Rakesh Kumar Sharma

Evaluation of radioprotective activities of Rhodiola imbricata Edgew – A high altitude plant

The present study reports the radioprotective properties of a hydro-alcoholic rhizome extract of Rhodiola imbricata (code named REC-7004), a plant native to the high-altitude Himalayas. The radioprotective effect, along with its relevant superoxide ion scavenging, metal chelation, antioxidant, anti-lipid peroxidation and anti-hemolytic activities was evaluated under both in vitro and in vivo conditions. Chemical analysis showed the presence of high content of polyphenolics (0.971 ± 0.01 mg% of quercetin). Absorption spectra analysis revealed constituents that absorb in the range of 220–290 nm, while high-performance liquid chromatography (HPLC) analysis confirmed the presence of four major peaks with retention times of 4.780, 5.767, 6.397 and 7.577 min. REC-7004 was found to lower lipid oxidation significantly (p < 0.05) at concentrations viz., 8 and 80 μg/ml respectively as compared to reduced glutathione, although the optimally protective dose was 80 μg/ml, which showed 59.5% inhibition of induction of linoleic acid degradation within first 24 h. The metal chelation activity of REC-7004 was found to increase concomitantly from 1 to 50 μg/ml. REC-7004 (10–50 μg/ml) exhibited significant metal chelation activity (p < 0.05), as compared to control, and maximum percentage inhibition (30%) of formation of iron-2,2′-bi-pyridyl complex was observed at 50 μg/ml, which correlated well with quercetin (34.9%), taken as standard. The reducing power of REC-7004 increased in a dose-dependent manner. The absorption unit value of REC-7004 was significantly lower (0.0183± 0.0033) as compared to butylated hydroxy toluene, a standard antioxidant (0.230± 0.091), confirming its high reducing ability. Superoxide ion scavenging ability of REC-7004 exhibited a dose-dependent increase (1–100 μg/ml) and was significantly higher (p < 0.05) than that of quercetin at lower concentrations (1–10 μg/ml), while at 100 μg/ml, both quercetin and REC-7004 scavenged over 90% superoxide anions. MTT assay in U87 cell line revealed an increase in percent survival of cells at doses between 25 and 125 μg/ml in case of drug + radiation group. In vivo evaluation of radio-protective efficacy in mice revealed that intraperitoneal administration of REC-7004 (maximally effective dose: 400 mg/kg b.w.) 30 min prior to lethal (10 Gy) total-body γ-irradiation rendered 83.3% survival. The ability of REC-7004 to inhibit lipid peroxidation induced by iron/ascorbate, radiation (250 Gy) and their combination [i.e., iron/ascorbate and radiation (250 Gy)], was also investigated and was found to decrease in a dose-dependent manner (0.05–2 mg/ml). The maximum percent inhibition of formation of MDA-TBA complex at 2 mg/ml in case of iron/ascorbate, radiation (250 Gy) and both i.e., iron/ascorbate with radiation (250 Gy) was 53.78, 63.07, and 51.76% respectively and were found to be comparable to that of quercetin. REC-7004 (1 μg/ml) also exhibited significant anti-hemolytic capacity by preventing radiation-induced membrane degeneration of human erythrocytes. In conclusion, Rhodiola renders in vitro and in vivo radioprotection via multifarious mechanisms that act in a synergistic manner.

Antioxidant activity of fractionated extracts of rhizomes of high-altitude Podophyllum hexandrum: Role in radiation protection

Whole extract of rhizomes of Podophyllum hexandrum has been reported earlier by our group to render whole-body radioprotection. High-altitude P. hexandrum (HAPH) was therefore fractionated using solvents of varying polarity (non-polar to polar) and the different fractions were designated as, n-hexane (HE), chloroform (CE), alcohol (AE), hydro-alcohol (HA) and water (WE). The total polyphenolic content (mg% of quercetin) was determined spectrophotometrically, while. The major constituents present in each fraction were identified and characterized using LC-APCI/MS/MS. In vitro screening of the individual fractions, rich in polyphenols and lignans, revealed several bioactivities of direct relevance to radioprotection e.g. metal-chelation activity, antioxidant activity, DNA protection, inhibition of radiation (250 Gy) and iron/ascorbate-induced lipid peroxidation (LPO). CE exhibited maximum protection to plasmid (pBR322) DNA in the plasmid relaxation assay (68.09% of SC form retention). It also showed maximal metal chelation activity (41.59%), evaluated using 2,2′-bipyridyl assay, followed by AE (31.25%), which exhibited maximum antioxidant potential (lowest absorption unit value: 0.0389± 0.00717) in the reducing power assay. AE also maximally inhibited iron/ascorbate-induced and radiation-induced LPO (99.76 and 92.249%, respectively, at 2000 μg/ml) in mouse liver homogenate. Under conditions of combined stress (radiation (250 Gy) + iron/ascorbate), at a concentration of 2000 μg/ml, HA exhibited higher percentage of inhibition (93.05%) of LPO activity. HA was found to be effective in significantly (p < 0.05) lowering LPO activity over a wide range of concentrations as compared to AE. The present comparative study indicated that alcoholic (AE) and hydro-alcoholic (HA) fractions are the most promising fractions, which can effectively tackle radiation-induced oxidative stress.

Radioprotective properties of apple polyphenols: an in vitro study.

Present study was undertaken to evaluate the radioprotective ability of total polyphenols extracted from edible portion (epicarp and mesocarp) of apple. Prior administration of apple polyphenols to murine thymocytes significantly countered radiation induced DNA damage (evaluated by alkaline halo assay) and cell death (trypan blue exclusion method) in a dose dependent manner maximally at a concentration of 2 and 0.2 mg/ml respectively. Apple polyphenols in a dose dependent fashion inhibited both radiation or Fenton reaction mediated 2-deoxyribose (2-DR) degradation indicating its ability to scavenge hydroxyl radicals and this activity was found to be unaltered in presence of simulated gastric juice. Similarly apple polyphenols in a dose dependent fashion scavenged DPPH radicals (maximum 69% at 1 mg/ml), superoxide anions (maximum 88% at 2 mg/ml), reduced Fe3 + to Fe2 + (maximum at 1 mg/ml) and inhibited Fenton reaction mediated lipid peroxidation (maximum 66% at 1.5 mg/ml) further establishing its antioxidative properties. Studies carried out with plasmid DNA revealed the ability of apple polyphenols to inhibit radiation induced single as well as double strand breaks. The results clearly indicate that apple polyphenols have significant potential to protect cellular system from radiation induced damage and ability to scavenge free radicals might be playing an important role in its radioprotective manifestation.

Enhanced Retention of Celecoxib-Loaded Solid Lipid Nanoparticles after Intra-Articular Administration

AbstractObjective: The objective of this study was to determine whether the retention of celecoxib in inflamed articular joints of arthritic rats could be enhanced by incorporation of the drug into solid lipid nanoparticles. Methods: Celecoxib-loaded solid lipid nanoparticles (SLN) were prepared by emulsification and high-pressure homogenisation, then characterised by particle size analysis and scanning electron microscopy. In vitro drug-release studies indicated that the nanoparticles exhibited sustained release of celecoxib and the release pattern followed quasi-Fickian diffusion. The biocompatibility of solid lipid nanoparticles was evaluated by histopathology of the rat joints after intra-articular injection in normal rats. Celecoxib and celecoxib-loaded SLN were labelled with 99mTc and the labelling parameters were optimised to obtain maximum labelling efficiency. The labelled complexes were administered intra-articularly and the pharmacokinetics and biodistribution were determined. Results: The nanoparticles showed no inflammatory infiltrates 3 and 7 days post-intra-articular injection, proving their biocompatibility and suitability for intra-articular use. Free celecoxib underwent rapid clearance from the inflamed articular joints into the systemic circulation, while the celecoxib-loaded SLN were associated with significantly lower blood levels compared with free celecoxib. Free celecoxib was found to have been extensively distributed to organs of the reticuloendothelial system such as the liver, lungs and spleen. In contrast, celecox-ib-loaded nanoparticles demonstrated significantly lower distribution to the reticuloendothelial organs. The articular concentrations of celecoxib-loaded nanoparticles in the inflamed joints were 16-fold higher at 4 hours post-injection and 15-fold higher at 24 hours post-injection than free celecoxib concentrations, indicating greater and prolonged retention in the inflamed articular joints. Conclusion: Celecoxib-loaded SLN with its greater intra-articular retention and sustained-release properties would be a beneficial delivery system for the effective treatment of arthritis and is expected to result in prolonged anti-arthritic activity of celecoxib.

Podophyllum hexandrum Offers Radioprotection by Modulating Free Radical Flux: Role of Aryl-Tetralin Lignans

We have evaluated the effect of variation in aryl-tetralin lignans on the radioprotective properties of Podophyllum hexandrum. Two fractionated fractions of P. hexandrum [methanolic (S1) and chloroform fractions (S2)], with varying aryl-tetralin lignan content were utilized for the present study. The peroxyl ion scavenging potentials of S1 and S2 were found to be comparable [i.e. 45.88% (S1) and 41% (S2)] after a 48 h interval in a time-dependent study, whereas in a 2 h study, S2 exhibited significant (P < 0.05) antioxidant activity in different metal ion + flux states. In the aqueous phase, S2 exhibited non-site-specific reactive oxygen species scavenging activity, i.e. 73.12% inhibition at 500 μg ml−1. S1 exhibited 58.40 ± 0.8% inhibition (at 0.025 μg ml−1) of the formation of reactive nitrite radicals, comparable to S2 (52.45 ± 0.825%), and also showed 45.01% site-specific activity (1000 μg ml−1), along with significant (P < 0.05) electron donation potential (50–2000 μg ml−1) compared to S2. Such activities of S1 could be attributed to the significantly (P < 0.05) higher levels of podophyllotoxin β-d-glucopyranoside (16.5 times) and demethyl podophyllotoxin glucoside (2.9 times) compared with S2. Together, these findings clearly prove that aryl-tetralin lignan content influences the radiation protective potential of the Podophyllum fractions to a great extent.

Simulators for Wireless Networks: A Comparative Study

Network simulation is the most predominant evaluation techniques in the area of computer Networks. The simulation models in a reliable and simple way can be developed by researchers using different simulators for evaluating the behavior and performance of protocols for different networks, simulations are a good example between cost and complexity by maintaining the accuracy of the results. This paper gives briefly survey on a performance comparison study of the different network simulators for wireless network. There are many simulators for wireless network, but among them five are the popular simulators these are: Qualnet/GlomoSim, OM-Net++, Ns-2, OPNET modeler and JSim with a real-world test bed. The information about each of the simulator abilities, their internal structure, development environment and their support for wireless network simulation has detailed discussed in this paper. This paper helps to identify one is best option among shown simulator for their best need. Finally, the paper contains survey, comparatively study and conclusion about making the suitable choice of network simulator supporting wireless network based on the numbers of surveys and papers.

Extremophiles: Sustainable Resource of Natural Compounds-Extremolytes

Microorganisms that thrive in extreme adverse environmental conditions are extremophiles. Examples of these conditions are temperature (>45°C; 500 atmosphere), pH (>8.0; 1.0 M NaCl), high concentrations of calcitrant, heavy metals, high levels of radiation exposure etc. The discovery of extremophiles has enabled the biotechnology industry to innovate corresponding bioproducts, extremolytes, for people’s benefit. The production of Taq DNA polymerase has revolutionized biotechnology research in many ways. Many thermostable enzymes including cellulase, lipase, amylase, and proteases have contributed significantly as industrial bioproducts. Extremophilic radioresistant bacteria and fungi can be used strategically for the development of radioprotective drugs to protect against radiation exposure. Further these extremophiles can be used to develop cryoprotectants. Categorically, the piezophilic microorganisms in the deep sea are a prominent source of specific bio-molecules that has ability to stabilize cell membrane blebbing by maintaining the membrane fluidity. Extremophiles are a sustainable resource for biotechnology industry, which needs to be explored. This chapter provides a comprehensive view of the extremophiles and their products with the possible implications in human interest.

Medical radiation countermeasures for nuclear and radiological emergencies: Current status and future perspectives.

Nuclear and radiological emergencies (NREs) occurred globally and recent incidences in India are indicating toward the need for comprehensive medical preparedness required both at incident site and hospitals. The enhanced threat attributed toward insurgency is another causative factor of worry. The response capabilities and operational readiness of responders (both health and non-health service providers) in contaminated environment need to be supported by advancement in R & D and technological efforts to develop prophylactics and radiation mitigators. It is essential to develop phase 1 alternatives of such drugs for unseen threats as a part of initial preparedness. At the incident site and hospital level, external decontamination procedures need to be standardized and supported by protective clothing and Shudika kits developed by INMAS. The medical management of exposure requires systematic approach to perform triage, resuscitation and curative care. The internal contamination requires decorporation agents to be administered based on procedural diagnostics. Various key issues pertaining to policy decisions, R & D promotion, community awareness, specialized infrastructure for NREs preparedness has been discussed. The present review is an attempt to provide vital information about the current status of various radiation countermeasures and future perspective(s) ahead.

Podophyllum hexandrum as a Potential Botanical Supplement for the Medical Management of Nuclear and Radiological Emergencies (NREs) and Free Radical-Mediated Ailments: Leads From In Vitro/In Vivo Radioprotective Efficacy Evaluation

ABSTRACT. Management of radiation-induced reactive oxygen/nitrogen species requires a holistic approach to mitigate the deleterious effects of free radicals. Flora of the Himalayas, which prevails under extreme climatic conditions, has been explored for its potential utility to develop radioprotective drugs. The Himalayan high altitude medicinal plant, Podophyllum hexandrum Royle, was selected on the basis of its unique properties, and a novel fractionated nonpolar extract (REC-2003) was prepared and evaluated for radioprotective efficacy, in vitro as well as in vivo. The free radical scavenging activity of REC-2003 was found to be > 75% (20 μg/ml) with maximum superoxide scavenging activity (57.56 ± 1.38%) recorded at 1 mg/ml concentration (tetrazolium-based estimation). More than 30% inhibition of nitric oxide radicals was observed at concentrations > 0.5 mg/ml, while hydroxyl radical scavenging activity (deoxy-D-ribose assay) exhibited a dose-dependent (100–600 μg/ml) increase. Significantly high (90%) protection to human erythrocytes was observed at 75 μg/ml, which was found to be the most optimized dose. Similarly, more than 90% inhibition was observed against lipid peroxidation (evaluated by estimating levels of malondialdehyde). The significant antihemolytic potential of REC-2003 could be attributed to its ability to scavenge free radicals, reduce peroxidative stress on lipid membranes, and render protection to DNA (evaluated using plasmid relaxation assay). All these activities holistically contributed toward the radioprotective ability. REC-2003 (8 mg/kg BW; intraperitoneal (i.p.), −30 min) rendered > 80% total-body protection in Swiss Albino Strain ‘A’ mice [against lethal radiation (10 Gy)] in a 30-day survival assay. Phytochemical characterization of the constituents of REC-2003 revealed the presence of polyphenolics (flavonoids). The characterized constituents also included the aryl-tetralin lignans like podophyllotoxin, its glycoside, 4′-demethyl derivative, and epi-podophyllotoxin. The optimized requisite single dose (8 mg/KgBW; i.p., −30 min) for obtaining significant radioprotection is reasonably low and establishes its future utility as a dietary supplement in the medical management of free radical-mediated diseases and specifically for rescue missions during nuclear and radiological emergencies (NREs).

Cytotoxic and radioprotective effects of Podophyllum hexandrum.

Podophyllum hexandrum, a herb thriving in Himalayas has already been reported to exhibit antitumor and radioprotective properties. Present study was undertaken to unravel the possible mechanism responsible for the cytotoxic and radioprotective properties of REC-2001, a fraction isolated from the rhizome of P. hexandrum using murine peritoneal macrophages and plasmid DNA as model systems. Cell death, levels of intracellular reactive oxygen species (ROS) and apoptosis were studied employing trypan blue exclusion assay, dichlorofluorescein diacetate and DNA fragmentation assay, respectively. Superoxide anions, hydroxyl radicals and DNA damage were estimated following nitroblue tetrazolium, 2-deoxyribose degradation and plasmid DNA relaxation assays, respectively. Pre-irradiation administration of REC-2001 to peritoneal macrophages in the concentration range of 25-200μg/ml significantly reduced radiation induced ROS generation, DNA damage, apoptosis and cell killing in comparison to radiation control group indicating radioprotective potential. Studies with plasmid DNA indicated the ability of REC-2001 to inhibit 20Gy induced single and double strand breaks further supporting the antioxidative potential. However, REC-2001 in a dose-dependent fashion induced cell death, ROS and DNA fragmentation indicating the cytotoxic nature. REC-2001, in presence of 100μM copper sulfate, generated significant amount of hydroxyl radicals and superoxide anions indicating ability to act as a pro-oxidant in presence of metal ions. The superoxide anion generation was found to be sensitive to metal chelators like EDTA and deferoxamine mesylate (DFR). These results suggest that the ability of REC-2001 to act as a pro-oxidant in presence of metal ions and antioxidant in presence of free radicals might be responsible for cytotoxic and radioprotective properties.