Citronellal as a Promising Candidate for Alzheimer's Disease Treatment: A Comprehensive Study on In Silico and In Vivo Anti-Acetylcholine Esterase Activity.

One of the primary therapeutic approaches for managing Alzheimer's disease (AD) involves the modulation of Acetylcholine esterase (AChE) activity to elevate acetylcholine (ACh) levels inside the brain. The current study employed computational chemistry approaches to evaluate the inhibitory effects of CTN on AChE. The docking results showed that Citronellal (CTN) and standard Donepezil (DON) have a binding affinity of -6.5 and -9.2 Kcal/mol, respectively, towards AChE. Further studies using molecular dynamics (MD) simulations were carried out on these two compounds. Binding free energy calculations and ligand-protein binding patterns suggested that CTN has a binding affinity of -12.2078. In contrast, DON has a much stronger binding relationship of -47.9969, indicating that the standard DON has a much higher binding affinity than CTN for AChE. In an in vivo study, Alzheimer-type dementia was induced in mice by scopolamine (1.5 mg/kg/day i.p) for 14 days. CTN was administered (25 and 50 mg/kg. i.p) along with scopolamine (SCO) administration. DON (0.5 mg/kg orally) was used as a reference drug. CTN administration significantly improved the mice's behavior as evaluated by the Morris water maze test, evident from decreased escape latency to 65.4%, and in the CPS test, apparent from reduced escape latency to 69.8% compared to the positive control mice. Moreover, CTN significantly increased the activities of antioxidant enzymes such as catalase and superoxide dismutase (SOD) compared to SCO. Furthermore, CTN administration significantly decreased SCO-induced elevated AChE levels in mice. These results were supported by histopathological and in silico molecular docking studies. CTN may be a potential antioxidant and neuroprotective supplement.

Molecular Simulation-based Combinatorial Modeling and Antioxidant Activities of Zingiberaceae Family Rhizomes.

OBJECTIVE: The main aim of this scientific report was to investigate a series of phytochemicals in silico and the pharmacology of four plants found at higher altitude in the ginger family, Zingiberaceae (incl. Costaceae) from North-East India, particularly Sikkim. First, the goal was to determine the biological activities of the four herbs (used under Zingiberaceae family) using antioxidant assays to identify the best species. Second, previously reported compounds in litero were subsequently screened for their anticancerous activities using in silico methods. MATERIALS AND METHODS: Using the methanolic extracts of herbs, quantitative detection of phytochemicals such as total phenols and total flavonoids was detected, and the free radical scavenging activity was also studied using 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay. Docking process was studied, using Discovery Studio version 3.5, to identify suitable molecules at the protein-binding sites through annealing and genetic simulation algorithms. Grids centered on active sites were obtained with spacing of 54 × 55 × 56, and 0.503 grid spacing was calculated. The methods adopted and used in this study were comparisons of Global and Local Search Methods to determine the parameters such as maximum number of 250,000 energy evaluations as well as generations of 27,000, followed by mutation and crossover rates of 0.02 and 0.80. The number of docking runs was set to 10. Molecular dynamics study was done to check the stability of the complex. RESULTS: Among all the genus of Zingiberaceae family investigated in this study, Curcuma angustifolia and Hedychium sp. exhibited the highest 537 ± 12.45; 292 ± 9.16 mg gallic acid equivalent/g total polyphenols and 38 ± 1.54; 75 ± 6.75 mg quercetin equivalent/g flavonoids, respectively. Depending on the concentration, the Hedychium sp. extract exerted the highest scavenging activity on DPPH radical (IC50 36.4 µg/mL). In silico result demonstrated that the synergetic effects of ß-phellandrene with other compounds might be responsible for its anticancerous activity. ß-phellandrene and farnesene epoxide showed bonding with Leu298, Ala302, Met336, Leu339, Leu343, Phe356, Ala302, Glu305, Met340, Leu343, Arg346, Phe356, Ile373, Ile376, Leu380, His475, Leu476, and Leu491. CONCLUSION: Based on the current available literature, this is the first study to understand the interaction of compounds found in the rhizomes of Zingiberaceae family. SUMMARY: The aqueous methanolic extract of Zingiberaceae family Curcuma angustifolia and Hedychium sp. has potent antioxidant activity as assessed by 2,2-diphenyl-1-picryl-hydrazyl assaysHedychium sp. is understood to possess more active compounds than other varietiesIn silico studies indicated synergetic effects of ß-phellandrene and other compounds for its anticancerous activity. Abbreviations used: CADD: Computer-aided drug designing; ROS: Reactive oxygen species; ADMET: Absorption, distribution, metabolism, and excretion-toxicity; FeCl3: Ferric chloride; DPPH: 2,2-diphenyl-1-picryl-hydrazyl; NaNO2: Sodium nitrite; TCA: Trichloroacetic acid; K2HPO4: Di-potassium hydrogen phosphate; H2O2: Hydrogen peroxide; KH2PO4: Potassium di-hydrogen phosphate, K2Fe (CN)6: Potassium ferricyanide; KOH: Potassium hydroxide; NaOH: Sodium hydroxide; Na2CO3: Sodium carbonate; CH3COONa: Sodium acetate; AlCl3: Aluminum chloride.

Purification and characterization of carboxylesterase from the seeds of Jatropha curcas.

Carboxylesterases are hydrolases which catalyze the hydrolysis of various types of esters. Carboxylesterase from the seeds of Jatropha curcas has been purified to homogeneity using ammonium sulfate fractionation, CM-cellulose chromatography, Sephadex G-100 chromatography and preparative polyacrylamide gel electrophoresis (PAGE). The homogeneity of the purified enzyme was confirmed by PAGE, iso-electrofocusing and SDS-PAGE. The molecular weight of the purified enzyme was determined by both gel-permeation chromatography on Sephadex G-150 and SDS-PAGE. The molecular weight determined by Sephadex G-150 chromatography and SDS-PAGE both in the presence and absence of 2-mercaptoethanol was 31 kDa. The isoelectric point of the purified enzyme was found to be 8.9. JCSE-I (J. curcas seed esterase-I) was classified as carboxylesterase on the basis of substrate and inhibitor specificity. The K(m) of JCSE-I with 1-naphthyl acetate, 1-naphthyl propionate, 1-naphthyl butyrate and 2-naphthyl acetate as substrates were found to be 0.0,794, 0.0,658, 0.0,567 and 0.1 mM, respectively. The enzyme exhibited an optimum temperature of 45 °C and an optimum pH of 6.5. The enzyme was stable up to 15 min at 65 °C. The enzyme was resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB) and inhibited by organophosphates (dichlorvos, parathion and phosphamidon).

Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization.

Two carboxylesterases (ME-III and ME-IV) have been purified to apparent homogeneity from the seeds of Mucuna pruriens employing ammonium sulfate fractionation, cation exchange chromatography on CM-cellulose, gel-permeation chromatography on Sephadex G-100 and preparative PAGE. The homogeneity of the purified preparations was confirmed by polyacrylamide gel electrophoresis (PAGE), gel-electrofocussing and SDS-PAGE. The molecular weights determined by gel-permeation chromatography on Sephadex G-200 were 20.89 kDa (ME-III) and 31.62 kDa (ME-IV). The molecular weights determined by SDS-PAGE both in the presence and absence of 2-mercaptoethanol were 21 kDa (ME-III) and 30.2 kDa (ME-IV) respectively, suggesting a monomeric structure for both the enzymes. The enzymes were found to have Stokes radius of 2.4 nm (ME-III) and 2.7 nm (ME-IV). The isoelectric pH values of the enzymes, ME-III and ME-IV, were 6.8 and 7.4, respectively. ME-III and ME-IV were classified as carboxylesterases employing PAGE in conjunction with substrate and inhibitor specificity. The K(m) of ME-III and ME-IV with 1-naphthyl acetate as substrate was 0.1 and 0.166 mM while with 1-naphthyl propionate as substrate the K(m) was 0.052 and 0.0454 mM, respectively. As the carbon chain length of the acyl group increased, the affinity of the substrate to the enzyme increased indicating hydrophobic nature of the acyl group binding site. The enzymes exhibited an optimum temperature of 45°C (ME-III) and 37°C (ME-IV), an optimum pH of 7.0 (ME-III) and 7.5 (ME-IV) and both the enzymes (ME-III and ME-IV) were stable up to 120 min at 35°C. Both the enzymes were inhibited by organophosphates (dichlorvos and phosphamidon), but resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB).

Coin-shaped epithelial lesions following an acute attack of erythema multiforme minor with confocal microscopy findings.

We report an interesting ocular finding of bilateral multiple coin-shaped epithelial lesions along with the confocal microscopy findings in a patient following an acute attack of erythema multiforme (EM) minor. A 30-year-old male presented with a history of watering and irritation in both eyes of three days duration. He was diagnosed to have EM minor and was on oral acyclovir. Slit-lamp examination revealed multiple coin-shaped epithelial lesions. Confocal microscopy showed a corresponding conglomerate of hyper-reflective epithelial lesions. The corneal lesions resolved over six weeks with oral steroids and acyclovir. An immunological mechanism is suspected.

A study of the efficacy of endoscopic cyclophotocoagulation for the treatment of refractory glaucomas.

BACKGROUND: Endoscopic cyclophotocoagulation (ECP) is a relatively new method of cyclodestruction which can be used in the management of refractory glaucomas. AIM: To evaluate the safety and efficacy of ECP in the management of refractory glaucomas. SETTINGS AND DESIGN: Prospective interventional non-comparative study. MATERIALS AND METHODS: Fifty eyes of 50 patients with refractory glaucoma, whose intraocular pressures (IOP) were not under control with maximal medical therapy underwent ECP, by the anterior, or pars plana route. IOP, best corrected visual acuity (BCVA), and the number of anti-glaucoma medications, were compared postoperatively to preoperative values. Success was defined as IOP pound 22 mmhg, with or without use of medications. STATISTICAL ANALYSIS USED: Student's t test and repeated measures ANOVA were used to evaluate change in IOP and Student's t test, for comparison of BCVA. Kaplan Meier survival curve was plotted. Wilcoxon signed rank test was used to evaluate reduction in medications. RESULTS: Patients were followed for an average of 12.27 months (3-21months). IOP decreased significantly from 32.58 +/- 9.16 mmHg to 13.96 +/- 7.71 mmHg at last follow-up ( P < 0.001, student's t test). BCVA was significantly improved in the postoperative period ( P < 0.001, student's t test). The average number of antiglaucoma medications decreased from 2.51 +/- 0.97 to 1.09 +/- 1.16 ( P < 0.001, Wilcoxon signed rank test). ECP had a success rate of 82.2%. CONCLUSION: Endoscopic cyclophotocoagulation is an effective procedure in this subset of refractory glaucomas.

In vivo confocal microscopy in different types of posterior polymorphous dystrophy.

Posterior polymorphous dystrophy is a rare corneal dystrophy, usually detected by chance. This case series describes the morphologic features in the three different types of posterior polymorphous dystrophy using confocal microscopy.

NK/T-cell lymphoma in AIDS.

A 42-year-old man diagnosed to be HIV positive and on highly active antiretroviral treatment (HAART), presented with double vision and gradual drooping of the left eyelid. He had left 3rd cranial nerve palsy and partial right lower-motor-neuron facial palsy. CT of the PNS revealed soft tissue filling the right maxillary sinus antruin. Further workup showed the mass to be an NK/T cell lymphoma.

Effect of Indian red scorpion (Mesobuthus tamulus concanesis, Pocock) venom on thyroxine and triiodothyronine in experimental acute myocarditis and its reversal by species specific antivenom.

Acute myocarditis was induced in dogs and rabbits by injection of scorpion (M. tamulus concanesis, earlier called Buthus tamulus) venom by s.c., i.m. or i.v. routes. A decrease in thyroxine (T4) levels was observed following i.v. injection of venom in dogs. In rabbits the venom (i.m.) did not elicit any change in T4 levels. Envenomation (s.c.) resulted in a reduction in triiodothyronine (T3) levels in dogs. Venom injection (s.c.) along with i.v. administration of the species specific antivenom (AScVS) did not cause any change in T3 and T4 levels in general. However an increase in T3 levels following AScVS was observed in envenomated dogs. The results suggest that scorpion envenomation caused an autonomic storm releasing massive amounts of catecholamines, angiotensin II, suppressed insulin secretion and reduced circulating T4 and T3 levels. Decrease in thyroid hormones results in fall in body temperature. Changes in the body temperature may increase the sensitivity of the scorpion venom and influence the course of toxicity.

Mutations in RPE65 cause autosomal recessive childhood-onset severe retinal dystrophy.

Autosomal recessive childhood-onset severe retinal dystrophy (arCSRD) designates a heterogeneous group of disorders affecting rod and cone photoreceptors simultaneously. The most severe cases are termed Leber congenital amaurosis (LCA), while the less aggressive forms are usually considered juvenile retinitis pigmentosa. Recently, mutations in the retinal-specific guanylate cyclase gene were found in patients with LCA. Disease genes implicated in other forms of arCSRD are expected to encode proteins present in the neuroretina or in the retinal pigment epithelium (RPE). The RPE, a monolayer of cells separating the vascular-rich choroid and the neuroretina, is in intimate contact with the outer segments of rods and cones via the microvilli surrounding the photoreceptors. The RPE expresses a tissue-specific and evolutionarily highly conserved 61 kD protein (RPE65) present at high levels in vivo. Although the function of RPE65 is not yet known, an important role in the RPE/photoreceptor vitamin-A cycle is suggested by the fact that RPE65 associates both with serum retinol-binding protein and with the RPE-specific 11-cis retinol dehydrogenase, an enzyme active in the synthesis of the visual pigment chromophore 11-cis retinal. Here we report that the analysis of RPE65 in a collection of about 100 unselected retinal-dystrophy patients of different ethnic origin revealed five that are likely to be pathogenic mutations, including a missense mutation (Pro363Thr), two point mutations affecting splicing (912 + 1G-->T and 65 + 5G-->A) and two small re-arrangements (ins144T and 831del8) on a total of nine alleles of five patients with arCSRD. In contrast to other genes whose defects have been implicated in degenerative retinopathies, RPE65 is the first disease gene in this group of inherited disorders that is expressed exclusively in the RPE, and may play a role in vitamin-A metabolism of the retina.

Colour of the nucleus as a marker of nuclear hardness, diameter and central thickness.

Hundred and thirty patients, aged above 40 years, with senile cataract were examined. Age and colour were selected as the probable preoperative indicators of nuclear hardness. The lens material collected after manual extracapsular extraction was washed and the nucleus isolated. The diameter and central thickness of the nucleus were measured; the mean diameter and mean central thickness were 7.13 mm +/- 0.76 and 3.05 mm +/- 0.48, respectively. The hardness of the nucleus was measured with a lens guillotine designed by us. Regression analysis was applied to the parameters measured and these were compared with the colour and age. The parameters measured had the following relationship: Colour vs hardness (r value = 0.7569) (p < 0.001) Colour vs diameter (r value = 0.3962) (p < 0.001) Colour vs central thickness (r value = 0.4785) (p < 0.001) Age vs hardness (r value = -0.0499) (p > 0.05) Age vs diameter (r value = 0.0987) (p > 0.05) Age vs central thickness (r value = 0.1700) (p > 0.05) The values showed that colour had a statistically significant relationship with all the 3 parameters (p < 0.001), while age had no significant relationship with the same parameters. The results indicated that colour can be used more reliably to predict physical characteristics of the cataractous lens nucleus, the preoperative knowledge of which would help the surgeon in planning small-incision surgery including phacoemulsification.

Xenobiotic metabolism in humans during early pregnancy: peroxidase-mediated oxidation and bioactivation of 2-aminofluorene.

Arylamines such as 2-aminofluorene (2-AF) are known teratogens and transplacental carcinogens in laboratory animal species. Although exposure of women to arylamines is likely to occur during pregnancy, how these chemicals are metabolized by the enzymes from the human conceptual tissues is currently unknown. Highly purified preparations of peroxidase isolated from human intrauterine conceptual tissues at 8 weeks of gestation were used to study in vitro metabolism of 2-AF. The oxidation of 2-AF was examined spectrophotometrically whereas the bioactivation was assessed from the covalent binding to protein and DNA using [3H] 2-AF. Using guaiacol as a model substrate, the purified preparations of peroxidase used exhibited a specific activity of 15-20 micromol/min/mg protein. 2-AF oxidation was found to be enzymatic in nature. Kinetic data obtained under optimal assay conditions yielded a Km = 41 microM for 2-AF, 8.33 microM for H2O2, and a Vmax=1.2 micromol 2-AF oxidized/min/mg protein. Under optimal assay conditions, the covalent binding of reactive intermediates to protein and DNA (nmol equivalent/min/mg enzyme/mg bovine serum albumin or calf thymus DNA) was observed at the rate of about 3.75 +/- 0.39 and 1.90 +/- 0.11 respectively. A significant decline in the rate of both oxidation and bioactivation of 2-AF was observed in the presence of classical peroxidase inhibitors, KCN and NaN3.

2-Aminofluorene bioactivation by human term placental peroxidase.

Earlier investigations from our laboratory demonstrated that human term placental peroxidase (HTPP) is capable of metabolism of xenobiotics and endogenous compounds. In this study, purified HTPP was found to bioactivate 2-aminofluorene (2-AF) in the presence of H2O2. 2-AF oxidation was studied spectrophotometrically while radiometry was employed to assess the bioactivation. The rate of oxidation and covalent binding to protein and DNA was dependent upon the pH of the reaction medium and the concentration of 2-AF, the enzyme, and H2O2. To observe maximal enzyme velocity of oxidation, the presence of 16.5 microM H2O2, 100 microM 2-AF, 37 micrograms of the enzyme protein/ml, and pH 7.2 was required. Under optimal assay conditions, the range of specific activity between 130 and 165 nmol of 2-AF oxidized/min/mg HTPP was observed. Using similar assay conditions, the magnitude of covalent binding of [3H]-2-AF to protein (BSA) and calf thymus DNA was found to be about 508 pmol bound/min/mg HTPP/mg BSA and 84 pmol bound/min/mg HTPP/mg DNA, respectively. Potassium cyanide and sodium azide, the known inhibitors of different peroxidases, significantly blocked both the oxidation and covalent binding of 2-AF in a dose dependent manner. These results strongly suggest that peroxidase may be one of the important pathways responsible for the bioactivation of arylamines in human term placenta.

Scorpion envenoming and the role of insulin.

Scorpion envenoming results in a severe autonomic storm with a massive release of catecholamines, increased angiotensin II and inhibition of insulin secretion. These hormonal alterations could be responsible for the pathogenesis of a variety of clinical manifestations. Under these conditions, scorpion envenoming essentially results in a syndrome of fuel-energy deficits and an inability to utilize the existing metabolic substrates by vital organs causing multi-organ system failure and death. Based on our animal experiments in which insulin administration reversed the metabolic and ECG changes induced by scorpion envenoming and treating the poisonous scorpion sting victims with insulin, we consider that insulin has a primary metabolic role in preventing and reversing the cardiovascular, haemodynamic, and neurological manifestations and pulmonary oedema induced by scorpion envenoming. The use of continuous infusion of regular crystalline insulin at the rate of 0.3 U/g glucose and glucose at the rate of 0.1 g/kg/hr, with supplementation of potassium as needed and maintenance of fluid electrolytes and acid-based balance, has become a routine protocol in our setting for treating the victims of scorpion envenoming.