Machine Learning and In Vitro Chemical Screening of Potential α-Amylase and α-Glucosidase Inhibitors from Thai Indigenous Plants

Diabetes mellitus is a major predisposing factor for cardiovascular disease and mortality. α-Amylase and α-glucosidase enzymes are the rate-limiting steps for carbohydrate digestion. The inhibition of these two enzymes is clinically used for the treatment of diabetes mellitus. Here, in vitro study and machine learning models were employed for the chemical screening of inhibiting the activity of 31 plant samples on α-amylase and α-glucosidase enzymes. The results showed that the ethanolic twig extract of Pinus kesiya had the highest inhibitory activity against the α-amylase enzyme. The respective ethanolic extract of Croton oblongifolius stem, Parinari anamense twig, and Polyalthia evecta leaf showed high inhibitory activity against the α-glucosidase enzyme. The classification analysis revealed that the α-glucosidase inhibitory activity of Thai indigenous plants was more predictive based on phytochemical constituents, compared with the α-amylase inhibitory activity (1.00 versus 0.97 accuracy score). The correlation loading plot revealed that flavonoids and alkaloids contributed to the α-amylase inhibitory activity, while flavonoids, tannins, and reducing sugars contributed to the α-glucosidase inhibitory activity. In conclusion, the ethanolic extracts of P. kesiya, C. oblongifolius, P. anamense, and P. evecta have the potential for further chemical characterization and the development of anti-diabetic recipes.     

In Vitro Anti-Osteoporosis Properties of Diverse Korean Drynariae rhizoma Phenolic Extracts

Drynariae rhizoma has been used to prevent bone loss that occurs with increasing age. However, the chemical compounds in extracts that act on bone metabolism in herbal medicine are poorly understood. This study aimed to investigate and compare the extraction efficacy of polyphenolic compounds, antioxidant activity, and in vitro anti-osteoporosis properties of water extract (DR-DW) and ethanol extract (DR-EtOH) from D. rhizoma. Total phenolics and flavonoids were better extracted with 70% EtOH, and this extraction method also resulted in higher antioxidant activity and in vitro anti-osteoporosis properties in these extracts. In particular, the contents of phloroglucinol, protocatechuic acid ethyl ester, 2-amino-3,4-dimethyl-benzoic acid, 3-(3,5-dimethyl-pyrazol-1-yl)-benzoic acid, chlorogenic acid, syringic acid, trans-ferulic acid, (−)-epigallocatechin, epigallocatechin gallate, quercetin dehydrate, luteolin and emodin in DR-EtOH were higher than those in DR-DW. These results indicated that DR-EtOH could be a good source of natural herbs with anti-osteoporosis properties.     

Synthesis and aldose reductase inhibitory activity of a new series of 5-[[2-(ω-carboxyalkoxy)aryl]methylene]-4-oxo-2-thioxothiazolidine derivatives

Anew series of 5-[[2-(ω-carboxyalkoxy)aryl]methylene]-4-oxo-2-thioxothiazolidine derivatives was synthesized and evaluated for their potency as aldose reductase inhibitors (ARIs). Their activities were examined in terms of their inhibitory effect on rat lens aldose reductase in vitro and in terms of the preventive effect on sorbitol accumulation in the sciatic nerve of streptozotocin (STZ)-induced diabetic rats in vivo. Of these compounds, some of the naphthylmethylene thiazolidine derivatives were comparable to Zenarestat in the inhibitory potency in vitro and in vivo. In particular, compound 30 was 1.5 times more potent than Zenarestat in the in vivo activity, and had an adequate potency for clinical development.     

Extracts,Anthocyanins and Procyanidins from Aronia melanocarpa as Radical Scavengers and Enzyme Inhibitors

Extracts, subfractions, isolated anthocyanins and isolated procyanidins B2, B5 and C1 from the berries and bark of Aronia melanocarpa were investigated for their antioxidant and enzyme inhibitory activities. Four different bioassays were used, namely scavenging of the diphenylpicrylhydrazyl (DPPH) radical, inhibition of 15-lipoxygenase (15-LO), inhibition of xanthine oxidase (XO) and inhibition of α-glucosidase. Among the anthocyanins, cyanidin 3-arabinoside possessed the strongest and cyanidin 3-xyloside the weakest radical scavenging and enzyme inhibitory activity. These effects seem to be influenced by the sugar units linked to the anthocyanidin. Subfractions enriched in procyanidins were found to be potent α-glucosidase inhibitors; they possessed high radical scavenging properties, strong inhibitory activity towards 15-LO and moderate inhibitory activity towards XO. Trimeric procyanidin C1 showed higher activity in the biological assays compared to the dimeric procyanidins B2 and B5. This study suggests that different polyphenolic compounds of A. melanocarpa can have beneficial effects in reducing blood glucose levels due to inhibition of α-glucosidase and may have a potential to alleviate oxidative stress.     

A Structure—Activity Relationship Study of the Inhibition of α-Amylase by Benzoic Acid and Its Derivatives

Phenolic acids are widely found in fruits and vegetables. The inhibitory effect of phenolic acids on α-amylase, a key enzyme for starch digestion, has attracted the attention of researchers. To further investigate the effects of different substituents on the benzene ring of phenolic acid on the inhibition of α-amylase activity, in vitro experiments and molecular docking were used. The structure-activity relationships of 17 phenolic acids with benzoic acid as the parent nucleus were analyzed by determining their half inhibitory concentration (IC₅₀) toward α-amylase. The results showed that 2,3,4-trihydroxybenzoic acid had the strongest inhibitory effect on α-amylase with an IC₅₀ value of 17.30 ± 0.73 mM. According to the structure-activity analysis, the hydroxyl group at the 2-position on the benzene ring had a strong positive effect on the inhibitory activity of α-amylase, while methoxylation at the 2-position and hydroxylation at the 5-position had a negative effect. Molecular docking revealed that hydrogen bonding and hydrophobic interactions were involved in the inhibition, with hydrogen bonding being the primary force. These findings provide a more comprehensive understanding of phenolic acids as inhibitors of α-amylase and provide new ideas for the design of dietary formulations for diabetic patients.     

Inhibitory effects of 5-benzylidene barbiturate derivatives on mushroom tyrosinase and their antibacterial activities

A series of novel 5-benzylidene barbiturate and thiobarbiturate derivatives were synthesized and evaluated as tyrosinase inhibitors and antibacterial agents. The results demonstrated that some compounds had more potent inhibitory activities than the parent compound 4-hydroxybenzaldehyde (IC₅₀ = 1.22 mM). Particularly, compounds 1a and 2a were found to be the most potent inhibitors with IC₅₀ value of 13.98 μM and 14.49 μM, respectively. The inhibition mechanism study revealed that these compounds were irreversible inhibitors. The circular dichroism spectra indicated that these compounds induced conformational changes of mushroom tyrosinase upon binding. In addition, these compounds exhibited selectively antibacterial activity against Staphylococcus aureus. All these results suggested that further development of such compounds may be of interest.     

Baicalein, 7,8-Dihydroxyflavone and Myricetin as Potent Inhibitors of Human Ornithine Decarboxylase

Background: Human ornithine decarboxylase (ODC) is a well-known oncogene, and the discovery of ODC enzyme inhibitors is a beneficial strategy for cancer therapy and prevention. Methods: We examined the inhibitory effects of a variety of flavone and flavonol derivatives on ODC enzymatic activity, and performed in silico molecular docking of baicalein, 7,8-dihydroxyflavone and myricetin to the whole dimer of human ODC to investigate the possible binding site of these compounds on ODC. We also examined the cytotoxic effects of these compounds with cell-based studies. Results: Baicalein, 7,8-dihydroxyflavone and myricetin exhibited significant ODC suppression activity with IC₅₀ values of 0.88 µM, 2.54 µM, and 7.3 µM, respectively, which were much lower than that of the active-site irreversible inhibitor α-DL-difluoromethylornithine (IC₅₀, the half maximal inhibitory concentration, of approximately 100 µM). Kinetic studies and molecular docking simulations suggested that baicalein, and 7,8-dihydroxyflavone act as noncompetitive inhibitors that are hydrogen-bonded to the region near the active site pocket in the dimer interface of the enzyme. Baicalein and myricetin suppress cell growth and induce cellular apoptosis, and both of these compounds suppress the ODC-evoked anti-apoptosis of cells. Conclusions: Therefore, we suggest that the flavone or flavonol derivatives baicalein, 7,8-dihydroxyflavone, and myricetin are potent chemopreventive and chemotherapeutic agents that target ODC.     

Phytochemical Compounds and Antioxidant Capacity of Tucum-Do-Cerrado (Bactris setosa Mart), Brazil’s Native Fruit

This study identified major phenolic compounds of the tucum-do-cerrado (Bactris setosa) peel, as well as antioxidant activity and total phytochemical compound concentration of different extracts of the peel and pulp of this fruit. Phenolic compounds of the different extracts of tucum-do-cerrado peel were identified and quantified using a high-performance liquid chromatography system coupled to a diode array detector (DAD). Total phytochemical compound content was determined by spectrophotometric assays and the antioxidant activity by ferric reducing antioxidant power and β-carotene/linoleic assays. Total phenolic, flavanols, total anthocyanins and yellow flavonoids concentration of tucum-do-cerrado were 122-, 14-, 264- and 61-fold higher in the peel than in the pulp, respectively. The aqueous, methanolic and ethanolic extracts of the tucum-do-cerrado peel exhibited higher antioxidant activity compared to its pulp. Flavanols, anthocyanins, flavones, phenolic acids and stilbenes were the main phenolic classes identified in the tucum-do-cerrado peel extracts. Results suggest that the antioxidant capacity and the phytochemical compound content of the tucum-do-cerrado are mainly associated with the peel. Although flavonoids are the main compounds identified in tucum-do-cerrado peel, other phenolics identified in minor amounts, such as phenolic acids and stilbenes, may be responsible for the high antioxidant capacity of the fruit.     

Graptopetalum paraguayense Extract Ameliorates Proteotoxicity in Aging and Age-Related Diseases in Model Systems

Declines in physiological functions are the predominant risk factors for age-related diseases, such as cancers and neurodegenerative diseases. Therefore, delaying the aging process is believed to be beneficial in preventing the onset of age-related diseases. Previous studies have demonstrated that Graptopetalum paraguayense (GP) extract inhibits liver cancer cell growth and reduces the pathological phenotypes of Alzheimer’s disease (AD) in patient IPS-derived neurons. Here, we show that GP extract suppresses β-amyloid pathology in SH-SYS5Y-APP₆₉₅ cells and APP/PS1 mice. Moreover, AMP-activated protein kinase (AMPK) activity is enhanced by GP extract in U87 cells and APP/PS1 mice. Intriguingly, GP extract enhances autophagy in SH-SYS5Y-APP₆₉₅ cells, U87 cells, and the nematode Caenorhabditis elegans, suggesting a conserved molecular mechanism by which GP extract might regulate autophagy. In agreement with its role as an autophagy activator, GP extract markedly diminishes mobility decline in polyglutamine Q35 mutants and aged wild-type N2 animals in C. elegans. Furthermore, GP extract significantly extends lifespan in C. elegans.     

Discovery of Natural Inhibitors of Cholinesterases from Hydrangea: In Vitro and In Silico Approaches

Alzheimer’s disease (AD) is a neurodegenerative disease conceptualized as a clinical-biological neurodegenerative construct where amyloid-beta pathophysiology is supposed to play a role. The loss of cognitive functions is mostly characterized by the rapid hydrolysis of acetylcholine by cholinesterases including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Moreover, both enzymes are responsible for non-catalytic actions such as interacting with amyloid β peptide (Aβ) which further leads to promote senile plaque formation. In searching for a natural cholinesterase inhibitor, the present study focused on two isocoumarines from hydrangea, thunberginol C (TC) and hydrangenol 8-O-glucoside pentaacetate (HGP). Hydrangea-derived compounds were demonstrated to act as dual inhibitors of both AChE and BChE. Furthermore, the compounds exerted selective and non-competitive mode of inhibition via hydrophobic interaction with peripheral anionic site (PAS) of the enzymes. Overall results demonstrated that these natural hydrangea-derived compounds acted as selective dual inhibitors of AChE and BChE, which provides the possibility of potential source of new type of anti-cholinesterases with non-competitive binding property with PAS.     

Potentially Bio-Accessible Metabolites from an Extract of Cornus mas Fruit after Gastrointestinal Digestion In Vitro and Gut Microbiota Ex Vivo Treatment

Targeting pancreatic lipase and α-amylase by digestion-derived fractions of ethanolic-aqueous (60%, v/v) extract from Cornus mas fruit (CM) in relation to the control and prevention of metabolic disorders, including diabetes, was the first purpose of the present study. Taking into consideration the significance of bio-accessibility of compounds, we attempted to identify metabolites of CM after gastrointestinal digestion in vitro, as well as their kinetic changes upon gut microbiota treatment. The digestion of extract was simulated with digestive enzymes in vitro and human gut microbiota ex vivo (1 h, 3 h, 6 h, 24 h), followed by chromatographic analysis using the UHPLC-DAD-MSⁿ method. The effect of fractions from gastrointestinal digestion in vitro on the activity of pancreatic lipase and α-amylase was studied with fluorescence-based assays. The gastric and intestinal fractions obtained after in vitro digestion of CM inhibited pancreatic lipase and α-amylase. Loganic acid as the main constituent of the extract was digested in the experimental conditions in contrast to cornuside. It was found in most analytes such as salivary, gastric, intestinal, and even colon (fecal slurry, FS) fractions. In all fractions, kaempferol hexoside and reduced forms of kaempferol, such as aromadendrin, and benzoic acid were assigned. The signals of tannins were detected in all fractions. Cornusiin A was tentatively assigned in the gastric fraction. The metabolites originating from kinetic analytes have been classified mainly as phenolic acids, hydrolyzable tannins, and flavonoids. Phenolic acids (protocatechuic acid, gallic acid), tannins (digalloylglucose, tri-O-galloyl-β-D-glucose), and flavonoids (aromadendrin, dihydroquercetin) were detected in the late phases of digestion in fecal slurry suspension. Cornuside was found in FS analyte after 3 h incubation. It was not detected in the samples after 6 and 24 h incubation with FS. In conclusion, cornuside, aromadendrin, and phenolic acids may be potentially bio-accessible compounds of CM. The presence of plants’ secondary metabolites in the intestinal fractions allows us to indicate them as responsible for decreasing glucose and lipid absorption.     

Effects of flavonoids on α-glucosidase activity: potential targets for glucose homeostasis

ObjectiveFlavonoids are naturally occurring compounds widely distributed in plants, which have hypoglycemic potential and have been described as glucosidase inhibitors. This study evaluated the effect of flavonoids on intestinal glucosidase activity after in vivo and in vitro treatment.MethodsFor the in vivo studies animals received quercetin by gavage and for the in vitro assays a segment of the small intestine was used. To obtain the oral glucose tolerance curve fasted normal rats were loaded with glucose plus flavonoids. The glycemia was measured by the glucose oxidase method.ResultsQuercetin reduced the effect of sucrase and maltase in the in vivo and in vitro treatments. It was observed in the in vitro studies that the maximum inhibitory effect of kaempferitrin was around 23% for maltase activity. Also, for the sucrose substrate the specific enzyme activity was significantly decreased. Aglycone, flavonoids, and kaempferol decreased significantly the maltase activity at all concentrations assayed. Finally, rutin reduced maltase-specific activity at all concentrations studied. According to the oral glucose tolerance curve, rutin reduced the serum glucose levels at 15, 30, and 60 min when administered by oral gavage 30 min before glucose overload in rats.ConclusionBased on these results, we can conclude that disaccharidases are targets of flavonoids in the regulation of glucose absorption and consequently glucose homeostasis.     

Association among Vitamin D,Retinoic Acid-Related Orphan Receptors,and Vitamin D Hydroxyderivatives in Ovarian Cancer

Vitamin D and its derivatives, acting via the vitamin D receptor (VDR) and retinoic acid-related orphan receptors γ and α (RORγ and RORα), show anticancer properties. Since pathological conditions are characterized by disturbances in the expression of these receptors, in this study, we investigated their expression in ovarian cancers (OCs), as well as explored the phenotypic effects of vitamin D hydroxyderivatives and RORγ/α agonists on OC cells. The VDR and RORγ showed both a nuclear and a cytoplasmic location, and their expression levels were found to be reduced in the primary and metastatic OCs in comparison to normal ovarian epithelium, as well as correlated to the tumor grade. This reduction in VDR and RORγ expression correlated with a shorter overall disease-free survival. VDR, RORγ, and RORα were also detected in SKOV-3 and OVCAR-3 cell lines with increased expression in the latter line. 20-Hydroxy-lumisterol3 (20(OH)L₃) and synthetic RORα/RORγ agonist SR1078 inhibited proliferation only in the OVCAR-3 line, while 20-hydroxyvitamin-D₃ (20(OH)D₃) only inhibited SKOV-3 cell proliferation. 1,25(OH)₂D₃, 20(OH)L₃, and SR1078, but not 20(OH)D₃, inhibited spheroid formation in SKOV-3 cells. In summary, decreases in VDR, RORγ, and RORα expression correlated with an unfavorable outcome for OC, and compounds targeting these receptors had a context-dependent anti-tumor activity in vitro. We conclude that VDR and RORγ expression can be used in the diagnosis and prognosis of OC and suggest their ligands as potential candidates for OC therapy.     

Prenylated derivatives of baicalein and 3,7-dihydroxyflavone: synthesis and study of their effects on tumor cell lines growth, cell cycle and apoptosis

Fourteen baicalein and 3,7-dihydroxyflavone derivatives were synthesized and evaluated for their inhibitory activity against the in vitro growth of three human tumor cell lines. The synthetic approaches were based on the reaction with prenyl or geranyl bromide in alkaline medium, followed by cyclization of the respective monoprenylated derivative. Dihydropyranoflavonoids were also obtained by one-pot synthesis, using Montmorillonite K10 clay as catalyst combined with microwave irradiation. In vitro screening of the compounds for cell growth inhibitory activity revealed that the presence of one geranyl group was associated with a remarkable increase in the inhibitory activity. Moreover, for the 3,7-dihydroxyflavone derivatives a marked increase in growth inhibitory effect was also observed for compounds with furan and pyran fused rings. The most active compounds were also studied regarding their effect on cell cycle profile and induction of apoptosis. Overall the results point to the relevant role of the prenylation of flavone scaffold in the growth inhibitory activity of cancer cells.     

Vitis amurensis Ruprecht root inhibited α-melanocyte stimulating hormone-induced melanogenesis in B16F10 cells

BACKGROUND/OBJECTIVES

The root of Vitis amurensis Ruprecht, a sort of wild-growing grape, has been used in oriental medicine for treatment of skin ailments; however, its dermatological activity is not sufficiently understood. The aim of this study was to investigate tyrosinase inhibitory and anti-melanogenic activities of V. amurensis Ruprecht root methanol extract (VARM) in B16F10 mouse melanoma cells and to attempt to isolate and identify the active compound issued from VARM.

MATERIALS/METHODS

Anti-melanogenic activity of VARM was analyzed in α-melanocyte stimulating hormone (MSH)-stimulated B16F10 cells through evaluation of antioxidative activity as well as inhibited tyrosinase activity and melanin contents compared with those of kojic acid and arbutin. After anti-melanogenic analysis of VARM, serial fractionation, nuclear magnetic resonance (NMR), and thin layer chromatorgraphy (TLC) were applied for identification of active compounds contained in VARM.

RESULTS

VARM significantly inhibited oxidative stress and tyrosinase activity and attenuated α-MSH-induced melanin production in B16F10 cells. For isolation of active compounds, VARM was fractionated using a series of organic solvents, including dichloromethane (CH₂Cl₂), ethyl acetate (EtOAc), and n-butanol (n-BuOH). Among fractions showing anti-melanogenic activity, the CH₂Cl₂ fraction induced the most potent attenuation of melanogenesis without cytotoxicity and the major compound in the CH₂Cl₂ fraction was identified as betulinic acid. Betulinic acid isolated from the CH₂Cl₂ fraction of VARM significantly attenuated α-MSH-induced melanogenesis in a dose dependent manner, which was stronger than that of arbutin used as a positive control.

CONCLUSIONS

These results indicate that VARM inhibits oxidative stress, tyrosinase activity, and α-MSH-induced melanogenesis in B16F10 cells, due primarily to the active compound, betulinic acid, in the CH₂Cl₂ fraction.     

In vitro PAI-1 inhibitory activity of oxalamide derivatives

A number of oxalamide derivatives have been synthesized and evaluated for PAI-1 inhibitory activity. In vitro PAI-1 inhibitory activities of oxalamide derivatives are evaluated by chromogenic assay. Few compounds have shown significant PAI-1 inhibitory activity.     

Design, synthesis and biological study of novel pyrido[2,3-d]pyrimidine as anti-proliferative CDK2 inhibitors

The design and synthesis of a small library of 4-aminopyrido[2,3-d]pyrimidine derivatives is reported. The potential activity of these compounds as CDK2/Cyclin A, CDK4/Cyclin D, EGFR and anti-tumor was evaluated by cytotoxicity studies in A431a, SNU638b, HCT116 and inhibition of CDK2-Cyclin A, CDK4/Cyclin D and EGFR enzyme activity in vitro. The anti-proliferative and CDK2-Cyclin A inhibitory activity of compounds 4c and 11a was significantly more active than roscovotine with IC₅₀ 0.3 and 0.09 μM respectively. Molecular modeling study, including fitting to a 3D-pharmacophore model, docking into cyclin dependant kinase2 (CDK2) active site and binding energy calculations were carried out and these studies suggested the same binding orientation inside the CDK2 binding pocket for these analogs compared to ATP.     

Synthesis and inhibitory activity towards human leukocyte elastase of new 7alpha-methoxy and 7alpha-chloro (2-acyloxymethyl) cephem derivatives

Some new cephem derivatives of types 4 and 5, viewed as analogues of type I esters in which the atomic sequence of the C-2 ester group is formally inverted, were synthesised and tested in vitro for their inhibitory activity towards human leukocyte elastase and porcine pancreatic elastase. An examination of the inhibition data obtained for the new type 4 and 5 derivatives, while exhibiting a considerable reduction in their activity against porcine pancreatic elastase, indicated that these compounds still maintain an appreciable inhibitory activity against human leukocyte elastase. On this basis the new type of C-2 substitution appears to contribute to the research of new, potentially interesting, cephalosporinic human leukocyte elastase inhibitors.     

Structure-based design, synthesis and preliminary anti-inflammatory activity of bolinaquinone analogues

As a part of our drug discovery efforts we developed a series of simplified derivatives of bolinaquinone (BLQ), a hydroxyquinone marine metabolite, showing potent anti-inflammatory activity. Thirteen new hydroxyquinone derivatives closely related to BLQ were synthesized and tested on mouse macrophage-like RAW 264.7 cell line in order to investigate their ability to modulate the production of Prostaglandin E₂ (PGE₂). This optimization process led to the identification of three strictly correlated compounds with comparable and higher inhibitory potency than BLQ on PGE₂ production. To evaluate the affinity of BLQ and its analogues for hsPLA₂, surface plasmon resonance (SPR) experiments were performed.     

Tyrosyl-tRNA synthetase inhibitors as antibacterial agents: synthesis, molecular docking and structure-activity relationship analysis of 3-aryl-4-arylaminofuran-2(5H)-ones

Thirty-five 3-aryl-4-arylaminofuran-2(5H)-one derivatives were designed, prepared and tested for their inhibitory activity against tyrosyl-tRNA synthetase. Out of these compounds, 3-(3-bromophenyl)-4-(3,5-dichlorophenylamino)furan-2(5H)-one (35) was the most active with IC₅₀ of 0.09 ± 0.02 μM. The structure-activity relationship revealed that introduction of chlorine atoms at both meta positions of aniline moiety significantly increased the enzyme inhibitory activity. The results of antibacterial assay revealed that the tested compounds showed good activity against Gram-positive bacteria, with 35 being the most potent with MIC₅₀ of 0.06 μg/mL against Staphylococcus aureus ATCC 25923. Molecular docking of 35 into S. aureus tyrosyl-tRNA synthetase active site was also performed. The inhibitor snugly fitting the active site may well explain its excellent inhibitory activity.