A fast and efficient method for screening and evaluation of hypoglycemic ingredients of Traditional Chinese Medicine acting on PTP1B by capillary electrophoresis.

As a pivotal enzyme that regulates dephosphorylation in cell activities and participates in the insulin signaling pathway, protein tyrosine phosphatase 1B (PTP1B) is considered to be an important target for the therapy of diabetes. In this work, a rapid and efficient inhibitor screening method of PTP1B was established based on capillary electrophoresis (CE), and used for screening and evaluating the inhibition effect of Traditional Chinese Medicine on PTP1B. Response Surface Methodology was used for optimizing the conditions of analysis. After method validation, the enzyme kinetic study and inhibition test were performed. As a result, the IC50 of PTP1B inhibitors Ⅳ and ⅩⅧ were consistent with reported values measured by a conventional method. It was found that the extracts of Astragalus membranaceus (Fisch) Bunge and Morus alba L. showed prominent inhibition on the activity of PTP1B, which were stronger than the positive controls. Meanwhile, on top of the excellent advantages of CE, the whole analysis time is less than 2 min. Thus, the results demonstrated that a fast and efficient screening method was successfully developed. This method could be a powerful tool for screening inhibitors from complex systems. It can also provide an effective basis for lead compound development in drug discovery.

Biological activities, identification, method development, and validation for analysis of polyphenolic compounds in Nymphaea rubra flowers and leaves by UHPLC-Q-cIM-TOF-MS and UHPLC-TQ-MS.

INTRODUCTION: Nymphaea rubra belongs to the Nymphaea family and is regarded as a vegetable used in traditional medicine to cure several ailments. These species are rich in phenolic acid, flavonoids, and hydrolysable tannin. OBJECTIVE: This study aimed to assess the biological activities of Nymphaea rubra flowers (NRF) and leaves (NRL) by identifying and quantifying their polyphenolic compounds using ultra-performance liquid chromatography coupled to quadrupole cyclic ion mobility time-of-flight mass spectrometry (UHPLC-Q-cIM-TOF-MS) and triple quadrupole mass spectrometry (UHPLC-TQ-MS). METHODOLOGY: NRF and NRL powder was extracted with methanol and fractionated using hexane, ethylacetate, and water. Antioxidant and α-glucosidase, and tyrosinase enzyme inhibitory activities were evaluated. The polyphenolic components of NRF and NRL were identified and quantified using UHPLC-Q-cIM-TOF-MS and UHPLC-TQ-MS. The method was validated using linearity, precision, accuracy, limit of detection (LOD), and lower limit of quantification (LLOQ). RESULTS: Bioactive substances and antioxidants were highest in the ethylacetate fraction of flowers and leaves. Principal component analysis showed how solvent and plant components affect N. rubra's bioactivity and bioactive compound extraction. A total of 67 compounds were identified, and among them 21 significant polyphenols were quantified. Each calibration curve had R2 > 0.998. The LOD and LLOQ varied from 0.007 to 0.09 µg/mL and from 0.01 to 0.1 µg/mL, respectively. NRF contained a significant amount of gallic acid (10.1 mg/g), while NRL contained abundant pentagalloylglucose (2.8 mg/g). CONCLUSION: The developed method is simple, rapid, and selective for the identification and quantification of bioactive molecules. These findings provide a scientific basis for N. rubra's well-documented biological effects.

Screening and identification of neuraminidase inhibitors from Baphicacanthus cusia by a combination of affinity ultrafiltration, HPLC-MS/MS, molecular docking, and fluorescent techniques.

Natural products provide a new opportunity for the discovery of neuraminidase (NA)inhibitors. In this study, an affinity ultrafiltration (AUF) coupled with HPLC-MS/MS method was firstly developed and optimized for screening of NA inhibitors from natural products. The critical factors influencing the interaction of enzyme-ligand (including sample concentration, enzyme concentration, incubation time and temperature, pH of the buffer, and dissociation solvents and time) were investigated and optimized by a one-factor-at-a-time design. The method was then applied to discover NA inhibitory compounds in stems and leaves of Baphicacanthus cusia. As a result, five active alkaloids were screened out and identifiedas 2,4(1H,3H)-quinazolinedione (1), 4(3H)-quinazolinone (2), 2(3H)-benzoxazolone (3), tryptanthrin (4), and indirubin (5) through analysis of their DAD profiles, MS/MS fragments, and comparison with reference substances. These active compounds were further evaluated for their NA inhibitory activity using a fluorescence-based NA inhibition assay. The result from the fluorescent assay revealed that all the five compounds(1-5) showed pronounced NA inhibitory activities with IC50values of 98.98, 64.69, 40.16, 69.44, and 144.73 µM, respectively. Finally, molecular docking of these five alkaloids with NA showed that hydrogen bond and π-cation interactions dominated within the binding sites with binding energies ranging between -5.7 to -7.9 kcal/mol, which was supported by the results of the AUF and the fluorescence-based enzyme assay. The developed AUF method is simple and efficient for screening potential NA inhibitors from stems and leaves of B. cusia.

A Study of the Chemical Composition and Biological Activity of Michelia macclurei Dandy Heartwood: New Sources of Natural Antioxidants, Enzyme Inhibitors and Bacterial Inhibitors.

The wood of Michelia macclurei Dandy (MD) is an excellent material that is widely used in the furniture, handicraft, and construction industries. However, less research has been conducted on the chemical composition and biological activity of heartwood, which is the main valuable part of the wood. This study aimed to investigate the chemical composition and biological activities of the heartwood of Michelia macclurei Dandy (MDHW) and to confirm the active ingredients. Triple quadrupole gas chromatography-mass spectrometry (GC-MS) was used to characterize the volatile components of MDHW, while ultra-performance liquid chromatography-mass spectrometry was used to analyze the non-volatile components (UPLC-MS). The total reducing power, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging assays, acetylcholinesterase and α-glucosidase inhibition assays, and an antimicrobial test of 4 gram bacteria were used to describe the in vitro bioactivities. The GC-MS analysis showed that the volatile components of MDHW were mainly fatty compounds and terpenoids, with sesquiterpenes and their derivatives dominating the terpene composition. ß-elemene was the main terpene component in the steam distillation (11.88%) and ultrasonic extraction (8.2%) methods. A total of 67 compounds, comprising 45 alkaloids, 9 flavonoids, 6 lignans, and others, were found by UPLC-MS analysis. The primary structural kinds of the non-volatile components were 35 isoquinoline alkaloids. Alkaloids were the predominant active constituent in all MDHW extracts, including crude extracts, alkaloid fractions, and non-alkaloid fractions. These extracts all demonstrate some biological effects in terms of antioxidant, enzyme inhibition, and bacterial inhibition. The findings of this study show that MDHW is abundant in chemical structure types, has great bioactivity assessment, and has the potential to be used to create natural antioxidants, products that postpone Alzheimer's disease and lower blood sugar levels and antibacterial agents.

An affinity interaction guided two-dimensional separation system for the screening of neuraminidase inhibitors from Reynoutria japonica Houtt. roots.

Discovering bioactive compounds from medicinal herbs is crucial for drug discovery. Ultrafiltration is often used in the screening of bioactive compounds from natural herbs because of its simple and rapid operations. However, the ultrafiltration results are often disturbed by the undissolved compounds and the non-target compounds, which reduces the accuracy of the results. Herein, an affinity interaction guided two-dimensional (2D) separation system was developed. Discovery of the potential neuraminidase (NA) inhibitors from the dried roots of Reynoutria japonica Houtt. (RRJ) was used as an example. Only the small molecules showing affinity interaction with NA could be screened by the affinity interaction guided 2D separation system. Firstly, the NA and crude extract were incubated to form a sample solution (containing NA-inhibitor complexes, NA, and three types of small molecules with different polarities) by affinity interaction. Then the sample solution was separated and detected by the 2D separation system. This aimed to reduce the interference of the undissolved compounds and non-target compounds, and pick out the NA-inhibitor complexes (NA-Is). The collected NA-Is were denatured to release small molecular inhibitors (Is) for LC-MS/MS analysis. Compared with the ultrafiltration, more obvious peak area differences were observed in the results, and four potential NA inhibitors were successfully identified. In all, we provided a simple strategy with better performance in the screening of natural bioactive compounds.

LC-ESI-MS/MS Characterization of Concentrated Polyphenolic Fractions from Rhododendron luteum and Their Anti-Inflammatory and Antioxidant Activities.

The high biological potential of polyphenols encourages the search for new natural sources of and biomedical applications for these compounds. Rhododendron luteum Sweet was previously reported to contain pharmaceutically active polyphenols. The present research investigates the polyphenolic fractions in R. luteum leaves, including a determination of the free and bound phenolic acid and flavonoid contents and their anti-inflammatory and antioxidant activities. LC-ESI-MS/MS (liquid chromatography/electrospray ionization triple quadrupole mass spectrometry) analysis revealed a great abundance of free (e.g., 5-O-caffeoylquinic acid, ferulic acid, protocatechuic acid, catechin, and dihydromyricetin) and bound (e.g., caffeic acid, p-coumaric, protocatechuic acid, myricetin, quercetin) phenolics. The R. luteum samples exhibited high anti-inflammatory potential in lipoxygenase (IC50: 0.33 ± 0.01-2.96 ± 0.06 mg dry extract (DE)/mL) and hyaluronidase (IC50: 78.76 ± 2.09 - 429.07 ± 31.08 µg DE/mL) inhibition capacity assays. Some samples also had the ability to inhibit cyclooxygenase 1 (IC50: 311.8 ± 10.95 µg DE/mL) and cyclooxygenase 2 (IC50: 53.40 ± 5.07; 608.09 ± 14.78 µg DE/mL). All fractions showed excellent antioxidant activity in the Oxygen Radical Absorbance Capacity (ORAC) assay (5.76-221.81 g Trolox/g DE), ABTS•+ radical scavenging ability (0.62 ± 0.03 - 5.09 ± 0.23 g Trolox/g DE), and moderate ion (Fe2+) chelating power. This paper expands our knowledge of the phytochemistry and pharmacological activity of R. luteum polyphenols. It reveals, for the first time, the presence of dihydromyricetin, afzelin, and laricitrin in the plant material. It indicates biologically active polyphenolic fractions that should be further investigated or which could be efficiently used in pharmaceutical, cosmetic, or nutraceutical applications.

Polarity Directed Appraisal of Pharmacological Potential and HPLC-DAD Based Phytochemical Profiling of Polygonum glabrum Willd.

The present study was designed to evaluate polarity-dependent extraction efficiency and pharmacological profiling of Polygonum glabrum Willd. Crude extracts of leaves, roots, stems, and seeds, prepared from solvents of varying polarities, were subjected to phytochemical, antioxidant, antibacterial, antifungal, antidiabetic, and cytotoxicity assays. Maximum extraction yield (20.0% w/w) was observed in the case of an acetone:methanol (AC:M) root extract. Distilled water:methanol (W:M) leaves extract showed maximum phenolic contents. Maximum flavonoid content and free radical scavenging potential were found in methanolic (M) seed extract. HPLC-DAD quantification displayed the manifestation of substantial quantities of quercetin, rutin, gallic acid, quercetin, catechin, and kaempferol in various extracts. The highest ascorbic acid equivalent total antioxidant capacity and reducing power potential was found in distilled water roots and W:M leaf extracts, respectively. Chloroform (C) seeds extract produced a maximum zone of inhibition against Salmonella typhimurium. Promising protein kinase inhibition and antifungal activity against Mucor sp. were demonstrated by C leaf extract. AC:M leaves extract exhibited significant cytotoxic capability against brine shrimp larvae and α-amylase inhibition. Present results suggest that the nature of pharmacological responses depends upon the polarity of extraction solvents and parts of the plant used. P. glabrum can be considered as a potential candidate for the isolation of bioactive compounds with profound therapeutic importance.

Based on Multi-Activity Integrated Strategy to Screening, Characterization and Quantification of Bioactive Compounds from Red Wine.

According to French Paradox, red wine was famous for the potential effects on coronary heart disease (CHD), but the specific compounds against CHD were unclear. Therefore, screening and characterization of bioactive compounds from red wine was extremely necessary. In this paper, the multi-activity integrated strategy was developed and validated to screen, identify and quantify active compounds from red wine by using ultra high performance liquid chromatography-fraction collector (UHPLC-FC), ultra fast liquid chromatography-quadrupole-time-of-flight/mass spectrometry (UFLC-Q-TOF/MS) and bioactive analysis. UHPLC-FC was employed to separate and collect the components from red wine, which was further identified by UFLC-Q-TOF/MS to acquire their structural information. Furthermore, the active fractions were tested for antioxidant activity, inhibitory activity against thrombin and lipase activities in vitro by the activity screening kit. As the results, there were 37 fractions had antioxidant activity, 22 fractions had thrombin inhibitory activity and 28 fractions had lipase inhibitory activity. Finally, 77 active components from red wine were screened and 12 ingredients out of them were selected for quantification based on the integration of multi-activity. Collectively, the multi-activity integrated strategy was helpful for the rapid and effective discovery of bioactive components, which provided reference for exploring the health care function of food.

Comprehensive evaluation of two Astragalus species (A. campylosema and A. hirsutus) based on biological, toxicological properties and chemical profiling.

Astragalus L. (Fabaceae) is an important genus with numerous species having various traditional medicinal uses making them of interest for scientific investigations to ascertain their therapeutic benefits. In the present study, the quantitative polyphenolic profiles of methanolic extracts from different parts (leaves, flowers, and roots) of two endemic Astragalus species growing in Turkey, i.e. A. campylosema Boiss. and A. hirsutus Vahl were determined, along with their antioxidant and enzyme inhibitory properties. A. campylosema and A. hirsutus extracts showed varying total phenolic (25.80-40.60 and18.59-29.46 mg GAE/g, respectively) and total flavonoid (11.21-105.91 and 16.06-131.91 mg RE/g, respectively) contents. HPLC-MS/MS revealed rutin to be the predominant phenolic compound in all the extracts of A. campylosema and leaf extract of A. hirsutus (133.53-752.42 µg g-1), while hyperoside was the major one in the flower and root extracts of A. hirsutus (2014.07 and 123.13 µg g-1, respectively). In DPPH and ABTS assays, radical scavenging capacity was demonstrated by all extracts of A. campylosema (47.13-48.10 and 87.03-115.36 mg TE/g, respectively) and A. hirsutus (17.82-38.67 and 47.84-57.29 mg TE/g, respectively). Reducing activity was also displayed by the extracts in CUPRAC and FRAP assays (A. campylosema: 83.06-135.20 and 59.15-90.19 mg TE/g, respectively; A. hirsutus: 53.02-83.42 and 31.25-43.25 mg TE/g, respectively). All extracts were also found to act as metal chelators (12.32-21.45 mg EDTAE/g) and exhibited total antioxidant capacity ranging from 1.16 to 1.60 mmol TE/g, in phosphomolybdenum assay. Acetyl- and butyryl-cholinesterase inhibitory effects were observed by all the extracts of the two species (1.56-4.99 mg GALAE/g). Anti-hyperpigmentation potential by inhibiting tyrosinase (54.55-67.35 mg KAE/g) was reported as well. Carbohydrate hydrolyzing enzymes, amylase and glucosidase were also inhibited (0.22-1.03 mmol ACAE/g). Overall, A. campylosema extracts showed relatively better antioxidant and enzyme inhibitory potentials compared to A. hirsutus extracts. Strikingly, A. hirsutus extracts was found to have higher AGE inhibition activity than A. campylosema. Although the cytotoxic effect of three different organs obtained from A. campylosema and A. hirsutus increased depending on the dose (from 10 to 200 µg/mL), it was found that both plant extracts did not show a genotoxic effect at the highest concentration of 200 µg/mL. Indeed, data amassed from this current scientific work showed the two selected Astragalus species to be rich in bioactive polyphenols that could be responsible for the various pharmacological activities and hence demands to be further explored for their possible applications as natural health promoting agents.

Development of a new nano arginase HPLC capillary column for the fast screening of arginase inhibitors and evaluation of their binding affinity.

A simple and rapid Nano LC method has been developed for the screening of arginase inhibitors. The method is based on the immobilization of biotinylated arginase on a neutravidin functionalized nano HPLC capillary column. The arginase immobilization step performed by frontal analysis is very fast and only takes a few minutes. The miniaturized capillary column of 170 nL (length 5 cm, internal diameter 75 µm) significantly decreased the required amount of used enzyme (25 pmol). This was of significance importance when working with less available or expensive purified enzyme. Non-selective adsorption of the organic monolith matrix was reduced (<6%) and the arginase efficient yield was high (92%). The resultant affinity capillary columns showed excellent repeatability and long lifetime. The arginase reaction product was achieved within 60 s and the immobilized arginase retained 97% of the initial activity beyond 90 days. This novel approach can thus be used for the fast evaluation of recognition assay induced bya series of inhibitor molecules (caffeic acid phenylamide, chlorogenic acid, piceatannol, nor-NOHA acetate) and plant extracts.

Tanacetum vulgare L. (Tansy) as an effective bioresource with promising pharmacological effects from natural arsenal.

The Tanacetum genus is a big treasure with the presence of biologically-active compounds and members of this genus are widely used for the treatment of several diseases in traditional medicine system. Considering this fact, we aimed to analyze the extracts from Tanacetum vulgare L. in case of chemical profiles and biological effects. Chemical characterization was performed by using UHPLC-HRMS technique and showed the presence of several phytochemical groups (107 compounds were identified, including phenolic acids, flavonoids, terpenoids and fatty acids. Biological abilities were examined by using antioxidant (DPPH, ABTS, FRAP, CUPRAC, metal chelating and phosphomolybdenum assays) and enzyme inhibition (tyrosinase, amylase, glucosidase and cholinesterase) properties. Pharmaco-toxicological investigations were also performed with the aim to identify limits of biocompatibility, anti-oxidant and neuromodulatory effects, in hypothalamic HypoE22 cells. A bioinformatic analysis was also carried to unravel the putative protein-targets for the observed biological effects. Generally, the tested hexane and hydroalcoholic extracts displayed stronger activities in antioxidant and enzyme inhibitory assays, when compared with water. In addition, multivariate analysis was performed to understand the differences in both solvents and plant parts and we clearly observed the separation of these parameters. The extracts (10 µg/mL) also stimulated DAT and inhibited TNFα and BDNF gene expression, in HypoE22 cells. In parallel, the extracts were also able to stimulate norepinephrine release from this cell line. By contrast, in the concentration range 50-100 µg/mL, the extracts reduced the HypoE22 viability, thus demonstrating cytotoxicity at concentrations 5-10 fold higher compared to those effective as neuromodulatory. Our observations manifested that T. vulgare has several beneficial effects and it can be used as a potential natural raw material for designing further health-promoting applications in nutraceutical, cosmeceutical, and pharmaceutical areas.

Xanthine Oxidase Inhibitors from Filipendula ulmaria (L.) Maxim. and Their Efficient Detections by HPTLC and HPLC Analyses.

Filipendula ulmaria is a plant commonly used for the treatment of several pathologies, such as diarrhoea, ulcers, pain, stomach aches, fevers, and gout. Our study focused on the use of F. ulmaria for the treatment of gout disease. We first studied the chemical composition of a methanolic extract of the aerial parts and demonstrated its xanthine oxidase (XO) inhibitory activity. Then, we performed a fractionation and evaluated the most XO inhibitory active fractions by UV measurement. Purification of some fractions allowed the determination of the inhibitory activity of pure compounds. We demonstrated that spiraeoside, a glycosylated flavonoid, possesses an activity around 25 times higher than allopurinol, used as a reference in the treatment of gout disease. In order to easily and quickly identify potent inhibitors in complex matrix, we developed a complementary strategy based on an HPLC method and an Effect Directed Assay (EDA) method combining HPTLC and biochemical assays. The HPLC method, capable of determining compounds exhibiting interactions with the enzyme, could be an efficient strategy for evaluating potent enzyme inhibitors in a complex mixture. This strategy could be applied for quantitative assays using LC/MS experiments.

Metabolomic profiling and biological properties of six Limonium species: novel perspectives for nutraceutical purposes.

The genus Limonium includes important halophyte plants containing a variety of bioactive compounds of therapeutic interest. In the present work, the untargeted phytochemical profiles of both aerial part and root extracts from six Limonium species namely, L. bellidifolium, L. globuliferum, L. gmelinii, L. lilacinum, L. sinuatum and L. iconicum from Turkey were determined. Furthermore, several biological activities (in vitro antioxidant and enzyme inhibitory effects) were investigated. Overall, significant amounts of total phenolics (43.64-238.18 mg g-1) and flavonoids (1.61-129.69 mg g-1) were recorded. Particularly, the root extracts of L. gmelinii, L. iconicum and L. globuliferum showed the highest total phenolic content (204.13-238.18 mg g-1), whilst the highest total flavonoid content was recorded in the root extracts of L. gmelinii (129.69 mg g-1). Overall, the tested extracts demonstrated potent radical scavenging activities in both DPPH (2,2- diphenyl-1-picrylhydrazyl) and ABTS (3-ethylbenzothiazoline-6-sulphonic acid) (90.10-507.94 mg g-1 and 163.39-1175.34 mg g-1, respectively). However, the highest scavenging potential (p < 0.05) was displayed by the root extracts of L. iconicum. Conversely, the metal chelating ability assay revealed that L. lilacinum root extract showed the highest activity (21.03 mg g-1). Interestingly, all the extracts were found to be active inhibitors of cholinesterases (AChE (acetylcholinesterase): 4.20-5.11 mg GALAE (galantamine equivalent) per g; BChE (butyrylcholinesterase): 3.89-10.75 mg GALAE per g), amylase (0.52-1.09 mmol ACAE (acarbose equivalent) per g) and tyrosinase (119.41-155.67 mg KAE (kojic acid equivalent) per g), unlike for glucosidase (2.31-2.41 mmol ACAE per g). Taken together, these findings demonstrated a diverse chemical profiles and biological of the extracts, to be potentially considered as phytotherapeutic or functional ingredients due to their antioxidant properties and inhibition of key enzymes involved in several diseases.

Rapid screening for natural lipase inhibitors from Alisma orientale combining high-performance thin-layer chromatography-bioautography with mass spectrometry.

Lipase inhibitors are an attractive class of hypolipidemic compounds, which inhibit the activity of human pancreatic lipase, thereby preventing the absorption of triglycerides in vivo. As a library of promising lead compounds for drug development, traditional Chinese medicine (TCM) has gained growing attention in quick discovery and identification of enzyme inhibitors of natural-origin. The purpose of this work was to discover unknown lipase inhibitors from Alisma orientale by the activity oriented analysis method thin-layer chromatography-bioautography, then use electrospray ionization mass spectrometry technology via the elution based TLC-MS interface to identify their structures. As a result, eleven natural lipase inhibitors from Alisma orientale extracts were identified based on molecular mass and fragment ions obtained by HPTLC-MS, and further confirmed by a series of complementary means including UV spectra, 1H NMR characteristic proton signals and polarity of compounds, eleven lipase inhibitors were tentatively assigned as triterpenoids: alisol B (m/z 495.50 [M + Na]+), alisol B 23-acetate (m/z 537.58 [M + Na]+), 11-deoxy-alisol B (m/z 479.50 [M + Na]+), 11-deoxy-alisol B 23-acetate (m/z 521.50 [M + Na]+), alisol A/epialisol A (m/z 513.50 [M + Na]+), 16-oxo-11-deoxy-alisol A (m/z 511.50 [M + Na]+), 16-oxo-alisol A (527.50 [M + Na] +), alisol C (m/z 509.58 [M + Na]+), alisol C 23-acetate (m/z 551.50 [M + Na]+), alisol M 23-acetate (m/z 567.50 [M + Na]+), and alismanol Q/neoalisol (m/z 493.42 [M + Na]+). The integrated approach is an efficient method for rapid screening lipase inhibitors from complex plant extracts and provides a reasonable and favorable basis for the identification and separation of other enzymatic system and other important compounds with therapeutic values.

Purification, identification, and computational analysis of xanthine oxidase inhibitory peptides from kidney bean.

Hyperuricemia is related to plenty of diseases, seriously damaging human health. Current clinical drugs used to treat hyperuricemia have many adverse effects. In this study, kidney bean hydrolysate (KBH) was found to exert high xanthine oxidase inhibitory (XOI) activity. Compared to KBH (50.31 ± 2.73%), XOI activities of three fractions (Mw <5 kDa, Mw <3 kDa, Mw  < 1 kDa) by ultrafiltration were higher and increased to 58.58 ± 3.57%, 59.34 ± 1.78%, and 55.05 ± 5.00%, respectively (P < 0.05). A total of 69 peptides were identified by HPLC-ESI-MS/MS and analyzed binding affinities with XO with the help of molecular docking. AVDSLVPIGR, DWYDIK, LDNLLR, ISPIPVLK, ISSLEMTR showed well binding affinities with XO and DWYDIK presented the highest XOI activity (68.63 ± 5.07%) among five synthetic peptides (P < 0.05). Additionally, visual analysis results indicated that DWYDIK was pushed into the hydrophobic channel and formed hydrogen bonds with pivotal amino acids of xanthine oxidase. Overall, KBH could be a promising candidate as anti- hyperuricemia functional food. PRACTICAL APPLICATION: This research initially revealed that kidney bean peptides could significantly inhibit the activity of xanthine oxidase, indicating kidney bean peptides could be a treatment for hyperuricemia. Kidney bean peptides may have commercial potentials as a safer alternative with few side effects to drugs.

Identification of new BACE1 inhibitors for treating Alzheimer's disease.

Alzheimer's disease (AD) is a type of brain disorder, wherein a person experiences gradual memory loss, state of confusion, hallucination, agitation, and personality change. AD is marked by the presence of extracellular amyloid plaques and intracellular neurofibrillary tangles (NFTs) and synaptic losses. Increased cases of AD in recent times created a dire need to discover or identify chemical compounds that can cease the development of AD. This study focuses on finding potential drug molecule(s) active against ß-secretase, also known as ß-site amyloid precursor protein cleaving enzyme 1 (BACE1). Clustering analysis followed by phylogenetic studies on microarray datasets retrieved from GEO browser showed that BACE1 gene has genetic relatedness with the RCAN1 gene. A ligand library comprising 60 natural compounds retrieved from literature and 25 synthetic compounds collected from DrugBank were screened. Further, 350 analogues of potential parent compounds were added to the library for the docking purposes. Molecular docking studies identified 11-oxotigogenin as the best ligand molecule. The compound showed the binding affinity of - 11.1 Kcal/mole and forms three hydrogen bonds with Trp124, Ile174, and Arg176. The protein-ligand complex was subjected to 25 ns molecular dynamics simulation and the potential energy of the complex was found to be - 1.24579e+06 Kcal/mole. In this study, 11-oxotigogenin has shown promising results against BACE1, which is a leading cause of AD, hence warrants for in vitro and in vivo validation of the same. In addition, in silico identification of 11-oxotigogenin as a potential anti-AD compound paves the way for designing of chemical scaffolds to discover more potent BACE1 inhibitors.Graphical abstract.

Structure-based drug design, synthesis and screening of MmaA1 inhibitors as novel anti-TB agents.

Tuberculosis is one of the leading causes of death across the world. The treatment regimens for tuberculosis are well established, but still the control of the disease faces many challenges such as lengthy treatment protocols, drug resistance and toxicity. In the present work, mycolic acid methyl transferase (MmaA1), a protein involved in the maturation of mycolic acids in the biochemical pathway of the Mycobacterium, was studied for novel drug discovery. The homology model of the MmaA1 protein was built and validated by using computational techniques. The MmaA1 protein has 286 amino acid residues consisting of 10 α-helices and 7 ß-sheets. The active site of the MmaA1 protein was identified using CASTp, SiteMap and PatchDock. Virtual screening studies were performed with two small molecule ligand databases: Asinex synergy and Diverse_Elite_Gold_Platinum databases having a total of 43,446 molecules and generated 1,30,814 conformers against the predicted and validated active site of the MmaA1 protein. Binding analysis showed that the residues ASP 19, PHE 22, TRP 30, TYR 32, TRP 74 and ALA 77 of MmaA1 protein have consistent interactions with the ligands. The hit ligands were further filtered by in silico ADME properties to eliminate potentially toxic molecules. Of the top 10 molecules, 3-(2-morpholinoacetamido)-N-(1,4-dihydro-4-oxoquinazolin-6-yl) benzamide was synthesised and screened for in vitro anti-TB activity against Mtb H37Rv using MABA assay. The compound and its intermediates exhibited good in vitro anti-TB activity which can be taken up for future lead optimisation studies. Structure based virtual screening study was performed using a validated homology model against small molecules from two virtual compound libraries. Synthesised the lead compound 3-(2-morpholinoacetamido)-N-(1,4-dihydro-4-oxoquinazolin-6-yl)benzamide obtained from virtual screening. In vitro activity against Mtb H37Rv has given a promising result.

Screening and characterization of aldose reductase inhibitors from Traditional Chinese medicine based on ultrafiltration-liquid chromatography mass spectrometry and in silico molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenqi Jiangtang granule (SJG) is an ancient Chinese herbal formula used for treatment of Diabetes mellitus and its complications. AIM OF THE STUDY: To establish an integrated approach for discovery of effective Aldose reductase inhibitors (ARIs) from SJG. MATERIALS AND METHODS: An integrated approach combining ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS) with in silico molecular docking was established for development of ARIs. AR enzyme was separated from the rabbit's crystalline lens. The inhibitory activities of these compounds were detected by UV spectrophotometry with DL-glyceraldehyde as a substrate. Furthermore, molecular docking was used to understand the binding mechanism of these screened compounds interacting with AR. RESULTS: After optimization of AR reaction system and ultrafiltration incubation system, 17 active ingredients were screened from SJG by UF-LC-MS technique. Among these potential AR inhibitors, ginsenoside Rd exhibited the strongest activity with IC50 value of 45.77 µM. Three of them, calycosin, gomisin J and schisandrin A were demonstrated to be potential inhibitors for the first time, with IC50 at 447.34 µM, 181.73 µM, and 429.00 µM, respectively. Most of the active compounds exhibited competitive inhibition against AR. The docking scores of saponins were higher than that of lignans, which was consistent with the verification results. CONCLUSION: The results indicated that TCM formula with clinical efficacy was indeed hopeful source for screening active ingredients, and the combination of UF-LC-MS and in silico molecular docking was a universal and promising approach for development of effective enzyme inhibitors.

A comparative study of the HPLC-MS profiles and biological efficiency of different solvent leaf extracts of two African plants: Bersama abyssinica and Scoparia dulcis.

In the present study, two medicinal plants from Africa, namely Bersama abyssinica Fresen. and Scoparia dulcis L., were extracted using ethyl acetate, methanol, and water. The antioxidant, enzyme (α-amylase, α-glucosidase, acetyl- and butyrylcholinesterase, lipase, and tyrosinase) inhibitory action, and phytochemical profiles of extracts of Bersama abyssinica and Scoparia dulcis were determined. The aqueous (180.62 and 61.81 mg gallic acid equivalent/g extract, for B. abyssinica and S. dulcis respectively) and methanol (75.21 and 57.81 mg rutin equivalent/g extract, for B. abyssinica and S. dulcis, respectively) extracts contained high concentrations of phenolic and flavonoids, respectively. The ethyl acetate extracts of both plants were potent inhibitors of α-glucosidase and tyrosinase. Several phytochemical groups were determined by HPLC-MS/MS. The study tend to suggest that B. abyssinica and S. dulcis are potential candidates for the development of novel therapeutical agents.

Development of a thin-layer chromatography bioautographic assay for neuraminidase inhibitors hyphenated with electrostatic field induced spray ionisation-mass spectrometry for identification of active Isatis indigotica root compounds.

The identification of neuraminidase inhibitors from natural products is a promising strategy in the field of anti-influenza research. In this study, a new thin-layer chromatography (TLC) bioautographic assay for the screening of neuraminidase inhibitors from natural products was developed. This TLC bioassay is based on the one-step reaction of neuraminidase with the sodium salt of 5­bromo­4­chloro­3-indolyl-α-d-N-acetylneuraminic acid (substrate) and the subsequent formation of blue coloured products. Neuraminidase inhibitory activity was shown by the development of white spots against the blue TLC background. The key factors affecting the assay (such as enzyme concentration, substrate concentration, incubation time, reaction time, and pH) were investigated and optimised by a combination of a one-factor-at-a-time design and a Box-Behnken design/response surface method. The developed TLC bioautographic method was applied to identify neuraminidase inhibitory compounds in the roots of Isatis indigotica. Eleven active compounds including six alkaloids, three lignans, one sterol, and one fatty acid were identified in situ by direct coupling with an electrostatic field induced spray ionisation-mass spectrometry approach through analysis of their MSn (n = 4) data or comparison with reference substances. The developed TLC bioautographic assay is simple, rapid, and efficient for screening potential neuraminidase inhibitors from natural products.