Identification of putative α-glucosidase inhibitors and antioxidants in Zingiber officinale rhizome using LCMS-based metabolomics and in silico molecular docking.

Metabolite profiling is required to reveal bioactive chemicals in ginger rhizome for supporting its traditional claim as anti-diabetic agent. This study aimed to evaluate α-glucosidase inhibitory (AGI) and antioxidant activities of the rhizome, to identify its putative α-glucosidase inhibitors, and to analyse the protein-ligand interaction of the inhibitors. The ginger extracts were tested to in vitro AGI assay and analysed using LCMS-based metabolomics to pinpoint the putative α-glucosidase inhibitors. The methanol extract exhibited the highest AGI activity (IC50 = 185.2 µg/mL) compared to the other extracts. This extract showed antioxidant activities with DPPH-IC50 and FRAP value of 125.0 µg/mL and 16.95 mmol TE/mgDW, respectively. The LCMS-based metabolomics revealed α-glucosidase inhibitors in the extract, namely 7-methoxycoumarin, supinine and 12-hydroxycorynoline. The presence of these compounds in ginger is being reported for the first time in this study. The activity of these compounds was supported by computational study using in silico molecular docking.

Identification of cancer inhibitors from Hystrix brachyura bezoar extracts using LC - MS multivariate data analysis and in silico evaluation on Bcl - 2, cyclin B/CDK1, VEGF and NM23 - H1 / Identificación de inhibidores de cáncer a partir de extractos de bezoar de Hystrix branchyura usando análisis multivariado LC - MS y evaluación in silico en Bcl - 2, cyclin B/CDK1, VEGF, y NM23 - H1

Hystrix brach yura bezoar is calcified undigested material found in the gastrointestinal tract known for various medicinal benefits including as an anticancer agent. However, the H. brachyura population has been declining due to its demand and is under Malaysian law pro tection. Therefore, present study aimed to identify bezoar anticancer active compounds through metabolomics and in - silico approaches. Five replicates of bezoar powder were subjected to extraction using different solvent ratios of methanol - water (100, 75, 5 0, 25, 0% v/v). Cytotoxicity and metabolite profiling using liquid chromatography - mass spectrometry were conducted. Putative compounds identified were subjected to in - silico analysis with targeted anticancer proteins namely, Bcl - 2, Cyclin B/CDK1 complex, V EGF and NM23 - H1. The correlation of LC - MS and cytotoxicity profile pinpointed two compounds, mangiferin and propafenone. In - silico study showed both compounds exerted good binding scores to all proteins with hydrophobic interaction dominating the ligand - pr otein complex binding, suggesting the ligands act as hydrophobes in the interactions.

El bezpar de Hystrix branchyura es material calcificado sin digerir encontr ados en el tracto gastrointestinal, conocido por sus variados beneficios médicos, incluyendo propiedades anticancerosas. De todas formas, la población de H. Branchyura ha ido declinando debido a su demanda y está bajo la protección de la ley de Malasia. Po r esto, este estudio busca identificar los componentes activos anticancerosos del bezoar mediante abordajes metabolómico e in silico. Cinco réplicas de polvo de bezoar fueron sometidos a extracción usando solventes con diferentes proporciones metanol - agua (100, 75, 50, 25, 0% v/v). Se hicieron perfiles de citotoxicidad y de metabolitos usando cromatografía líquida - espectrometría de masa ( LC - MS ). Se identificaron compuestos putativos yse sometieron a a nálisis in silico, buscando las proteínas anticancerosas B cl - 2, complejo Cyclin B/CDK1, VEGF, y NM23 - H1. La correlación LC - MS y el perfil de citotoxicidad identificaron dos compuestos: mangiferina y propafenona. El estudio in silico mostró que ambos compuestos tenían buenos índices de enlace con todas las proteín as con interacción hidrofóbica dominando el enlace complejo proteína - ligando, sugeriendo que los ligandos actúan como hidrófobos en las interacciones

In Vitro Anti-Diabetic, Anti-Inflammatory, Antioxidant Activities and Toxicological Study of Optimized Psychotria malayana Jack Leaves Extract.

Psychotria malayana Jack (Family: Rubiaceae, local name: Salung) is a traditional herb used to treat diabetes. A previous study by our research group demonstrated that P. malayana methanolic and water extract exhibits significant potential as an effective agent for managing diabetes. Further research has been performed on the extraction optimization of this plant to enhance its inhibitory activity against α-glucosidase, a key enzyme associated with diabetes, and to reduce its toxicity. The objectives of this study are to evaluate the anti-diabetic, anti-inflammatory, and antioxidant properties of the optimized P. malayana leaf extract (OE), to evaluate its toxicity using a zebrafish embryo/larvae model, and to analyze its metabolites. The anti-diabetic effects were assessed by investigating α-glucosidase inhibition (AGI), while the inflammation inhibitory activity was performed using the soybean lipoxygenase inhibitory (SLOXI) test. The assessment of antioxidant activity was performed utilizing FRAP and DPPH assays. The toxicology study was conducted using the zebrafish embryo/larvae (Danio rerio) model. The metabolites present in the extracts were analyzed using GC-MS and LC-MS. OE demonstrated significant AGI and SLOXI activities, represented as 2.02 and 4.92 µg/mL for IC50 values, respectively. It exhibited potent antioxidant activities as determined by IC50 values of 13.08 µg/mL (using the DPPH assay) and 95.44 mmol TE/mg DW (using the FRAP assay), and also demonstrated an LC50 value of 224.29 µg/mL, which surpasses its therapeutic index of 111.03. OE exhibited a higher therapeutic index compared to that of the methanol extract (13.84) stated in the previous state of the art. This suggests that OE exhibits a lower level of toxicity, making it safer for use, and has the potential to be highly effective in its anti-diabetic activity. Liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) demonstrated the presence of several constituents in this extract. Among them, several compounds, such as propanoic acid, succinic acid, D-tagatose, myo-inositol, isorhamnetin, moracin M-3'-O-ß-D-glucopyranoside, procyanidin B3, and leucopelargonidin, have been reported as possessing anti-diabetic and antioxidant activities. This finding offers great potential for future research in diabetes treatment.

Impact of GSK199 and GSK106 binding on protein arginine deiminase IV stability and flexibility: A computational approach.

Protein arginine deiminase IV (PAD4) is a potential target for diseases including rheumatoid arthritis and cancers. Currently, GSK199 is a potent, selective yet reversible PAD4 inhibitor. Its derivative, GSK106, on the other hand, was reported as an inactive compound when tested against PAD4 assay. Although they had similar skeleton, their impact towards PAD4 structural and flexibility is unknown. In order to fill the research gap, the impact of GSK199 and GSK106 binding towards PAD4 stability and flexibility is investigated via a combination of computational methods. Molecular docking indicates that GSK199 and GSK106 are capable to bind at PAD4 pocket by using its back door with -10.6 kcal/mol and -9.6 kcal/mol, respectively. The simulations of both complexes were stable throughout 100 ns. The structure of PAD4 exhibited a tighter packing in the presence of GSK106 compared to GSK199. The RMSF analysis demonstrates significant changes between the PAD4-GSK199 and PAD4-GSK106 simulations in the regions containing residues 136, 160, 220, 438, and 606. The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis shows a marked difference in binding free energies, with -11.339 kcal/mol for the PAD4-GSK199 complex and 1.063 kcal/mol for the PAD4-GSK106 complex. The hydrogen bond analysis revealed that the GSK199 and GSK106 binding to PAD4 are assisted by six hydrogen bonds and three hydrogen bonds, respectively. The visualisation of the MD simulations revealed that GSK199 remained in the PAD4 pocket, whereas GSK106 shifted away from the catalytic site. Meanwhile, molecular dockings of benzoyl arginine amide (BAEE) substrate have shown that BAEE is able to bind to PAD4 catalytic site when GSK106 was present but not when GSK199 occupied the site. Overall, combination of computational approaches successfully described the behaviour of binding pocket of PAD4 structure in the presence of the active and inactive compounds.

In Vitro neurotoxicity and myotoxicity of Malaysian Naja sumatrana and Naja kaouthia venoms: Neutralization by monovalent and Neuro Polyvalent Antivenoms from Thailand.

Naja sumatrana and Naja kaouthia are medically important elapids species found in Southeast Asia. Snake bite envenoming caused by these species may lead to morbidity or mortality if not treated with the appropriate antivenom. In this study, the in vitro neurotoxic and myotoxic effects N. sumatrana and N. kaouthia venoms from Malaysian specimens were assessed and compared. In addition, the neutralizing capability of Cobra Antivenom (CAV), King Cobra Antivenom (KCAV) and Neuro Polyvalent Antivenom (NPAV) from Thailand were compared. Both venoms produced concentration-dependent neurotoxic and myotoxic effects in the chick biventer cervicis nerve-muscle preparation. Based on the time to cause 90% inhibition of twitches (i.e. t90) N. kaouthia venom displayed more potent neurotoxic and myotoxic effects than N. sumatrana venom. All three of the antivenoms significantly attenuated venom-induced twitch reduction of indirectly stimulated tissues when added prior to venom. When added after N. sumatrana venom, at the t90 time point, CAV and NPAV partially restored the twitch height but has no significant effect on the reduction in twitch height caused by N. kaouthia venom. The addition of KCAV, at the t90 time point, did not reverse the attenuation of indirectly stimulated twitches caused by either venom. In addition, none of the antivenoms, when added prior to venom, prevented attenuation of directly stimulated twitches. Differences in the capability of antivenoms, especially NPAV and CAV, to reverse neurotoxicity and myotoxicity indicate that there is a need to isolate and characterize neurotoxins and myotoxins from Malaysian N. kaouthia and N. sumatrana venoms to improve neutralization capability of the antivenoms.

GC-MS- and NMR-Based Metabolomics and Molecular Docking Reveal the Potential Alpha-Glucosidase Inhibitors from Psychotria malayana Jack Leaves.

Psychotria malayana Jack leaf, known in Indonesia as "daun salung", is traditionally used for the treatment of diabetes and other diseases. Despite its potential, the phytochemical study related to its anti-diabetic activity is still lacking. Thus, this study aimed to identify putative inhibitors of α-glucosidase, a prominent enzyme contributing to diabetes type 2 in P. malayana leaf extract using gas chromatography-mass spectrometry (GC-MS)- and nuclear magnetic resonance (NMR)-based metabolomics, and to investigate the molecular interaction between those inhibitors and the enzyme through in silico approach. Twenty samples were extracted with different solvent ratios of methanol-water (0, 25, 50, 75, and 100% v/v). All extracts were tested on the alpha-glucosidase inhibition (AGI) assay and analyzed using GC-MS and NMR. Multivariate data analysis through a partial least square (PLS) and orthogonal partial square (OPLS) models were developed in order to correlate the metabolite profile and the bioactivity leading to the annotation of the putative bioactive compounds in the plant extracts. A total of ten putative bioactive compounds were identified and some of them reported in this plant for the first time, namely 1,3,5-benzenetriol (1); palmitic acid (2); cholesta-7,9(11)-diene-3-ol (3); 1-monopalmitin (4); ß-tocopherol (5); α-tocopherol (6); 24-epicampesterol (7); stigmast-5-ene (8); 4-hydroxyphenylpyruvic acid (10); and glutamine (11). For the evaluation of the potential binding modes between the inhibitors and protein, the in silico study via molecular docking was performed where the crystal structure of Saccharomyces cerevisiae isomaltase (PDB code: 3A4A) was used. Ten amino acid residues, namely ASP352, HIE351, GLN182, ARG442, ASH215, SER311, ARG213, GLH277, GLN279, and PRO312 established hydrogen bond in the docked complex, as well as hydrophobic interaction of other amino acid residues with the putative compounds. The α-glucosidase inhibitors showed moderate to high binding affinities (-5.5 to -9.4 kcal/mol) towards the active site of the enzymatic protein, where compounds 3, 5, and 8 showed higher binding affinity compared to both quercetin and control ligand.

Characterization of α-Glucosidase Inhibitors from Psychotria malayana Jack Leaves Extract Using LC-MS-Based Multivariate Data Analysis and In-Silico Molecular Docking.

Psychotria malayana Jack has traditionally been used to treat diabetes. Despite its potential, the scientific proof in relation to this plant is still lacking. Thus, the present study aimed to investigate the α-glucosidase inhibitors in P.malayana leaf extracts using a metabolomics approach and to elucidate the ligand-protein interactions through in silico techniques. The plant leaves were extracted with methanol and water at five various ratios (100, 75, 50, 25 and 0% v/v; water-methanol). Each extract was tested for α-glucosidase inhibition, followed by analysis using liquid chromatography tandem to mass spectrometry. The data were further subjected to multivariate data analysis by means of an orthogonal partial least square in order to correlate the chemical profile and the bioactivity. The loading plots revealed that the m/z signals correspond to the activity of α-glucosidase inhibitors, which led to the identification of three putative bioactive compounds, namely 5'-hydroxymethyl-1'-(1, 2, 3, 9-tetrahydro-pyrrolo (2, 1-b) quinazolin-1-yl)-heptan-1'-one (1), α-terpinyl-ß-glucoside (2), and machaeridiol-A (3). Molecular docking of the identified inhibitors was performed using Auto Dock Vina software against the crystal structure of Saccharomyces cerevisiae isomaltase (Protein Data Bank code: 3A4A). Four hydrogen bonds were detected in the docked complex, involving several residues, namely ASP352, ARG213, ARG442, GLU277, GLN279, HIE280, and GLU411. Compound 1, 2, and 3 showed binding affinity values of -8.3, -7.6, and -10.0 kcal/mol, respectively, which indicate the good binding ability of the compounds towards the enzyme when compared to that of quercetin, a known α-glucosidase inhibitor. The three identified compounds that showed potential binding affinity towards the enzymatic protein in molecular docking interactions could be the bioactive compounds associated with the traditional use of this plant.

Alpha-Glucosidase Inhibitory Effect of Psychotria malayana Jack Leaf: A Rapid Analysis Using Infrared Fingerprinting.

The plant Psychotria malayana Jack belongs to the Rubiaceae family and is known in Malaysia as "meroyan sakat/salung". A rapid analytical technique to facilitate the evaluation of the P. malayana leaves' quality has not been well-established yet. This work aimed therefore to develop a validated analytical technique in order to predict the alpha-glucosidase inhibitory action (AGI) of P. malayana leaves, applying a Fourier Transform Infrared Spectroscopy (FTIR) fingerprint and utilizing an orthogonal partial least square (OPLS). The dried leaf extracts were prepared by sonication of different ratios of methanol-water solvent (0, 25, 50, 75, and 100% v/v) prior to the assessment of alpha-glucosidase inhibition (AGI) and the following infrared spectroscopy. The correlation between the biological activity and the spectral data was evaluated using multivariate data analysis (MVDA). The 100% methanol extract possessed the highest inhibitory activity against the alpha-glucosidase (IC50 2.83 ± 0.32 µg/mL). Different bioactive functional groups, including hydroxyl (O-H), alkenyl (C=C), methylene (C-H), carbonyl (C=O), and secondary amine (N-H) groups, were detected by the multivariate analysis. These functional groups actively induced the alpha-glucosidase inhibition effect. This finding demonstrated the spectrum profile of the FTIR for the natural herb P. malayana Jack, further confirming its medicinal value. The developed validated model can be used to predict the AGI of P. malayana, which will be useful as a tool in the plant's quality control.

Cytotoxic xanthones isolated from Calophyllum depressinervosum and Calophyllum buxifolium with antioxidant and cytotoxic activities.

The stem bark of Calophyllum depressinervosum and Calophyllum buxifolium were extracted and examined for their antioxidant activities, together with cytotoxicity towards human cancer cells. The methanol extract of C. depressinervosum exhibited good DPPH and NO scavenging effects. The strongest BCB inhibition and FIC effects were shown by dichloromethane and ethyl acetate extracts of both species. Overall, DPPH, FRAP and FIC assays showed strong correlation with TPC. For cytotoxicity, hexane extract of C. depressinervosum possessed the strongest anti-proliferative activities towards SNU-1 cells while the hexane extract of C. buxifolium showed the strongest activity towards LS-174T and K562 cells with the IC50 values ranging from 7 to 17 µg/mL. The purification of plant extracts afforded eight xanthones, ananixanthone (1), caloxanthone B (2), caloxanthone I (3), caloxanthone J (4) xanthochymone B (5), thwaitesixanthone (6), 1,3,5,6-tetrahydroxyxanthone (7) and dombakinaxanthone (8). All the xanthones, except 1 were reported for the first time from both Calophyllum species. The xanthones were examined for their cytotoxic effect against K562 leukemic cells. Compounds 1 and 2 showed strong cytotoxicity with the IC50 values of 2.96 and 1.23 µg/mL, respectively. The molecular binding interaction of 2 was further investigated by performing molecular docking study with promising protein receptor Src kinase.

Identification of α-glucosidase inhibitors from Clinacanthus nutans leaf extract using liquid chromatography-mass spectrometry-based metabolomics and protein-ligand interaction with molecular docking.

The present study used in vitro and in silico techniques, as well as the metabolomics approach to characterise α-glucosidase inhibitors from different fractions of Clinacanthus nutans. C. nutans is a medicinal plant belonging to the Acanthaceae family, and is traditionally used to treat diabetes in Malaysia. n-Hexane, n-hexane: ethyl acetate (1:1, v/v), ethyl acetate, ethyl acetate: methanol (1:1, v/v), and methanol fractions were obtained via partitioning of the 80% methanolic crude extract. The in vitro α-glucosidase inhibitory activity was analyzed using all the fractions collected, followed by profiling of the metabolites using liquid chromatography combined with mass spectrometry. The partial least square (PLS) statistical model was developed using the SIMCA P+14.0 software and the following four inhibitors were obtained: (1) 4,6,8-Megastigmatrien-3-one; (2) N-Isobutyl-2-nonen-6,8-diynamide; (3) 1',2'-bis(acetyloxy)-3',4'-didehydro-2'-hydro-ß, ψ-carotene; and (4) 22-acetate-3-hydroxy-21-(6-methyl-2,4-octadienoate)-olean-12-en-28-oic acid. The in silico study performed via molecular docking with the crystal structure of yeast isomaltase (PDB code: 3A4A) involved a hydrogen bond and some hydrophobic interactions between the inhibitors and protein. The residues that interacted include ASN259, HID295, LYS156, ARG335, and GLY209 with a hydrogen bond, while TRP15, TYR158, VAL232, HIE280, ALA292, PRO312, LEU313, VAL313, PHE314, ARG315, TYR316, VAL319, and TRP343 with other forms of bonding.

Toxicity study on Clinacanthus nutans leaf hexane fraction using Danio rerio embryos.

Clinacanthus nutans, an herbal shrub belonging to the Acanthaceae family, is traditionally used as a functional food to treat various ailments in Malaysia and Indonesia. Although the polar fraction of this plant shows non-toxic effect, the toxicity of the non-polar extract is not reported so far. The present study aimed to assess the toxic effect and determine the lethal concentration of this non-polar fraction using zebrafish embryos. The n-hexane fraction was partitioned from the crude extract of C. nutans obtained using 80% methanolic solution. After spawning of the adult male and female zebrafish, the eggs were collected, transferred into a 96-well plate and incubated with the n-hexane fraction at concentrations of 15.63 µg/ml, 31.25 µg/ml, 62.5 µg/ml, 125 µg/ml, 250 µg/ml and 500 µg/ml in 2% DMSO. The survival and sublethal endpoint were assessed, the mortality and hatchability rates were calculated based on microscopic observation, while the heartbeat rate was measured using DanioScope software. The median lethal concentration (LC50) of the C. nutans n-hexane fraction, which was determined using probit analysis, was calculated to be 75.49 µg/mL, which is harmful. Moreover, gas chromatography-mass spectrometry (GC-MS) analysis revealed the presence of palmitic acid, phytol, hexadecanoic acid, 1-monopalmitin, stigmast-5-ene, pentadecanoic acid, heptadecanoic acid, 1-linolenoylglycerol and stigmasterol in the n-hexane fraction.

Identification of α-glucosidase inhibitors from Clinacanthus nutans leaf extract using liquid chromatography-mass spectrometry-based metabolomics and protein-ligand interaction with molecular docking / 药物分析学报

The present study used in vitro and in silico techniques, as well as the metabolomics approach to char-acterise α-glucosidase inhibitors from different fractions of Clinacanthus nutans. C. nutans is a medicinal plant belonging to the Acanthaceae family, and is traditionally used to treat diabetes in Malaysia. n-Hexane, n-hexane: ethyl acetate (1:1, v/v), ethyl acetate, ethyl acetate: methanol (1:1, v/v), and methanol fractions were obtained via partitioning of the 80％ methanolic crude extract. The in vitro α-glucosidase inhibitory activity was analyzed using all the fractions collected, followed by profiling of the metabolites using liquid chromatography combined with mass spectrometry. The partial least square (PLS) statistical model was developed using the SIMCA P +14.0 software and the following four inhibitors were obtained:(1) 4,6,8-Megastigmatrien-3-one; (2) N-Isobutyl-2-nonen-6,8-diynamide; (3) 1′,2′-bis(acetyloxy)-3′,4′-didehydro-2′-hydro-β, ψ-carotene; and (4) 22-acetate-3-hydroxy-21-(6-methyl-2,4-octadienoate)-olean-12-en-28-oic acid. The in silico study performed via molecular docking with the crystal structure of yeast isomaltase (PDB code: 3A4A) involved a hydrogen bond and some hydrophobic interactions be-tween the inhibitors and protein. The residues that interacted include ASN259, HID295, LYS156, ARG335, and GLY209 with a hydrogen bond, while TRP15, TYR158, VAL232, HIE280, ALA292, PRO312, LEU313, VAL313, PHE314, ARG315, TYR316, VAL319, and TRP343 with other forms of bonding.

Characterization of α-Glucosidase Inhibitors from Clinacanthus nutans Lindau Leaves by Gas Chromatography-Mass Spectrometry-Based Metabolomics and Molecular Docking Simulation.

BACKGROUND: Clinacanthus nutans (C. nutans) is an Acanthaceae herbal shrub traditionally consumed to treat various diseases including diabetes in Malaysia. This study was designed to evaluate the α-glucosidase inhibitory activity of C. nutans leaves extracts, and to identify the metabolites responsible for the bioactivity. METHODS: Crude extract obtained from the dried leaves using 80% methanolic solution was further partitioned using different polarity solvents. The resultant extracts were investigated for their α-glucosidase inhibitory potential followed by metabolites profiling using the gas chromatography tandem with mass spectrometry (GC-MS). RESULTS: Multivariate data analysis was developed by correlating the bioactivity, and GC-MS data generated a suitable partial least square (PLS) model resulting in 11 bioactive compounds, namely, palmitic acid, phytol, hexadecanoic acid (methyl ester), 1-monopalmitin, stigmast-5-ene, pentadecanoic acid, heptadecanoic acid, 1-linolenoylglycerol, glycerol monostearate, alpha-tocospiro B, and stigmasterol. In-silico study via molecular docking was carried out using the crystal structure Saccharomyces cerevisiae isomaltase (PDB code: 3A4A). Interactions between the inhibitors and the protein were predicted involving residues, namely LYS156, THR310, PRO312, LEU313, GLU411, and ASN415 with hydrogen bond, while PHE314 and ARG315 with hydrophobic bonding. CONCLUSION: The study provides informative data on the potential α-glucosidase inhibitors identified in C. nutans leaves, indicating the plant's therapeutic effect to manage hyperglycemia.

Synthesis and in vitro Bioactivity Evaluation of New Galactose and Fructose Ester Derivatives of 5-Aminosalicylic Acid.

Inflammatory bowel disease (IBD) is the main risk factor for developing colorectal cancer which is common in patients of all ages. 5-Aminosalicylic acid (5-ASA), structurally related to the salicylates, is highly active in the treatment of IBD with minor side effects. In this study, the synthesis of galactose and fructose esters of 5-ASA was planned to evaluate the role of glycoconjugation on the bioactivity of the parent drug. The antibacterial activity of the new compounds were evaluated against two Gram-negative and two Gram-positive species of bacteria, with a notable effect observed against Staphylococcus aureus and Escherichia coli in comparisons with the 5-ASA. Cytotoxicity testing over HT-29 and 3T3 cell lines indicated that the toxicity of the new products against normal cells was significantly reduced compared with the original drug, whereas their activity against cancerous cells was slightly decreased. The anti-inflammatory activity test in RAW264.7 macrophage cells indicated that the inhibition of nitric oxide by both of the monosaccharide conjugated derivatives was slightly improved in comparison with the non-conjugated drug.