Nirmalendu Das

Nanoencapsulation of quercetin enhances its dietary efficacy in combating arsenic-induced oxidative damage in liver and brain of rats.

AIMS This study was performed to evaluate the therapeutic efficacy of nanocapsulated flavonoidal quercetin (QC) in combating arsenic-induced reactive oxygen species (ROS)-mediated oxidative damage in hepatocytes and brain cells in a rat model. MAIN METHODS Hepatic and neuronal cell damage in rats was made by a single injection (sc) of sodium arsenite (NaAsO(2), 13 mg/kg b. wt. in 0.5 ml of physiological saline). A single dose of 500 microl of quercetin suspension (QC) (QC 8.98 micromol/kg) or 500 microl of nanocapsulated QC (NPQC) (QC 8.98 micromol/kg) was given orally to rats at 90 min prior to the arsenite injection. KEY FINDINGS Inorganic arsenic depositions (182+/-15.6 and 110+/-12.8 ng/g protein) were found in hepatic and neuronal mitochondrial membranes. Antioxidant levels in hepatic and neuronal cells were reduced significantly by arsenic. NPQC prevented the arsenite-induced reduction in antioxidant levels in the liver and brain. Arsenic induced a substantial decrease in liver and brain cell membrane microviscosities, and NPQC treatment resulted in a unique protection against the loss. A significant correlation between mitochondrial arsenic and its conjugated diene level was observed both in liver and brain cells for all experimental rats. SIGNIFICANCE Arsenic-specific antidotes are used against arsenic-induced toxicity. However, the target site is poorly recognized and therefore achieving an active concentration of drug molecules can be a challenge. Thus, our objective was to formulate NPQC and to investigate its therapeutic potential in an oral route against arsenite-induced hepatic and neuronal cell damage in a rat model.

Targeting of liposomal andrographolide to L. donovani-infected macrophages in vivo.

Despite the rapid development in medicinal and pharmaceutical technology, the targeting of drugs to phagocytic cells in macrophage-related diseases still remains a major unsolved problem. By using the mannosyl-fucosyl receptors on macrophages, attempts were made to target antileishmanial drugs encapsulated in mannosylated or fucosylated liposomes to treat experimental leishmaniasis in the hamster model. Mannosylated liposomes were found to be more potent in delivering antileishmanial drugs to phagocytic cells. Liposomes loaded with an indigenous drug, andrographolide, a labdane diterpenoid isolated from Indian medicinal plant Andrographis paniculata, were prepared and tested against experimental leishmaniasis in a hamster model. Mannosylated liposomes loaded with the drug were found to be most potent in reducing the parasitic burden in the spleen as well as in reducing the hepatic and renal toxicity. In addition, mannosylated drug-loaded liposome-treated animals showed a normal blood picture and splenic tissue histoarchitecture when compared with those treated with free drug or regular liposomal drug. Such a drug-vehicle formulation may be considered for clinical trials.Despite the rapid development in medicinal and pharmaceutical technology, the targeting of drugs to phagocytic cells in macrophage-related diseases still remains a major unsolved problem. By using the mannosyl-fucosyl receptors on macrophages, attempts were made to target antileishmanial drugs encapsulated in mannosylated or fucosylated liposomes to treat experimental leishmaniasis in the hamster model. Mannosylated liposomes were found to be more potent in delivering antileishmanial drugs to phagocytic cells. Liposomes loaded with an indigenous drug, andrographolide, a labdane diterpenoid isolated from Indian medicinal plant Andrographis paniculata, were prepared and tested against experimental leishmaniasis in a hamster model. Mannosylated liposomes loaded with the drug were found to be most potent in reducing the parasitic burden in the spleen as well as in reducing the hepatic and renal toxicity. In addition, mannosylated drug-loaded liposome-treated animals showed a normal blood picture and splenic tissue histoarchitecture when compared with those treated with free drug or regular liposomal drug. Such a drug-vehicle formulation may be considered for clinical trials.

Nanocapsulated curcumin: Oral chemopreventive formulation against diethylnitrosamine induced hepatocellular carcinoma in rat

Toxic outcome of chemical therapeutics as well as multidrug resistance are two serious phenomena for their inacceptance in cancer chemotherapy. Antioxidants like curcumin (Cur) have gained immense importance for their excellent anticarcinogenic activities and minimum toxic manifestations in biological system. However, Cur is lipophilic and thus following oral administration hardly appears in blood indicating its potential therapeutic challenge in cancer therapy. Nanocapsulated Cur has been used as a drug delivery vector to focus the effectiveness of these vesicles against hepatocellular carcinoma. The theme of work was to evaluate effectiveness in oral route of polylactide co-glycolide (PLGA) Nanocapsulated curcumin (Nano Cur) against diethylnitrosamine (DEN) induced hepatocellular carcinoma (HCC) in rat. Nano Cur of average diameter 14nm and encapsulation efficiency of 78% were prepared. Fourier Transform Infra Red (FTIR) analysis revealed that there is no chemical interaction between drug and the polymer. Three i.p. injections of the chemical hepatocarcinogen DEN at 15days interval causes hepatotoxicity, the generation of reactive oxygen species (ROS), lipid peroxidation, decrease in plasma membrane microviscosity and depletion of antioxidant enzyme levels in liver. Nano Cur (weekly oral treatment for 16weeks at 20mg/kg b.wt) in DEN induced HCC rats exerted significant protection against HCC and restored redox homeostasis in liver cells. Nanocapsulated Cur caused cancer cell apoptosis as visualized by ApoBrdU analysis. Histopathological analysis confirmed the pathological improvement in the liver. Nano Cur was found to be a potential formulation in oral route in combating the oxidative damage of hepatic cells and eliminating DEN induced hepatocellular cancer cells in rat whereas identical amount of free Cur treatment was found almost ineffective.

Neuroprotective Role of Nanoencapsulated Quercetin in Combating Ischemia-Reperfusion Induced Neuronal Damage in Young and Aged Rats

Cerebral stroke is the leading cause of death and permanent disability among elderly people. In both humans and animals, cerebral ischemia damages the nerve cells in vulnerable regions of the brain, viz., hippocampus, cerebral cortex, cerebellum, and hypothalamus. The present study was conducted to evaluate the therapeutic efficacy of nanoencapsulated quercetin (QC) in combating ischemia-reperfusion-induced neuronal damage in young and aged Swiss Albino rats. Cerebral ischemia was induced by occlusion of the common carotid arteries of both young and aged rats followed by reperfusion. Nanoencapsulated quercetin (2.7 mg/kg b wt) was administered to both groups of animals via oral gavage two hours prior to ischemic insults as well as post-operation till day 3. Cerebral ischemia and 30 min consecutive reperfusion caused a substantial increase in lipid peroxidation, decreased antioxidant enzyme activities and tissue osmolality in different brain regions of both groups of animals. It also decreased mitochondrial membrane microviscosity and increased reactive oxygen species (ROS) generation in different brain regions of young and aged rats. Among the brain regions studied, the hippocampus appeared to be the worst affected region showing increased upregulation of iNOS and caspase-3 activity with decreased neuronal count in the CA1 and CA3 subfields of both young and aged rats. Furthermore, three days of continuous reperfusion after ischemia caused massive damage to neuronal cells. However, it was observed that oral treatment of nanoencapsulated quercetin (2.7 mg/kg b wt) resulted in downregulation of iNOS and caspase-3 activities and improved neuronal count in the hippocampal subfields even 3 days after reperfusion. Moreover, the nanoformulation imparted a significant level of protection in the antioxidant status in different brain regions, thus contributing to a better understanding of the given pathophysiological processes causing ischemic neuronal damage.

Nanoparticulated Quercetin in Combating Age Related Cerebral Oxidative Injury

Reactive oxygen species e.g. O(2)(*-), H(2)O(2) and *OH generated by the induction of oxidative stress exert a potential threat on the activity of endogenous antioxidant enzymes and substantially influence the aging process and age-dependant neuropathology. Chemical antioxidant is almost ineffective in protecting neuronal cells from oxidative damage as Blood Brain Barrier exists in between blood and brain interstitial fluid that restricts undegradable influx from the circulation into cerebral region. Quercetin (QC), a flavonoidal antioxidant is known as a potent antioxidant for its polyphenolic configuration. Formulation of QC in polylactide nanocapsule has been done and the efficacy of this vesicular flavonoid has been tested against cerebral ischemia induced oxidative damage in young and old rat brains. Antioxidant potential of QC loaded in nanocapsule (QC 7.2 mmol/kg b.wt., size 50 nm) was investigated by an in vivo model of cerebral ischemia and reperfusion on Sprague Dawley young (2 months, b.wt. 160-180 g) and aged (20 months, b.wt. 415-440 g) rats. Diene level, the index of lipid peroxidation and GSSG/GSH ratio were found to be higher in normal aged, compared to normal young rat brain. Endogenous antioxidants activities were lower in aged rat brain compared to young. Further reduction of these antioxidants were observed in aged rat brain by the induction of cerebral ischemia - reperfusion. Nanocapsule encapsulated QC treatment resulted a significant protection to endogenous antioxidant enzymes against ischemia induced oxidative damage in neuronal cells of young and old rats.

Mannosylated liposomal flavonoid in combating age-related ischemia-reperfusion induced oxidative damage in rat brain

Active oxygen species alter the activities of the enzymes involved in the defence against free radicals and substantially influence the aging process and age-dependent neuropathology. Unilamellar liposomes were used to deliver flavonoidal antioxidant quercetin (QC) to rat brain. Antioxidant potential of QC loaded in mannosylated (QC 7.2 micromol/kg b.wt.) liposomes (50 nm) was investigated by an in vivo model of cerebral ischemia and reperfusion on Sprague Dawley young (2 months old, b.wt. 160-180 g) and aged (20 months old, b.wt. 415-440 g) rats. Animals were made ischemic for 30 min by bilateral clamping of the common carotid artery followed by a 30 min cerebral reperfusion by withdrawing the clamping. Diene level and (GSSG/GSH) ratio were found to be higher in normal aged, compared to normal young rat brain. Superoxide dismutase, catalase, glucose-6-phosphate dehydrogenase, glutathione reductase and glutathione S-transferase activities were lower in normal aged rat brain. Further reduction of these antioxidant enzymes was observed in aged rat brain by the induction of cerebral ischemia and reperfusion. Mannosylated liposomally encapsulated QC treatment resulted in a significant preservation of the activities of antioxidant enzymes and a marked inhibition of cellular edema formation in neuronal cells of young and old rats.

The use of nano-quercetin to arrest mitochondrial damage and MMP-9 upregulation during prevention of gastric inflammation induced by ethanol in rat.

Gastric ulcer is a multifaceted process that involves reactive oxygen species (ROS) generation, extracellular matrix degradation and mitochondrial damage. Mitochondria play a crucial role for homeostasis of ROS and cell survival. In our study, we investigated the efficacy and mechanism of polymeric nanocapsuled quercetin (NQC) over the free quercetin (QC) molecule in prevention of ethanol-induced gastric ulcer in rat. NQC possessed significantly higher efficacy (~20 fold) than free QC while preventing gastric ulcers. Our data show that prior administration of NQC and/or QC significantly blocked synthesis and secretion of matrix metalloproteinase (MMP)-9 as well as infiltration of inflammatory cells and oxidative damage in rat gastric tissues. As compared to free QC, NQC protected much better the mitochondrial integrity and size along with mitochondrial functions by controlling succinate dehydrogenase and NADH oxidase in rat gastric tissues. In addition, both free QC and NQC down regulated PARP-1 as well as apoptosis during protection against ethanol-induced gastric ulcer. Herein, the effect of NQC was greater than QC on expression of enzymes like cyclooxygenase and nitric oxidase synthase (NOS)-2. We conclude that NQC with greater bioavailability offers significantly higher potency in downregulating MMP-9 and NOS-2 as well as oxidative stress in blocking ethanol-induced gastric ulcer.

Hepatoprotective Activity of Liposomal Flavonoid against Arsenite-Induced Liver Fibrosis

Arsenic, the environmental metalloid toxicant, is known to induce oxidative damage to liver and produce hepatic fibrosis. The theme of our study was to optimize and evaluate the therapeutic efficacy of galactosylated liposomal flavonoidal antioxidant, quercetin (QC), in combating arsenic-induced hepatic fibrogenesis. The rats of the hepatic damage group were injected s.c. a single dose of sodium arsenite (NaAsO2) (100.06 μM/kg b. wt. in 0.5 ml of physiological saline). Hepatocytes and stellate cells were separated. Mitochondrial membranes were isolated from all those separated cells. Oxidative damage was monitored at different isolated subcellular parts of different hepatic cells. Liver fibrosis was also induced by the injection of NaAsO2. Galactosylated liposomal QC injection before NaAsO2 treatment checked fibrogenesis completely by protecting the liver from oxidative attack in cellular and subcellular levels. The maximal protections from hepatocellular and fatty metamorphosis, necrosis, Kupffer cell hyperplasia, fibrosis, and in the deposition of collagen contents were observed and reconfirmed by our histopathological and histochemical analysis when rats were treated with galactosylated liposomal QC before NaAsO2 injection. Application of galactosylated liposomal QC may be a potent therapeutic approach for NaAsO2-induced fibrogenesis through a complete protection against oxidative attack in cellular and subcellular parts of rat liver.

Quercetin in vesicular delivery systems: evaluation in combating arsenic-induced acute liver toxicity associated gene expression in rat model.

Arsenic, the environmental toxicant causes oxidative damage to liver and produces hepatic fibrosis. The theme of our study was to evaluate the therapeutic efficacy of liposomal and nanocapsulated herbal polyphenolic antioxidant quercetin (QC) in combating arsenic induced hepatic oxidative stress, fibrosis associated upregulation of its gene expression and plasma TGF beta (transforming growth factor beta) in rat model. A single dose of arsenic (sodium arsenite-NaAsO(2), 13 mg/kgb.wt) in oral route causes the generation of reactive oxygen species (ROS), arsenic accumulation in liver, hepatotoxicity and decrease in hepatic plasma membrane microviscosity and antioxidant enzyme levels in liver. Arsenic causes fibrosis associated elevation of its gene expression in liver, plasma TGF ss (from normal value 75.2+/-8.67 ng/ml to 196.2+/-12.07 ng/ml) and release of cytochrome c in cytoplasm. Among the two vesicular delivery systems formulated with QC, polylactide nanocapsules showed a promising result compared to liposomal delivery system in controlling arsenic induced alteration of those parameters. A single dose of 0.5 ml of nanocapsulated QC suspension (QC 2.71 mg/kg b.wt) when injected to rats 1h after arsenic administration orally protects liver from arsenic induced deterioration of antioxidant levels as well as oxidative stress associated gene expression of liver. Histopathological examination also confirmed the pathological improvement in liver. Nanocapsulated plant origin flavonoidal compound may be a potent formulation in combating arsenic induced upregulation of gene expression of liver fibrosis through a complete protection against oxidative attack in hepatic cells of rat liver.

Liposomal antioxidants in combating ischemia-reperfusion injury in rat brain.

Liposome-encapsulated antioxidants have been tested in vivo to prevent oxidative attack during cerebral ischemia and reperfusion. Oxidative stress is a causal factor in the neuropathogenesis of ischemic-reperfusion injury. From the therapeutic point of view free chemical antioxidants were almost ineffective to protect cerebral tissues from those oxidative attacks. Thus an attempt has been made to prevent the oxidative damage due to the cerebral ischemic insult by the introduction of chemical antioxidants, ascorbic acid and alpha-tocopherol either encapsulated or intercalated in small unilamellar liposomes. The effectiveness of antioxidant-loaded liposomes was tested against an experimental in vivo rat model of global cerebral ischemia. Oxidative free radical attack on cerebral tissues by the ischemic insult and brief reperfusion was accounted for by the amount of diene production per unit of tissue protein. Diene production in ischemic reperfused rat brain increases almost twofold over that of the normal rats. Prevention of excess diene production has been attributed to rats when they were treated either with L-ascorbic acid-encapsulated liposomes or alpha-tocopherol intercalated liposomes 2 hours prior to the cerebral ischemic insult. Complete restriction of excess diene generation has also been achieved when a mixture of alpha-tocopherol and L-ascorbic acid-encapsulated liposomes were injected 3 hours before the ischemic infraction.