PRODUCTION OF THERAPEUTIC METHOTREXATE DEGRADING ENZYME AND STUDIES ON ITS NANOCOMPLEXES WITH HUMAN SERUM ALBUMIN

Objective: To improve the parent strain of Variovorax paradoxus for the production of methotrexate (MTX) degrading enzyme and to study the serum stability, release kinetics and functionality of the nano complexes of the enzyme with human serum albumin (HSA)Methods: The activity of the enzyme was quantified by using the extinction coefficient of 8300 for the substrate, MTX. The mutant strain of V. paradoxus was isolated by exposing the cells to the UV light (302 nm) so that 50 % of the cells were killed. The enzyme was purified on DEAE-cellulose, and sephadex-G-100 columns and the purity of the enzyme was checked on 10 % SDS-polyacrylamide gel. The enzyme-HSA nano complexes were prepared by adopting desolvation-crosslinking method and their size was determined by using transmission electron microscope.Results: MTX degrading enzymes are required to avoid the toxicity of the MTX during the treatment of cancer. The enzyme from V. paradoxus converts the MTX into non-toxic glutamate and 4-amino-N [10]-methylpteroate, and the culture utilizes the derived glutamate. Improving the production of this enzyme will be beneficial due to its therapeutic application. Different carbon and nitrogen sources didn't improve the production of this enzyme from the parent strain of M. verrucaria. The strain improvement was carried out by using UV radiation to improve the yields of the enzyme. The mutant strain produced around 6 times higher levels of the enzyme compared to the parent suggesting its advantage for the industrial production of the enzyme. Since this enzyme is of microbial origin, it was complexed to the safe carrier, HSA and these complexes showed their size in the nano-range. The nano complexes showed longer stability compared to the native enzyme in the serum, and the enzyme was readily released from the complex suggesting the protective role of the carrier, HSA. The nano complexes showed the higher degradation of MTX in the serum compared to the native enzyme suggesting their better functionality compared to the native enzyme.Conclusion: Usage of mutant strain will be advantageous for the industrial production of the enzyme since it produces higher levels of enzyme compared to the parent strain. Enzyme-HSA nanocomputers will be a better choice for the therapeutic applications since they show better serum stability and functionality compared to the native enzyme.Keywords: Methotrexate, Variovorax paradoxus, Glutamate, 4-amino-N[10]-methylpteroate, Strain improvement, UV radiation, Human serum albumin, Nanocomplexes, Native enzym

Study of physicochemical and antioxidant synergy efficacy of betel leaf dried paste powder

The present study analyses the drying kinetics and changes in qualities during hot air drying of betel leaf paste for the manufacturing of betel leaf paste powder. A comparative study was conducted to evaluate the changes in different properties, including physico-chemical properties, proximate composition and other functional properties. The betel leaf paste dried powders have longer shelf life than raw leaves. Drying of betel leaf paste was conducted in three different levels, i.e., 50, 70 and 80 °C, till the samples reached a constant weight. The samples dried at 80 °C and 70 °C, showed the lowest water activity 0.1, whereas the sample dried at 50 °C, showed the highest water activity of 0.15. The values of protein, fat, crude fibre and ash were observed a little higher in betel leaf powder dried at 70 °C, than in other drying temperatures. This increase in the composition may be the result of higher dry matter in dried powder and less drying time of paste and minimum degradation in nutritional value at 70°C. However, the highest carbohydrate content was observed in betel leaf powder dried at 50 °C. The alcoholic extract of the oven-dried powder exhibited the DPPH radical scavenging activity (at 50 µg/mL) of 41.45, 46.32, and 44.35 at temperatures of 50, 70 and 80 °C respectively. The amount of phenolic content in the ethanolic extract was found to be 307, 322, and 316 mg/g in oven-dried betel leaf paste powder at 50, 70 and 80 °C respectively

LIPASE INHIBITORS FROM PLANTS AND THEIR MEDICAL APPLICATIONS

Obesity and its related disorders have become a major concern across the world. However, there are only few medications for treating obesity. Reducing the fat absorption through the inhibition of pancreatic lipase has become most favorable strategy for treating obesity since pancreatic lipase is a safe target and its inhibition does not alter the central pathways. However, the only available pancreatic lipase inhibitor for the treatment of obesity is orlistat and it is derived from lipstatin which is produced by a microbe, Streptomyces toxytricini. Many pancreatic lipase inhibitors are reported from the plant sources and they can be classified in to saponins, phenols, terpenes, glycosides, alkaloids, carotenoids and polysaccharides. Plant pancreatic lipase inhibitors are reported to show the antiobesity effects in the animal models. However, there is no plant inhibitor in the clinical use. This review describes the different lipase inhibitors from plant sources and their effects on the obesity and its related parameters.Keywords: Obesity, Pancreatic lipase inhibitor, Orlistat, Saponins, Phenols, Terpenes, Glycosides, Alkaloids, Carotenoids, Polysaccharides

Current Status of Assessment of Experimental Skills in Science Education: A Perspective from India

Experimental skills are very important for science students, and it is necessary to visualize them by evaluating student achievement. It is appropriate to acquire experimental skills by carrying out science experiments in labs. Testing the students' experimental skills involves assessing their capacity to use their knowledge in practical ways. Although it is obvious that practical abilities in science are important, it is unclear exactly what these skills are and how they might be most efficiently and accuraters be assisted in understanding the function of practical work and necessary to embrace more recent ally evaluated. Engaging student in high-quality practical work at a younger age can aid in the development of important skills, a thorough knowledge of scientific inquiry, and conceptual comprehension. It is crucial that teacheternative teaching and evaluation techniques. This article reviews how experimental skills are currently assessed and the importance of evaluating these skills in science. Keywords: Experimental Skills; Scientific Thinking; Visualization of Skills; Rubric; Assessment DOI: 10.7176/JEP/15-4-02 Publication date:March 31st 202

SCREENING OF ACTINOMYCETES ISOLATED FROM NATURAL ECOSYSTEM (PERIYAR TIGER RESERVE, KERALA) FOR LIPASE INHIBITORY ACTIVITIES

Objectives: The present study was undertaken to screen pancreatic lipase inhibitory activity of crude extracts of actinomycetes species isolated from the soil sediments of Periyar Tiger Reserve, Kerala. The identified lipase inhibitory activity was partially purified, and the selected isolate was identified by 16S rRNA sequencing. Methods: The preliminary screening for the extracellular lipase inhibitory activities of actinomycetes isolates was performed by inoculating the culture to the test tubes containing inoculation media in submerged condition. The lipase inhibitory activities were again evaluated based on secondary screening on the production media and the strain which produced consistent highest lipase inhibitory activity was selected for further studies. The crude extract from the selected strain was subjected to solvent extraction and partially purified by plain silica gel column (mesh size 100–200 μm; column 300 mm×18 mm) and eluted with different solvents in the increasing order of polarity and all the solvent fractions were checked for lipase inhibitory activity. Results: Based on the secondary screening on the production of media, DDE6 strain showed highest lipase inhibitory activity (96.2%) and was selected for further studies. The dichloromethane extract showed the highest lipase inhibitory activity (96.38%) when compared to other solvents. The partial 16S rRNA sequence analysis of DDE6 isolate confirmed the strain as Curtobacterium oceanosedimentum. Conclusion: Many research publications have reported the isolation of Curtobacterium species and its type strains. However, this is the first time to report its potential to produce lipase inhibitor metabolite under submerged fermentation conditions. Further, studies are needed to be conducted to characterize the active principle of lipase inhibition as well as to elucidate the structure of the extracted compound