New insight on porcine carboxylesterases expression and activity in lung tissues.

Over the course of the last twenty years, there has been a growing recognition of the pig's potential as a valuable model for studying human drug metabolism. This study aimed to investigate the expression, enzymatic activity, inhibitory susceptibility, and cellular localization of carboxylesterases (CES) in porcine lung tissue not yet explored. Our results showed that CESs hydrolysis activity followed Michaelis-Menten kinetics in both cytosolic and microsomal fractions of porcine lung tissues (N = 8), with comparable hydrolysis rates for tested substrates, namely 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD). We also determined the CESs hydrolysis activity in a representative sample of the porcine liver that, as expected, displayed higher activity than the lung ones. The study demonstrated variable levels of enzyme activities and interindividual variability in both porcine lung fractions. Inhibition studies used to assess the CESs' involvement in the hydrolysis of pNPA, 4-MUA, and FD suggested that CESs may be the enzymes primarily involved in the metabolism of ester compounds in the pig lung tissue. Overall, this study provides insight into the distribution and diversity of CES isoforms involved in substrate hydrolysis across different cellular fractions (cytosol and microsomes) in porcine lungs.

Investigating the promising SARS-CoV-2 main protease inhibitory activity of secoiridoids isolated from Jasminum humile; in silico and in Vitro assessments with structure-activity relationship.

The proteolytic enzyme 3 C-like protease (3Clpro or Mpro) is considered the most important target for SARS-CoV-2 which could be attributed to its crucial role in viral maturation and/or replication. Besides, natural phytoconstituents from plant origin are always promising lead compounds in the drug discovery area. Herein, the previously isolated and identified seven compounds from Jasminum humile (J. humile) were examined in vitro and in silico against the SARS-CoV-2 Mpro. First, the Vero E6 cells were utilized to pursue the potential of the investigated compounds (both in fractions and individual isolates) using the MTT assay. The total extract (T1) displayed the most significant activity against SARS-CoV-2 with IC50 = 29.36 µg/mL. Besides, the fractions (Fr1 and Fr3) showed good activity against the SARS-CoV-2 with IC50 values of 70.42, and 73.09 µg/mL, respectively. Then, the SARS-CoV-2 Mpro inhibitory assay was utilized to emphasize the inhibitory potential of the investigated isolates. MJN, JMD, and IJM candidates displayed prominent Mpro inhibitory potentials with IC50 = 30.44, 30.24, and 56.25 µM, respectively. Moreover, molecular docking of the identified seven compounds against the Mpro of SARS-CoV-2 showed that the five secoiridoids achieved superior results. MJN, JSM, IJM, and JMD showed higher affinities towards the Mpro target compared to the co-crystallized antagonist. Furthermore, the most active complexes (MJN, JSM, IJM, and JMD-Mpro) were subjected to MD simulations run for 150 ns and MM-GBSA calculations, compared to the co-crystallized inhibitor (O6K-Mpro). Finally, the SAR study clarified that JMD achieved the best anti-SARS-CoV-2 Mpro activity followed by MJN.Communicated by Ramaswamy H. Sarma.

HIGHLIGHTSSeven isolates from J. humile, besides different extracts, were examined both in vitro and in silico.Anti-SARS-CoV-2 using the MTT assay and anti-SARS-CoV-2 Mpro inhibitory assay were performed.Compounds MJN, JMD, and IJM displayed prominent SARS-CoV-2 Mpro inhibition.Molecular docking, molecular dynamics simulations, and MM-GBSA calculations were carried out.SAR study was conducted on the isolated compounds.

Andrographolide and Deoxyandrographolide Inhibit Protease and IFN-Antagonist Activities of Foot-and-Mouth Disease Virus 3Cpro.

Foot-and mouth-disease (FMD) caused by the FMD virus (FMDV) is highly contagious and negatively affects livestock worldwide. The control of the disease requires a combination of measures, including vaccination; however, there is no specific treatment available. Several studies have shown that plant-derived products with antiviral properties were effective on viral diseases. Herein, antiviral activities of andrographolide (AGL), deoxyandrographolide (DAG), and neoandrographolide (NEO) against FMDV serotype A were investigated using an in vitro cell-based assay. The results showed that AGL and DAG inhibited FMDV in BHK-21 cells. The inhibitory effects of AGL and DAG were evaluated by RT-qPCR and exhibited EC50 values of 52.18 ± 0.01 µM (SI = 2.23) and 36.47 ± 0.07 µM (SI = 9.22), respectively. The intracellular protease assay revealed that AGL and DAG inhibited FMDV 3Cpro with IC50 of 67.43 ± 0.81 and 25.58 ± 1.41 µM, respectively. Additionally, AGL and DAG significantly interfered with interferon (IFN) antagonist activity of the 3Cpro by derepressing interferon-stimulating gene (ISGs) expression. The molecular docking confirmed that the andrographolides preferentially interacted with the 3Cpro active site. However, NEO had no antiviral effect in any of the assays. Conclusively, AGL and DAG inhibited FMDV serotype A by interacting with the 3Cpro and hindered its protease and IFN antagonist activities.

Design, synthesis, and SAR studies of novel 4-methoxyphenyl pyrazole and pyrimidine derivatives as potential dual tyrosine kinase inhibitors targeting both EGFR and VEGFR-2.

Guided by the pharmacophoric features of both EGFR and VEGFR-2 antagonists, two novel series of 4-methoxyphenyl pyrazole and pyrimidine derivatives [(4a-c) and (5a-c, 6, 7a-c, 8, 9, 10, 11a,c, 12, 13a-c, 14a-c, and 15a,b)], respectively, were designed and synthesized as dual EGFR/VEGFR-2 inhibitors. Interestingly, compound 12 showed very strong antiproliferative effects towards all the five studied cell lines (HepG-2, MCF-7, MDA-231, HCT-116, and Caco-2) with IC50 values of 3.74, 7.81, 4.85, 2.96, and 9.27 µM, respectively. Also, it achieved the highest inhibitory activities against both EGFR and VEGFR-2 as well (IC50 = 0.071 and 0.098 µM) compared to the two reference drugs, erlotinib (IC50 = 0.063 µM) and sorafenib (IC50 = 0.041 µM), respectively. Moreover, four compounds (4a, 7a, 7c, and 12) were selected for further evaluation through cell cycle analysis and Annexin V-based flow cytometry assay in the HepG-2 cell line. In addition, deep computational studies including molecular docking, physicochemical properties, profiling pharmacokinetics, ADMET studies, and toxicity predictions were performed for the designed compounds to evaluate the prospective drug candidates. Finally, analyzing the structure-activity relationship (SAR) of the new derivatives gives us a lot of interesting promising results which could help medicinal chemists to design more potent drug candidates soon as well.

Characterization and Optimization of the Tyrosinase Inhibitory Activity of Vitis amurensis Root Using LC-Q-TOF-MS Coupled with a Bioassay and Response Surface Methodology.

Vitis amurensis roots have been reported to have the potential for skin whitening through the evaluation of melanogenesis and tyrosinase inhibitory activities. In this study, V. amurensis roots were utilized to quickly select whitening ingredients using LC-Q-TOF-MS coupled with tyrosinase inhibitory assay, and to optimize the extraction process for use as a skin whitening functional material by response surface methodology. Results showed that V. amurensis roots exhibited tyrosinase inhibitory effects by two stilbene oligomers, ε-viniferin (1) and vitisin B (2), as predicted by LC-Q-TOF-MS coupled with bioassay. The optimal extraction conditions (methanol concentration 66%, solvent volume 140 mL, and extraction time 100 min) for skin whitening ingredients were established with the yields 6.20%, and tyrosinase inhibitory activity was 87.27%. The relationship between each factor and its corresponding response was confirmed by Pearson's correlation analysis. The solvent volume showed clear linear relationship with yields, and methanol concentration had a strong linear relationship with tyrosinase inhibitory activity for compounds 1 and 2, as well as their combination. Overall, LC-Q-TOF-MS coupled with bioassay was proved to have the potential to effectively find new active constituents, as well as known active constituents; vitisin B can be proposed as a new natural potential whitening agent.

Phytosterol supplements do not inhibit dipeptidyl peptidase-4.

BACKGROUND AND AIMS: Several commercially available phytosterol supplements are promoted for their cholesterol-lowering effects. However, limited information is available about their potential anti-hyperglycaemic effects. This study aimed to evaluate the dipeptidyl peptidase-4 (DPP-4) inhibitory effects of phytosterol supplements in silico and in vitro to determine their potential for anti-diabetic activity. METHODS: Docking studies were carried out in silico to evaluate the potential for interactions between three major phytosterol compounds (stigmasterol, ß-sitosterol, campesterol) and the DPP-4 enzyme, the enzyme that is inhibited by the anti-diabetic gliptins. Gas chromatography-tandem mass spectrometry (GC-MS/MS) was used to analyse three different supplements for phytosterol content. DPP-4 inhibitory activity was tested in vitro for these phytosterol supplements and two major phytosterol standards. RESULTS: In silico calculations predicted free binding energies for DPP-4 with the phytosterols to be: stigmasterol -8.78 kcal/mol; ß-sitosterol -8.70 kcal/mol; campesterol -8.40 kcal/mol. These binding energies indicated a potential for significant DPP-4 inhibition. However, these results were not supported by the in vitro studies. Stigmasterol and ß-sitosterol had an IC50 > 50 mg/ml (maximum tested concentration) and the Thompson's Cholesterol Manager® and Mega Strength Beta Sitosterol® supplements gave an IC50 > 100 mg/ml (maximum tested concentration). Blackmores Cholesterol Health® gave an IC50 value of 40 mg/ml which was attributed to ß-carotene content. CONCLUSIONS: Phytosterol supplements do not appear to offer any anti-diabetic activity potential via pathways that involve the inhibition of DPP-4.

Antidiabetic Activity of an Ayurvedic Formulation Chaturmukha Rasa: A Mechanism Based Study.

OBJECTIVES: The objective of this study was to evaluate antidiabetic activity of Chaturmukha rasa based on streptozotocin induced diabetes model, alpha amylase inhibitory activity, alpha Glucosidase inhibitory activity and inhibition of sucrase. METHODS: Chaturmukha rasa was prepared as per Ayurvedic formulary. Antidiabetic activity was measured in experimentally streptozotocin induced rats. The dose was taken as 45 mg/kg, i.p. The antidiabetic activity of Chaturmukha rasa was compared Triphala Kwatha, a marketed formulation. Further In vitro ά-Amylase Inhibitory Assay, In vitro salivary amylase Inhibitory Assay, In vitro α-Glucosidase Inhibitory Assay and In vitro Sucrase Inhibitory Assay was performed with respect to Chaturmukha rasa. The IC50 value was calculated for all the above activity. RESULTS: Streptozotocin with Acarbose showed significant decrease in blood glucose level whereas streptozotocin with Triphala kwatha showed more decrease in blood glucose level than Streptozotocin with Acarbose. The combination of Streptozotocin + Triphala kwatha + Chaturmukha rasa showed a significant decrease in blood glucose level on 21st day. In vitro ά-Amylase Inhibitory Assay the Chaturmukha rasa showed IC50 value 495.94 µl when compared with Acarbose 427.33 µl, respectively. In the α-Glucosidase Inhibitory Assay Chaturmukha rasa showed IC50 value 70.93 µl when compared with Acarbose 102.28 µl, respectively. In vitro Sucrase Inhibitory Assay Chaturmukha rasa showed IC50 value 415.4 µl when compared with Acarbose 371.43 µl, respectively. CONCLUSION: This study supports that Chaturmukha rasa may inhibit diabetes by inhibition of salivary amylase or alpha Glucosidase or sucrase. This may be the mechanism by which Chaturmukha rasa inhibits diabetes. Further this study supports the usage of Chaturmukha rasa for the management of diabetes.

Novel indolizine derivatives lowers blood glucose levels in streptozotocin-induced diabetic rats: A histopathological approach.

BACKGROUND: Diabetes mellitus is a deadly disorder in human which induce chronic complications. The streptozotocin (STZ)-induced diabetes in rat is the most common animal model of human diabetes. The present study investigated the effects of novel indolizine derivatives (1-16) on plasma blood glucose concentrations in STZ-diabetic rats. METHODS: In vitro experiments were performed on 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide free radicals, α-glucosidase enzyme and in vivo studies on normal, oral glucose loaded and STZ-induced diabetic rats. RESULTS: Among all synthetic derivatives, compound 12 showed good inhibitory profile against DPPH, superoxide free radicals and α-glucosidase enzyme with half maximal inhibitory concentration (IC50) values of 56.2, 33.5 and 26.5 µg/mL, respectively. The lethal dosage of indolizine derivatives was found to be above 1000 mg/kg body weight (b.w.). From the in vivo studies, it can be determined that the compound 12 depicted pronounced protective hypoglycemic effects in normal, glucose-loaded and STZ-induced diabetic rats with respect to the standard. Furthermore, 21 days of successive treatment with compound 12 in diabetic rats exhibited better recovery of body weight and considerable variations in biochemical parameters as that of the standard drug. Moreover, the histopathological section of pancreas and testes justifies the rehabilitation and regeneration of islets, acini and Sertoli cells in animals treated with compound 12. CONCLUSION: Our data suggest that the indolizine derivatives can be a benchmarks for designing potent oral antidiabetic agents.

Citrus bioflavonoids dipeptidyl peptidase-4 inhibition compared with gliptin antidiabetic medications.

This study compared dipeptidyl peptidase-4 (DPP-4) inhibitory activity of citrus bioflavonoid nutraceuticals compared with three gliptins. Citrus bioflavonoid standards and three commercially available citrus bioflavonoid supplements (Thompson's Super Bioflavonoid Complex®(SB), Ethical Nutrients Bioflavonoids Plus Vitamin C®(EN), and Country Life Citrus Bioflavonoids and Rutin®(CB)) were considered in this study. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis was undertaken to identify and quantitate the citrus bioflavonoids present in each supplement. The DPP-4 inhibitory activity was determined by fluorometric assay. All of the tested individual citrus flavonoids demonstrated DPP-4 inhibitory activity, with IC50 values ranging from 485 µM (rutin) to 5700 µM (hesperitin and eriodictyol). Similarly, the flavonoid supplements had IC50 values of 16.9 mg/mL (EN), 3.44 mg/mL (SB) and 2.72 mg/mL (CB). These values compare with gliptin IC50 values of 0.684 µM (sitagliptin), 0.707 µM (saxagliptin) and 2.286 µM (vildagliptin). The supplement flavonoid content varied from 11.98% (CB) to 5.26% (EN) and 14.51% (SB) of tablet mass, corresponding to daily flavonoid doses of around 300, 150 and 400 mg, respectively, with CB and SB containing rutin at levels of 7.0% and 7.5% of tablet mass, respectively. While our data demonstrated that citrus bioflavonoid based supplements do possess DPP-4 inhibitory activity, they are several orders of magnitude less potent than gliptins. Further studies using higher concentrations of citrus bioflavonoids, as well as investigations into antioxidant properties which may add additional benefit are warranted.

Presence and inter-individual variability of carboxylesterases (CES1 and CES2) in human lung.

Lungs are pharmacologically active organs and the pulmonary drug metabolism is of interest for inhaled drugs design. Carboxylesterases (CESs) are enzymes catalyzing the hydrolysis of many structurally different ester, amide and carbamate chemicals, including prodrugs. For the first time, the presence, kinetics, inhibition and inter-individual variations of the major liver CES isozymes (CES1 and CES2) were investigated in cytosol and microsomes of human lungs from 20 individuals using 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD) as substrates the rates of hydrolysis (Vmax) for pNPA and 4-MUA, unlike FD, were double in microsomes than in cytosol. In these cellular fractions, the Vmax of pNPA, as CES1 marker, were much greater (30-50-fold) than those of FD, as a specific CES2 marker. Conversely, the Km values were comparable suggesting the involvement of the same enzymes. Inhibition studies revealed that the FD hydrolysis was inhibited by bis-p-nitrophenylphosphate, phenylmethanesulfonyl fluoride, and loperamide (specific for CES2), whereas the pNPA and 4-MUA hydrolysis inhibition was limited. Inhibitors selective for other esterases missed having any effect on above-mentioned activities. In cytosol and microsomes of 20 lung samples, inter-individual variations were found for the hydrolysis of pNPA (2.5-5-fold), FD or 4-MUA (8-15-fold). Similar variations were also observed in CES1 and CES2 gene expression, although determined in a small number (n = 9) of lung samples. The identification of CES1 and CES2 and their variability in human lungs are important for drug metabolism and design of prodrugs which need to be activated in this organ.

Chiral resolution, absolute configuration, and bioactivity of a new racemic asarone derivative from the rhizome of Acorus tatarinowii.

A new asarone-derived racemate (1) was isolated from the rhizome of Acorus tatarinowii. The structure of 1 was established by comprehensive spectroscopic analyses, and it was successfully resolved by chiral HPLC, demonstrating that it is racemic. The absolute configurations of 1a [(-)-acortatarone A] and 1b [(+)-acortatarone A] were determined using quantum chemical calculations. Compounds 1a and 1b were the first cases of asarone derivatives with the 5,7-dialkyl-6-aryl-8-oxabicyclo[3.2.1]oct-3-en-2-one core. The α-glucosidase inhibitory and acetylcholinesterase (AChE) inhibitory activities of 1 were evaluated, and it exhibited α-glucosidase inhibitory activity with potency close to that of the positive control (acarbose).

Chemical Constituents of Smilax china L. Stems and Their Inhibitory Activities against Glycation, Aldose Reductase, α-Glucosidase, and Lipase.

The search for natural inhibitors with anti-diabetes properties has gained increasing attention. Among four selected Smilacaceae family plants, Smilax china L. stems (SCS) showed significant in vitro anti-glycation and rat lens aldose reductase inhibitory activities. Bioactivity-guided isolation was performed with SCS and four solvent fractions were obtained, which in turn yielded 10 compounds, including one phenolic acid, three chlorogenic acids, four flavonoids, one stilbene, and one phenylpropanoid glycoside; their structures were elucidated using nuclear magnetic resonance and mass spectrometry. All solvent fractions, isolated compounds, and stem extracts from plants sourced from six different provinces of South Korea were next tested for their inhibitory effects against advanced glycation end products, as well as aldose reductase. α-Glucosidase, and lipase assays were also performed on the fractions and compounds. Since compounds 3, 4, 6, and 8 appeared to be the superior inhibitors among the tested compounds, a comparative study was performed via high-performance liquid chromatography with photodiode array detection using a self-developed analysis method to confirm the relationship between the quantity and bioactivity of the compounds in each extract. The findings of this study demonstrate the potent therapeutic efficacy of SCS and its potential use as a cost-effective natural alternative medicine against type 2 diabetes and its complications.

Combined Simulation and Mutation Studies to Elucidate Selectivity of Unsubstituted Amphetamine-like Cathinones at the Dopamine Transporter.

The dopamine and serotonin transporter proteins (DAT, SERT) play a vital role in behavior and mental illness. Although their substrate transport has been studied extensively, the molecular basis of their selectivity is not completely understood yet. In this study, we exploit molecular dynamics simulations combined with mutagenesis studies to shed light on the driving factors for DAT-over-SERT selectivity of a set of cathinones. Results indicate that these compounds can adopt two binding modes of which one is more favorable. In addition, free energy calculations indicated the substrate binding site (S1) as the primary recognition site for these ligands. By simulating DAT with SERT-like mutations, we hypothesize unsubstituted cathinones to bind more favorably to DAT, due to a Val152 offering more space, as compared to the bulkier Ile172 in SERT. This was supported by uptake inhibition measurements, which showed an increase in activity in SERT-I172V.

A new label-free screen for steroid 5α-reductase inhibitors using LC-MS.

Steroid 5α-reductase (S5αR) plays an important role in metabolizing testosterone into active androgen dihydrotestosterone (DHT) which is involved in many androgen dependent disorders, such as androgenic alopecia, benign prostatic hyperplasia and acne. The method for screening for S5αR inhibition is key in finding new antagonists. In this study, the label-free S5αR inhibitory assay using LC-MS was developed. S5αR type 1 enzyme was obtained from LNCaP prostate cancer cells. The enzymatic assay was optimised for enzyme-substrate (testosterone) concentration, NADPH-cofactor concentration, solvent tolerance, enzyme activity stability and incubation time. The developed assay was validated by measuring the signal to background ratio (S/B), the signal to noise ratio (S/N), the signal window (SW) and the zeta factor Z' in accordance with published bioassay guidelines. The enzymatic reaction was performed in 96-well plates and DHT formation was determined by LC-MS. S/B, S/N, SW and Z' factor were well above acceptable criteria and the reproducibility was good using Z' factor other 3days and further validated by dutasteride and finasteride inhibition. The method was successfully applied to quantify S5αR inhibitory activity of some Thai herbal extracts. Two plant extracts, Impatiens balsamina L. and Curcuma longa L. showed IC50 at 5.4±0.2 and 9.0±1.2µgmL-1 and are therefore promising sources of new S5αR inhibitors. The assay has high selectability and reproducibility and suited to medium throughput screening required by phytochemistry.