Abul Kalam Azad Mandal

Potential neuroprotective properties of epigallocatechin-3-gallate (EGCG)

Neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) enforce an overwhelming social and economic burden on society. They are primarily characterized through the accumulation of modified proteins, which further trigger biological responses such as inflammation, oxidative stress, excitotoxicity and modulation of signalling pathways. In a hope for cure, these diseases have been studied extensively over the last decade to successfully develop symptom-oriented therapies. However, so far no definite cure has been found. Therefore, there is a need to identify a class of drug capable of reversing neural damage and preventing further neural death. This review therefore assesses the reliability of the neuroprotective benefits of epigallocatechin-gallate (EGCG) by shedding light on their biological, pharmacological, antioxidant and metal chelation properties, with emphasis on their ability to invoke a range of cellular mechanisms in the brain. It also discusses the possible use of nanotechnology to enhance the neuroprotective benefits of EGCG.

Identification of differentially expressed genes in dormant (banjhi) bud of tea (Camellia sinensis (L.) O. Kuntze) using subtractive hybridization approach.

Growth regulation associated with dormancy is an essential element in plants life cycle that leads to changes in expression of large number of genes. Forward and reverse suppression subtractive hybridization (SSH) libraries were developed to identify and characterize the genes associated with bud (banjhi) dormancy in tea (Camellia sinensis (L.) O. Kuntze). Efficiency of subtraction was confirmed by comparing the abundance of β-actin gene. A total of 17 and 45 unique sequences were obtained from forward and reverse SSH library respectively. Many of the differentially regulated genes have unknown (41.1% and 26.7%) or hypothetical functions (11.7% and 2.2%) in forward and reverse SSH library respectively, while others have a role in cell growth and metabolism. Further, semi-quantitative RT-PCR was carried out for selected genes to validate the quality of ESTs from SSH library. Gene Ontology analysis identified a greater association of these ESTs in cellular metabolic pathways and their relevance to bud dormancy. Based on the EST data, the putative role of identified genes from tea is discussed in relation to dormancy, which includes various metabolic and signalling pathways. We demonstrated that SSH is an efficient tool for enriching up- and down-regulated genes related to bud dormancy in tea. This study represents an attempt to investigate banjhi dormancy in tea under field conditions, and the findings indicate that there is a potential to develop new approaches to modulate dormancy in this species.

Susceptibility Against Grey Blight Disease-Causing Fungus Pestalotiopsis sp. in Tea ( Camellia sinensis (L.) O. Kuntze) Cultivars Is Influenced by Anti-oxidative Enzymes

Reactive oxygen species (ROS) production is the first level of response by a host during stress. Even though the ROS are toxic to cell, when present in a limited amount, they act as a signalling molecule for the expression of defence-related genes and later are scavenged by either enzymatic or non-enzymatic mechanisms of the host. The different anti-oxidative enzymes like glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APO), peroxidase (POD) and polyphenol oxidase (PPO) were estimated, and their activities were compared between infected and healthy leaves of the tolerant and susceptible cultivars of tea. The infected leaves of the susceptible cultivars registered higher amount of enzyme activity when compared with the tolerant cultivars. The study reveals that the more anti-oxidative enzymes, the more susceptible the cultivar will be.

Positive regulation of biochemical parameters by tea polyphenol encapsulated solid lipid nanoparticles at in vitro and in vivo conditions.

Tea polyphenols (TPPs) comprise preventive and therapeutic potentials against cancer, cardiovascular and neurological disorders. Chemical instability of TPP which leads to low bioavailability is the major constrain to its use as therapeutic agent. The authors prepared TPP encapsulated solid lipid nanoparticles (TPP-SLNs) to increase its stability and bioefficacy. Comparison of Fourier transformed infrared spectra of unloaded SLN, free TPP and TPP-SLN indicated encapsulation of TPP. Sustained release of TPP from TP-SLN was observed. TPP-SLN showed prolonged free radical scavenging activity compared with free TPP indicating protection of TPP. TPP-SLN showed activation of Caspases-9 and -3 cascades in breast cancer cell line (Michigan cancer foundation (MCF)-7) at in vitro conditions. Biochemical parameters were altered in Ehrlich ascetic carcinoma (EAC) cell bearing mice compared with normal (uninduced) mice which were ameliorated significantly by oral feeding of TPP-SLN. Oral administration (pre- and post-treated) of TPP-SLN in EAC bearing mice resulted in significant increase of plasma haemoglobin, glucose, superoxide dismutase and catalase when compared with EAC bearing control mice. Other biochemical parameters (cholesterol, bilirubin, triglyceride, urea, total protein, alanine aminotransferase, alkaline phosphatase and aspertate transaminase were significantly decreased on oral administration (pre- and post-treated) of TPP-SLN in EAC bearing mice.

Molecular cloning and characterization of nucleoside diphosphate kinase 1 cDNA in tea

Nucleoside diphosphate kinase (NDPK) operates in the homeostasis of cellular nucleoside triphosphate (NTP) pools and the cytosolic NDPK1 is the main NDPK isoform in plants, accounting for more than 70 % of total NDPK activity in plant. A full length cDNA (697 bp), designated as CsNDPK1 was cloned from Camellia sinensis (L.) O. Kuntze leaves. Sequence analysis of CsNDPK1 shows several motifs, binding and catalytic sites which are highly conserved among other NDPKs. Southern blot analysis revealed that tea genome has two copies of CsNDPK1. Transcription pattern analysis indicated that CsNDPK1 is expressed in all tissues examined, but expressed more in buds than in other organs.

Inhibition of Al(III)-Induced Aβ42 Fibrillation and Reduction of Neurotoxicity by Epigallocatechin-3-Gallate Nanoparticles

RATIONAL Accumulation of amyloid beta fibrils is the pathological hallmark of Alzheimers disease. Epigallocatechin-3-gallate (EGCG) has shown to possess potent anti-amyloidogenic, metal chelation and antioxidant properties. However, its therapeutic potential is limited in-vivo due to its poor bioavailability and stability. Therefore, the present study aims to evaluate the neuroprotective role of EGCG nanoparticles (nanoEGCG) against Al(III)-induced Aβ42 fibrillation in-vitro. METHOD NanoEGCG was synthesized and its physiochemical characterization was performed. In-vitro release profiles and stability of nanoEGCG in simulated gastro-intestinal fluids, along with its antioxidant and metal chelation potential was evaluated. The anti-amyloidogenic potential of nanoEGCG on Aβ42 secondary structure and its morphology was evaluated via induction with Al(III) and nanoEGCG treatment. Further, the effect of Aβ42 on cellular toxicity was also assessed. RESULT NanoEGCG with 96% encapsulation efficiency and a hydrodynamic diameter of 300 nm with spherical to slightly ellipsoid shape was synthesized. EGCG release from the nanoparticle occurred in a sustained manner and was stable when released in simulated gastro-intestinal fluids. The antioxidant and metal chelation potential of nanoEGCG over time was better than its free form. Effective inhibition of both Aβ42 and Al(III) induced Aβ42 fibrillation with nanoEGCG treatment was noted. This was achieved through the generation of soluble Aβ42 amorphous aggregates instead of insoluble Aβ42 oligomers and fibril generation. Significant reduction in cellular toxicity was also noted when treated with nanoEGCG. CONCLUSION In conclusion, this study strengthens the hypothesis that EGCG nanoparticles can inhibit Al(III)-induced Aβ42 fibrillation and its neurotoxicity in-vitro.

Improved bioavailability and pharmacokinetics of tea polyphenols by encapsulation into gelatin nanoparticles

The authors prepared surface modified (with polyelectrolyte layers), tea polyphenols (TPP) encapsulated, gelatin nanoparticles (TPP-GNP) and characterised them. The size of the spherical nanoparticles was ∼50 nm. Number of polyelectrolyte layers and incubation time influenced the encapsulation efficiency (EE); highest EE was noted in nanoparticles with six polyelectrolyte layers (TPP-GNP-6L) incubated for 4 h. TPP released from TPP-GNP-6L in simulated biological fluids indicated protection and controlled release of TPP due to encapsulation. Mathematical modelling indicated anomalous type as a predominant mode of TPP release. TPP-GNP-6L exhibited enhanced pharmacokinetics in rabbit model compared with free TPP. The area under the concentration-time curve and mean residence time were significantly higher in TPP-GNP-6L compared with free TPP which provide an evidence of higher bioavailability of TPP due to encapsulation. The authors demonstrated that encapsulation of TPP into GNPs favoured slow and sustained release of TPP with improved pharmacokinetics and bioavailability thereby can prolong the action of TPP.

Biochemical and molecular studies on the resistance mechanisms in tea [Camellia sinensis (L.) O. Kuntze] against blister blight disease

Tea (Camellia sinensis) plantations are exposed to biotic and abiotic stresses. Among the biotic factors, blister blight (BB), caused by Exobasidium vexans, affects the quality and quantity of the product and demands high fungicide application. A long term solution for disease resistance would require the knowledge of the basic molecular and biochemical changes occurring in plant as an attempt to resist the pathogen and limit the spread of the disease which can further help in developing resistant cultivars using biotechnological tools. Thus, gene expression studies using the cDNA based suppressive subtractive hybridization library, characterization of genes for pathogenesis related (PR) proteins [chitinase (CsCHIT), glucanase (CsGLUC), phenylalanine ammonia lyase (CsPAL)] and genes in flavonoid pathway were accessed in the BB resistant and susceptible cultivars, SA6 and TES34, respectively. Further, biochemical analysis of PR and antioxidant enzymes (POX, APX, SOD) involved in BB resistance have been carried out to investigate the potential molecular and biochemical changes. Various stages of pathogen development had varied impact on PR protein, flavonoid pathway and anti-oxidative enzymes and indicates the possible role of reactive oxygen species, lignins, flavonoids, anthocyanins and other synthesized compounds in acting as antimicrobial/antifungal agents in tea cultivars.

EGCG Nanoparticles Attenuate Aluminum Chloride Induced Neurobehavioral Deficits, Beta Amyloid and Tau Pathology in a Rat Model of Alzheimer’s Disease

Rational: Alzheimer’s disease (AD) is a neurodegenerative pathology characterized by the presence of neuritic plaques and neurofibrillary tangles. Aluminum has been reported to play an important role in the etiology and pathogenesis of this disease. Hence, the present study aimed to evaluate the neuroprotective role of epigallocatechin-gallate (EGCG) loaded nanoparticles (nanoEGCG) against aluminum chloride (AlCl3) induced neurobehavioral and pathological changes in AD induced rats. Method: 100 mg/kg body weight AlCl3 was administered orally for 60 days, which was followed by 10 mg/kg body weight free EGCG and nanoEGCG treatment for 30 days. Morris water maze, open field and novel object recognition tests were employed for neurobehavioral assessment of the rats. This was followed by histopathological assessment of the cortex and the hippocampus in the rat brain. For further validation biochemical, immunohistochemistry and western blot assays were carried out. Result: Aluminum exposure reduced the exploratory and locomotor activities in open field and significantly reduced the memory and learning curve of rats in Morris water maze and novel object recognition tests. These neurobehavioral impairments were significantly attenuated in nanoEGCG treated rats. Histopathological assessment of the cortex and hippocampus of AlCl3 induced rat brains showed the presence of both neuritic plaques and neurofibrillary tangles. In nanoEGCG treated rats this pathology was absent. Significant increase in biochemical, immunohistochemical and protein levels was noted in AlCl3 induced rats. While these levels were greatly reduced in nanoEGCG treated rats. Conclusion: In conclusion, this study strengthens the hypothesis that EGCG nanoparticles can reverse memory loss, neuritic plaque and neurofibrillary tangles formation.

Fabrication of PLA-PEG Nanoparticles as Delivery Systems for Improved Stability and Controlled Release of Catechin

The purpose of this study was to develop an oral delivery system for the controlled release of catechin and evaluate the antioxidant potential and stability of catechin loaded PLA/PEG nanoparticles (CATNP). Nanoparticles were synthesized using a double emulsion solvent evaporation method. The fabricated nanoparticles were relatively small with a hydrodynamic diameter of 300 nm and an encapsulation efficiency of 95%. SEM image analysis showed uniform sized and spherically shaped nanoparticles. In vitro release profiles indicated a slow and sustained release of catechin from the nanoparticle. Stability of the nanoparticle in simulated gastric and intestinal fluids is maintained due to the PEG coating on the nanoparticles, which effectively protected catechin against gastrointestinal enzyme activity. Enhanced inhibition action of free radicals and metal chelation potential was noted when catechin was encapsulated in these polymeric nanoparticles. The reports obtained from this study would provide an opportunity for designing an oral delivery system aimed at inhibiting oxidative stress in the human body.