Nabanita Halder

Lens aldose reductase inhibiting potential of some indigenous plants.

Cataract is the leading cause of blindness worldover. Diabetes is one of the major risk factors for cataractogenesis and aldose reductase (AR) has been reported to play an important role in sugar-induced cataract. In the present study, the AR inhibitory activity of Ocimum sanctum (OS), Withania somnifera (WS), Curcuma longa (CL), Azadirachta indica (AI) were studied together with their effect on sugar-induced cataractogenic changes in rat lenses in vitro. Aqueous extracts of the plants, procured from Dabur, India, were reconstituted with double distilled water to make various dilutions. AR inhibitory activity of these extracts and their anticataract potentials were evaluated in vitro in rat lenses. AR inhibitory activity of the aqueous extract of different plants was calculated considering the AR activity of normal rat lenses as 100%. The concentration of the plant extract that showed maximum AR inhibitory activity was selected to further study its effect on galactose-induced lens swelling and polyol accumulation in vitro. All the four plants were found to inhibit lens AR activity but to different extent. From dose-response curve, OS was found to be the most effective AR inhibitor followed by CL, AI and WS. The IC(50) values of OS, CL, AI and WS were calculated to be 20, 55, 57 and 89 microg/ml, respectively. OS showed a significant inhibition (38.05%) in polyol accumulation followed by CL and AI (28.4 and 25.04%, respectively). WS did not show any effect on polyol level in rat lenses. None of the plant extracts showed any significant effect on lens water content.OS possesses a significant anticataract activity in vitro and its anticataract potential could be related with its AR inhibitory effect.

Green Tea (Camellia sinensis) Protects against Selenite-Induced Oxidative Stress in Experimental Cataractogenesis

Cataract is the leading cause of blindness worldwide. It is a multifactorial disease primarily associated with oxidative stress produced by free radicals. The protection offered by various antioxidants in cataract development is well established. Polyphenolic compounds present in green tea (Camellia sinensis) are reported to possess antioxidant property in various pathological conditions. The present study was undertaken to evaluate the anticataract potential of green tea leaf (GTL) extract in the development of lens opacification. Enucleated rat lenses were randomly divided into normal, control and treated groups and incubated for 24 h at 37°C. Oxidative stress was induced by sodium selenite in the culture medium of the two groups (except the normal group). The medium of the treated group was additionally supplemented with GTL extract. After incubation, lenses were subjected to glutathione and malondialdehyde estimation. Enzyme activity of superoxide dismutase, catalase and glutathione peroxidase was also measured in different sets of the experiment. In vivo cataract was induced in 9-day-old rat pups of both control and treated groups by a single subcutaneous injection of sodium selenite. The treated pups were injected GTL extract intraperitoneally prior to selenite challenge and continued for 2 consecutive days thereafter. Cataract incidence was evaluated on 16th postnatal day by slit lamp examination. There was positive modulation of biochemical parameters in the organ culture study. Green tea was also found to reduce the incidence of selenite cataract in vivo. The results suggest that green tea possesses significant anticataract potential and acts primarily by preserving the antioxidant defense system.

Ocimum sanctum Modulates Selenite-Induced Cataractogenic Changes and Prevents Rat Lens Opacification

Purpose: To study the effect of Ocimum sanctum (OS) on selenite-induced morphological and biochemical changes in isolated rat lenses as well as on cataract incidence in rat pups. Methods: Transparent rat lenses were divided into normal, selenite-only, and four treated groups. Selenite-only and treated group lenses were subjected to oxidative stress in vitro by incorporating sodium selenite (100 μ M) in the culture medium. The effect of OS (70, 140, 280, and 560 μ g/ml) was studied on the levels of reduced glutathione (GSH) and thiobarbituric acid reacting substances (TBARS) in selenite-challenged lenses. The lowest concentration of OS offering significant modulation on these two parameters was determined. Subsequently, the effect of prior and cotreatment with the lowest effective concentration of OS was studied on TBARS, GSH, and on lens antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSHPx), catalase (CAT), and glutathione-S-transferase (GST). Changes in lens protein profiles under different incubation conditions were analyzed by SDS gel-electrophoresis. In vivo, cataract was induced by a single subcutaneous injection of sodium selenite (25 μ mole/kg b.w.) to 9-day-old rat pups. The anticataract effect of OS (5 and 10 mg/kg b.w.) injected intraperitoneally 4 hr prior to selenite challenge was evaluated by the presence of lens nuclear opacity in rat pups on the 16th postnatal day. Insolubilization of lens proteins post–selenite injection was monitored for 4 days. Results: The lenses in the selenite-only group developed cortical opacities in 24 hr. OS showed different degrees of positive modulation in selenite-induced morphological as well as biochemical changes. The lowest effective dose of OS that significantly modulated glutathione and thiobarbituric acid reacting substances was found to be 140 μ g/ml. At this dose, a significant increase in antioxidant enzyme levels and preservation of normal lens protein profile was observed. OS at the dose of 70 μ g/ml did not show any significant protection with respect to either morphology or biochemistry of lenses. In vivo, 5 and 10 mg/kg of OS reduced the incidence of selenite cataract by 20% and 60%, respectively, and prevented protein insolubilization as well. Conclusions: Aqueous extract of OS possesses potential anticataract activity against selenite-induced experimental cataractogenesis. The protective effect was supported by restoration of the antioxidant defense system and inhibition of protein insolubilization of rat lenses as well.

Development and validation of a LC–MS/MS method for homocysteine thiolactone in plasma and evaluation of its stability in plasma samples

The present study demonstrates the development and validation of a sensitive method for the quantification of homocysteine thiolactone (HCTL) in human plasma using the technique of LC-MS/MS. The gradient elution of HCTL was achieved within 5min using ZIC HILIC column having acetonitrile with 0.1% formic acid and water with 0.1% formic acid. The method was validated for the linearity, sensitivity, accuracy, precision, recovery, matrix effect and stability. A good linearity was found within a range of 0.5-32.5nmol/ml. Quantification was performed using multiple reaction monitoring (MRM) mode based on the molecular/fragment ion transitions for HCTL (118/56) and homatropine (276.1/142.2) as internal standard. Generally, HCTL levels in plasma were found to be highly unstable. In order to verify the stability of the HCTL levels in plasma for a longer period, the samples were extracted immediately and stored at -86°C. Using the above method it was found to be stable for a period of 1 month. The method was well applied for quantification of HCTL in plasma of healthy human volunteers.

Stability of latanoprost in generic formulations using controlled degradation and patient usage simulation studies.

Abstract Purpose: To evaluate the stability of latanoprost in generic formulations by using controlled degradation and patient usage simulation studies Methods: Standard latanoprost was subjected to controlled degradation studies. Latanoprost content was assessed by using MRM, and generated Degradation Products (DP) were analysed by using the Information Dependent Acquisition (IDA) protocol of positive ESI-LC-MS/MS. Latanoprost content and formation of DP were assessed in generic formulations and were compared with Xalatan® in a controlled patient usage simulation studies. The last few drops of latanoprost, present in containers used by patients were also evaluated. Results: Extreme pH conditions, oxidation, light and heat were found to be the significant factors for high degree of latanoprost degradation. Systematic analysis of 7 selected generics revealed that the latanoprost content varied from 90–330%. Concentration of the latanoprost in Xalatan was found to be 97% of the label claim. Degradation studies showed the formation of 3 novel and 3 already known impurities. Upon simulated patient usage, 2 of the generic formulations showed significant degradation of latanoprost. Generic formulations having thermally sealed gas tight packing showed good stability during patient usage. Overage of latanoprost was observed in generics with other than thermal sealing. Latanoprost bottles used by patients showed concentrations ranging from 20 to 250% of label claim (144% median). Conclusion: This study revealed the presence of overage of latanoprost in some generic formulations and formation of degradation products. Packaging with gas tight containers may be one of the important factors for latanoprost stability, along with its storage at low temperature during patient usage.

Ocimum sanctum extracts attenuate hydrogen peroxide induced cytotoxic ultrastructural changes in human lens epithelial cells

Hydrogen peroxide (H2O2) is the major oxidant involved in cataract formation. The present study investigated the effect of an aqueous leaf extract of Tulsi (Ocimum sanctum) against H2O2 induced cytotoxic changes in human lens epithelial cells (HLEC). Donor eyes of the age range 20–40 years were procured within 5–8 h of death. After several washings with gentamicin (50 mL/L) and betadine (10 mL/L), clear transparent lenses (n = 6 in each group) were incubated in Dulbeccos modified Eagles medium (DMEM) alone (normal) or in DMEM containing 100 µm of H2O2 (control) or in DMEM containing both H2O2 (100 µm) and 150 µg/mL of Ocimum sanctum extract (treated) for 30 min at 37 °C with 5% CO2 and 95% air. Following incubation, the semi‐hardened epithelium of each lens was carefully removed, fixed and processed for electron microscopic studies. Thin sections (60–70 mm) were contrasted with uranyl acetate and lead citrate and viewed under a transmission electron microscope. Normal epithelial cells showed intact, euchromatic nucleus with few small vacuoles (diameter 0.58 ± 0.6 µm) in well‐demarcated cytoplasm. After treatment with H2O2, they showed pyknotic nuclei with clumping of chromatin and ill‐defined edges. The cytoplasm was full of vacuoles (diameter 1.61 ± 0.7 µm). The overall cellular morphology was typical of dying cells. Treatment of cells with Ocimum sanctum extract protected the epithelial cells from H2O2 insult and maintained their normal architecture. The mean diameter of the vacuoles was 0.66 ± 0.2 µm. The results indicate that extracts of O. sanctum have an important protective role against H2O2 injury in HLEC by maintaining the normal cellular architecture. The protection could be due to its ability to reduce H2O2 through its antioxidant property and thus reinforcing the concept that the extracts can penetrate the HLEC membrane. Copyright

Involvement of Renin-Angiotensin System in Retinopathy of Prematurity - A Possible Target for Therapeutic Intervention

Objective Examining the Retinal Renin Angiotensin System (RRAS) in the ROP neonates and analyzing the possibility of modulating the RRAS to prevent the progression in Oxygen Induced Retinopathy (OIR) model. Method Vitreous of ROP patients (n = 44, median age 5.5 months) was quantified for RRAS components, VEGF, HIF-1α and compared with age matched control. The involvement of RRAS in ROP was tested in the rat model of OIR and compared with normoxia. Expressions of RAS components, VEGF and HIF-1α in retina were analyzed using qPCR and retinal structure and function was also analyzed. Effect of Angiotensin Converting Enzyme Inhibitor (ACEI) and Angiotensin Receptor Blocker (ARB) was evaluated and compared with Bevacizumab which served as a positive control. Drug penetration into retina was confirmed by liquid chromatography coupled ESI-tandem mass spectroscopy (LC-MS/MS). Results Multifold increase in the expression of RAS components in human vitreous and rat retina showed their involvement in ROP. ERG & fundus studies in OIR revealed the altered function of retina and were successfully prevented by ARB (telmisartan), ACEI (lisinopril) and bevacizumab. Retinal analysis revealed the presence of ACEI and ARB in their therapeutic levels. Conclusion This study for the first time demonstrates the upregulated level of RAS components in human ROP vitreous and further that the pharmacological intervention in RRAS can functionally and structurally preserve retina against the progression of ROP in the OIR model.

Awareness assessment of harmful effects of mercury in a health care set-up in India A survey-based study

Mercury, one of the most toxic heavy metals, is ubiquitous in environment. The adverse health impact of mercury on living organisms is well known. The health care facilities are one of the important sources of mercury release into the atmosphere as mercury items are extensively used in hospitals. To assess the awareness about mercury toxicity and the knowledge of proper handling and disposal of mercury-containing items in health care set–up, a questionnaire-based survey was carried out amongst doctors (n = 835), nurses (n = 610) and technicians (n = 393) in government hospitals, corporate hospitals and primary health care centres in the Indian states of Delhi, Uttar Pradesh and Haryana. The study was conducted using a tool-containing pretested structured multiple-choice questionnaire. Analysis of the results using STATA 11.1 software highlighted that overall awareness was more in corporate sector. However, percentage range of knowledge of respondents irrespective of health care sector was only between 20 and 40%. Despite the commitment of various hospitals to be mercury free, mercury containing-thermometer/sphygmomanometer are still preferred by health professionals. The likely reasons are availability, affordability, accuracy and convenience in use. There is an urgent need for source reduction, recycling and waste minimization. Emphasis must be laid on mercury alternative products, education and training of health personnel and public at large, about correct handling and proper clean up of spills.

Microwave assisted synthesis for A2E and development of LC-ESI–MS method for quantification of ocular bisretinoids in human retina

PURPOSE To develop a microwave assisted method for the rapid synthesis of A2E and also to develop a method to quantify N-retinylidene-N-retinylethanolamine(A2E), all-trans retinal dimer (ATRD), A2-glycerophospho ethanolamine (A2GPE), dihydropyridine phosphatidyl ethanolamine (A2DHPE) and monofuran A2E (MFA2E) in age matched retina. METHODS The development of microwave assisted synthesis of A2E, its purification and characterization for its utility in quantification in human retina. The semi-quantitative method development using LC-ESI-MS, LC-ESI-MS/MS and LC-APCI-MS/MS from pooled macula and peripheral retina for the bisretinoid analysis has been done. RESULTS Maximum A2E conversion using microwave assisted process took place at 80°C for 45min with a yield of 55.01%. Highly sensitive and specific mass spectrometric method was developed using reverse phase C-18 separation with positive electrospray ionization and positive atmospheric phase chemical ionization of tandom mass spectrometry. A gradient mobile phase separation was achieved using water and methanol with 0.1% TFA. Multiple reaction monitoring acquisition for ESI and APCI was performed at ATRD m/z 551.2/522.2, A2GPE m/z 746.4/729.5, A2DHPEm/z 594.4/576.5, MFA2E m/z 608.2/591.2, A2E m/z 592.4/418.2. Method was validated using LC-ESI-SIM mode to determine selectivity, linearity, sensitivity, precision and accuracy. CONCLUSION An attempt towards optimization of the synthetic procedure of A2E was made so as to reduce the lengthy reaction time without compromising the yield. Developed method was capable enough for the detection of low level of bisretinids in retina.

Involvement of nucleoside transporters in the transcorneal permeation of topically instilled substrates in rabbits in-vivo

Abstract The objective of the current study was to characterize and evaluate the functional importance of the Nucleoside Transporters (NTs) in the cornea of the rabbits. Reverse transcriptase polymerase chain reaction (RT‐PCR) was used for the molecular characterization of the NTs. Their functionality was evaluated using two substrates, ribavirin and cytarabine. Dipyridamole was used as a blocker for the study. All the treatments were given topically. Molecular characterization of NTs revealed presence of ent1, ent2, ent3 and cnt3 in the cornea. The concentration vs time profile for cytarabine in Aqueous Humor (AH) exhibited a statistically significant (p < 0.05) drop at 1 h with blocker pretreatment. The mean AUC0–2 between the treatments was also differing in a significant (p < 0.05) manner. The concentration vs time profile for ribavirin in AH also showed a significant (p < 0.05) decrease in its concentration at 1 h with blocker pretreatment. Dipyridamole was able to block ribavirins entry with as low as 40 nM concentration while complete blockade was achieved at 8 mM and above. When cytarabine and ribavirin were co‐administered, ribavirin at a concentration of 6.5 mM significantly inhibited (p < 0.05) the transcorneal permeation of cytarabine up to 80%. To conclude, this study showed the presence and functional importance of NTs in the transcorneal uptake of nucleoside substrates. This study further revealed the presence of concentration dependent competitive inhibition among substrates for their transcorneal permeation. Graphical abstract Figure. No caption available.