Automated optimization of the solubility of a hyper-stable α-amylase.

Most successes in computational protein engineering to date have focused on enhancing one biophysical trait, while multi-trait optimization remains a challenge. Different biophysical properties are often conflicting, as mutations that improve one tend to worsen the others. In this study, we explored the potential of an automated computational design strategy, called CamSol Combination, to optimize solubility and stability of enzymes without affecting their activity. Specifically, we focus on Bacillus licheniformis α-amylase (BLA), a hyper-stable enzyme that finds diverse application in industry and biotechnology. We validate the computational predictions by producing 10 BLA variants, including the wild-type (WT) and three designed models harbouring between 6 and 8 mutations each. Our results show that all three models have substantially improved relative solubility over the WT, unaffected catalytic rate and retained hyper-stability, supporting the algorithm's capacity to optimize enzymes. High stability and solubility embody enzymes with superior resilience to chemical and physical stresses, enhance manufacturability and allow for high-concentration formulations characterized by extended shelf lives. This ability to readily optimize solubility and stability of enzymes will enable the rapid and reliable generation of highly robust and versatile reagents, poised to contribute to advancements in diverse scientific and industrial domains.

In-vitro and in-vivo antidiabetic activity of aerial parts of Aitchisonia rosea supported by phytochemical and GC-MS analysis.

Medicinal plants contain a wide variety of bioactive phytoconstituents which can serve as new therapeutic agents for several diseases. This study examines the antidiabetic potential of Aitchisonia rosea in alloxan-induced diabetic rats and identifies its bioactive phytoconstituents using GC-MS. In vitro, antidiabetic potential was established using the α-amylase inhibition assay. In vivo, antidiabetic potential was investigated by employing the oral glucose tolerance test (OGTT). GC-MS analysis was used to identify the bioactive phytoconstituents. The in vitro and in vivo tests showed that the aqueous extract of A. rosea possesses better antidiabetic potential. The α-amylase inhibition assay highlighted an IC50 value of 134.87µg/ml. In an oral glucose tolerance test, rats given an aqueous A. rosea extract significantly lowered their blood sugar levels significant reduction in the blood glucose concentration was observed in the oral glucose tolerance test in rats treated with the aqueous A. rosea extract. GC-MS investigation revealed many phytoconstituents, with serverogenin acetate and cycloheptasiloxane tetradecamethyl being important antidiabetic agents. This study found anti-diabetic properties in A. rosea extract. The phytochemical and GC-MS investigation also found serverogenin acetate and cycloheptasiloxane tetradecamethyl, which could be used to develop new antidiabetic drugs.

Preparation of a Novel Oat ß-Glucan-Chromium(III) Complex and Its Hypoglycemic Effect and Mechanism.

This study synthesized a novel oat ß-glucan (OBG)-Cr(III) complex (OBG-Cr(III)) and explored its structure, inhibitory effects on α-amylase and α-glucosidase, and hypoglycemic activities and mechanism in vitro using an insulin-resistant HepG2 (IR-HepG2) cell model. The Cr(III) content in the complex was found to be 10.87%. The molecular weight of OBG-Cr(III) was determined to be 7.736 × 104 Da with chromium ions binding to the hydroxyl groups of OBG. This binding resulted in the increased asymmetry and altered spatial conformation of the complex along with significant changes in morphology and crystallinity. Our findings demonstrated that OBG-Cr(III) exhibited inhibitory effects on α-amylase and α-glucosidase. Furthermore, OBG-Cr(III) enhanced the insulin sensitivity of IR-HepG2 cells, promoting glucose uptake and metabolism more efficiently than OBG alone. The underlying mechanism of its hypoglycemic effect involved the modulation of the c-Cbl/PI3K/AKT/GLUT4 signaling pathway, as revealed by Western blot analysis. This research not only broadened the applications of OBG but also positioned OBG-Cr(III) as a promising Cr(III) supplement with enhanced hypoglycemic benefits.

High-confidence structural annotation of substances via multi-layer molecular network reveals the system-wide constituent alternations in milk interfered with diphenylolpropane.

The spectral database-based mass spectrometry (MS) matching strategy is versatile for structural annotating in ingredient fluctuation profiling mediated by external interferences. However, the systematic variability of MS pool attributable to aliasing peaks and inadequacy of present spectral database resulted in a substantial metabolic feature depletion. An amended procedure termed multiple-charges overlap peaks extraction algorithm (MCOP) was proposed involving identifying collision-trigged dissociation precursor ions through iteratively matching mass features of fragmentations to expand the spectral reference library. We showcased the versatility and utility of established strategy in an investigation centered on the stimulation of milk mediated by diphenylolpropane (BPA). MCOP enabled efficient unknown annotations at metabolite-lipid-protein level, which elevated the accuracy of substance annotation to 85.3% after manual validation. Arginase and α-amylase (|r| > 0.75, p < 0.05) were first identified as the crucial issues via graph neural network-based virtual screening in the abnormal metabolism of urea triggered by BPA, resulting in the accumulation of arginine (original: 1.7 µg kg-1 1.7 times) and maltodextrin (original: 6.9 µg kg-1 2.9 times) and thus, exciting the potential dietary risks. Conclusively, MCOP demonstrated generalisation and scalability and substantially advanced the discovery of unknown metabolites for complex matrix samples, thus deciphering dark matter in multi-omics.

Structural and functional insights of starch processing α-amylase from hyperthermophilic archaeon Pyrococcusabyssi.

The genomic screening of hyper-thermophilic Pyrococcus abyssi showed uncharacterized novel α-amylase sequences. Homology modelling analysis revealed that the α-amylase from P. abyssi consists of an N-terminal GH57 catalytic domain, α-amylase central, and C-terminal domain. Current studies emphasize in-silico structural and functional analysis, recombinant expression, characterization, structural studies through CD spectroscopy, and ligand binding studies of the novel α-amylase from P. abyssi. The soluble expression of PaAFG was observed in the E. coli Rosetta™ (DE3) pLysS strain upon incubation overnight at 18 °C in an orbital shaker. The optimum temperature and pH of the PaAFG were observed at 90 °C in 50 mM phosphate buffer pH 6. The Km value for PaAFG against wheat starch was determined as 0.20 ± 0.053 mg while the corresponding Vmax value was 25.00 ± 0.67 µmol min-1 mg-1 in the presence of 2 mM CaCl2 and 12.5 % glycerol. The temperature ramping experiments through CD spectroscopy reveal no significant change in the secondary structures and positive and negative ellipticities of the CD spectra showing the proper folding and optimal temperature of PaAFG protein. The RMSD and RMSF of the PaAFG enzyme determined through molecular dynamic simulation show the significant protein's stability and mobility. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various industrial applications.

Purification of exopolysaccharide produced from Lactobacillus spp. using ionic-liquid as adjuvant in alcohol/salt-based aqueous two-phase system for its antidiabetic properties.

Diabetes is a chronic, metabolic disease characterized by hyperglycemia resulting from either insufficient insulin production or impaired cellular response to insulin. Exopolysaccharides (EPS) produced by Lactobacillus spp. demonstrated promising therapeutic potential in terms of their anti-diabetic properties. Extraction and purification of EPS produced by Lactobacillus acidophilus and Limosilactobacillus reuteri were performed using ethanol precipitation, followed by alcohol/salt based aqueous two-phase system (ATPS). The purification process involved ethanol precipitation followed by an alcohol/salt-based ATPS. The study systematically investigated various purification parameters in ATPS, including ethanol concentration, type and concentration of ionic liquid, type and concentration of salt and pH of salt. Purified EPS contents from L. acidophilus (63.30 µg/mL) and L. reuteri (146.48 µg/mL) were obtained under optimum conditions of ATPS which consisted of 30 % (w/w) ethanol, 25 % (w/w) dipotassium hydrogen phosphate at pH 10 and 2 % (w/w) 1-butyl-3-methylimidazolium octyl sulfate. The extracted EPS content was determined using phenol sulphuric acid method. In α-amylase inhibition tests, the inhibitory rate was found to be 92.52 % (L. reuteri) and 90.64 % (L. acidophilus), while in α-glucosidase inhibition tests, the inhibitory rate was 73.58 % (L. reuteri) and 68.77 % (L. acidophilus), based on the optimized parameters selected in ATPS. These results suggest that the purified EPS derived from the postbiotics of Lactobacillus spp. hold promise as potential antidiabetic agents.

Biphasic water-oil systems for functional augmentation of probiotic Lactobacillus acidophilus nanoencapsulated in luteolin-Fe3+ shells.

Single-cell nanoencapsulation (SCNE) has great potential in the enhancement of therapeutic effects of probiotic microbes. However, the material scope has been limited to water-soluble compounds to avoid non-biocompatible organic solvents that are harmful to living cells. In this work, the SCNE of probiotic Lactobacillus acidophilus with water-insoluble luteolin and Fe3+ ions is achieved by the vortex-assisted, biphasic water-oil system. The process creates L. acidophilus nanoencapsulated in the luteolin-Fe3+ shells that empower the cells with extrinsic properties, such as resistance to lysozyme attack, anti-ROS ability, and α-amylase-inhibition activity, as well as sustaining viability under acidic conditions. The proposed protocol, embracing water-insoluble flavonoids as shell components in SCNE, will be an advanced add-on to the chemical toolbox for the manipulation of living cells at the single-cell level.

Understanding Antidiabetic Potential of Oligosaccharides from Red Alga Dulse Devaleraea inkyuleei Xylan by Investigating α-Amylase and α-Glucosidase Inhibition.

In this study, the α-glucosidase (maltase-glucoamylase: MGAM) and α-amylase inhibitory properties elicited by xylooligosaccharides (XOSs) prepared from dulse xylan were analysed as a potential mechanism to control postprandial hyperglycaemia for type-2 diabetes prevention and treatment. Xylan was purified from red alga dulse powder and used for enzymatic hydrolysis using Sucrase X to produce XOSs. Fractionation of XOSs produced xylobiose (X2), ß-(1→3)-xylosyl xylobiose (DX3), xylotriose (X3), ß-(1→3)-xylosyl-xylotriose (DX4), and a dulse XOS mixture with n ≥ 4 xylose units (DXM). The different fractions exhibited moderate MGAM (IC50 = 11.41-23.44 mg/mL) and α-amylase (IC50 = 18.07-53.04 mg/mL) inhibitory activity, which was lower than that of acarbose. Kinetics studies revealed that XOSs bound to the active site of carbohydrate digestive enzymes, limiting access to the substrate by competitive inhibition. A molecular docking analysis of XOSs with MGAM and α-amylase clearly showed moderate strength of interactions, both hydrogen bonds and non-bonded contacts, at the active site of the enzymes. Overall, XOSs from dulse could prevent postprandial hyperglycaemia as functional food by a usual and continuous consumption.

Synthesis, structural, molecular docking, and in vitro biological activities of Cu-doped ZnO nanomaterials.

Copper-doped ZnO nanoparticles with the formula Zn1-x(Cu)O, where x = 0.0, 0.03, 0.05, and 0.07 were produced using the co-precipitation process. Physical, chemical, and structural properties were properly examined. Powdered X-ray diffraction (P-XRD) patterns revealed the formation of hexagonal wurtzite crystal structure in all samples, through atomic substitutional incorporation in the Cu-doped ZnO lattice. The presence of Cu ions and their dissolution in the host ZnO crystal structure was supported by FT-IR spectra. HR-TEM images were used to assess the average size, morphology, and shape regularity of the synthesized samples. The form and homogeneity of the ZnO changed when Cu ions were substituted, as evidenced by FE-SEM/EDX analysis. The presence of copper signals in the Cu-doped samples indicates that the doping was successful. The decrease in zeta potential with an increased copper doping percentage designates that the nanoparticles (NPs) are more stable, which could be attributed to an increase in the ionic strength of the aqueous solution. The synthesized NPs were evaluated for their substantial in vitro antioxidant properties. In addition, the antimicrobial efficacy of the materials was tested against pathogenic microorganisms. Regarding the anti-diabetic activity, the 7Cu ZnO sample showed the highest inhibitory effect on the α-amylase enzyme. No variations were observed in the activities of the acetylcholinesterase enzyme (AChE) and proteinase enzymes with ZnO and samples doped with different concentrations of Cu. Therefore, further studies are recommended to reveal the in-vitro anti-diabetic activity of the studied doped samples. Finally, molecular docking provided valuable insights into the potential binding interactions of Cu-doped ZnO with α-amylase, FabH of E. coli, and Penicillin-binding proteins of S. aureus. These outcomes suggest that the prepared materials may have an inhibitory effect on enzymes and hold promise in the battle against microbial infections and diabetes.

The antihyperglycemic potential of pyrazolobenzothiazine 1, 1-dioxide novel derivative in mice using integrated molecular pharmacological approach.

Diabetes Mellitus is a metabolic disease characterized by elevated blood sugar levels caused by inadequate insulin production, which subsequently leads to hyperglycemia. This study was aimed to investigate the antidiabetic potential of pyrazolobenzothiazine derivatives in silico, in vitro, and in vivo. Molecular docking of pyrazolobenzothiazine derivatives was performed against α-glucosidase and α-amylase and compounds were selected based on docking score, bonding interactions and low root mean square deviation (RMSD). Enzyme inhibition assay against α-glucosidase and α-amylase was performed in vitro using p-nitrophenyl-α-D-glucopyranoside (PNPG) and starch substrate. Synthetic compound pyrazolobenzothiazine (S1) exhibited minimal conformational changes during the 100 ns MD simulation run. S1 also revealed effective IC50 values for α-glucosidase (3.91 µM) and α-amylase (8.89 µM) and an enzyme kinetic study showed low ki (- 0.186 µM, - 1.267 µM) and ki' (- 0.691 µM, - 1.78 µM) values with the competitive type of inhibition for both enzymes α-glucosidase and α-amylase, respectively. Moreover, studies were conducted to check the effect of the synthetic compound in a mouse model. A low necrosis rate was observed in the liver, kidney, and pancreas through histology analysis performed on mice. Compound S1 also exhibited a good biochemical profile with lower sugar level (110-115 mg/dL), increased insulin level (25-30 µM/L), and low level of cholesterol (85 mg/dL) and creatinine (0.6 mg/dL) in blood. The treated mice group also exhibited a low % of glycated haemoglobin (3%). This study concludes that S1 is a new antidiabetic-agent that helps lower blood glucose levels and minimizes the complications associated with type-II diabetes.

Enzymatic Assembly of Chitosan-Based Network Polysaccharides and Their Encapsulation and Release of Fluorescent Dye.

We prepared network polysaccharide nanoscopic hydrogels by crosslinking water-soluble chitosan (WSCS) with a carboxylate-terminated maltooligosaccharide crosslinker via condensation. In this study, the enzymatic elongation of amylose chains on chitosan-based network polysaccharides by glucan phosphorylase (GP) catalysis was performed to obtain assembly materials. Maltoheptaose (Glc7) primers for GP-catalyzed enzymatic polymerization were first introduced into WSCS by reductive amination. Crosslinking of the product with the above-mentioned crosslinker by condensation was then performed to produce Glc7-modified network polysaccharides. The GP-catalyzed enzymatic polymerization of the α-d-glucose 1-phosphate monomer from the Glc7 primers on the network polysaccharides was conducted, where the elongated amylose chains formed double helices. Enzymatic disintegration of the resulting network polysaccharide assembly successfully occurred by α-amylase-catalyzed hydrolysis of the double helical amyloses. The encapsulation and release of a fluorescent dye, Rhodamine B, using the CS-based network polysaccharides were also achieved by means of the above two enzymatic approaches.

Profile of Phenolic Compounds and Antioxidant Activity of Celery (Apium graveolens) Juices Obtained from Pulp after α-Amylase Treatment from Aspergillus oryzae.

The purpose of this study was to determine the content of certain phenolic compounds, antioxidant activity, pressing efficiency, extract content, and sugars in celeriac juices obtained from the pulp after α-amylase treatment from Aspergillus oryzae. The test material consisted of peeled and unpeeled celery pulp kept at a temperature of 25 °C with and without the enzyme for a period of 30 and 60 min. The juices obtained from them were analyzed for the content of selected phenolic acids and flavonoids using the UPLC-PDA-ESI-MS/MS method, for antioxidant activity measured using the ABTS˙+ and DPPH˙ method, and for the total polyphenol content using the F-C method. Additionally, the juice pressing efficiency, the extract content using the refractometer method, and the sugar content using the HPLC method were checked. Significantly higher antioxidant activity, pressing yield, and average content of caffeic acid glucoside, quinic acid, kaempferol-3,7-di-O-glucoside, and chrysoeriol-7-O-apiosylglucoside were obtained in juices from peeled celery. Maceration of the pulp with amylase resulted in a significant reduction in antioxidant activity compared to control samples. An is-total increase of 17-41% in total flavonoid content was observed in all juices tested after treatment with the enzyme for 30 and 60 min, and the phenolic acid content increased by 4-41% after treatment of the pulp with amylase for 60 min. The 60 min holding of the pulp at 25 °C, including with the enzyme, was shown to decrease the antioxidant activity and the content of quinic acid, ferulic acid, and chrysoriol-7-O-apiose-glucoside in the juices tested compared to the samples held for 30 min, while the content of other phenolic acids and flavonoids increased. In addition, after 60 min of enzymatic maceration, the pressing yield of the juices increased.

Site-Directed Mutagenesis: Improving the Acid Resistance and Thermostability of Bacillus velezensis α-Amylase and Its Preliminary Feed Application.

The current study aimed to improve the acid resistance and thermostability of Bacillus velezensis α-amylase through site-directed mutagenesis, with a specific focus on its applicability to the feed industry. Four mutation sites, P546E, H572D, A614E, and K622E, were designed in the C domain of α-amylase, and three mutants, Mut1 (E), Mut2 (ED), and Mut3 (EDEE), were produced. The results showed that the specific activity of Mut3 was 50 U/mg higher than the original α-amylase (Ori) after incubation at 40 °C for 4 h. Compared to Ori, the acid resistance of Mut3 showed a twofold increase in specific activity at pH 2.0. Moreover, the results of preliminary feed hydrolysis were compared between Ori and Mut3 by designing three factors, three levels of orthogonal experiment for enzymatic hydrolysis time, feed quantity, and amount of amylase. It was observed that the enzymatic hydrolysis time and feed quantity showed an extremely significant difference (p < 0.01) in Mut3 compared to Ori. However, the amount of enzyme showed significant (p < 0.05) improvement in the enzymatic hydrolysis in Mut3 as compared to Ori. The study identified Mut3 as a promising candidate for the application of α-amylase in the feed industry.

Influences of superfine-grinding and enzymolysis separately assisted with carboxymethylation and acetylation on the in vitro hypoglycemic and antioxidant activities of oil palm kernel expeller fibre.

The synergistic effects of ultrafine grinding and enzymolysis (cellulase and Laccase hydrolysis) alone or combined with carboxymethylation or acetylation on the hypoglycemic and antioxidant activities of oil palm kernel fibre (OPKEF) were studied for the first time. After these synergistic modifications, the microstructure of OPKEF became more porous, and its soluble fibre and total polyphenols contents, and surface area were all improved (P < 0.05). Superfine-grinding and enzymolysis combined with carboxymethylation treated OPKEF exhibited the highest viscosity (13.9 mPa∙s), inhibition ability to glucose diffusion (38.18%), and water-expansion volume (3.58 mL∙g-1). OPKEF treated with superfine-grinding and enzymolysis combined with acetylation showed the highest surface hydrophobicity (50.93) and glucose adsorption capacity (4.53 µmol∙g-1), but a lower α-amylase-inhibition ability. Moreover, OPKEF modified by superfine-grinding and enzymolysis had the highest inhibiting activity against α-amylase (25.78%). Additionally, superfine-grinding and enzymolysis combined with carboxymethylation or acetylation both improved the content and antioxidant activity of OPEKF's bounding polyphenols (P < 0.05).

Pain and salivary biomarkers of stress in temperomandibular disorders were affected by maxillary splints.

BACKGROUND: Longitudinal intervention studies on treatment options in temporomandibular dysfunction (TMD) including self reports and salivary biomarkers of stress are rare and the exact therapeutic function of occlusal splints widely unknown. METHODS: We examined the therapeutic effects of a Michigan splint with occlusal relevance in patients with TMD using a placebo-controlled, delayed-start design. Two intervention groups received a Michigan splint, while one of them had a placebo palatine splint for the first 3 weeks. We collected pain intensities (at rest and after five occlusal movements), salivary measures associated with stress (cortisol and alpha-amylase) and self-reported psychological distress (stress, anxiety, catastrophizing) at baseline and 3 and 7 weeks after onset of intervention. RESULTS: At baseline, we observed increased pain intensity and psychological distress in TMD patients compared to 11 matched healthy controls. Baseline anxiety was linked to movement pain intensity through stress. Over therapy reductions in pain intensity and morning cortisol were more pronounced in those patients starting immediately with the Michigan splint, while psychological distress decreased similarly in both groups. CONCLUSION: Our results suggest that perceived stress plays a role for the association between anxiety and TMD pain and underlines the need for an interdisciplinary perspective on the pathogenesis and therapy of TMD in a setting where psychotherapeutic knowledge is still scarce or rarely applied.

Phytochemical Profiling and Assessment of Antidiabetic Activity of Curcuma Angustifolia Rhizome Methanolic Extract: An In Vitro and In Silico Analysis.

Curcuma angustifolia Roxb. is a plant with medicinal potential, traditionally used to treat different diseases. The present study aimed to determine the antidiabetic activity of C. angustifolia rhizome in vitro and in silico. The methanolic extract of C. angustifolia rhizome was analyzed by FTIR and GC-MS to determine the phytochemicals present. The antidiabetic potential of the extract was evaluated by different assays in vitro. The extract inhibited both α-amylase and α-glucosidase enzymes and the glucose diffusion through the dialysis membrane in a concentration-dependent manner with IC50 values of 530.39±0.09, 293.75±0.11, and 551.74±0.3 µg/ml respectively. The methanolic extract also improved yeast cell's ability to take up glucose across plasma membranes and the adsorption of glucose. The findings were supported by molecular docking studies. The results showed that the methanol extract of C. angustifolia rhizome has significant antidiabetic activity and thus can be also studied to isolate the potential compound with antidiabetic activities.

Studies on α-amylase inhibition by acarbose and quercetin using fluorescence, circular dichroism, docking, and dynamics simulations.

This study looked at the effects of acarbose (ACA) and quercetin (QUE) on α-amylase activity, employing QUE and ACA to measure enzyme activity. The study observed that both drugs suppressed α-amylase activity, with greater inhibition reported at higher concentrations. The use of tryptophan residues as an intrinsic fluorescence probe permitted the observation of conformational changes in α-amylase, with CD measurements utilized to explore the secondary structure in the presence of QUE and ACA. Docking studies revealed an effective interaction between α-amylase, quercetin and acarbose, with a higher binding energy. Finally, a trajectory analysis was done to establish the stability and volatility of these complexes. These findings have potential significance for the development of new α-amylase-related therapeutics.

Aloe juvenna Brandham & S.Carter as α-Amylase Inhibitor and Hypoglycaemic Agent with Anti-inflammatory Properties for Diabetes Management.

Despite Aloe's traditional use, Aloe juvenna Brandham & S.Carter is poorly characterized. Other Aloes are known for their antidiabetic activity. This study describes the antidiabetic potentials and phytoconstituents of the A. juvenna leaves methanolic extract (AJME). Twenty-six phytoconstituents of AJME were described using HPLC/MS-MS. Lupeol and vitexin were isolated using column chromatography. The antidiabetic activity of AJME was investigated using an in vivo high-fat diet/streptozotocin-induced diabetic rat model and in vitro α-glucosidase and α-amylase inhibitory activity assays. AJME demonstrated its α-amylase inhibitory activity (IC50=313±39.9 ppm) with no effect on α-glucosidase. In vivo, AJME dose-dependently improved hyperglycaemia in a high-fat diet/streptozotocin-induced diabetic rat model. Notably, the higher dose (1600 mg/kg) of AJME significantly downregulated serum interleukin-6, tumor necrosis factor-α, and matrix metalloproteinase-1 genes, suggesting its anti-inflammatory effect. These findings indicate AJME's potential as a significant antidiabetic agent through its α-amylase inhibition, hypoglycaemic, and anti-inflammatory properties.

Chemical Profiling and Biological Activities on Nepalese Medicinal Plant Extracts and Isolation of Active Fraction of Nyctanthes arbor-tristis.

The importance of medicinal plants for the treatment of different diseases is high from the aspects of the pharmaceutical industry and traditional healers. The present study involves nine different medicinal plants, namely, Neolamarckia cadamba, Nyctanthes arbor-tristis, Pogostemon benghalensis, Equisetum debile, Litsea monopetala, Spilanthes uliginosa, Desmostachya bipinnata, Mallotus philippensis, and Phoenix humilis, collected from Chitwan district of Nepal for biochemical analysis followed by the isolation of active plant fractions from the bioactive plant extract. The methanolic extracts of roots, barks, seeds, seed cover, and the other aerial parts of plants were used for the phytochemical analysis and biological activities. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay was adopted to evaluate the antioxidant activity. Antibacterial activity was evaluated using the agar well diffusion method. The antidiabetic activity was studied by the α-amylase enzyme inhibition assay. The highest antioxidant activity was observed in extracts of Nyctanthes arbor-tristis followed by Mallotus philippensis (seed cover), Pogostemon benghalensis, Litsea monopetala, Phoenix humilis, and Neolamarckia cadamba with IC50 values of 27.38 ± 1.35, 32.08 ± 2.81, 32.75 ± 2.13, 33.82 ± 1.07, 40.14 ± 0.93, and 50.44 ± 3.75 µg/mL, respectively. The highest antidiabetic activity was observed in extracts of Phoenix humilis followed by Desmostachya bipinnata and Pogostemon benghalensis with IC50 values of 95.69 ± 6.97, 99.24 ± 12.6, and 106.3 ± 12.89 µg/mL, respectively. The mild α-amylase enzyme inhibition was found in extracts of Nyctanthes arbor-tristis, Spilanthes uliginosa Swartz, Litsea monopetala, and Equisetum debile showing IC50 values of 110.4 ± 7.78, 115.98 ± 10.24, 149.83 ± 8.3, and 196.45 ± 6.04 µg/mL, whereas Mallotus Philippensis (seed cover), Mallotus philippensis (seed), and Desmostachya bipinnata showed weak α-amylase inhibition with IC50 values of 208.87 ± 1.76, 215.41 ± 2.09, and 238.89 ± 9.27 µg/mL, respectively. The extract of Nyctanthes arbor-tristis showed high zones of inhibition against S. aureus (ATCC 25923) and E. coli (ATCC 25922) of ZOI 26 and 22 mm, respectively. The chemical constituents isolated from the active plant Nyctanthes arbor-tristis were subjected to GCMS analysis where the major chemical compounds were 11,14,17-eicosatrienoic acid and methyl ester. These results support the partial scientific validation for the traditional uses of these medicinal plants in the treatment of diabetes and infectious diseases by the people living in different communities of Chitwan, Nepal.

LC-MS/MS, GC-MS and molecular docking analysis for phytochemical fingerprint and bioactivity of Beta vulgaris L.

The plants that we consume in our daily diet and use as a risk preventer against many diseases have many biological and pharmacological activities. In this study, the phytochemical fingerprint and biological activities of Beta vulgaris L. leaf extract, which are widely consumed in the Black Sea region, were investigated. The leaf parts of the plant were dried in an oven at 35 °C and then ground into powder. The main constituents in B. vulgaris were identified by LC-MS/MS and GC-MS analyses. Phenolic content, betaxanthin and betacyanin levels were investigated in the extracts obtained using three different solvents. The biological activity of the extract was investigated by anti-microbial, anti-mutagenic, anti-proliferative and anti-diabetic activity tests. Anti-diabetic activity was investigated by in vitro enzyme inhibition and in-silico molecular docking was performed to confirm this activity. In the LC-MS analysis of B. vulgaris extract, a major proportion of p_coumaric acid, vannilin, protecatechuic aldehyde and sesamol were detected, while the major essential oils determined by GC-MS analysis were hexahydrofarnesyl acetone and phytol. Among the solvents used, the highest extraction efficiency of 2.4% was obtained in methanol extraction, and 36.2 mg of GAE/g phenolic substance, 5.1 mg/L betacyanin and 4.05 mg/L betaxanthin were determined in the methanol extract. Beta vulgaris, which exhibited broad-spectrum anti-microbial activity by forming a zone of inhibition against all tested bacteria, exhibited anti-mutagenic activity in the range of 35.9-61.8% against various chromosomal abnormalities. Beta vulgaris extract, which did not exhibit mutagenic, sub-lethal or lethal effects, exhibited anti-proliferative activity by reducing proliferation in Allium root tip cells by 21.7%. 50 mg/mL B. vulgaris extract caused 58.9% and 55.9% inhibition of α-amylase and α-glucosidase activity, respectively. The interactions of coumaric acid, vanniline, hexahydrofarnesyl acetone and phytol, which are major compounds in phytochemical content, with α-amylase and α-glucosidase were investigated by in silico molecular docking and interactions between molecules via various amino acids were determined. Binding energies between the tested compounds and α-amylase were obtained in the range of - 4.3 kcal/mol and - 6.1 kcal/mol, while for α-glucosidase it was obtained in the range of - 3.7 kcal/mol and - 5.7 kcal/mol. The biological activities of B. vulgaris are closely related to the active compounds it contains, and therefore studies investigating the phytochemical contents of plants are very important. Safe and non-toxic plant extracts can help reduce the risk of various diseases, such as diabetes, and serve as an alternative or complement to current pharmaceutical practices.