)-β-D-glucopyranoside] (3), and oleanolic acid 3-O-[β-D-glucopyranosyl-(1 → 4)-6-O-methyl-β-D-glucuronopyranoside] ester (5). Other reported known compounds include two triterpene glycosides: oleanolic acid 3-O-[β-D-glucopyranosyl-(1 → 4)-6-O-methyl-β-D-glucuronopyranoside]-28-O-β-D-glucopyranosyl ester (1) and oleanolic acid 3-O-[β-D-glucopyranosyl-(1 → 4)-β-D-glucuronopyranoside] (4); and a triterpene acid, oleanolic acid (6). The structures of these compounds were elucidated by spectroscopic means. The isolated compounds were tested against human cervical cancer (HeLa), colorectal cancer (Caco-2) and breast cancer (MCF-7) cell lines using the in vitro 3-[4,5-dimethylthiazole-2-yl] 3,5-diphenyltetrazolium bromide (MTT) assay, with vincristine as positive control. The cytotoxicity assay showed that compounds 3 and 5 exhibited significant inhibitory effects on the HeLa cell line, with IC50 values of 7.42 ± 0.34 μM and 10.27 ± 1.26 μM; and moderate effects on MCF-7 (IC50 values, 36.67 ± 1.23 μM and 43.83 ± 0.65 μM) and Caco-2 (IC50 values, 35.83 ± 0.55 μM and 39.03 ± 4.38 μM, respectively) cell lines. They were also more selectively cytotoxic than vincristine against the cancer cell lines, when compared with cytotoxicity against the normal lung cell line MRC5.

In vitro inhibition of human cytochrome P450 enzymes by licoisoflavone B from Glycyrrhiza uralensis Fisch. ex DC.

Glycyrrhiza uralensis Fisch. ex DC, one of the 3 pharmacopeial species of licorice and widely used in dietary supplements, can inhibit certain cytochrome P450 (CYP) enzymes. Thereby, G. uralensis preparations have the potential to cause pharmacokinetic drug interactions when consumed along with prescription medicines. One compound (1.34 mg dry weight) responsible for inhibiting CYP2B6, CYP2C8, and CYP2C9 was isolated using bioactivity-guided fractionation from 250 g dried roots, stolons, and rhizomes. The enzyme kinetics and mechanisms of inhibition were determined using human liver microsomes, recombinant enzymes, and UHPLC-MS/MS-based assays. Identified as licoisoflavone B, this compound displayed reversible inhibition of CYP2C8 with an IC50 value of 7.4 ± 1.1 µM and reversible inhibition of CYP2C9 with an IC50 value of 4.9 ± 0.4 µM. The enzyme kinetics indicated that the mechanism of inhibition was competitive for recombinant CYP2C8, with a Ki value of 7.0 ± 0.7 μM, and mixed-type inhibition for recombinant CYP2C9, with a Ki value of 1.2 ± 0.2 μM. Licoisoflavone B moderately inhibited CYP2B6 through a combination of irreversible and reversible mechanisms with an IC50 value of 16.0 ± 3.9 µM.

Nutritional Composition and Pharmacological Activity of Musa balbisiana Colla Seed: An Insight into Phytochemical and Cellular Bioenergetic Profiling.

Musa balbisiana Colla belongs to the family Musaceae which is well-known for its nutritional and pharmacological properties. Here, we have analysed the phytochemical content and evaluated the nutritional, antioxidant, anti-glycation, α-amylase, and α-glucosidase inhibition potential. Moreover, for the first time, we have studied the bioenergetic profiles of the bioactive fractions of M. balbisiana seeds extract against oxidative stress-related mitochondrial and cellular dysfunction using XFe24 extracellular flux analyzer. M. balbisiana seeds have high nutritional values with significant levels of carbohydrates, starch, protein, and minerals (Ca, Na, Mg, Cu, Fe, and Zn). Bioactivity-guided fractionation of the methanolic extract of M. balbisiana seeds revealed that the ethyl acetate fraction (EAF) showed the highest antioxidant, anti-glycation, and phytochemical content as compared to other fractions. Moreover, the EAF showed a lower α-amylase inhibition and a higher α-glucosidase inhibitory activity. Most importantly, our GC-MS analyses of EAF revealed the presence of unique and previously unreported 14 phytochemical compounds. A strong correlation between the biological activities and total phenolic/tannin content was observed. In addition, the bioactive fraction of M. balbisiana seeds (EAF) improved the bioenergetic profiles of free fatty acid-induced oxidative stress with a concomitant increase in ATP production, and respiratory and glycolytic capacity. Altogether, our findings suggest that M. balbisiana seeds can be used as a natural supplement to boost antioxidant levels and combat oxidative stress and non-enzymatic glycation.