Molecular docking and inhibition studies of α-amylase activity by labdane diterpenes from Alpinia nigra seeds

Wide varieties of synthetic drugs are available for combating type 2 diabetes but are not free of associated side effects. Commercially available antidiabetic drugs are known to be potent inhibitors of α-amylase which reduce postprandial hyperglycaemia. Here, we have investigated Alpinia nigra seed extracts and the isolated two labdane diterpenes (I and II) for the α-amylase inhibitory activity. These labdane type diterpenes showed more promising inhibitory effects (IC₅₀ value 24.3 ± 2.05 and 15.167 ± 0.52 μM for compound I and II, respectively) against α-amylase than the standard inhibitor, acarbose. For both compounds, the mode of enzymatic inhibition was found to be non-competitive with K _i 13.303 ± 0.065 and 12.19 ± 0.099 μM, respectively. Molecular docking studies revealed that both I and II bind the human pancreatic α-amylase in the active site cleft similar to the acarbose. Among all the compounds under investigation, acarbose and compound II were found to have the highest MolDock and re-rank score. Further molecular dynamic simulation studies also supports the docking results obtained for both I and II. This is the first report on α-amylase inhibitory effect of the two labdane diterpenes with their potential candidature as future antidiabetic drugs of herbal origin.     

Interactions between dopamine D1 receptors and γ-aminobutyric acid mechanisms in substantia nigra pars reticulata of the rat: neurochemical and behavioral studies

RATIONALE: Several studies have shown that dopamine D1 agonists act on forebrain dopamine terminal regions to exert many of their behavioral effects. Yet, there is also a large number of D1 receptors in the substantia nigra pars reticulata (SNr), and these receptors are located mainly on terminals of gamma-aminobutyric acid (GABA)-ergic striatonigral neurons. OBJECTIVE: The present studies were undertaken to determine the behavioral and neurochemical effects of local administration of the D1 agonist SKF 82958 and to study the interactions between D1 and GABA mechanisms in SNr. METHODS: Microdialysis methods were used to characterize the effect of SKF 82958 on extracellular GABA, and several experiments studied the effects of nigral D1 stimulation on motor activity and investigated the behavioral significance of D1/GABA interactions in SNr. RESULTS: Local infusion of 10(-6) M SKF 82958 increased extracellular levels of SNr GABA, and this effect was blocked by co-infusion of the D1 antagonist SCH 23390. Bilateral SNr injections of SKF 82958 increased locomotor activity, and this effect was blocked by the GABA-A antagonist bicuculline. Intranigral bicuculline reduced motor activity, while the GABA-A agonist muscimol increased various motor activities in a manner similar to SKF 82958. CONCLUSIONS: The present results suggest that the D1 agonist SKF 82958 acts on D1 receptors in SNr to increase extracellular levels of GABA, and the increase in motor activity produced by nigral D1 stimulation is dependent on stimulation of GABA-A receptors. D1/GABA interactions in SNr are important for the modulation of basal ganglia output, which may have important implications for Parkinson's disease.     

Salvianolic acid B suppresses maturation of human monocyte-derived dendritic cells by activating PPARγ

BACKGROUND AND PURPOSE

Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a Chinese medicinal herb, is known to be effective in the prevention of atherosclerosis. Here, we tested the hypothesis that the anti-atherosclerotic effect of Sal B might be mediated by suppressing maturation of human monocyte-derived dendritic cells (h-monDC).

EXPERIMENTAL APPROACH

h-monDC were derived by incubating purified human monocytes with GM-CSF and IL-4. h-monDC were pre-incubated with or without Sal B and stimulated by oxidized low-density lipoprotein (ox-LDL) in the presence or absence of PPARγ siRNA. Expression of h-monDC membrane molecules (CD40, CD86, CD1a, HLA-DR) were analysed by FACS, cytokines were measured by elisa and the TLR4-associated signalling pathway was determined by Western blotting.

KEY RESULTS

Ox-LDL promoted h-monDC maturation, stimulated CD40, CD86, CD1a, HLA-DR expression and IL-12, IL-10, TNF-α production; and up-regulated TLR4 signalling. These effects were inhibited by Sal B. Sal B also triggered PPARγ activation and promoted PPARγ nuclear translocation, attenuated ox-LDL-induced up-regulation of TLR4 and myeloid differentiation primary-response protein 88 and inhibited the downstream p38-MAPK signalling cascade. Knocking down PPARγ with the corresponding siRNA blocked these effects of Sal B.

CONCLUSIONS AND IMPLICATIONS

Our data suggested that Sal B effectively suppressed maturation of h-monDC induced by ox-LDL through PPARγ activation.     

Differential inhibition of hepatic and duodenal sulfation of (−)-salbutamol and minoxidil by mefenamic acid

Objective: The aim of this investigation was to determine whether mefenamic acid and salicylic acid inhibit the sulfation of (−)-salbutamol and minoxidil in the human liver and duodenum, and if so, to ascertain whether the 50% inhibitory concentration (IC₅₀) estimates are different in the two tissues. Methods: Sulfotransferase activities were measured for 10 mM (−)-salbutamol and 5 mM minoxidil, and the concentration of 3′-phosphoadenosine-5′-phosphosulphate-[³⁵S] was 0.4 μM. Results: The IC₅₀ estimates for (−)-salbutamol and minoxidil sulfation of mefenamic acid were 72 ± 5.4 nM and 1.5 ± 0.6 μM (liver), respectively, and 161 ± 23 μM and 420 ± 18 μM (duodenum), respectively. The figures for the liver were significantly lower (P < 0.0001) than those for the duodenum. The IC₅₀ estimates for (−)-salbutamol sulfation of salicylic acid were 93 ± 11 μM (liver) and 705 ± 19 μM (duodenum, P < 0.0001). Salicylic acid was a poor inhibitor of minoxidil sulfation. Conclusion: The IC₅₀ estimates for (−)-salbutamol sulfation of mefenamic acid and salicylic acid are lower than their unbound plasma concentrations after standard dosing, suggesting that mefenamic acid and salicylic acid should inhibit the hepatic sulfation of (−)-salbutamol in vivo. Received: 11 November 1999 / Accepted in revised form: 28 April 2000     

Responses of rat substantia nigra dopamine-containing neurones to (–)-HA-966 in vitro

1. Extracellular single unit recording techniques were used to compare the effects of (-)-3-amino-1-hydroxypyrrolidin-2-one ((–)-HA-966) and (±)-baclofen on the activity of dopamine-containing neurones in 300 μm slices of rat substantia nigra. Electrophysiological data were compared with the outcome of in vitro binding experiments designed to assess the affinity of (–)-HA-966 for γ-aminobutyric acid (GABA_B) receptors.
2. Bath application of (–)-HA-966 produced a concentration-dependent inhibition of dopaminergic neuronal firing (EC₅₀=444.0 μM; 95% confidence interval: 277.6 μM–710.1 μM, n=27) which was fully reversible upon washout from the recording chamber. Although similar effects were observed in response to (±)-baclofen, the direct-acting GABA_B receptor agonist proved to be considerably more potent than (–)-HA-966 (EC₅₀=0.54 μM; 95% confidence interval: 0.44 μM–0.66 μM, n=29) in vitro.
3. Low concentrations of chloral hydrate (10 μM) were without effect on the basal firing rate of nigral dopaminergic neurones but significantly increased the inhibitory effects produced by concomitant application of (–)-HA-966.
4. The inhibitory effects of (–)-HA-966 were completely reversed in the presence of the GABA_B receptor antagonists, CGP-35348 (100 μM) and 2-hydroxysaclofen (500 μM). Bath application of CGP-35348 alone increased basal firing rate. However, the magnitude of the excitation (9.2±0.3%) was not sufficient to account for the ability of the antagonist to reverse fully the inhibitory effects of (–)-HA-966.
5. (–)-HA-966 (0.1–1.0 mM) produced a concentration-dependent displacement of [^†H]-GABA from synaptic membranes in the presence of isoguvacine (40 μM). However, the affinity of the drug for GABA_B binding sites was significantly less than that of GABA (0.0005 potency ratio) and showed no apparent stereoselectivity.
6. These results indicate that while (–)-HA-966 appears to act as a direct GABA_B receptor agonist in vitro, its affinity for this receptor site is substantially less than that of GABA or baclofen and unlikely to account for the depressant actions of this drug which occur at levels approximately ten fold lower in vivo.

     

γ-Hydroxybutyric Acid (GHB) Pharmacokinetics and Pharmacodynamics: Semi-Mechanistic and Physiologically Relevant PK/PD Model

An overdose of γ-hydroxybutyric acid (GHB), a drug of abuse, results in fatality caused by severe respiratory depression. In this study, a semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model was developed to characterize monocarboxylate transporter 1 (MCT1)-mediated transport of GHB, as well as effects of GHB on respiration frequency, for IV doses of 200, 600, and 1500 mg/kg in rats. The proposed PK/PD model for GHB consists of nonlinear metabolism of GHB in the liver, MCT1-mediated renal reabsorption with physiologically relevant concurrent fluid reabsorption, MCT1-mediated uptake into the brain, and direct effects of binding of GHB to GABA_B receptors on the PD parameter, respiration frequency. Michaelis-Menten affinity constants for metabolism, renal reabsorption, and uptake into and efflux from the brain were fixed to the observed in vitro values. The IC ₅₀ value for the effect of GHB on respiration frequency was fixed to a reported value for binding of GHB to GABA_B receptors. All physiological parameters were fixed to the reported values for a 300-g rat. The model successfully captured the GHB PK/PD data and was further validated using the data for a 600-mg/kg dose of GHB after IV bolus administration. Unbound GHB brain ECF/blood partition coefficient (Kp _u,u ) values obtained from the model agreed well with values calculated using experimental ECF concentrations obtained with brain microdialysis, demonstrating the physiological relevance of this model. Sensitivity analysis indicated that the PK/PD model was stable. In conclusion, we developed a semi-mechanistic and physiologically relevant PK/PD model of GHB using in vitro drug-transporter kinetics and in vivo PK/PD data in rats.     

Hepatoprotective activity of berberine is mediated by inhibition of TNF-α, COX-2, and iNOS expression in CCl(4)-intoxicated mice

This study investigated the protective effects of isoquinoline alkaloid berberine on the CCl(4)-induced hepatotoxicity in mice. Berberine was administered as a single dose at 5 and 10mg/kg intraperitoneally (i.p.), 1h before CCl(4) (10%, v/v in olive oil, 2ml/kg) injection and mice were euthanized 24h later. The rise in serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in CCl(4)-intoxicated mice was markedly suppressed by berberine in a concentration-dependent manner. The decrease in hepatic activity of superoxide dismutase (Cu/Zn SOD) and an increase in lipid peroxidation were significantly prevented by berberine. Histopathological changes were reduced and the expression of tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) was markedly attenuated by berberine 10mg/mg. The results of this study indicate that berberine could be effective in protecting the liver from acute CCl(4)-induced injury. The hepatoprotective mechanisms of berberine may be related to the free radical scavenging and attenuation of oxidative/nitrosative stress, as well as to the inhibition of inflammatory response in the liver.     

Stimulation of central cholinergic neurons by(-)clausenamide in vitro

Ｓｔｉｍｕｌａｔｉｏｎｏｆｃｅｎｔｒａｌｃｈｏｌｉｎｅｒｇｉｃｎｅｕｒｏｎｓｂｙ（－）ｃｌａｕｓｅｎａｍｉｄｅｉｎｖｉｔｒｏ１ＤＵＡＮＷｅｎＺｈｅｎ，ＺＨＡＮＧＪｕｎＴｉａｎ２（ＤｅｐａｒｔｍｅｎｔｏｆＰｈａｒｍａｃｏｌｏｇｙ，Ｉｎｓｔｉｔｕｔｅ...     

The substantia nigra pars reticulata mediates the enhancement of startle by the dopamine D<Subscript>1</Subscript> receptor agonist SKF 82958 in rats

Rational and objectives Several studies have shown that the substantia nigra pars reticulata (SNr) is a critical site of action mediating dopamine agonist effects on motor behaviors. Because dopaminergic and GABAergic mechanisms may interact in the SNr, we tested the contribution of both dopamine and GABA receptors in the SNr on the enhancement of startle by the dopamine D₁ agonist SKF 82958.Methods Male Sprague-Dawley rats were implanted with cannulae into the SNr and 1 week later infused with either the D₁ antagonist SCH 23390 (0.1, 1 µg) or the GABA_A antagonist bicuculline (0.1 µg), followed by a systemic challenge with the D₁ agonist SKF 82958 (1 mg/kg). Other rats were infused with the GABA_A agonist muscimol (0.1 µg) or SKF 82958 (0.1, 1, 5 µg).Results Both SCH 23390 and bicuculline infused into the SNr completely blocked the enhancement of startle by systemic SKF 82958. Muscimol infused into the SNr produced a significant increase in startle by itself, whereas SKF 82958 had no effect.Conclusions These results suggest that activation of D₁ receptors in the SNr is necessary for the enhancement of startle by SKF 82958, but that activation of these receptors alone is not sufficient to increase startle. These results also suggest that GABA transmission in the SNr may be involved in the enhancement of startle by SKF 82958. Based on these data, we propose that activation of striatonigral neurons by D₁ receptor agonists facilitates GABA release in the SNr to produce the observed enhancement of startle.     

Pharmacokinetic interaction between ϵ-acetamidocaproic acid (AACA) and cimetidine in indomethacin-induced acute gastric ulcer and control rats: inhibition of active renal secretion of AACA by cimetidine

1. After both the intravenous and oral administration of zinc acexamate [ZAC; ion-pairing between zinc and ?-acetamidocaproic acid (AACA)] and cimetidine together, the areas under the curve (AUCs) of AACA were significantly greater [by 28.2 and 98.9% after the intravenous and oral administration, respectively, for control rats and 13.5 and 16.9% for indomethacin-induced acute gastric ulcer (IAGU) rats, respectively] than those of ZAC alone due to the significantly slower renal clearance (CL_R). The significantly greater AUCs of AACA after both the intravenous and oral administration of ZAC and cimetidine together in control and IAGU rats could have been due to the inhibition of active renal tubular secretion of AACA by cimetidine.

2. After the intravenous and oral administration of both drugs together, the AUCs of cimetidine in control and IAGU rats were not different compared with those with cimetidine alone.

     

Pharmacokinetic interaction between ϵ-acetamidocaproic acid (AACA) and cimetidine in indomethacin-induced acute gastric ulcer and control rats: inhibition of active renal secretion of AACA by cimetidine

After both the intravenous and oral administration of zinc acexamate [ZAC; ion-pairing between zinc and ?-acetamidocaproic acid (AACA)] and cimetidine together, the areas under the curve (AUCs) of AACA were significantly greater [by 28.2 and 98.9% after the intravenous and oral administration, respectively, for control rats and 13.5 and 16.9% for indomethacin-induced acute gastric ulcer (IAGU) rats, respectively] than those of ZAC alone due to the significantly slower renal clearance (CL(R)). The significantly greater AUCs of AACA after both the intravenous and oral administration of ZAC and cimetidine together in control and IAGU rats could have been due to the inhibition of active renal tubular secretion of AACA by cimetidine. After the intravenous and oral administration of both drugs together, the AUCs of cimetidine in control and IAGU rats were not different compared with those with cimetidine alone.     

Potent inhibition of human sulfotransferase 1A1 by 17α-ethinylestradiol: role of 3'-phosphoadenosine 5'-phosphosulfate binding and structural rearrangements in regulating inhibition and activity

Sulfotransferase (SULT) 1A1 is the major drug/xenobiotic-conjugating SULT isoform in human liver because of its broad substrate reactivity and high expression level. SULT1A1 sulfates estrogens with low micromolar K(m) values consistent with its affinity for sulfation of many small phenolic compounds. Binding studies showed the unexpected ability of 17α-ethinylestradiol (EE2) to bind and inhibit SULT1A1 activity toward p-nitrophenol and β-naphthol at low nanomolar concentrations, whereas EE2 was not sulfated until significantly higher concentrations were reached. EE2 had a K(i) of 10 nM for inhibiting p-nitrophenol and β-naphthol sulfation and inhibited 17β-estradiol (E2) sulfation in intact human MCF-7 breast cancer cells with a K(i) of 19 nM. In contrast, the K(m) for EE2 sulfation by SULT1A1 was 700 nM. The K(d) for EE2 binding of pure SULT1A1 was 0.5 ± 0.15 μM; however, the K(d) for EE2 binding to the SULT1A1-PAP complex was >100-fold lower (4.3 ± 1.7 nM). The K(d) for E2 binding to SULT1A1 changed from 2.3 ± 0.9 to 1.2 ± 0.56 μM in the presence of PAP. Docking studies with E2 indicate that E2 binds in a competent orientation in the resolved structure of SULT1A1 in the both presence and absence of 3'-phosphoadenosine 5'-phosphosulfate (PAPS). However, EE2 binds in a catalytically competent orientation in the absence of PAPS but in a noncompetent orientation via formation of a charge interaction with Tyr108 if PAPS is bound first. In conclusion, EE2 is a potent inhibitor, but not a substrate, of SULT1A1 at low nanomolar concentrations, indicating the possibility of drug-drug interactions during contraceptive therapy.     

Anticancer activity and concurrent analysis of ursolic acid, β-sitosterol and lupeol in three different Hibiscus species (aerial parts) by validated HPTLC method

The genus Hibiscus contains about 275 species of flowering plants widely grown in the tropics and sub-tropics. The available literature revealed that several Hibiscus species exhibited excellent anticancer activity against several cancer cells like lung, breast, and liver. This motivated the authors to explore the anticancer property of other Hibiscus species (Hibiscus calyphyllus, H. deflersii and H. micranthus) along with development of a validated HPTLC method for the concurrent analysis of three anticancer biomarkers (ursolic acid, β-sitosterol and lupeol) in different Hibiscus species. The anticancer activity of various fractions (petroleum ether, toluene, dichloromethane, ethyl acetate and n-butanol) of all the Hibiscus species (aerial parts) were evaluated in vitro against HepG2 and MCF-7 cell lines using MTT assay. The HPTLC analysis was carried out using chloroform and methanol as mobile phase (97:3; v/v) on 20?×?10 cm glass-backed silica gel 60F254 plates and analyzed different phytoconstituents present in all fractions at λ?=?575?nm wavelength. Of the tested fractions of H. calyphyllus, H. deflersii and H. micranthus, HdP (H. deflersii petroleum ether fraction) exhibited the most potent cytotoxic effect on HepG2 and MCF-7 (IC50: 14.4 and 11.1?μg/mL, respectively) cell lines. Using the developed HPTLC method a compact and intense peak of ursolic acid, β-sitosterol and lupeol were obtained at R_f?=?0.22, 0.39 and 0.51, respectively. The LOD/LOQ (ng) for ursolic acid, β-sitosterol and lupeol were found as 42.30/128.20, 13.20/40.01 and 31.57/95.68, respectively in the linearity range 100–1200?ng/spot. The obtained result showed maximum presence of ursolic acid, β-sitosterol and lupeol (5.50, 11.85 and 7.47?μg/mg, respectively) in HdP which also supported its strong anticancer effect. Our data suggest that H. deflersii petroleum ether fraction (HdP) can be further subjected to the isolation of active cytotoxic phytoconstituents and establishment of their mechanism of action. The maiden developed HPTLC method for concurrent analysis of anticancer biomarkers may be further employed in the in process quality control of herbal formulation containing the said biomarkers.     

Biophysical properties of Na(v) 1.8/Na(v) 1.2 chimeras and inhibition by µO-conotoxin MrVIB

BACKGROUND AND PURPOSE

Voltage-gated sodium channels are expressed primarily in excitable cells and play a pivotal role in the initiation and propagation of action potentials. Nine subtypes of the pore-forming α-subunit have been identified, each with a distinct tissue distribution, biophysical properties and sensitivity to tetrodotoxin (TTX). Na_v1.8, a TTX-resistant (TTX-R) subtype, is selectively expressed in sensory neurons and plays a pathophysiological role in neuropathic pain. In comparison with TTX-sensitive (TTX-S) Na_vα-subtypes in neurons, Na_v1.8 is most strongly inhibited by the µO-conotoxin MrVIB from Conus marmoreus. To determine which domain confers Na_v1.8 α-subunit its biophysical properties and MrVIB binding, we constructed various chimeric channels incorporating sequence from Na_v1.8 and the TTX-S Na_v1.2 using a domain exchange strategy.

EXPERIMENTAL APPROACH

Wild-type and chimeric Na_v channels were expressed in Xenopus oocytes, and depolarization-activated Na⁺ currents were recorded using the two-electrode voltage clamp technique.

KEY RESULTS

MrVIB (1 µM) reduced Na_v1.2 current amplitude to 69 ± 12%, whereas Na_v1.8 current was reduced to 31 ± 3%, confirming that MrVIB has a binding preference for Na_v1.8. A similar reduction in Na⁺ current amplitude was observed when MrVIB was applied to chimeras containing the region extending from S6 segment of domain I through the S5-S6 linker of domain II of Na_v1.8. In contrast, MrVIB had only a small effect on Na⁺ current for chimeras containing the corresponding region of Na_v1.2.

CONCLUSIONS AND IMPLICATIONS

Taken together, these results suggest that domain II of Na_v1.8 is an important determinant of MrVIB affinity, highlighting a region of the α-subunit that may allow further nociceptor-specific ligand targeting.     

Transport of saquinavir across human brain-microvascular endothelial cells by poly(lactide-co-glycolide) nanoparticles with surface poly-(γ-glutamic acid)

Poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) with surface poly-(γ-glutamic acid) (γ-PGA) were applied to enhance the transport of saquinavir (SQV) across the blood-brain barrier (BBB). PLGA NPs encapsulated SQV and grafted with γ-PGA to form drug carriers (γ-PGA/SQV-PLGA NPs) for crossing through a monolayer of human brain-microvascular endothelial cells (HBMECs) regulated with human astrocytes. The results revealed that a lower molecular weight of γ-PGA yielded a higher grafting efficiency of γ-PGA on PLGA NPs. In addition, γ-PGA with a low molecular weight accelerated the dissolution of SQV from γ-PGA/SQV-PLGA NPs. A higher grafting efficiency (more didecyl dimethylammonium bromide) and a lower molecular weight of γ-PGA increased the permeability of SQV across the BBB, in general. When the grafting efficiency was 85.2% at 6 kDa of γ-PGA, γ-PGA/SQV-PLGA NPs reached about 6 times the permeability of free SQV (the maximal permeability). γ-PGA could also promote the endocytosis of NPs and expression of ornithine decarboxylase by HBMECs. γ-PGA/SQV-PLGA NPs are efficacious nanoparticulate carriers in delivering antiretroviral drug across the BBB.