Natural Enzyme Inhibitors Research Articles

Grape Pomace as a Source of Phenolics for the Inhibition of Starch Digestion Enzymes: A Comparative Study and Standardization of the Efficacy

The increase in the incidence of hyperglycemia and diabetes poses the challenge of finding cost-effective natural inhibitors of starch digestion enzymes. Among natural compounds, phenolics have been considered as promising candidates. The aims of this study were as follows: (a) to investigate the effectiveness of the inhibition of different winemaking byproducts towards intestinal brush border α-glucosidase and pancreatic α-amylase in vitro; (b) to calculate an efficacy index relative to the standard acarbose for the phenolic pool of winemaking byproducts, as well as for isolated phenolic compounds and for the phenolic pools of different plants studied in the literature, in order to rank winemaking byproducts with respect to the reference drug and other natural alternatives. Among winemaking byproducts, red grape skins showed the highest inhibitory activities towards both α-glucosidase and α-amylase, which were, on average, 4.9 and 2.6 µg acarbose equivalents/µg total phenolics (µg Ac eq/µg GAE), respectively. A correlation was observed between the total phenolic contents of red grape skins and their inhibitory effectiveness, which is useful for standardizing the efficacy of phenolic extracts obtained from different winemaking processes. In general, the inhibitory activity of the phenolic pool of grape skins was higher than those of isolated phenolic compounds, namely anthocyanins and monomeric and polymeric flavanols and flavonols, probably due to synergistic effects among compounds. Hence, bioactive phenolic fractions could be produced with the focus on functionality rather than purity, in line with the principles of sustainable processing. Based on the efficacy index developed to compare different phenolic compounds and phenolic-rich plants studied in the literature as starch digestion enzyme inhibitors, red grape skins proved to be cost-effective candidates.

Assessing Algerian Ilex aquifolium L.’s phenolic compounds, antioxidant and enzyme inhibitory properties to find a new natural remedy for Alzheimer’s, diabetes and skin diseases

This research explored the impact of methanolic extracts from the fruits and leaves of Ilex aquifolium L., a rare Algerian species, on several enzymes, focusing on tyrosinase, α-amylase, α-glucosidase, and cholinesterases, as well as their antioxidant potential in vitro assays, including phosphomolybdenum, DPPH, ABTS, CUPRAC, FRAP, and MCA. HPLC-DAD analysis identified chlorogenic acid, rosmarinic acid, hesperidin, and quercetin as predominant phenolic components. Both extracts exhibited notable antioxidant properties, up to 250.08 mg TE/g for fruits and 237.08 mg TE/g for leaf extracts. Enzyme inhibitory assays demonstrated significant inhibition against AChE and BChE with maximum activities of 2.34 mg GALAE/g in leaves and 4.32 mg GALAE/g in fruits. Additionally, considerable tyrosinase inhibition was observed in leaves at 39.69 mg KAE/g. These findings highlight I. aquifolium L. as a valuable source for investigating natural antioxidants and enzyme inhibitors, particularly for possible cosmetics and food additives applications.

Influence of Harvesting Stage on Phytochemical Composition, Antioxidant, and Antidiabetic Activity of Immature Ceratonia siliqua L. Pulp from Béni Mellal-Khénifra Region, Morocco: In Silico, In Vitro, and In Vivo Approaches.

Ceratonia siliqua L. is a medicinal plant that has long been used in traditional Moroccan medicine to treat many diseases. This study aimed to assess the impact of the stages of the immature phase of carob pulp (M1, M2, M3, M4, and M5) on phytochemical composition, antioxidant activity, and antidiabetic activity of Ceratonia siliqua L. The identification of the phenolic profile by HPLC-UV/MS-MS and the study of the antidiabetic effect by in silico, in vitro, and in vivo studies were carried out for extracts with high contents of phenolic compounds from immature wild carob pulp from the communes of Timoulit (TM), Bin Elouidane (AW), and Ouaouizerth (TG) in the province of Azilal in the Béni Mellal-Khénifra region. The results revealed a gradual increase in total sugar content over the pulp's ripening period, reaching a value of 2134 ± 56.23 mg GE/100 g fresh weight (FW) for TG. The three locations showed peak values for total polyphenol content (TPC), total flavonoid content (TFC), and total condensed tannin (TCT) at the M2 stage. AW had the highest concentrations of TPC (3819 ± 226.4 mg GAE/100 g FM), TFC (1034 ± 57.08 mg QE/100 g FM), and TCT (1472 ± 28.46 mg CE/100 g FM). The DPPH assay (7892 ± 296.1 mg TE/100 g FM) and the FRAP assay (278.2 ± 7.85 mg TE/100 g FM) both demonstrated that the TG zone is a highly potent antioxidant zone. In contrast, the AW site exhibited a markedly elevated value of 725.4 ± 103.6 mg TE/100 g FM in the ABTS assay. HPLC-UV-MS/MS analysis showed that the methanolic extracts of immature carob pulp (MEICP) from the three areas contained several different chemical compounds. The most prevalent were 3-O-p-coumaroyl-5-O-caffeoylquinic acid, quercetin 3-methyl ether, gallic acid, and galloylquinic acid. Immature carob pulp extract (ICPE) from AW showed the strongest in vitro inhibition of pancreatic α-amylase (IC50 = 0.405 µg/mL) and TG extracts were most potent against intestinal α-glucosidase (IC50 = 0.063 µg/mL). In vivo, AW, TG, and TM extracts significantly reduced postprandial glycemia in rats, with AW having the greatest effect. These results highlight the antidiabetic potential of ICPE. The 3-O-p-Coumaroyl-5-O-caffeoylquinic acid showed better affinity for α-amylase compared to acarbose and interacted significantly with several amino acid residues of the enzyme. Similarly, this molecule and 3,4-Dicaffeoylquinic acid demonstrated a strong affinity for α-glucosidase, suggesting their potential as natural inhibitors of enzymes involved in carbohydrate metabolism. Most of the compounds are not substrates of P-glycoprotein and exhibited high intestinal absorption. Furthermore, the majority of these compounds did not act as inhibitors or substrates of CYP450 enzymes, reinforcing their suitability for development as oral medications. These results underscore the potential of immature carob pulp as a promising antidiabetic agent.

Assessment and comparison of phytochemical constituents and biological activities of endemic Odonterrhena floribunda (Brassicaceae) from Türkiye

ABSTRACT This study aimed to evaluate the phenolic profile, antioxidant, antibacterial, and antidiabetic properties of ethanolic and methanolic extracts of the different parts of the endemic Odontarrhena floribunda. The antioxidant activities of O. floribunda were determined with 1,1-diphenyl-1-picrylhydrazyl (DPPH) and the copper-reducing antioxidant capacity (CUPRAC) assays. The strongest antioxidant activity was recorded in methanolic root extract (IC50:1.68±0.25 μg/mL) for DPPH and ethanolic root (λ450:0.68±0.20) extract for CUPRAC. Antibacterial activities of the extracts were displayed by the Resazurin Microplate Method (REMA). All plant extracts analyzed demonstrated stronger effects on Acinetobacter baumannii (ATCC 02026) than those of the standard compound. The in vitro α-amylase and α-glucosidase inhibitory of the plant extract were assessed using the Iodine method. Except for the methanol leaf extract, all other extracts showed strong α-glucosidase inhibitory activity (0.88–2.13 mmolTE/g). These results show that this endemic O. floribunda is a potential natural antioxidant, antibacterial, and enzyme inhibitor source.

Assessment of fat-and-oil products quality conformity (safflower oil)

The study aims to analyse the qualitative characteristics of safflower oil, including its fatty acid profile, and determine the acid and peroxide numbers. The study also investigated the levels of heavy metals in the oil and assessed its potential as an inhibitor of α-amylase and α-glucosidase enzymatic activity. In this study, standardised methods were used to analyse the fatty acid composition of safflower oil using a standard mixture of 37 fatty acid methyl esters (Supelco™ 37 Component FAME Mix), which ensured high accuracy in determining the fatty acid composition. The physical properties of safflower oil, such as density (0.94) and specific gravity (0.917), depend on the fatty acid composition and temperature. The refractive index, determined at 1.469, was used to estimate changes in the oil’s unsaturation and viscosity (45.6 cP). Colour parameters (lightness 45.24, red-green scale -2.87, yellowblue scale 21.04) confirm the authenticity of the oil. The results of gas chromatographic analysis confirmed that the composition of the samples did not meet the requirements of the standard. The study also included the evaluation of the inhibitory activity of safflower oil against the enzymes α-amylase and α-glucosidase, where one of the samples showed significant inhibition of α-amylase activity and the other α-glucosidase activity, indicating their potential use as natural inhibitors. The safety control of heavy metal content showed that the levels of lead, cadmium and other toxic elements in the oil are well below the permissible limits, confirming its safety for consumption. These data indicate that, although some safflower oil samples do not meet the standards for fatty acid composition, its potential as a natural enzyme inhibitor and safety in terms of heavy metal content opens up prospects for further research and possible optimisation of production processes

Study of an inhibitory effect of plant polyphenolic compounds against digestive enzymes using bench-working experimental evidence predicted by molecular docking and dynamics

The substantial nutritional content and diversified biological activity of plant-based nutraceuticals are due to polyphenolic chemicals. These chemicals are important and well-studied plant secondary metabolites. Their protein interactions are extensively studied. This relationship is crucial for the logical development of functional food and for enhancing the availability and usefulness of polyphenols. This study highlights the influence of protein types and polyphenols on the interaction, where the chemical bindings predominantly consist of hydrophobic interactions and hydrogen bonds. The interaction between polyphenolic compounds (PCs) and digestive enzymes concerning their inhibitory activity has not been fully studied. Therefore, we have examined the interaction of four digestive enzymes (α-amylase, pepsin, trypsin, and α-chymotrypsin) with four PCs (curcumin, diosmin, morin, and 2′,3′,4′-trihydroxychalcone) through in silico and in vitro approaches. In vitro plate assays, enzyme kinetics, spectroscopic assays, molecular docking, and simulations were performed. We observed all these PCs have significant docking scores and preferable interaction with the active site of the digestive enzymes, resulting in the reduction of enzyme activity. The enzyme-substrate binding mechanism was determined using the Lineweaver Burk plot, indicating that the inhibition occurred competitively. Among four PCs diosmin and morin has the highest interaction energy over digestive enzymes with IC50 value of 1.13 ± 0.0047 and 1.086 ± 0.0131 μM. Kinetic studies show that selected PCs inhibited pepsin, trypsin, and chymotrypsin competitively and inhibited amylase in a non-competitive manner, especially by 2′,3′,4′-trihydroxychalcone. This study offers insights into the mechanisms by which the selected PCs inhibit the enzymes and has the potential to enhance the application of curcumin, diosmin, morin, and 2′,3′,4′-trihydroxychalcone as natural inhibitors of digestive enzymes.

Mentha longifolia ssp. longifolia Essential Oil Components as Novel Carbonic Anhydrase Isoform II and IX Inhibitors: Biological and Molecular Docking Studies

Background:Medicinal plant oils are used in the treatment of various human diseases due to their phytochemical components. Recently, enzyme inhibition studies have been increasing in cosmetics, the food industry, and especially pharmaceuticals.Objective:The main goal of this study is to focus on a specific interaction between the essential oil components of Mentha longifolia ssp. longifolia and carbonic anhydrase (CA) enzyme in vitro and in silico.Methods:The chemical composition of the essential oil was identified by gas chromatography coupled with mass spectrometry (GC-MS). The CA inhibitory activity of M. longifolia essential oil was investigated by using esterase activity for the first time in this study. Molecular docking was performed separately for two different CA isoforms (CA-II and CA-IX).Results:Among fourteen components identified, piperitone (27.14%), 2-acetylcyclopentanone (21.05%), p-menthan-3-one (13.90%), menthan (6.60%), and piperitone oxide (6.52%) were defined as the major compounds. The essential oil showed remarkable inhibitory activity against CA with an IC50 value of 0.010 mg/mL. According to the molecular docking analysis, caryophyllene oxide (-6.5 kcal/mol for CAIX isoform, -6.8 kcal/mol for CA-II isoform) and trans-caryophyllene (-6.3 kcal/mol for CA-IX isoform, - 6.7 kcal/mol for CA-II isoform) molecules showed the best inhibitory activity in two different CA isoforms. In this study, it was determined that all molecules are bioavailable by ADMET analyses.Conclusion:The results of this study are valuable for the development of natural and new CA enzyme inhibitors without side effects in the treatment of diseases, such as glaucoma, obesity, and epilepsy.

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. β-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.

Phytochemical composition, antioxidant, anti-tyrosinase, anti-cholinesterase, and anti-inflammatory activities of Rhynchanthus beesianus rhizome extracts

Rhynchanthus beesianus is a medicine food homology plant, and its rhizome is a food spice and traditional Chinese medicine used to treat inflammation-related disorders. Still, little research has been done on its phytochemicals and biological activities. Therefore, this study firstly reported the phytochemical analysis by UHPLC-Q-Orbitrap-MS, antioxidant, anti-enzymatic, and anti-inflammatory effects of R. beesianus rhizome water extract (WE) and 70% ethanol extract (EE). For phytochemical analysis, WE and EE had high total phenolic (25.57–28.19 mg GAE/g extract) and flavonoid (10.57–28.08 mg RE/g extract) contents, and further UHPLC-Q-Orbitrap-MS test identified sixty-one compounds, including twelve phenolic compounds and six flavonoids. In the antioxidant assay, WE and EE showed significant DPPH (IC50: 83.93 ± 5.90 and 69.48 ± 1.57 μg/mL, respectively) and ABTS (IC50: 73.46 ± 0.69 and 40.72 ± 0.67 μg/mL, respectively) radical scavenging ability, especially the efficacy of EE was superior or equivalent to that of positive control BHT. Moreover, the WE and EE exhibited poor inhibition on acetylcholinesterase (IC50: 3.31 ± 0.16 and 3.53 ± 0.14 mg/mL, respectively), butyrylcholinesterase (IC50: 1.29 ± 0.02 and 0.75 ± 0.08 mg/mL, respectively), and tyrosinase (IC50: 5.91 ± 0.63 and 21.30 ± 1.52 mg/mL, respectively). Interestingly, EE showed remarkable inhibition on NO, PGE2, IL-6, and TNF-α produced by LPS stimulation without cytotoxicity. It reduced ROS generation and down-regulated mRNA and protein levels of iNOS and COX-2 in LPS-stimulated RAW264.7 macrophages. Besides, EE decreased LPS-stimulated MAPKs (ERK, p38, and JNK) phosphorylation and inhibited NF-κB p65 nuclear translocation by suppressing the phosphorylation and degradation of IκBα. Therefore, R. beesianus can be used as a promising natural antioxidant, enzyme inhibitor, and anti-inflammatory agent with great exploitation potential in functional food and pharmaceuticals.

Microorganism-Derived Molecules as Enzyme Inhibitors to Target Alzheimer's Diseases Pathways.

Alzheimer's disease (AD) is the most common form of dementia. It increases the risk of other serious diseases and causes a huge impact on individuals, families, and socioeconomics. AD is a complex multifactorial disease, and current pharmacological therapies are largely based on the inhibition of enzymes involved in the pathogenesis of AD. Natural enzyme inhibitors are the potential sources for targeting AD treatment and are mainly collected from plants, marine organisms, or microorganisms. In particular, microbial sources have many advantages compared to other sources. While several reviews on AD have been reported, most of these previous reviews focused on presenting and discussing the general theory of AD or overviewing enzyme inhibitors from various sources, such as chemical synthesis, plants, and marine organisms, while only a few reviews regarding microbial sources of enzyme inhibitors against AD are available. Currently, multi-targeted drug investigation is a new trend for the potential treatment of AD. However, there is no review that has comprehensively discussed the various kinds of enzyme inhibitors from the microbial source. This review extensively addresses the above-mentioned aspect and simultaneously updates and provides a more comprehensive view of the enzyme targets involved in the pathogenesis of AD. The emerging trend of using in silico studies to discover drugs concerning AD inhibitors from microorganisms and perspectives for further experimental studies are also covered here.

Fijian medicinal plants and their role in the prevention of Type 2 diabetes mellitus.

Medicinal plants (MPs) are natural sources of active compounds with potential therapeutic benefits in alleviating various illnesses for decades. Fijian people also are using these MPs for the management/prevention of Type 2 diabetes mellitus (T2DM) and associated complications. However, till date, none of these Fijian MP's antidiabetic potential have been explored or evaluated. Here, we investigated the antidiabetic potential of Fijian MPs scientifically. Phytochemicals such as polyphenols were detected to inhibit the activity of α-amylase and α-glucosidase, the two key carbohydrate enzymes linked to T2DM. Therefore, in the present study, the total phenolic content (TPC), α-amylase and α-glucosidase inhibitory activity of five Fijian MPs: Vobo (Mussaenda raiateensis, MR), Vula walu (Blechnum orientale, BO), Gasau (Miscanthus floridulus, MF), Molikaro (Citrus limon, CL) and Beki ni sina (Dicranopteris caudate, DC) collected from mainland region of Vitilevu, Fiji Islands, were evaluated in vitro. The hydromethanolic (ME) and dichloromethane (DM) extracts of these selected MPs were investigated. The ME extracts of BO (0.102 ± 0.009 mM CE) and DC (0.098 ± 0.09 mM Catechin Equivalence [CE]) showed a higher TPC compared with the control [vanillic acid (0.052 ± 0.003 mM CE, *P value < 0.05)]. However, the TPC of MF, MR and CL were found in the range of 0.020 ± 0.009 to 0.009 ± 0.01 mM CE. The ME extracts of MF and MR inhibited α-glucosidase significantly in comparison with acarbose as evidenced from the IC50 values (IC50 of MF = 1.58 ± 0.03 ng/µl; IC50 of MR = 1.87 ± 0.43 ng/µl and IC50 of acarbose = 3.34 ± 0.15 ng/µl). Moreover, DM extracts of MR (IC50 = 1.31 ± 0.29 ng/µl) also showed significantly higher α-glucosidase inhibitory activity. In contrary, MR (IC50 = 16.18 ± 0.16 ng/µl) and CL (IC50 = 9.21 ± 0.51 ng/µl) also showed significant α-amylase inhibitory activity in ME and DM extracts, respectively. These, results suggest that Fijian MPs could be a potential source of natural inhibitors of enzymes involved in carbohydrate digestion and thus may possibly be used in managing T2DM.

A First Look at the Inhibitory Potential of Urospatha sagittifolia (Araceae) Ethanolic Extract for Bothrops atrox Snakebite Envenomation.

Bothrops atrox snakebites are a relevant problem in the Amazon basin. In this biodiverse region, the ethnomedicinal approach plays an important role as an alternative to antivenom therapy. Urospatha sagittifolia (Araceae) is a plant used for this purpose; however, its neutralizing properties have not been scientifically accessed. To fill this gap, we investigated the ability of U. sagittifolia to modulate the catalytic activity of Bothrops atrox venom, and their toxic consequences, such as local damage and lethality. The venom profile of B. atrox was assessed by chromatography and electrophoresis. Inhibition of the three main enzymatic and medically important toxins from the venom was evaluated using synthetic substrates and quantified by chromogenic activity assays. Additionally, the neutralization of lethality, hemorrhage and edema were investigated by in vivo assays. The possible interactions between venom proteins and plant molecules were visualized by polyacrylamide gel electrophoresis. Finally, the phytochemical constituents present in the ethanolic extract were determined by qualitative and quantitative analyses. The ethanolic extract reduced the activity of the three main enzymes of venom target, achieving ranges from 19% to 81% of inhibition. Our in vivo venom neuralizations assays showed a significant inhibition of edema (38.72%) and hemorrhage (42.90%). Additionally, lethality was remarkably counteracted. The highest extract ratio evaluated had a 75% survival rate. Our data support the biomedical value of U. sagittifolia as a source of natural enzyme inhibitors able to neutralize catalytically active B. atrox venom toxins and their toxic effects.

Comparative Content, Biological and Anticancer Activities of Heracleum humile Extracts Obtained by Ultrasound-Assisted Extraction Method.

As the safety and effectiveness of synthetic drugs remain in doubt, researchers are trying to develop natural medicines from medicinal plants. Herein, ethyl acetate, methanol and water extracts from the Heracleum humile plant were obtained by an ultrasonic-assisted extraction process and the aim was to evaluate some biological effects of the extracts due to the limited data on the pharmacological properties of Heracleum humile in the literature. Weak antibacterial activity was observed on tested bacterial species. The minimum inhibitory concentration and the minimum bactericidal concentration values ranged from 250 to 500 μg/mL. In addition, cytotoxic activity was determined using the MTT test. The strongest findings were determined for ethyl acetate extract on the MDA-MB-231 cell lines at the 48th hour (IC50 :97.94 μg/mL), followed by the MCF-7 cell lines at the 24th hour (IC50 :103.9 μg/mL). All extracts of Heracleum humile contained mainly flavonoids, phenolic acids and their derivatives, i. e., well-known compounds that possess numerous biological activities such as antioxidant, anti-inflammatory, anticancer, antimicrobial etc. The study results could provide important information that Heracleum humile could be a potential candidate as a natural enzyme inhibitor. It can be concluded that these extracts could be useful in the elementary step of improving novel plant-derived multifunctional pharmaceuticals.

Chemical characterization, comprehensive antioxidant capacity, and enzyme inhibitory potential of leaves from Pistacia terebinthus L. (Anacardiaceae)

Pistacia terebinthus, Anacardiaceae, is used in traditional medicine and its fruits are consumed as coffee in Turkey. However, the research on the chemical and biological activity of P. terebinthus leaves is almost lack. In the present study, we aimed to evaluate the comprehensive antioxidant capacity (DPPH, ABTS, reducing power and metal chelating activity) and enzyme inhibitory effects (against AChE, BChE, α-amylase, α-glucosidase, and tyrosinase) as well as phytochemical content of the leaves extracts from P. terebinthus. The phytochemical composition in extracts were analysed using UHPLC-MS. Total phenolics and flavonoids contents of the extracts were 65.51–210.54 mg GAE/g and 54.94–169.89 mg RE/g, respectively. The highest level of total phenolic content was found in the methanol extract while the maximum total flavonoid content observed in ethyl acetate extract. The antioxidant results showed that methanol extract (ABTS: 3.29 mmol TE/g, DPPH: 2.06 mmol TE/g, CUPRAC: 2.67 mmol TE/g, FRAP: 1.62 mmol TE/g, Metal chelating activity: 65.65 mg EDTA/g) exhibited promising antioxidant capacity. The methanol extract also demonstrated important enzyme inhibitory effects. The results suggest that the extracts of P. terebinthus leaves has the potential to be used as good candidate of natural antioxidants and enzyme inhibitors.

Investigation of effective natural inhibitors for starch hydrolysing enzymes from Simaroubaceae plants by molecular docking analysis and comparison with in-vitro studies

The present study aims to find the effective natural enzyme inhibitors against alpha-amylase and alpha-glucosidase from the array of compounds identified in plants of the Simaroubaceae family using molecular docking and ADME/Toxicity studies. Among the 218 compounds docked against seven enzymes, buddlenol-A and citrusin-B showed the best binding energies (kcal/mol) of -7.830 and -7.383 against human salivary alpha-amylase and pancreatic alpha-amylase respectively. The other two compounds 9-hydroxycanthin-6-one and bruceolline-B had the best binding energy of -6.461 and -7.576 against N-terminal and C-terminal maltase glucoamylase respectively. Whereas the binding energy of prosopine (-6.499) and fisetinidol (-7.575) was considered as the best against N-terminal and C-terminal sucrase-isomaltase respectively. Picrasidine-X showed the best binding energy (-7.592) against yeast alpha-glucosidase. The study revealed that the seven compounds which showed the best binding energy against respective enzymes are considered as the ‘lead hit compounds’. Even though the ‘lead hit compounds’ are not obeying all the laws of ADMET, the drug-likeness properties of 9-hydroxycanthin-6-one, fisetinidol, picrasidine-X, and prosopine were considerable. Also, kaempferol-3-O-pentoside was the recent compound identified from the Simarouba glauca plant extract found to be one among the top five lead hit compounds against four enzymes. This study provides valuable insight into the direction of developing natural compounds as potential starch hydrolysing enzyme inhibitors for managing type 2 diabetes.

Transcriptomic and Drug Discovery Analyses Reveal Natural Compounds Targeting the KDM4 Subfamily as Promising Adjuvant Treatments in Cancer.

KDM4 proteins are a subfamily of histone demethylases that target the trimethylation of lysines 9 and 36 of histone H3, which are associated with transcriptional repression and elongation respectively. Their deregulation in cancer may lead to chromatin structure alteration and transcriptional defects that could promote malignancy. Despite that KDM4 proteins are promising drug targets in cancer therapy, only a few drugs have been described as inhibitors of these enzymes, while studies on natural compounds as possible inhibitors are still needed. Natural compounds are a major source of biologically active substances and many are known to target epigenetic processes such as DNA methylation and histone deacetylation, making them a rich source for the discovery of new histone demethylase inhibitors. Here, using transcriptomic analyses we determined that the KDM4 family is deregulated and associated with a poor prognosis in multiple neoplastic tissues. Also, by molecular docking and molecular dynamics approaches, we screened the COCONUT database to search for inhibitors of natural origin compared to FDA-approved drugs and DrugBank databases. We found that molecules from natural products presented the best scores in the FRED docking analysis. Molecules with sugars, aromatic rings, and the presence of OH or O- groups favor the interaction with the active site of KDM4 subfamily proteins. Finally, we integrated a protein-protein interaction network to correlate data from transcriptomic analysis and docking screenings to propose FDA-approved drugs that could be used as multitarget therapies or in combination with the potential natural inhibitors of KDM4 enzymes. This study highlights the relevance of the KDM4 family in cancer and proposes natural compounds that could be used as potential therapies.

Engineering Tissue Inhibitors of Metalloproteinases Using Yeast Surface Display.

Yeast surface display (YSD) has been extensively used for protein design, engineering, and directed evolution in the past two decades. Here, we describe methods for directed evolution of tissue inhibitors of metalloproteinase (TIMP), the natural inhibitors of matrix metalloproteinases (MMPs), through design and generation of a combinatorial library of TIMP mutants and screening the targeted TIMP library of variants toward MMP binding using YSD. This protocol can be adopted to other natural enzyme inhibitors and similar protein binders such as antibodies.

Phytochemical Constituents and Biological Activities of the Unexplored Plant Rhinanthus angustifolius subsp. grandiflorus

In the present study, a total of 12 extracts of Rhinanthus angustifolius subsp. grandiflorus, an understudied hemiparasitic species, were obtained using different extraction techniques, namely, homogenizer-assisted extraction (HAE), maceration (MAC), soxhlet (SOX), infusion, and solvents (ethyl acetate, methanol, ethanol, and water), and were evaluated for their in vitro antioxidant and enzyme-inhibiting properties. Additionally, untargeted profiling based on high-resolution mass spectrometry targeted different phytochemical classes, namely, polyphenols, terpenoids, and alkaloids. The highest total phenolic and flavonoid contents were detected using methanol as the extraction solvent. Multivariate statistics following the untargeted profiling revealed that the extraction solvent had a hierarchically higher impact than the extraction method when considering the recovery of bioactive compounds. The methanolic extracts displayed the highest radical-scavenging antioxidant capacity, as provided by CUPRAC and FRAP assays. On the other hand, the water extracts (MAC and HAE) and the infusion extract showed the highest activity as metal chelators (25.66–27.51 mg EDTAE/g). Similarly, the water extract obtained by HAE and the infusion extract revealed the highest phosphomolybdenum activity (3.92 ± 0.14 and 3.71 ± 0.01 mmol TE/g, respectively). The different extracts also exhibited different enzyme inhibition potentials. For instance, HAE and MAC ethanolic extracts inhibited only α-amylase (0.69 ± 0.01 and 0.70 ± 0.01 mmol ACAE/g), while all the other extracts showed a dual inhibition against both carbohydrate-hydrolyzing enzymes tested (i.e., α-amylase: 0.07–0.69 mmol ACAE/g; α-glucosidase: 0.03–1.30 mmol ACAE/g). Nevertheless, the other extracts inhibited acetyl-, butyryl-cholinesterases, or both; MAC–water extract displayed no inhibition against the enzymes. Additionally, all the studied extracts were found to inhibit tyrosinase, ranging from 10.62 to 52.80 mg KAE/g. In general, the water extracts showed weaker inhibition towards the enzymes than the other extracts. This study demonstrated that R. angustifolius is an excellent source of natural antioxidants and enzyme inhibitors that could be further investigated and exploited for pharmaceutical purposes.

Investigation of in vitro anti-hyperpigmentation, antidiabetic, neuroprotective and antioxidant potential of Medicago murex WILLD. (Fabaceae)

Background and Aims: Most of the Medicago species are considered good quality forage crops due to their rich protein content. This study reports antioxidant properties, total phenolic and flavonoid content, enzyme (acetylcholinesterase (AChE), tyrosinase, α-amylase and α-glucosidase) inhibition activities of methanol (MeOH), ethyl acetate (EA) and, aqueous (AQ) extracts of Medicago murex (M. murex). Methods: The antioxidant activity of the different extracts of M. murex were evaluated using FRAP and CUPRAC assays. The contents of total phenolic and flavonoid were determined by the Folin–Ciocalteu and the Aluminium chloride (AlCl3) colorimetric methods. Also, the enzyme inhibition activities were shown spectrophotometrically for AChE, α-amylase, α-glucosidase and tyrosinase enzymes. Results: Total phenolic and flavonoid contents of different extracts were determined between 83.70-163.93 mg GAE/g extract and 24.48-26.05 μg QE/g extract, respectively. AQ extracts were the most active extracts in FRAP while being the best inhibitor of the AChE enzyme. On the other hand, EA extracts were the most active extracts in CUPRAC methods and the best inhibitors for other enzymes. Conclusion: Considering the findings, M. murex appears to be an important source of natural antioxidants and enzyme inhibitors, and it provides important baseline data for the food and pharmaceutical industries. 

Kinetic and thermodynamic insights into the inhibitory mechanism of TMG-chitotriomycin on Vibrio campbellii GH20 exo-β-N-acetylglucosaminidase

We investigated the inhibition kinetics of VhGlcNAcase, a GH20 exo-β-N-acetylglucosaminidase (GlcNAcase) from the marine bacterium Vibrio campbellii (formerly V. harveyi) ATCC BAA-1116, using TMG-chitotriomycin, a natural enzyme inhibitor specific for GH20 GlcNAcases from chitin-processing organisms, with p-nitrophenyl N-acetyl-β-d-glucosaminide (pNP-GlcNAc) as the substrate. TMG-chitotriomycin inhibited VhGlcNAcase with an IC50 of 3.0 ± 0.7 μM. Using Dixon plots, the inhibition kinetics indicated that TMG-chitotriomycin is a competitive inhibitor, with an inhibition constant Ki of 2.2 ± 0.3 μM. Isothermal titration calorimetry experiments provided the thermodynamic parameters for the binding of TMG-chitotriomycin to VhGlcNAcase and revealed that binding was driven by both favorable enthalpy and entropy changes (ΔH° = −2.5 ± 0.1 kcal/mol and −TΔS° = −5.8 ± 0.3 kcal/mol), resulting in a free energy change, ΔG°, of −8.2 ± 0.2 kcal/mol. Dissection of the entropic term showed that a favorable solvation entropy change (−TΔSsolv° = −16 ± 2 kcal/mol) is the main contributor to the entropic term.