Insights into the nature of ichthyotoxins from the Chrysochromulina leadbeateri blooms in Northern Norwegian fjords.

In May-June 2019, the microalga Chrysochromulina leadbeateri caused a massive fish-killing event in several fjords in Northern Norway, resulting in the largest direct impact ever on aquaculture in northern Europe due to toxic algae. Motivated by the fact that no algal toxins have previously been described from C. leadbeateri, we set out to investigate the chemical nature and toxicity of secondary metabolites in extracts of two strains (UIO 393, UIO 394) isolated from the 2019 bloom, as well as one older strain (UIO 035) isolated during a bloom in Northern Norway in 1991. Initial LC-DAD-MS/MS-based molecular networking analysis of the crude MeOH extracts of the cultivated strains showed that their profiles of small organic molecules, including a large number of known lipids, were very similar, suggesting that the same class of toxin(s) were likely the causative agents of the two harmful algal bloom (HAB) events. Next, bioassay-guided fractionation using the RTgill-W1 cell line and metabolomics analysis pointed to a major compound affording [M + H]+ ions at m/z 1399.8333 as a possible toxin, corresponding to a compound with the formula C67H127ClO27. Moreover, our study unveiled a series of minor analogues exhibiting distinct patterns of chlorination and sulfation, together defining a new family of compounds, which we propose to name leadbeaterins. Remarkably, these suspected toxins were detected in situ in samples collected during the 2019 bloom close to Tromsø, thereby consistent with a role in fish kills. The elemental compositions of the putative C. leadbeateri ichthyotoxins strongly indicate them to be long linear polyhydroxylated polyketides, structurally similar to sterolysins reported from a number of dinoflagellates.

Antifungal Potential of Secondary Metabolites Derived from Arcangelisia flava (L.) Merr.: An Analysis of In Silico Enzymatic Inhibition and In Vitro Efficacy against Candida Species.

Considering the escalating resistance to conventional antifungal medications, it is critical to identify novel compounds that can efficiently counteract this challenge. The purpose of this research was to elucidate the fungicidal properties of secondary metabolites derived from Arcangelisia flava, with a specific focus on their efficacy against Candida species. This study utilized a combination approach comprising laboratory simulations and experiments to discern and evaluate the biologically active constituents present in the dichloromethane extract of A. flava. The in vitro experiments demonstrated that compounds 1 (palmatine) and 2 (fibraurin) exhibited antifungal properties. The compounds exhibited minimum inhibitory concentrations (MICs) ranging from 15.62 to 62.5 µg/mL against Candida sp. Moreover, compound 1 demonstrated a minimum fungicidal concentration (MFC) of 62.5 µg/mL against Candida glabrata and C. krusei. In contrast, compound 2 exhibited an MFC of 125 µg/mL against both Candida species. Based on a molecular docking study, it was shown that compounds 1 and 2 have a binding free energy of -6.6377 and -6.7075 kcal/mol, respectively, which indicates a strong affinity and specificity for fungal enzymatic targets. This study utilized pharmacophore modeling and Density Functional Theory (DFT) simulations to better understand the interaction dynamics and structural properties crucial for antifungal activity. The findings underscore the potential of secondary metabolites derived from A. flava to act as a foundation for creating novel and highly efficient antifungal treatments, specifically targeting fungal diseases resistant to existing treatment methods. Thus, the results regarding these compounds can provide references for the next stage in antifungal drug design. Further investigation is necessary to thoroughly evaluate these natural substances' clinical feasibility and safety characteristics, which show great potential as antifungal agents.

Bioassay-Guided Fractionation of Pittosporum angustifolium and Terminalia ferdinandiana with Liquid Chromatography Mass Spectroscopy and Gas Chromatography Mass Spectroscopy Exploratory Study.

Bioprospecting native Australian plants offers the potential discovery of latent and novel bioactive compounds. The promising cytotoxic and antibacterial activity of methanolic extracts of Pittosporum angustifolium and Terminalia ferdinandiana led to further fractionation and isolation using our laboratory's bioassay-guided fractionation protocol. Hence, the aim of this study was to further evaluate the bioactivity of the fractions and subfractions and characterize bioactive compounds using liquid chromatography mass spectroscopy (LC-MS/MS) and gas chromatography MS (GC-MS). Compounds tentatively identified in P. angustifolium Fraction 1 using LC-ESI-QTOF-MS/MS were chlorogenic acid and/or neochlorogenic acid, bergapten, berberine, 8'-epitanegool and rosmarinic acid. GC-MS analysis data showed the presence of around 100 compounds, mainly comprising carboxylic acids, sugars, sugar alcohols, amino acids and monoalkylglycerols. Furthermore, the fractions obtained from T. ferdinandiana flesh extracts showed no cytotoxicity, except against HT29 cell lines, and only Fraction 2 exhibited some antibacterial activity. The reduced bioactivity observed in the T. ferdinandiana fractions could be attributed to the potential loss of synergy as compounds become separated within the fractions. As a result, the further fractionation and separation of compounds in these samples was not pursued. However, additional dose-dependent studies are warranted to validate the bioactivity of T. ferdinandiana flesh fractions, particularly since this is an understudied species. Moreover, LC-MS/GC-MS studies confirm the presence of bioactive compounds in P. angustifolium Fraction 1/subfractions, which helps to explain the significant acute anticancer activity of this plant. The screening process designed in this study has the potential to pave the way for developing scientifically validated phytochemical/bioactivity information on ethnomedicinal plants, thereby facilitating further bioprospecting efforts and supporting the discovery of novel drugs in modern medicine.

Naphthoquinone Derivatives from Angustimassarina populi CF-097565 Display Anti-Tumour Activity in 3D Cultures of Breast Cancer Cells.

Cancer is one of the leading causes of death worldwide, with breast cancer being the second cause of cancer-related mortality among women. Natural Products (NPs) are one of the main sources for drug discovery. During a screening campaign focused on the identification of extracts from Fundación MEDINA's library inhibiting the proliferation of cancer cell lines, a significant bioactivity was observed in extracts from cultures of the fungus Angustimassarina populi CF-097565. Bioassay-guided fractionation of this extract led to the identification and isolation of herbarin (1), 1-hydroxydehydroherbarin (4) plus other three naphthoquinone derivatives of which 3 and 5 are new natural products and 2 is herein described from a natural source for the first time. Four of these compounds (1, 3, 4 and 5) confirmed a specific cytotoxic effect against the human breast cancer cell line MCF-7. To evaluate the therapeutic potential of the compounds isolated, their efficacy was validated in 3D cultures, a cancer model of higher functionality. Additionally, an in-depth study was carried out to test the effect of the compounds in terms of cell mortality, sphere disaggregation, shrinkage, and morphology. The cell profile of the compounds was also compared to that of known cytotoxic compounds with the aim to distinguish the drug mode of action (MoA). The profiles of 1, 3 and 4 showed more biosimilarity between them, different to 5, and even more different to other known cytotoxic agents, suggesting an alternative MoA responsible for their cytotoxicity in 3D cultures.

Bioassay-guided identification of antithrombotic compounds from Cnidoscolus aconitifolius (Mill.) I. M. Jhonst.: molecular docking, bioavailability, and toxicity prediction.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Conventional antithrombotic therapy has reported hemorrhagic accidents. Ethnobotanical and scientific reports point to Cnidoscolus aconitifolius as an antithrombotic adjuvant. Previously, C. aconitifolius leaves ethanolic extract displayed antiplatelet, anticoagulant, and fibrinolytic activities. This work aimed to identify compounds from C. aconitifolius with in vitro antithrombotic activity through a bioassay-guided study. Antiplatelet, anticoagulant, and fibrinolytic tests guided the fractionation. Ethanolic extract was subjected to a liquid-liquid partitioning, followed by vacuum liquid, and size exclusion chromatography to obtain the bioactive JP10B fraction. The compounds were identified through UHPLC-QTOF-MS, and their molecular docking, bioavailability, and toxicological parameters were determined computationally. Kaempferol-3-O-glucorhamnoside and 15(S)-HPETE were identified; both showed affinity for antithrombotic targets, low absorption, and safety for human consumption. Further in vitro and in vivo evaluations will better understand their antithrombotic mechanism. This bioassay-guided fractionation demonstrated that C. aconitifolius ethanolic extract has antithrombotic compounds.Communicated by Ramaswamy H. Sarma.

2,4-Di-tert-butylphenol and 7-hydroxy-3-(2-methylpropyl)-2,3,6,7,8,8a-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione: two natural products from Serratia marcescens Ha1 and their herbicidal activities.

BACKGROUND: Weeds are one of the critical factors that negatively affect crop yield and quality. Microbial herbicides are a research hotspot for novel herbicides owing to their environmental safety and lack of weed resistance. In the current study, the active ingredients of Serratia marcescens Ha1, a new microbial herbicide, were investigated for their effectiveness against agricultural weeds using bioassay-guided fractionation. RESULTS: The results revealed that petroleum ether and ethyl acetate extracts of S. marcescens Ha1 had high herbicidal activity. Forty-nine compounds were identified from the petroleum ether extract, including 2,4-di-tert-butylphenol (DB; C14 H22 O, 38.82%), ethyl 14-methyl-hexadecanoate, 1-nonadecene, and [1,1'-biphenyl]-2,3'-diol, 3,4',5,6'-tetrakis. Of these, DB showed significant inhibitory effects on root and shoot growth in Amaranthus retroflexus, with half-maximal inhibitory concentration (IC50 ) values of 389.17 and 832.44 mg L-1 , respectively. In addition, 7-hydroxy-3-(2-methylpropyl)-2,3,6,7,8,8a-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (HPD) was identified as the major active ingredient in the ethyl acetate extract of S. marcescens Ha1 using bioassay-guided fractionation, with IC50 values of 439.86 and 476.95 mg L-1 against A. retroflexus shoot and root growth, respectively. Scanning electron microscopy indicated that DB and HPD exert destructive effects on A. retroflexus root, and the damage is gradually aggravated with increasing treatment time and concentration. CONCLUSION: The S. marcescens Ha1 extract and its active compounds DB and HPD exhibit significant herbicidal activity, which could be utilized further for the development of microbial herbicides. © 2023 Society of Chemical Industry.

Actinomycins from Soil-Inhabiting Streptomyces as Sources of Antibacterial Pigments for Silk Dyeing.

Actinobacteria produce a broad spectrum of bioactive substances that are used in the pharmaceutical, agricultural, and biotechnology industries. This study investigates the production of bioactive substances in Streptomyces, isolated from soil under five tropical plants, focusing on their potential as natural antibacterial dyes for silk fabrics. Out of 194 isolates, 44 produced pigments on broken rice as a solid substrate culture. Eight antibacterial pigmented isolates from under Magnolia baillonii (TBRC 15924, TBRC 15927, TBRC 15931), Magnolia rajaniana (TBRC 15925, TBRC 15926, TBRC 15928, TBRC 15930), and Cinnamomum parthenoxylon (TBRC 15929) were studied in more detail. TBRC 15927 was the only isolate where all the crude extracts inhibited the growth of the test organisms, Staphylococcus epidermidis TISTR 518 and S. aureus DMST 4745. The bioactive compounds present in TBRC 15927 were identified through LC-MS/MS analysis as belonging to the actinomycin group, actinomycin D (or X1), X2, and X0ß. Also, the ethyl acetate crude extract exhibited non-toxicity at an IC50 value of 0.029 ± 0.008 µg/mL on the mouse fibroblast L-929 assay. From the 16S rRNA gene sequence analysis, TBRC 15927 had 100% identity with Streptomyces gramineus JR-43T. Raw silk dyed with the positive antimicrobial TBRC 15927 extract (8.35 mg/mL) had significant (>99.99%) antibacterial properties. Streptomyces gramineus TBRC 15927 is the first actinomycin-producing strain reported to grow on broken rice and shows promise for antibacterial silk dyeing.

Anti-Inflammatory Effect of Ebractenoid F, a Major Active Compound of Euphorbia ebracteolata Hayata, through Inhibition of Nuclear Factor-κB Activation.

Euphorbia ebracteolata Hayata (Euphorbiaceae family) is a perennial plant that is widely distributed in Korea, Japan, and China. Its roots contain bioactive diterpenes that have anti-inflammatory properties. However, the anti-inflammatory mechanisms are not yet fully understood. This study aimed to identify the most active anti-inflammatory compound from the roots of E. ebracteolata Hayata, using bioassay-guided fractionation and a combinative method of high-speed countercurrent chromatography (HSCCC) and preparative high-performance liquid chromatography (HPLC). Then, we investigated its anti-inflammatory mechanism in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Ebractenoid F was identified as the most potent bioactive compound of E. ebracteolata Hayata. Ebractenoid F significantly decreased nitric oxide (NO) production and nuclear factor-κB (NF-κB) activation induced by LPS in RAW 264.7 macrophages. Moreover, ebractenoid F decreased the degradation of inhibitory κB-α, the nuclear translocation of the p65 and p50 subunits of NF-κB, and the expression of NF-κB downstream genes. Furthermore, ebractenoid F inhibited the phosphorylation of Akt and mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK) and c-Jun NH2 terminal kinase (JNK), in LPS-stimulated RAW 264.7 cells. In conclusion, ebractenoid F exerts the most potent anti-inflammatory effect by suppressing NF-κB-mediated NO production in LPS-stimulated RAW 264.7 cells. Ebractenoid F may be a useful therapeutic compound for the prevention or treatment of inflammation-associated diseases.

Antiproliferative activity of compounds isolated from the root bark of Lannea acida in multiple myeloma cell lines.

Lannea acida A. Rich. is a native plant of West Africa used in traditional medicine against diarrhea, dysentery, rheumatism, and women infertility. Eleven compounds were isolated from the dichloromethane root bark extract using various chromatographic techniques. Among those, nine compounds have not been previously reported, i.e. one cardanol derivative, two alkenyl 5-hydroxycyclohex-2-en-1-ones, three alkenyl cyclohex-4-ene-1,3-diols, two alkenyl 7-oxabicyclo[4.1.0]hept-4-en-3-ols, and one alkenyl 4,5-dihydroxycyclohex-2-en-1-one, together with two known cardanols. The structure of the compounds was elucidated using NMR, HRESIMS, ECD, IR, and UV. Their antiproliferative activity was evaluated in three multiple myeloma cell lines: RPMI 8226, MM.1S, and MM.1R. Two compounds showed activity in all cell lines with IC50 values < 5 µM. Further investigations are needed to understand the mechanism of action.

α-Amylase inhibitory potential of Thunbergia mysorensis leaves extract and bioactive compounds by in vitro and computational approach.

In this study, we aim to evaluate the anti-diabetic potential of Thunbergia mysorensis leaves methanolic extract (MeL) using inhibitory assays for α-glucosidase (AG), α-amylase (AM) (carbohydrate digestive enzymes) and aldose reductase (AR) (an enzyme involved in the polyol pathway responsible for glycation). In addition to antidiabetic studies, antioxidant studies were also performed due to the fact that reactive oxygen species (ROS) are produced by various pathways under diabetic conditions. Hyperglycemia induces ROS by activating the glycation reaction and the electron transport chain in mitochondria. The MeL effectively inhibited the enzymes (AG IC50: 27.86 ± 1.0, AM IC50: 12.00 ± 0.0, AR IC50: 4.50 ± 0.09 µg/mL) and showed effective radical ion scavenging activity during the antioxidant assay (DPPH EC50: 30.10 ± 0.75, ABTS EC50: 27.25 ± 1.00, Superoxide EC50: 35.00 ± 1.50 µg/mL). Using activity-guided repeated fractionation on a silica gel column chromatography, two compounds including 3,4-dimethoxy benzoic acid (DMBA) (101 mg) and 3,4-dimethoxy cinnamic acid (DMCA) (87 mg) with potent anti-diabetic activity were extracted from the MeL of T. mysorensis leaves. Both DMBA (IC50 AG: 27.00 ± 1.05, IC50 AM: 12.15 ± 0.10, IC50 AR: 4.86 ± 0.30 µg/mL) and DMCA (IC50 AG: 27.25 ± 0.98, IC50 AM: 12.50 ± 0.20, IC50 AR: 5.00 ± 1.00 µg/mL) were subjected for enzyme inhibition. Since both compounds significantly inhibited AM, enzyme kinetics for AM inhibition was performed. The compounds also showed effective antioxidant potential (DPPH EC50: 30.50 ± 0.99, ABTS EC50: 27.86 ± 0.16, Superoxide EC50: 36.10 ± 0.24 µg/mL), and DMCA (DPPH EC50: 31.00 ± 1.00, ABTS EC50: 28.00 ± 0.25, Superoxide EC50: 36.25 ± 0.37 µg/mL). Further, to elucidate the role of DMBA and DMCA in enzyme inhibition and stability at the molecular level, both compounds were subjected for in silico enzyme inhibitory studies using molecular docking simulation, molecular dynamics (MD) simulation, and binding free energy calculations. Compared to AR and AG, AM was the most significantly inhibited enzyme (DMBA: -6.6 and DMCA: -7.8 kcal/mol), and compounds combined with AM were subjected to MD simulation. Both compounds were stable in the binding pocket of AM till 100 ns and chiefly use Van der Waal's energy to bind. Compared to the controls, both DMBA and DMCA had a higher efficiency in the inhibition of target enzymes in vitro and in silico. The presence of DMBA and DMCA is more likely to be associated with the potential of MeL in antihyperglycemic activity. This bio-computational study indicates DMBA and DMCA as potential lead inhibitors of AM and could be used as effective anti-diabetic drugs in the near future.

Inhibitory effect of gallic acid from Thunbergia mysorensis against α-glucosidase, α-amylase, aldose reductase and their interaction: Inhibition kinetics and molecular simulations.

In this exploration, we assessed the antihyperglycaemic properties of methanol extract of flowers of Thunbergia mysorensis (MeT) against α-glucosidase, α-amylase and aldose reductase enzymes for the effective management of postprandial hyperglycemia. Hyperglycemia occurs when the body lacks enough insulin or is unable to correctly utilize it. MeT inhibited both the carbohydrate digestive enzymes (α-glucosidase and α-amylase) and aldose reductase, which are vital for the therapeutic control of postprandial hyperglycaemia. MeT was also found to have significant antioxidant activity. Using several spectroscopic approaches, the primary active component found in MeT was identified as gallic acid. With low Ki values, gallic acid significantly inhibited α-glucosidase (30.86 µg/mL) and α-amylase (6.50 µg/mL). Also, MeT and gallic acid both inhibited aldose reductase effectively, corresponding to an IC50 value of 3.31 and 3.05 µg/mL. Our findings imply that the presence of polyphenol compounds (identified via HPLC analysis) is more likely to be responsible for the antihyperglycaemic role exhibited by MeT via the inhibition of α-glucosidase and the polyol pathway. Further, gallic acid interacted with the key residues of the active sites of α-glucosidase (-6.4 kcal/mol), α-amylase (-5.8 kcal/mol) and aldose reductase (-5.8 kcal/mol) as observed in the protein-ligand docking. It was also predicted that gallic acid was stable inside the binding pockets of the target enzymes during molecular dynamics simulation. Overall, gallic acid derived from MeT via bioassay-guided isolation emerges as a natural antidiabetic drug and can be taken into in vivo and clinical studies shortly.Communicated by Ramaswamy H. Sarma.

Cola rostrata K. Schum. constituents induce cytotoxicity through reactive oxygen species generation and mitochondrial membrane depolarisation.

Aim: While the traditional use of Cola rostrata in treating illnesses and diseases has not been reported, the presence of cytotoxic principles has been reported in phylogenetically and biogeographically related species within the Cola genus. This study, therefore, evaluated the cytotoxic potential of extracts of the plant, and the associated cellular and molecular mechanisms. Methods: Activity-based fractionation of the extracts was carried out and cytotoxicity was assessed in the human cervical cancer cell line, HeLa, and the transformed human lung cell line, MRC5-SV2, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay complemented with brightfield imaging. The 2',7'-dichlorofluorescein diacetate (DCFDA) assay was used to assess induction of cellular reactive oxygen species (ROS), while flow cytometry of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-1)-stained cells assessed the loss of mitochondrial membrane potential (∆ΨM). Gas chromatography-mass spectrometry (GC-MS) analysis was carried out on an active fraction. Results: Extracts of the fruit epicarp and leaf were cytotoxic against the cell lines. Half-maximal inhibitory concentration (IC50) values for the 48 h cytotoxicity of the ethanol extract of the epicarp against HeLa and MRC5-SV2 cells were 48.0 µg/mL ± 12.1 µg/mL and 40.4 µg/mL ± 7.2 µg/mL, respectively, while fractions from second-level partitioning of the hexane fraction of the leaf extract elicited cytotoxicity with IC50 values ranging from 12.8 µg/mL ± 1.0 µg/mL to 39.6 µg/mL ± 7.2 µg/mL in both cell lines, following 48 h treatment. GC-MS revealed the presence of seventeen compounds in a hexane fraction of the leaf extract, including even- and odd-chain fatty acids, the most abundant of which were n-hexadecanoic acid, decanoic acid 10-(2-hexylcyclopropyl); and octadecanoic acid. The mechanisms of cytotoxicity of most active fractions involved generation of ROS and mitochondrial membrane depolarisation. Conclusions: The findings show that C. rostrata is rich in cytotoxic phytochemicals which could be isolated for developing new anti-cancer agents.

Metabolite Profiling and Bioassay-Guided Fractionation of Zataria multiflora Boiss. Hydroethanolic Leaf Extracts for Identification of Broad-Spectrum Pre and Postharvest Antifungal Agents.

Hydroethanolic leaf extracts of 14 Iranian Zataria multiflora Boiss. populations were screened for their antifungal activity against five plant pathogenic fungi and metabolically profiled using a non-targeted workflow based on UHPLC/ESI-QTOFMS. Detailed tandem mass-spectrometric analyses of one of the most active hydroethanolic leaf extracts led to the annotation of 68 non-volatile semi-polar secondary metabolites, including 33 flavonoids, 9 hydroxycinnamic acid derivatives, 14 terpenoids, and 12 other metabolites. Rank correlation analyses using the abundances of the annotated metabolites in crude leaf extracts and their antifungal activity revealed four O-methylated flavones, two flavanones, two dihydroflavonols, five thymohydroquinone glycoconjugates, and five putative phenolic diterpenoids as putative antifungal metabolites. After bioassay-guided fractionation, a number of mono-, di- and tri-O-methylated flavones, as well as three of unidentified phenolic diterpenoids, were found in the most active subfractions. These metabolites are promising candidates for the development of new natural fungicides for the protection of agro-food crops.

Bioassay Guided Fractionation Protocol for Determining Novel Active Compounds in Selected Australian Flora.

A large variety of unique and distinct flora of Australia have developed exceptional survival methods and phytochemicals and hence may provide a significant avenue for new drug discovery. This study proposes a bioassay guided fractionation protocol that maybe robust and efficient in screening plants with potential bioactive properties and isolating lead novel compounds. Hence, five native Australian plants were selected for this screening process, namely Pittosporum angustifolium (Gumbi gumbi), Terminalia ferdinandiana (Kakadu plum, seeds (KPS), and flesh (KPF)), Cupaniopsis anacardioides (Tuckeroo, seeds (TKS) and flesh (TKF)), Podocarpus elatus (Illawarra plum, seeds (IPS) and flesh (IPF)) and Pleiogynium timoriense (Burdekin plum, seeds (BPS) and flesh (BPF)). The methanolic extracts of the plants samples were analysed for Total phenolic content (TPC) and antioxidant capacity measure by FRAP. The highest values were found in the KPF which were 12,442 ± 1355 mg GAE/ 100 g TPC and 16,670 ± 2275 mg TXE/100 g antioxidant capacity. Extracts of GGL was deemed to be most potent with complete cell inhibition in HeLa and HT29, and about 95% inhibition in HuH7 cells. Comparative activity was also seen for KPS extract, where more than 80% cell inhibition occurred in all tested cell lines. Dose-dependent studies showed higher SI values (0.72-1.02) in KPS extracts than GGL (0.5-0.73). Microbial assays of the crude extracts were also performed against five bacterial strains commonly associated with causing food poisoning diseases were selected (Gram positive-Staphylococcus aureus and Gram negative-Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa bacteria). KPF extracts were effective in suppressing microbial growth of all tested bacterial strains except for P. aeruginosa, while TKS and TKF were only slightly effective against S. aureus. Due to the potential of the GGL crude extract to completely inhibit the cells compared to KPS, it was further fractionated and tested against the cell lines. HPLC phenolic profiling of the crude extracts were performed, and numerous peak overlaps were evident in the fruit extracts. The KPF extracts demonstrated the strongest peaks which was coherent with the fact that it had the highest TPC and antioxidant capacity values. A high occurrence of t-ferulic acid in the GGL extracts was found which may explain the cytotoxic activity of GGL extracts. Peaks in KPS and KPF extracts were tentatively identified as gallic acid, protocatechuic acid, 4-hydroxybenzoic acid and syringic acid and possibly ellagic acid. HPLC time-based fractionation of the GGL extract (F1-F5) was performed and Dose dependent cytotoxic effects were determined. It was construed that F1, having the highest SI value for HeLa, HT29 and HuH7 (1.60, 1.41 and 1.67, respectively) would be promising for further fractionation and isolation process.

Daidzein is the in vivo active compound of Puerariae Lobatae Radix water extract for muscarinic receptor-3 inhibition against overactive bladder.

Background: In the previous study, Puerariae Lobatae Radix (named Gegen in Chinese) water extract attenuated M3 receptor agonist carbachol-induced detrusor contraction after 3-week oral administration in a hypertension-associated OAB (overactive bladder) model. This research aimed to investigate the active ingredients from Gegen water extract against OAB. Methods: Bioassay-guided fractionation was performed by using preparative HPLC for fast isolation of fractions followed by screening their ex vivo activity through carbachol-induced bladder strip contraction assay. Chemicals in each active fraction were analyzed by HPLC-UV. Urine metabolites were quantified by LC-MS/MS after sub-acute administration. Thermal shift assay with the recombinant human M3 receptor protein was performed, and molecular docking analysis was used for molecular modelling of M3 receptor inhibition. Results: Bioassay-guided fractionation results for isolating M3 receptor inhibitors indicated that four compounds were identified as active ingredients of Gegen water extract, and their inhibition potency on carbachol-induced detrusor contraction was ranked in descending order according to their inhibition concentrations as follows: genistein > daidzein > biochanin A >> puerarin. Daidzein in urine reached an ex vivo effective concentration to inhibit detrusor contraction, but others did not. Daidzein concentration-dependently increased the melt temperature (Tm) of recombinant human M3 receptor protein with a positive binding (ΔTm = 2.12 °C at 100 µg/ml). Molecular docking analysis showed that daidzein can potently bind to the ligand binding pocket of the M3 receptor via hydrogen bonding. Conclusion: Puerarin and its derivatives were pro-drugs, and daidzein was their in vivo active form via M3 receptor inhibition for treating OAB.

Bioactive Compounds, Antioxidant, Anti-Inflammatory, Anti-Cancer, and Toxicity Assessment of Tribulus terrestris-In Vitro and In Vivo Studies.

Tribulus terrestris L. belongs to the family Zygophyllaceae and integral part of various ancient medicinal systems including Chinese, Indian, and European to combat various health ailments. The aim of the present study was to assess the phytochemical constituents, in vitro antioxidant activity using DPPH, FRAP, and H2O2 assays, in vitro anticancer activity using MTT assay, and in vitro and in vivo anti-inflammatory properties of T. terrestris. The acute and sub-acute toxicity of extracts exhibiting most biological potential was examined using murine models. Liquid-liquid partitioning followed by RP-HPLC sub-fraction of crude extract was performed. After that, ESI-MS/MS analysis was done for the timid identification of bioactive metabolites responsible for bioactivities of sub-fractions and HPLC analysis to quantify the compounds using external standards. Among all extracts, T. terrestris methanol extract was noted to hold maximum phenolic (341.3 mg GAE/g) and flavonoid (209 mg QE/g) contents, antioxidant activity in DPPH (IC50 71.4 µg/mL), FRAP (35.3 mmol/g), and H2O2 (65.3% inhibition) assays, anti-inflammatory activities in vitro at 400 µg/mL (heat-induced hemolysis, % inhibition 68.5; egg albumin denaturation, % inhibition 75.6%; serum albumin denaturation, % inhibition 80.2), and in vivo at 200 mg/kg (carrageenan-induced paw edema, % inhibition 69.3%; formaldehyde-induced paw edema, % inhibition 71.3%) and anticancer activity against breast cancer cell (MCF-7) proliferation (IC50 74.1 µg/mL). Acute and sub-acute toxicity studies recorded with no change in body weight, behavior, hematological, serum, and histopathological parameters in treated rats with T. terrestris methanol extracts when compared to control group. Fraction B obtained through liquid-liquid partitioning resulted in more bioactive potential as compared to the parent methanol extract. RP-HPLC analysis of fraction B resulted with four sub-fractions (TBTMF1-TBTMF4), wherein TBTMF3 delineated notable bioactive capabilities as compared to other fractions and parent methanol extract. ESI-MS/MS analysis of TBTMF3 resulted with tentative identification of myricetin, rutin, liquitrigenin, physcion, and protodioscin. It can be stated that T. terrestris is a potential bearing herb and findings of current study further verify the claims made in ancient medicinal systems. However, after investigation of each identified compound, it must be considered for drug discovery.

Bioassay-Guided Isolation of 2-[p-(2-Carboxyhydrazino)phenoxy]-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol from Oroxylum indicum and the Investigation of Its Molecular Mechanism Action of Apoptosis Induction.

The leaf crude extract of Oroxylum indicum (L.) Kurz induces genomic DNA fragmentation, comet formation, and the inhibition of cell proliferation in the prostate cancer cell line PC3, as assessed by agarose gel electrophoresis, comet assay and MTT assay, respectively. The bioactive compound was purified through bioassay-guided fractionation using preparative HPLC and MTT assay. The light brown and water-soluble compound was characterized using 1H and 13C nuclear magnetic resonance (NMR), Fourier transform infrared (FT-IR), and electrospray ionization (ESI) mass spectrometry. The compound was identified as a glycosylated hydroquinone derivative, 2-[p-(2-Carboxyhydrazino)phenoxy]-6-(hydroxymethyl) tetrahy-dro-2H-pyran-3,4,5-triol (molecular formula, C13H18N2O8; molecular mass = 330). The identified phytocompound has not been reported earlier elsewhere. Therefore, the common name of the novel anticancer phytocompound isolated from Oroxylum indicum in this current study is oroxyquinone. The half-maximal inhibitory concentration (IC50) of oroxyquinone on PC3 cells was 58.9 µM (95% CI = 54.5 to 63.7 µM). Treatment of PC3 cells with oroxyquinone induced genomic DNA fragmentation and chromatin condensation, increased in the annexin-V positive cells, arrested the cell cycle at S phases, and inhibited the cell migration; as assessed by comet assay, DAPI staining, flow cytometry and a wound healing assay, respectively. On the investigation of the molecular mechanism of the induction of apoptosis, the results indicated that oroxyquinone induced caspase-3 and PARP independent apoptosis but through the p38 pathway and the localization of AIF into the nucleus. The present study identifies a novel anticancer molecule and provides scientific evidence supporting the therapeutic potency of Oroxylum indicum for ethnomedicinal uses.

An exploration of angiotensin-converting enzyme (ACE) inhibitory peptides derived from gastrointestinal protease hydrolysate of milk using a modified bioassay-guided fractionation approach coupled with in silico analysis.

An improved bioassay-guided fractionation was performed to effectively screen angiotensin-I converting enzyme inhibitory (ACEI) peptides from milk protein hydrolysate. The aqueous normal phase liquid chromatography, namely hydrophilic interaction liquid chromatography (HILIC), was used as a format of solid-phase extraction (SPE) short column for the first fractionation, then the HILIC-SPE fraction with the best ACEI activity (IC50 = 61.75 ± 5.74 µg/mL; IC50 = half-maximal inhibitory concentration) was obtained when eluted by 95% acetonitrile + 0.1% formic acid (fraction F1). The best HILIC-SPE fraction was further fractionated using reversed-phase (RP)-SPE short column. The best RP-SPE fraction was obtained when eluted by 20% acetonitrile + 0.1% formic acid (fraction P3) with an ACEI activity of IC50 36.22 ± 1.18 µg/mL. After the 2-step fractionation, the IC50 value of fraction P3 significantly decreased by 8.92-fold when compared with the crude hydrolysate. Several peptides were identified from fraction P3 using liquid chromatography-tandem mass spectrometry. The in silico analysis of these identified sequences based on the BIOPEP database predicted that HLPLPLL (HL-7) was the most active peptide against angiotensin-converting enzyme (ACE). The HL-7 derived from ß-casein showed a potent ACEI activity (IC50 value is 16.87 ± 0.3 µM). The contents of HL-7 in the gastrointestinal protease hydrolysate and RP-SPE fraction originated from 1 mg of milk proteins were quantified using a multiple reaction monitoring mode upon liquid chromatography-tandem mass spectrometry analysis to give 19.86 ± 1.14 pg and 14,545.8 ± 572.9 pg, respectively. Besides, the kinetic study indicated that HL-7 was a competitive inhibitor and the result was rationalized using the docking simulation. The study demonstrated an efficient screening of ACEI peptides from commercially available milk powders using a simple SPE process instead of a sophisticated instrument such as HPLC. Moreover, the potent ACEI peptide HL-7 uncovered by this method could be a natural ACE inhibitor.

Antioxidant, antibacterial, antifungal activities and gas chromatographic fingerprint of fractions from the root bark of Afzelia africana.

BACKGROUND: Afzelia africana is a tropical plant with numerous ethno-medicinal benefits. The plant has been used for the treatment of pain, hernia, fever, malaria, inflammation and microbial infections. OBJECTIVES: To perform bioassay-guided fractionation, antioxidant and antimicrobial activities of the bark of Afzelia africana. METHODS: Column chromatography fractionation, antioxidant activity (% (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1,1-diphenyl picrylhydrazyl (DPPH) scavenging activity))), antimicrobial activity (microbroth dilution: Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), MBC/MIC ratio), and synergistic activities (Checkerboard assay: Fraction Inhibitory Concentration Index (FICI)). RESULTS: Bioassay-guided fractionation of A. africana produced four fractions that displayed promising free radical scavenging activities in the ABTS (54-93)% and the DPPH (35-76)% assays in the ranking order of F1(93-54)>F4(81-58)>F2(74-58)>F3(72-55) and F3(77-42)>F1(64-46)>F4(55-44)>F2(47-35) respectively at a concentration range of 1.0-0.01 mg/mL. The fraction F1 (MBC: 2.5-5.0 mg/mL) and F4 (MBC: 1.25-10.0 mg/mL) exhibited broad spectrum of superior bactericidal effects than F2 (MBC≥100.0 mg/mL) and F3 (MBC: 12.5-100.0 mg/mL) against Staphylococcus mutans, Staphylococcus aureus, Escherichia coli, fluconazole-resistant Candida albicans, methicillin-resistant S. aureus, Bacillus subtilis, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella typhi, and Candida albicans (standard strain). The two most active fractions (F1 and F4) reported synergistic effects (FICI≤0.5) against S. typhi whilst the F4 reported additional synergism against E. coli, K. pneumonia, and S. typhi when combined with ciprofloxacin. Furthermore, the two fractions reported synergistic effects against Escherichia coli, Klebsiella pneumonia, Salmonella typhi, and Pseudomonas aeruginosa when combined with tetracycline whilst F1 reported antifungal synergism against fluconazole resistant Candida albicans when combined with fluconazole and ketoconazole. CONCLUSION: The study has confirmed the antioxidant, antimicrobial and synergistic uses of A. africana for the treatment of both infectious and non-infectious disease.

Active Flavonoids from Colubrina greggii var. greggii S. Watson against Clinical Isolates of Candida spp.

Candida albicans is the most commonly implicated agent in invasive human fungal infections. The disease could be presented as minimal symptomatic candidemia or can be fulminant sepsis. Candidemia is associated with a high rate of mortality and high healthcare and hospitalization costs. The surveillance programs have reported the distribution of other Candida species reflecting the trends and antifungal susceptibilities. Previous studies have demonstrated that C. glabrata more frequently presents fluconazole-resistant strains. Extracts from Mexican plants have been reported with activity against pulmonary mycosis, among them Colubrina greggii. In the present study, extracts from the aerial parts (leaves, flowers, and fruits) of this plant were evaluated against clinical isolates of several species of Candida (C. albicans, C. glabrata, C. parapsilosis, C. krusei, and C. tropicalis) by the broth microdilution assay. Through bioassay-guided fractionation, three antifungal glycosylated flavonoids were isolated and characterized. The isolated compounds showed antifungal activity only against C. glabrata resistant to fluconazole, and were non-toxic toward brine shrimp lethality bioassay and in vitro Vero cell line assay. The ethyl acetate and butanol extracts, as well as the fractions containing the mixture of flavonoids, were more active against Candida spp.