Prunin from Poncirus trifoliata (L.) Rafin Inhibits Aldose Reductase and Glucose-Fructose-Mediated Protein Glycation and Oxidation of Human Serum Albumin.

Diabetes complications are associated with aldose reductase (AR) and advanced glycation end products (AGEs). Using bioassay-guided isolation by column chromatography, 10 flavonoids and one coumarin were isolated from Poncirus trifoliata Rafin and tested in vitro for an inhibitory effect against human recombinant AR (HRAR) and rat lens AR (RLAR). Prunin, narirutin, and naringin inhibited RLAR (IC50 0.48-2.84 µM) and HRAR (IC50 0.68-4.88 µM). Docking simulations predicted negative binding energies and interactions with the RLAR and HRAR binding pocket residues. Prunin (0.1 and 12.5 µM) prevented the formation of fluorescent AGEs and nonfluorescent Nε-(carboxymethyl) lysine (CML), as well as the fructose-glucose-mediated protein glycation and oxidation of human serum albumin (HSA). Prunin suppressed the formation of the ß-cross-amyloid structure of HSA. These results indicate that prunin inhibits oxidation-dependent protein damage, AGE formation, and AR, which may help prevent diabetes complications.

Dual regulatory effects of neferine on amyloid-ß and tau aggregation studied by in silico, in vitro, and lab-on-a-chip technology.

Alzheimer's disease (AD) is characterized by the presence of two critical pathogenic factors: amyloid-ß (Aß) and tau. Aß and tau become neurotoxic aggregates via self-assembly, and these aggregates contribute to the pathogenesis of AD. Therefore, there has been growing interest in therapeutic strategies that simultaneously target Aß and tau aggregates. Although neferine has attracted attention as a suitable candidate agent for alleviating AD pathology, there has been no study investigating whether neferine affects the modulation of Aß or tau aggregation/dissociation. Herein, we investigated the dual regulatory effects of neferine on Aß and tau aggregation/dissociation. We predicted the binding characteristics of neferine to Aß and tau using molecular docking simulations. Next, thioflavin T and atomic force microscope analyses were used to evaluate the effects of neferine on the aggregation or dissociation of Aß42 and tau K18. We verified the effect of neferine on Aß fibril degradation using a microfluidic device. In addition, molecular dynamics simulation was used to predict a conformational change in the Aß42-neferine complex. Moreover, we examined the neuroprotective effect of neferine against neurotoxicity induced by Aß and tau and their fibrils in HT22 cells. Finally, we foresaw the pharmacokinetic properties of neferine. These results demonstrated that neferine, which has attracted attention as a potential treatment for AD, can directly affect Aß and tau pathology.

In Vitro Human Monoamine Oxidase Inhibition and Human Dopamine D4 Receptor Antagonist Effect of Natural Flavonoids for Neuroprotection.

Natural flavone and isoflavone analogs such as 3',4',7-trihydroxyflavone (1), 3',4',7-trihydroxyisoflavone (2), and calycosin (3) possess significant neuroprotective activity in Alzheimer's and Parkinson's disease. This study highlights the in vitro human monoamine oxidase (hMAO) inhibitory potential and functional effect of those natural flavonoids at dopamine and serotonin receptors for their possible role in neuroprotection. In vitro hMAO inhibition and enzyme kinetics studies were performed using a chemiluminescent assay. The functional effect of three natural flavonoids on dopamine and serotonin receptors was tested via cell-based functional assays followed by a molecular docking simulation to predict interactions between a compound and the binding site of the target protein. A forced swimming test was performed in the male C57BL/6 mouse model. Results of in vitro chemiluminescent assays and enzyme kinetics depicted 1 as a competitive inhibitor of hMAO-A with promising potency (IC50 value: 7.57 ± 0.14 µM) and 3 as a competitive inhibitor of hMAO-B with an IC50 value of 7.19 ± 0.32 µM. Likewise, GPCR functional assays in transfected cells showed 1 as a good hD4R antagonist. In docking analysis, these active flavonoids interacted with a determinant-interacting residue via hydrophilic and hydrophobic interactions, with low docking scores comparable to reference ligands. The post-oral administration of 1 to male C57BL/6 mice did not reduce the immobility time in the forced swimming test. The results of this study suggest that 1 and 3 may serve as effective regulators of the aminergic system via hMAO inhibition and the hD4R antagonist effect, respectively, for neuroprotection. The route of administration should be considered.

Effects of Icariin and Its Metabolites on GPCR Regulation and MK-801-Induced Schizophrenia-Like Behaviors in Mice.

Icariin, a major bioactive compound found in the Epimedium genus, has been reported to exert protective effects against neurodegenerative disorders. In the current study, we aimed to investigate the regulatory effect of icariin and its active metabolites (icariside II and icaritin) against prime G-protein-coupled receptor targets, considering their association with neuronal disorders. Icariside II exhibited selective agonist activity towards the dopamine D3 receptor (D3R), with half-maximal effective concentrations of 13.29 µM. Additionally, they effectively inhibited the specific binding of radioligands to D3R. Molecular docking analysis revealed that icariside II potentially exerts its agonistic effect through hydrogen-bonding interaction with Asp110 of the D3R, accompanied by negative binding energy. Conversely, icaritin demonstrated selective antagonist effects on the muscarinic acetylcholine M2 receptor (M2R). Radioligand binding assay and molecular docking analysis identified icaritin as an orthosteric ligand for M2R. Furthermore, all three compounds, icariin and its two metabolites, successfully mitigated MK-801-induced schizophrenia-like symptoms, including deficits in prepulse inhibition and social interaction, in mice. In summary, these findings highlight the potential of icariin and its metabolites as promising lead structures for the discovery of new drugs targeting cognitive and neurodegenerative disorders.

An Arylbenzofuran, Stilbene Dimers, and Prenylated Diels-Alder Adducts as Potent Diabetic Inhibitors from Morus bombycis Leaves.

Morus bombycis has a long history of usage as a treatment for metabolic diseases, especially, diabetes mellitus (DM). Thus, we aimed to isolate and evaluate bioactive constituents derived from M. bombycis leaves for the treatment of DM. According to bioassay-guided isolation by column chromatography, eight compounds were obtained from M. bombycis leaves: two phenolic compounds, p-coumaric acid (1) and chlorogenic acid methyl ester (2), one stilbene, oxyresveratrol (3), two stilbene dimers, macrourin B (4) and austrafuran C (6), one 2-arylbenzofuran, moracin M (5), and two Diels-Alder type adducts, mulberrofuran F (7) and chalcomoracin (8). Among the eight isolated compounds, the anti-DM activity of 3-8 (which possess chemotaxonomic significance in Morus species) was evaluated by inhibition of α-glucosidase, protein tyrosine phosphatase 1B (PTP1B), human recombinant aldose reductase (HRAR), and advanced glycation end-product (AGE) formation as well as by scavenging peroxynitrite (ONOO-), which are crucial therapeutic targets of DM and its complications. Compounds 4 and 6-8 significantly inhibited α-glucosidase, PTP1B, and HRAR enzymes with mixed-type and non-competitive-type inhibition modes. Furthermore, the four compounds had low negative binding energies in both enzymes according to molecular docking simulation, and compounds 3-8 exhibited strong antioxidant capacity by inhibiting AGE formation and ONOO- scavenging. Overall results suggested that the most active stilbene-dimer-type compounds (4 and 6) along with Diels-Alder type adducts (7 and 8) could be promising therapeutic and preventive resources against DM and have the potential to be used as antioxidants, anti-diabetic agents, and anti-diabetic complication agents.

Icariside II, a Prenyl-Flavonol, Alleviates Inflammatory and Neuropathic Pain by Inhibiting T-Type Calcium Channels and USP5-Cav3.2 Interactions.

Cav3.2 channels play an important role in the afferent nociceptive pathway, which is responsible for both physiological and pathological pain transmission. Cav3.2 channels are upregulated during neuropathic pain or peripheral inflammation in part due to an increased association with the deubiquitinase USP5. In this study, we investigated nine naturally occurring flavonoid derivatives which we tested for their abilities to inhibit transiently expressed Cav3.2 channels and their interactions with USP5. Icariside II (ICA-II), one of the flavonols studied, inhibited the biochemical interactions between USP5 and Cav3.2 and concomitantly and effectively blocked Cav3.2 channels. Molecular docking analysis predicts that ICA-II binds to the cUBP domain and the Cav3.2 interaction region. In addition, ICA-II was predicted to interact with residues in close proximity to the Cav3.2 channel's fenestrations, thus accounting for the observed blocking activity. In mice with inflammatory and neuropathic pain, ICA-II inhibited both phases of the formalin-induced nocifensive responses and abolished thermal hyperalgesia induced by injection of complete Freund's adjuvant (CFA) into the hind paw. Furthermore, ICA-II produced significant and long-lasting thermal anti-hyperalgesia in female mice, whereas Cav3.2 null mice were resistant to the action of ICA-II. Altogether, our data show that ICA-II has analgesic activity via an action on Cav3.2 channels.

Ursonic acid from Artemisia montana exerts anti-diabetic effects through anti-glycating properties, and by inhibiting PTP1B and activating the PI3K/Akt signaling pathway in insulin-resistant C2C12 cells.

Artemisia is one of the largest genera in the plant family Asteraceae and has long been used in traditional medicine for its antitussive, analgesic, antihypertensive, antitoxic, antiviral, antimalarial, and anti-inflammatory properties. However, the anti-diabetic activity of Artemisia montana has not been broadly studied. The goal of this study was to determine whether extracts of the aerial parts of A. montana and its main constituents inhibit protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase activities. We isolated nine compounds from A. montana including ursonic acid (UNA) and ursolic acid (ULA), which significantly inhibited PTP1B with IC50 values of 11.68 and 8.73 µM, respectively. In addition, UNA showed potent inhibitory activity against α-glucosidase (IC50 = 61.85 µM). Kinetic analysis of PTP1B and α-glucosidase inhibition revealed that UNA was a non-competitive inhibitor of both enzymes. Docking simulations of UNA demonstrated negative binding energies and close proximity to residues in the binding pockets of PTP1B and α-glucosidase. Molecular docking simulations between UNA and human serum albumin (HSA) revealed that UNA binds tightly to all three domains of HSA. Furthermore, UNA significantly inhibited fluorescent AGE formation (IC50 = 4.16 µM) in a glucose-fructose-induced HSA glycation model over the course of four weeks. Additionally, we investigated the molecular mechanisms underlying the anti-diabetic effects of UNA in insulin-resistant C2C12 skeletal muscle cells and discovered that UNA significantly increased glucose uptake and decreased PTP1B expression. Further, UNA increased GLUT-4 expression level by activating the IRS-1/PI3K/Akt/GSK-3 signaling pathway. These findings clearly demonstrate that UNA from A. montana shows great potential for treatment of diabetes and its complications.

Bromophenols from Symphyocladia latiuscula (Harvey) Yamada as Novel Cholecystokinin 2 Receptor Antagonists.

BACKGROUND: Cholecystokinin (CCK) is one of the most abundant peptides in the central nervous system and is believed to function as a neurotransmitter as well as a gut hormone with an inverse correlation of its level to anxiety and depression. Therefore, CCK receptors (CCKRs) could be a relevant target for novel antidepressant therapy. METHODS: In silico target prediction was first employed to predict the probability of the bromophenols interacting with key protein targets based on a model trained on known bioactivity data and chemical similarity considerations. Next, we tested the functional effect of natural bromophenols from Symphyocladia latiuscula on the CCK2 receptor followed by a molecular docking simulation to predict interactions between a compound and the binding site of the target protein. RESULTS: Results of cell-based functional G-protein coupled receptor (GPCR) assays demonstrate that bromophenols 2,3,6-tribromo-4,5-dihydroxybenzyl alcohol (1), 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (2), and bis-(2,3,6-tribromo-4,5-dihydroxybenzyl) ether (3) are full CCK2 antagonists. Molecular docking simulation of 1‒3 with CCK2 demonstrated strong binding by means of interaction with prime interacting residues: Arg356, Asn353, Val349, His376, Phe227, and Pro210. Simulation results predicted good binding scores and interactions with prime residues, such as the reference antagonist YM022. CONCLUSIONS: The results of this study suggest bromophenols 1-3 are CCK2R antagonists that could be novel therapeutic agents for CCK2R-related diseases, especially anxiety and depression.

Antioxidant and Antineuroinflammatory Mechanisms of Kaempferol-3-O-ß-d-Glucuronate on Lipopolysaccharide-Stimulated BV2 Microglial Cells through the Nrf2/HO-1 Signaling Cascade and MAPK/NF-κB Pathway.

Aglycone- and glycoside-derived forms of flavonoids exist broadly in plants and foods such as fruits, vegetables, and peanuts. However, most studies focus on the bioavailability of flavonoid aglycone rather than its glycosylated form. Kaempferol-3-O-ß-d-glucuronate (K3G) is a natural flavonoid glycoside obtained from various plants that have several biological activities, including antioxidant and anti-inflammatory effects. However, the molecular mechanism related to the antioxidant and antineuroinflammatory activity of K3G has not yet been demonstrated. The present study was designed to demonstrate the antioxidant and antineuroinflammatory effect of K3G against lipopolysaccharide (LPS)-stimulated BV2 microglial cells and to evaluate the underlying mechanism. Cell viability was determined by MTT assay. The inhibition rate of reactive oxygen species (ROS) and the production of pro-inflammatory mediators and cytokines were measured by DCF-DA assay, Griess assay, enzyme-linked immunosorbent assay (ELISA), and western blotting. K3G inhibited the LPS-induced release of nitric oxide, interleukin (IL)-6, and tumor necrosis factor-α (TNF-α) as well as the expression of prostaglandin E synthase 2. Additionally, K3G reduced the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and nuclear factor-kappa B (NF-κB) related proteins. Mechanistic studies found that K3G downregulated phosphorylated mitogen-activated protein kinases (MAPKs) and upregulated the Nrf2/HO-1 signaling cascade. In this study, we demonstrated the effects of K3G on antineuroinflammation by inactivating phosphorylation of MPAKs and on antioxidants by upregulating the Nrf2/HO-1 signaling pathway through decreasing ROS in LPS-stimulated BV2 cells.

Green perilla leaf extract ameliorates long-term oxidative stress induced by a high-fat diet in aging mice.

BACKGROUND/OBJECTIVES: Oxidative stress is caused by an imbalance between harmful free radicals and antioxidants. Long-term oxidative stress can lead to an "exhausted" status of antioxidant defense system triggering development of metabolic syndrome and chronic inflammation. Green perilla (Perilla frutescens) is commonly used in Asian cuisines and traditional medicine in southeast Asia. Green perilla possesses numerous beneficial effects including anti-inflammatory and antioxidant functions. To investigate the potentials of green perilla leaf extract (PE) on oxidative stress, we induced oxidative stress by high-fat diet (HFD) in aging mice. MATERIALS/METHODS: C57BL/6J male mice were fed HFD continuously for 53 weeks. Then, mice were divided into three groups for 12 weeks: a normal diet fed reference group (NDcon), high-fat diet fed group (HDcon), and high-fat diet PE treated group (HDPE, 400 mg/kg of body weight). Biochemical analyses of serum and liver tissues were performed to assess metabolic and inflammatory damage and oxidative status. Hepatic gene expression of oxidative stress and inflammation related enzymes were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: PE improved hepatopathology. PE also improved the lipid profiles and antioxidant enzymes, including hepatic glutathione peroxidase (GPx) and superoxide dismutase (SOD) and catalase (CAT) in serum and liver. Hepatic gene expressions of antioxidant and anti-inflammatory related enzymes, such as SOD-1, CAT, interleukin 4 (IL-4) and nuclear factor erythroid 2-related factor (Nrf2) were significantly enhanced by PE. PE also reduced the levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA) in the serum and liver; moreover, PE suppressed hepatic gene expression involved in pro-inflammatory response; Cyclooxygenase-2 (COX-2), nitric oxide synthase (NOS), interleukin 1 beta (IL-1ß), and interleukin 6 (IL-6). CONCLUSIONS: This research opens opportunities for further investigations of PE as a functional food and possible anti-aging agent due to its attenuative effects against oxidative stress, resulting from HFD and aging in the future.

Phytoestrogen Coumestrol Selectively Inhibits Monoamine Oxidase-A and Amyloid ß Self-Aggregation.

Pueraria lobata leaves contain a variety of phytoestrogens, including flavonoids, isoflavonoids, and coumestan derivatives. In this study, we aimed to identify the active ingredients of P. lobata leaves and to elucidate their function in monoamine oxidase (MAO) activation and Aß self-aggregation using in vitro and in silico approaches. To the best of our knowledge, this is the first study to elucidate coumestrol as a selective and competitive MAO-A inhibitor. We identified that coumestrol, a coumestan-derivative, exhibited a selective inhibitory effect against MAO-A (IC50 = 1.99 ± 0.68 µM), a key target protein for depression. In a kinetics analysis with 0.5 µg MAO-A, 40-160 µM substrate, and 25 °C reaction conditions, coumestrol acts as a competitive MAO-A inhibitor with an inhibition constant of 1.32 µM. During an in silico molecular docking analysis, coumestrol formed hydrogen bonds with FAD and pi-pi bonds with hydrophobic residues at the active site of the enzyme. Moreover, based on thioflavin-T-based fluorometric assays, we elucidated that coumestrol effectively prevented self-aggregation of amyloid beta (Aß), which induces an inflammatory response in the central nervous system (CNS) and is a major cause of Alzheimer's disease (AD). Therefore, coumestrol could be used as a CNS drug to prevent diseases such as depression and AD by the inhibition of MAO-A and Aß self-aggregation.

6-Formyl Umbelliferone, a Furanocoumarin from Angelica decursiva L., Inhibits Key Diabetes-Related Enzymes and Advanced Glycation End-Product Formation.

Over the years, great attention has been paid to coumarin derivatives, a set of versatile molecules that exhibit a wide variety of biological activities and have few toxic side effects. In this study, we investigated the antidiabetic potential of 6-formyl umbelliferone (6-FU), a novel furanocoumarin isolated from Angelica decursiva. Numerous pharmacological activities of 6-FU have been previously reported; however, the mechanism of its antidiabetic activity is unknown. Therefore, we examined the action of 6-FU on a few candidate-signaling molecules that may underlie its antidiabetic activity, including its inhibition of protein tyrosine phosphatase 1B (PTP1B), α-glucosidase, human recombinant aldose reductase (HRAR), and advanced glycation end-product (AGE) formation (IC50 = 1.13 ± 0.12, 58.36 ± 1.02, 5.11 ± 0.21, and 2.15 ± 0.13 µM, respectively). A kinetic study showed that 6-FU exhibited mixed-type inhibition against α-glucosidase and HRAR and competitive inhibition of PTP1B. Docking simulations of 6-FU demonstrated negative binding energies and close proximity to residues in the binding pockets of those enzymes. We also investigated the molecular mechanisms underlying 6-FU's antidiabetic effects. 6-FU significantly increased glucose uptake and decreased PTP1B expression in insulin-resistant C2C12 skeletal muscle cells. Moreover, 6-FU (0.8-100 µM) remarkably inhibited the formation of fluorescent AGEs in glucose-fructose-induced human serum albumin glycation over the course of 4 weeks. The findings clearly indicate that 6-FU will be useful in the development of multiple target-oriented therapeutic modalities for the treatment of diabetes and diabetes-related complications.

Inhibition Mechanism of Components Isolated from Morus alba Branches on Diabetes and Diabetic Complications via Experimental and Molecular Docking Analyses.

Previously, we reported the anti-diabetic effect of Morus alba root bark and the compounds therein. In our continuous study of other parts of this plant, the ability of the branch of Morus alba to inhibit α-glucosidase, protein tyrosine phosphatase 1B (PTP1B), and advanced glycation end products (AGEs) formation was evaluated. Moreover, there are no previous studies that have performed enzyme kinetics and molecular docking analyses, along with assessments of peroxynitrite (ONOO-) inhibitory activities. Since the Morus alba branch exhibited favorable inhibitory effects, repeated column chromatography was performed to obtain eight compounds, including four flavonoids (1, 3, 6, 8), one arylbenzofuran (2), one stilbene (5), one Diels-Alder-type adduct (7), and one sterol (4). Among them, compounds 1-3 and 5-7 were mixed-type inhibitors of α-glucosidase, sharing the same catalytic residues with acarbose and the same allosteric sites with (Z)-3-bytylidenephthalide. On the other hand, kuwanon C (1) and oxyresveratrol (5) interacted with residues of the allosteric site (α3 and α6 helices) of PTP1B, indicating their use as non-competitive inhibitors. Interestingly, kuwanon G (7) directly bound the catalytic site, or interrupted the binding between the substrate and the active site, as a mixed-type inhibitor. Moreover, most of the compounds exhibited greater activity against AGE formation and ONOO- than positive controls. The IC50 values required to inhibit ONOO- using compounds 1, 3, 5, 6, and 7 were reported for the first time, and range from 1.08 to 12.92 µM. Based on the structure-activity relationship, the presence of hydroxyl, resorcinol, and prenyl moieties was important in the prevention of diabetes' pathological mechanisms, and these findings have been further supported by molecular docking analysis. These computational and experimental results will be useful in the development of therapeutic candidates to prevent/treat diabetes and its complications.

In Vitro and In Silico Characterization of Kurarinone as a Dopamine D1A Receptor Antagonist and D2L and D4 Receptor Agonist.

Alterations in the expression and/or activity of brain G-protein-coupled receptors (GPCRs) such as dopamine D1R, D2LR, D3R, and D4R, vasopressin V1AR, and serotonin 5-HT1AR are noted in various neurodegenerative diseases (NDDs). Since studies have indicated that flavonoids can target brain GPCRs and provide neuroprotection via inhibition of monoamine oxidases (hMAOs), our study explored the functional role of kurarinone, an abundant lavandulated flavonoid in Sophora flavescens, on dopamine receptor subtypes, V1AR, 5-HT1AR, and hMAOs. Radioligand binding assays revealed considerable binding of kurarinone on D1R, D2LR, and D4R. Functional GPCR assays unfolded the compound's antagonist behavior on D1R (IC50 42.1 ± 0.35 µM) and agonist effect on D2LR and D4R (EC50 22.4 ± 3.46 and 71.3 ± 4.94 µM, respectively). Kurarinone was found to inhibit hMAO isoenzymes in a modest and nonspecific manner. Molecular docking displayed low binding energies during the intermolecular interactions of kurarinone with the key residues of the deep orthosteric binding pocket and the extracellular loops of D1R, D2LR, and D4R, validating substantial binding affinities to these prime targets. With appreciable D2LR and D4R agonism and D1R antagonism, kurarinone might be a potential compound that can alleviate clinical symptoms of Parkinson's disease and other NDDs.

Isoliquiritigenin, a potent human monoamine oxidase inhibitor, modulates dopamine D1, D3, and vasopressin V1A receptors.

Isoliquiritigenin (= 4,2',4'-Trihydroxychalcone) (ILG) is a major constituent of the Glycyrrhizae Rhizoma that has significant neuroprotective functions. In the present study, we re-examined the potential of ILG to inhibit human monoamine oxidase (hMAO) in vitro and established its mechanism of inhibition through a kinetics study and molecular docking examination. ILG showed competitive inhibition of hMAO-A and mixed inhibition of hMAO-B with IC50 values of 0.68 and 0.33 µM, respectively, which varied slightly from the reported IC50 values. Since ILG has been reported to reduce dopaminergic neurodegeneration and psychostimulant-induced toxicity (both of which are related to dopamine and vasopressin receptors), we investigated the binding affinity and modulatory functions of ILG on dopamine and vasopressin receptors. ILG was explored as an antagonist of the D1 receptor and an agonist of the D3 and V1A receptors with good potency. An in silico docking investigation revealed that ILG can interact with active site residues at target receptors with low binding energies. These activities of ILG on hMAO and brain receptors suggest the potential role of the compound to ameliorate dopaminergic deficits, depression, anxiety, and associated symptoms in Parkinson's disease and other neuronal disorders.

Structural Bases for Hesperetin Derivatives: Inhibition of Protein Tyrosine Phosphatase 1B, Kinetics Mechanism and Molecular Docking Study.

In the present study, we investigated the structure-activity relationship of naturally occurring hesperetin derivatives, as well as the effects of their glycosylation on the inhibition of diabetes-related enzyme systems, protein tyrosine phosphatase 1B (PTP1B) and α-glycosidase. Among the tested hesperetin derivatives, hesperetin 5-O-glucoside, a single-glucose-containing flavanone glycoside, significantly inhibited PTP1B with an IC50 value of 37.14 ± 0.07 µM. Hesperetin, which lacks a sugar molecule, was the weakest inhibitor compared to the reference compound, ursolic acid (IC50 = 9.65 ± 0.01 µM). The most active flavanone hesperetin 5-O-glucoside suggested that the position of a sugar moiety at the C-5-position influences the PTP1B inhibition. It was observed that the ability to inhibit PTP1B is dependent on the nature, position, and number of sugar moieties in the flavonoid structure, as well as conjugation. In the kinetic study of PTP1B enzyme inhibition, hesperetin 5-O-glucoside led to mixed-type inhibition. Molecular docking studies revealed that hesperetin 5-O-glucoside had a higher binding affinity with key amino residues, suggesting that this molecule best fits the PTP1B allosteric site cavity. The data reported here support hesperetin 5-O-glucoside as a hit for the design of more potent and selective inhibitors against PTP1B in the search for a new anti-diabetic treatment.

Monoamine Oxidase Inhibition by Major Tanshinones from Salvia miltiorrhiza and Selective Muscarinic Acetylcholine M4 Receptor Antagonism by Tanshinone I.

Monoamine oxidases (MAOs) and muscarinic acetylcholine receptors (mAChRs) are considered important therapeutic targets for Parkinson's disease (PD). Lipophilic tanshinones are major phytoconstituents in the dried roots of Salvia miltiorrhiza that have demonstrated neuroprotective effects against dopaminergic neurotoxins and the inhibition of MAO-A. Since MAO-B inhibition is considered an effective therapeutic strategy for PD, we tested the inhibitory activities of three abundant tanshinone congeners against recombinant human MAO (hMAO) isoenzymes through in vitro experiments. In our study, tanshinone I (1) exhibited the highest potency against hMAO-A, followed by tanshinone IIA and cryptotanshinone, with an IC50 less than 10 µM. They also suppressed hMAO-B activity, with an IC50 below 25 µM. Although tanshinones are known to inhibit hMAO-A, their enzyme inhibition mechanism and binding sites have yet to be investigated. Enzyme kinetics and molecular docking studies have revealed the mode of inhibition and interactions of tanshinones during enzyme inhibition. Proteochemometric modeling predicted mAChRs as possible pharmacological targets of 1, and in vitro functional assays confirmed the selective M4 antagonist nature of 1 (56.1% ± 2.40% inhibition of control agonist response at 100 µM). These findings indicate that 1 is a potential therapeutic molecule for managing the motor dysfunction and depression associated with PD.

In Vitro and In Silico Characterization of G-Protein Coupled Receptor (GPCR) Targets of Phlorofucofuroeckol-A and Dieckol.

Phlorotannins are polyphenolic compounds in marine alga, especially the brown algae. Among numerous phlorotannins, dieckol and phlorofucofuroeckol-A (PFF-A) are the major ones and despite a wider biological activity profile, knowledge of the G protein-coupled receptor (GPCR) targets of these phlorotannins is lacking. This study explores prime GPCR targets of the two phlorotannins. In silico proteocheminformatics modeling predicted twenty major protein targets and in vitro functional assays showed a good agonist effect at the α2C adrenergic receptor (α2CAR) and an antagonist effect at the adenosine 2A receptor (A2AR), δ-opioid receptor (δ-OPR), glucagon-like peptide-1 receptor (GLP-1R), and 5-hydroxytryptamine 1A receptor (5-TH1AR) of both phlorotannins. Besides, dieckol showed an antagonist effect at the vasopressin 1A receptor (V1AR) and PFF-A showed a promising agonist effect at the cannabinoid 1 receptor and an antagonist effect at V1AR. In silico molecular docking simulation enabled us to investigate and identify distinct binding features of these phlorotannins to the target proteins. The docking results suggested that dieckol and PFF-A bind to the crystal structures of the proteins with good affinity involving key interacting amino acid residues comparable to reference ligands. Overall, the present study suggests α2CAR, A2AR, δ-OPR, GLP-1R, 5-TH1AR, CB1R, and V1AR as prime receptor targets of dieckol and PFF-A.

Neuroprotective Effect of Aurantio-Obtusin, a Putative Vasopressin V1A Receptor Antagonist, on Transient Forebrain Ischemia Mice Model.

Traditional Chinese medicines (TCMs) have been a rich source of novel drug discovery, and Cassia seed is one of the common TCMs with numerous biological effects. Based on the existing reports on neuroprotection by Cassia seed extract, the present study aims to search possible pharmacological targets behind the neuroprotective effects of the Cassia seeds by evaluating the functional effect of specific Cassia compounds on various G-protein-coupled receptors. Among the four test compounds (cassiaside, rubrofusarin gentiobioside, aurantio-obtusin, and 2-hydroxyemodin 1-methylether), only aurantio-obtusin demonstrated a specific V1AR antagonist effect (71.80 ± 6.0% inhibition at 100 µM) and yielded an IC50 value of 67.70 ± 2.41 µM. A molecular docking study predicted an additional interaction of the hydroxyl group at C6 and a methoxy group at C7 of aurantio-obtusin with the Ser341 residue as functional for the observed antagonist effect. In the transient brain ischemia/reperfusion injury C57BL/6 mice model, aurantio-obtusin attenuated the latency time that was reduced in the bilateral common carotid artery occlusion (BCCAO) groups. Likewise, compared to neuronal damage in the BCCAO groups, treatment with aurantio-obtusin (10 mg/kg, p.o.) significantly reduced the severity of damage in medial cornu ammonis 1 (mCA1), dorsal CA1, and cortex regions. Overall, the findings of this study highlight V1AR as a possible target of aurantio-obtusin for neuroprotection.

Discovery of Flazin, an Alkaloid Isolated from Cherry Tomato Juice, As a Novel Non-Enzymatic Protein Glycation Inhibitor via in Vitro and in Silico Studies.

Both overproduced reactive oxygen species/reactive nitrogen species and hyperglycemic conditions accompany a significant increase in protein glycation and nitration that contribute to the initiation and progression of diabetic complications and neuronal disorders. In this study, 19 compounds, including steroidal saponins, alkaloids, cerebroside, phenolic compounds, sterols, and nucleosides, were isolated from cherry tomato (Solanum lycopersicum var. cerasiforme) juice, of which flazin showed good inhibition on monosaccharide-induced non-enzymatic bovine pancreas insulin and bovine serum albumin (BSA) glycation. Molecular dynamics simulations revealed that flazin continuously interacts with Phe1, Val2, Tyr26, and Lys29 insulin residues, which play a key role in insulin glycation/dimerization. In addition, depending upon the flazin dose, this blocked the tyrosine nitration of BSA via scavenging peroxynitrite anions. Taken together, our novel findings suggest that flazin could be a lead compound for the treatment of diabetes and neuronal disorders via the inhibition of non-enzymatic protein glycation and the elimination of peroxynitrite.