DNSA Method Research Articles

Production of itaconic acid from sweet potato (Ipomoea batata) peel using naturally occurring fungi in solid state fermentation

This study evaluates the potentials of sweet potato peel as a substrate for itaconic acid production in solid state fermentation using naturally occurring fungi. Sweet potato peel was analyzed for proximate composition using standard methods. Fungi were obtained from the peel by solid state fermentation and identified using microscopic and molecular methods. Fermentation for itaconic acid production was done using isolated fungus and Aspergillus niger (ATCC 16888) was used as control. Optimization of fermentation parameters was carried out using standard procedures. Assay for itaconic acid and total titratable acidity (TTA) were analyzed using standard methods. Reducing sugar was obtained using DNSA method. Proximate composition of the peel reveals carbohydrate content to be 79%, protein, 5% and ash, 4%. Fungus isolated was identified as Aspergillus flavus L-2482/2012. Highest itaconic acid production by A. flavus was 7.74±0.00 mg/ml on day 5 at 30 g substrate concentration. Using 2 ml of 1 x 107 spores/ml, A. flavus and A. niger produced 6.97±0.13 and 6.67±0.09 mg/ml respectively. Optimum temperature for itaconic acid production was 30°C for A. niger and A. flavus. TTA ranged between 0.04±0.00 to 0.21±0.01 and 0.05±0.00 to 0.26±0.00 mg/ml for A. niger and A. flavus respectively. The highest reducing sugar, 0.51±0.00 mg/ml was obtained at substrate concentration 40 mg/ml on day 5 by A. flavus while A. niger had reducing sugar value of 0.37±0.00 mg/ml. In conclusion, A. flavus L-2482/2012. has great potentials for itaconic acid production using sweet potato peel in solid state fermentation under optimized conditions.

Biobutanol Production from Rice Husk

Abstract: This research aimed to study biobutanol production using Lignocellulosic renewable substrate Rice Husk, carried out using Clostridium acetobutylicum MTCC 11274 via the Separate hydrolysis and fermentation (SHF) process. The simple sugars were exposed using a delignification process with different concentrations of Ammonia solution and some were hydrolyzed using conc. H2SO4 after getting treated with ammonia. The sugars were estimated using DNSA Method after which samples were fermented. The concentration of Biobutanol was estimated using GC-MS showing 2.2 gm/L in one of the tested samples.

Isolation, Screening, Characterization and Maintenance of Amylase Producing Bacteria from Soil

Abstract: Amylases are amongst most widely used enzymes in industries such as food, fermentation, starch processing, textile, and paper. In the present investigation, bacteria were isolated from soil samples of different areas of Padmashri Vikhe Patil College, Pravaranagar, Maharashtra, India screened for the production of amylase. Soil samples were serially diluted in normal saline and plated on sterile nutrient agar plates. The colonies obtained from higher dilutions were subjected to Gram staining, biochemical reactions, and starch hydrolysis test. Bacterial colony yielding positive starch hydrolysis test were subjected to Amylase activity test by DNSA method. Characteristic feature of the strain indicates that it belongs to the genus Bacillus and will be later used for further characterization. Maximum yield of amylase was obtained after 48hrs of incubation. The isolated amylase producing organism was maintain by using agar slant & glycerol stock method.

SCREENING OF PECTINASE PRODUCING BACTERIA FROM SOIL AND PRODUCTION, PARTIAL PURIFICATION OF ENZYME

Bacterial pectinase enzyme is very much important at industrial level for different processes This present study focus on bacterial pectinase. Pectinase producing bacterial spp.is screen out from soil. About seven cultures of bacterial spp. were isolated from farmland soil sample using pectin agar plates. The selected isolates showed the zone of hydrolysis to confirmed the production of pectinase from the isolates. Among them the culture S4 showed prominent zone of hydrolysis surrounding the colony. Pectinase activity of each isolate were checked at an interval of 24 hrsusing DNSA method to access the enzyme efficiency produced by respective isolates. Keeping the ideal growth conditions the highest pectinase activity obtained among isolates was 11.43 U/ ml, by culture S4.Optimum condition for pectinase production was found at 72hrs. incubation period, 37˚C temperature and alkaline pH of 10 could be the optimum cultural conditions for bacterial strain S4. Bacterial strain S4 gave 40.85 U/ml partially purified enzyme.

Antioxidant and Enzymes Inhibitory Activities of Leaf Extracts of Plant Species Traditionally Used for Medicinal and Spiritual Purposes in Nepal

ABSTRACT The flavonoids and phenolics contents, antioxidant potential, and enzymes (α-amylase, lipase, elastase, tyrosinase, acetylcholinesterase, and butyrylcholinesterase) inhibition potential of leaf extracts from Elaeocarpus sphaericus, Santalum album, Pterocarpus santalinus, and Ziziphus budhensis collected from Ramechhap District of Nepal were evaluated. DPPH assay was used to determine the antioxidant potential, and the DNSA method was used to examine α-amylase inhibition activity. P-NPB, L-DOPA, AAAPVN, acetylthiocholine iodide, and butyrylthiocholine iodide were used as respective substrates for the determination of lipase, tyrosinase, elastase, acetylcholinesterase, and butyrylcholinesterase inhibition activities. The crude extract of P. santalinus showed inhibitory potential against all enzymes, whereas E. sphaericus, S. album, and Z. budhensis extracts showed moderate inhibition activity. However, S. album did not inhibit lipase, elastase, and tyrosinase.

Effect of temperature and duration of thermal pasteurization on polyphenol oxidase (PPO) enzyme activity, total plate count (TPC), physicochemical and organoleptic properties of Cavendish banana fruit juice (Musa cavendishii)

This research aims to determine the effect of temperature and duration of thermal pasteurization process in Cavendish banana juice. Cavendish banana is an abundant cultivated banana that has not been used properly, which is a climacteric fruit that has increased ethylene production after harvesting. Cavendish bananas have great potential as fruit juice. Pasteurization was carried out at temperatures of 55, 60, 65, 70 and 75 ℃ for 5, 10, and 15 minutes. Pasteurization with higher temperature and time can increase the total dissolved sugar and viscosity as well as decrease the pH value and PPO enzyme activity. The reducing sugar content of Cavendish banana juice was determined using the DNSA method with the greatest value at 55 ℃ for 5 minutes, which is 9.53%. Meanwhile, the content of phenol and flavonoid was determined using the Krishnan and Sinija method. The highest value of phenol and flavonoid content at 60 ℃ for 5 minutes, was 0.1728 mg GAE/g and 0.1583 mg QE/g sample. Antioxidant activity was determined using the DPPH method and the greatest percentage of inhibition at 60 ℃ for 5 minutes, which is 61.60%. Vitamin C levels were identified by the UV-Vis spectrophotometry method with the greatest value at 55 ℃ for 5 minutes, which is 1.1409 mg /100 g. The Total Plate Count (TPC) of Cavendish banana juice decreased after pasteurization with the smallest total number of bacteria at 75 ℃ for 15 minutes, with a value of 3.2×101 CFU/ml. Pasteurization process at 75 ℃ for 15 minutes can reduce the activity of PPO enzyme as much as 97%. In the organoleptic test, preferred level of Cavendish banana juice to all attributes of the average hedonic scale with the category of the likes.

Isolation, Screening and Production of Bacterial Cellulase from Cow Dung

Cellulases are the most important industrial enzyme due to their potential application in various industries including pulp and paper, textile, laundry, biofuel production, food and feed industry, brewing and agriculture. The present study was carried out to isolate and characterize cellulase producing microorganisms from different cow dung samples. Four different cow dung samples were collected from variety of cows namely Gir, Holstein, Jersey and Desi and isolation and screening was done to check cellulase producing microorganisms. The obtained 11 isolates were screened for their cellulase activity by using CMC (carboxymethyl cellulose) agar medium. In the current study, 10 cellulase producing isolates were obtained and were characterized morphologically from which 8 isolates were found Gram negative and 2 were found Gram positive. All the 10 cellulase producers were further confirmed for their cellulase producing ability by performing turbidity test. Out of these 10 isolates, GN4 and HN2 with optical density 0.35 and 0.28, respectively were found to be best cellulase producer and were selected for cellulase enzyme production and other further studies. Botssh the isolates were tested for their enzymatic activities by performing DNSA method and protein estimation by Lowry’s method. Cellulase activities were found to be 7.33 µg/ml/min and 3.66 µg/ml/min and the total protein concentrations were found to be 40 µg/ml and 32 µg/ml for GN4 and HN2, respectively. Thus, dungs cow dung can be considered as the excellent source for cellulase producing bacteria.

Antidiabetic profiling, cytotoxicity and acute toxicity evaluation of aerial parts of Phragmites karka (Retz.)

Ethnopharmacological relevancePhragmites karka (Retz.) of family Poaceae is a pristine tropical plant that is well known to the local healers for ailments of diabetes, fever, diarrhea and CNS depression but lacks the scientific evidence behind its traditional usage. Hence, we explicated this plant to find the scientific basis of its traditional utilization. Aim of the studyThe current study aims to find out the antidiabetic potential, toxicity after oral administration and in vitro cytotoxic activity of aerial parts of the plant on HeLa cells. MethodsThe plant was extracted with methanol by maceration and the crude extract was then subjected to solvent partitioning with modified Kupchan method for preparing several fractions. Phytochemical screening and total phenolic content of the plant was first determined through established procedures. Acute toxicity of the plant was studied by orally administering a single high dose (5000 mg/kg) of drug. Cytotoxicity of the methanolic plant extract was determined by measuring the percentage of cell viability on human cervical cancer cell lines, HeLa. In vitro antidiabetic activity was determined through iodine starch and DNSA (3,5-dinitrosalicylic acid) method of α-amylase inhibition. Finally, in vivo oral glucose tolerance test and alloxan induced antidiabetic activity test was performed at 150 and 300 mg/kg body weight doses of plant extract to confirm the in vivo antidiabetic activity. ResultsNo mortality was demonstrated by Phragmites karka in the acute toxicity test. However, signs of cellular toxicity was observed and histopathological studies on major organs exhibited necrosis in liver and kidney. In vitro cytotoxicity assay revealed the death of HeLa cells by DCM (dichloromethane) and n-hexane fractions of plant extract at 100 and 10 μg/mL concentrations. The IC50 value of the fractions were later evaluated by MTT assay (316.1 and 96.7 μg/mL for n-hexane and DCM fractions, respectively). In the iodine starch and DNSA method of α-amylase enzyme inhibitory activity test, substantial inhibition of enzyme was observed with the IC50 values of 2.05 and 2.08 mg/mL, respectively. In the in vivo antidiabetic activity test, considerable reduction in blood glucose level of diabetic mice was detected in both oral glucose tolerance test and alloxan induced antidiabetic activity test. In addition, the microscopic examination of pancreas showed noticeable recovery of pancreatic β cells and the blood lipid profile analysis represented a significant (p < 0.05) reduction of total cholesterol, LDL (low density lipoprotein) and triglyceride level in plant extract treated mice. ConclusionResults of this study reveals that the Phragmites karka extract is toxic at cellular level after oral administration and cytotoxic when tested on HeLa cells. The plant also evidenced hypoglycemic property, possibly through the inhibition of α-amylase enzyme and recovered the pancreatic beta cells along with the improvement of lipid profile of diabetic mice. However, robust studies on this plant is required to isolate the bioactive compounds, elucidate structures and evaluate their mechanism of actions in support of our findings. ClassificationToxicology and Safety, Quality Traditional Medicine.

Extracts of Leaves of Six Locally Available Plants from Bagmati Province of Nepal as Potent Inhibitors of Alpha-amylase, Lipase, Tyrosinase, Elastase, and Cholinesterases

Background: Medicinal plants and their products are gaining global popularity due to their several health benefits. However, the biological activities of the vast majority of medicinal plant species have not been explored yet. In this study, we evaluated the enzyme inhibitory potential of six medicinal plant species involved in digestion, skin-related problems, and neurological problems. Methods: The 80% methanol extracts of leaves of six locally available plants from the Bagmati province of Nepal were analyzed for their flavonoids content, phenolics content, antioxidant activity, and enzymes inhibitory potential. Antioxidant activity was analyzed by the DPPH assay. Alpha-amylase inhibition was carried out by the DNSA method. Lipase, tyrosinase, elastase, acetylcholinesterase, and butyrylcholinesterase inhibitions were analyzed by using p-NPB, L-DOPA, AAAPVN, acetylthiocholine, and butyrylthiocholine as a respective substrate. Results: Among the analyzed plants species, Artocarpus heterophyllus displayed highest α-amylase (IC50=6.28 ± 0.01 μg/mL), lipase (IC50= 475.14 ± 3.17 μg/mL), elastase (IC50= 72.75 ± 3.41 μg/mL) and acetylcholinesterase (IC50= 68.66 ± 1.71 μg/mL) inhibition, whereas, Actinidia deliciosa displayed highest tyrosinase inhibition (IC50=139.87 ± 0.72 μg/mL) and butyrylcholinesterase inhibition (IC50= 18.32 ± 0.44 μg/mL). Furthermore, Jasminum humile showed no inhibitory tendencies against elastase and Lygodium japonicum showed no inhibition towards lipase and elastase at the given concentration range. Conclusions: Our study revealed that A. deliciosa and A. heterophyllus are the potential source of α- amylase, lipase, elastase, acetylcholinesterase, and butyrylcholinesterase inhibitors. Based on our findings, we concluded that the analyzed plant species are of great scientific interest to the pharmaceutical, cosmetics, and food industries.

Platinum nanoparticle-carbon nanotubes dispersed in gum Arabic-corn flour composite-enzymes for an electrochemical sucrose sensing in commercial juice

The aim of the present study was to fabricate a rapid analytical platform for the analysis of saccharide in foods using new bio-composite of nanomaterials in the natural polysaccharides, gum Arabic (GA) and corn flour (CF). The hydrogel was reinforced by metal nanoparticles (PtNPs) anchored with carboxyl-functionalized carbon nanotubes (MWCNTs). The GA-CF composite was investigated for the possibility of co-immobilization of Invertase (Inv) and glucose oxidase (GOx). Physical immobilization of Inv-GOx in the form of thin film resulted in high enzyme retention efficiency (Inv-96.5% and GOx-92%) with excellent storage stability, up to 6 months at room temperature, for both enzymes. The resulting bioactive films were studied for various physicochemical parameters including reproducibility, pH, nanomaterial loading capacity, operational stability, and shelf life. The morphological characterization of the nanocomposite was examined by transmission electron microscopy, and it revealed that the diameter of PtNPs changed from ~ 3.0 to 21 nm due to outer surface coating after immobilization. Uniform dispersion of carbon nanowire network embedded in bi-enzymes/GA-CF composite film was also observed under scanning electron microscopy. Entrapment of MWCNTs and PtNPs in GA-CF carrier resulted in an improved catalytic efficiencies and loading capacity of both enzymes due to larger surface area for entrapment. Screen-printed carbon electrodes (SPCEs) were modified with this novel enzyme-nanocomposite matrix and an electrochemical biosensor (Inv-GOx-MWCNTs-AgNPs/SPCE) was thus developed. The biosensor exhibited excellent sensitivity, good reproducibility, long-term stability, fast amperometric response to sucrose with wide linear range of 1 × 10−4 to 1 × 10−9 mol L−1 with a correlation coefficient of 0.99, and better limit of detection (1 × 10−9 mol L−1) as compared to previous sensors. The sucrose analytical capabilities of the sensor were tested on commercial fruit, vegetable, and mix juice samples, and the validity of the measurement was confirmed by standard DNSA method.

Isolation, characterization and purification of xylanase producing bacteria from sea sediment

Xylan is the most important natural hemicellulose which has several industrial applications such as food, textile, bleaching of cellulose pulp, seed germination, degumming and agro waste treatment. Micro-organisms play a major role in the production of xylanase enzyme. In the present research, an attempt has been made to produce the xylanase enzyme from bacterial strains. The sea sediment was collected from Kovalam beach, Chennai (Latitude: 8.4004° North, Longitude: 76.9787° East) and serial dilution was done. The serially diluted sample was plated on xylan agar medium. The isolates were characterized and identified based on morphological, biochemical and physiological characters. The zones of hydrolysis for twelve xylan utilizing bacterial isolates were obtained. Of the 12 bacterial isolates, the species obtained from the serial dilution of 10−8 showed higher activity. The single colonies from the xylan agar plate was isolated and grown in nutrient broth supplemented with xylan in shake flask. The culture medium was centrifuged and the supernatant was used as crude enzyme. The enzyme partially purified to homogeneity by a combination of Ammonium-sulphate precipitation and dialyzed using culture supernatant as crude enzyme. The activity of the enzyme xylanase was assayed by DNSA method. The enzyme was optimally active at temperature 55 ºC and pH 9.0. The enzyme showed 95%, 90%, and 85% thermal stability at 55 °C, 60 °C and 65 °C. The enzyme was stable over a broad pH range of 8.0–10.0.

IN VITRO ANTI-DIABETIC ACTIVITY OF MICROENCAPSULATED AND NON-ENCAPSULATED ASTAXANTHIN

Objective: Diabetes is a long term condition which indicates the high blood pressure. The symptoms indicates, polyuria (frequent urination), they will become increasingly thirsty (polydipsia) and hungry (polyphagia). Many drugs has been discovered for curing diabetes. Recent studies reported that the administration of astaxanthin reduces the blood pressure in the diabetic patient. Astaxanthin is a powerful antioxidant found in wide variety of aquatic living organism which has wide applications in pharmacological studies.Methods: In vitro antidiabetic study of both encapsulated and non-encapsulated astaxanthin such as DNSA method, starch-iodine color assay method and α glycosidase enzymes assay was carried out.Results: The results of the present study indicated that both encapsulated and non-encapsulated astaxanthin shows higher antidiabetic activity in all the method. Each test samples possess the best activity when compared to standard drug acarbose.Conclusion: The present study, it is concluded that both non-encapsulated and encapsulated astaxanthin exhibit good antidiabetic activity.

NERIUM OLEANDER LINN. IN VITRO ALPHA AMYLASE INHIBITORY POTENTIAL OF STEM AND ROOT EXTRACTS

Objective: Extraction and evaluation of the antidiabetic activity of extracts from stem and roots of Nerium oleander (Apocynaceae) Linn.Methods: Stem and roots of N. oleander were collected, dried and extracted by using well-established methods for alkaloids, flavonoids, steroids and crude extracts in polar and non-polar solvents. Evaluation of their antidiabetic activity was done with salivary alpha-amylase and starch as a substrate using chromogenic DNSA (2,4-Di nitro Salicylic Acid) method and Starch-iodine method. All experiments were performed in 3 different sets each in triplicates. The data are expressed as mean±SEM (standard error of the mean).Results: The highest inhibition for stem was found in its free flavonoid extract at the concentration of 1.5 mg/ml, with percent inhibition 48.35±1.36 % and an IC50 value of 1.774 g/ml while in case of root, highest inhibition was obtained at 1.5 mg/ml of pet ether extract, with % inhibition 52±0.40 % and IC50 value 1.583 g/ml and at 1.5 mg/ml of methanol extract, with % inhibition 42.12±1.12 % and an IC50 value 1.729 g/ml. 8 (5 of stem and 3 of root) out of 14 tested extracts have shown good inhibitory potential. Extracts of the stem were found to be more potent than root extracts.Conclusion: Though stem extracts were found to be a more potent hypoglycemic agent than root extracts, however, extracts of both parts have good antidiabetic potential and both might be fruitful in managing the postprandial hyperglycemia.

Synthesis, Characterization, Docking and Biological Evaluation of Tetra Hydro Imidazo [1, 2-a] Pyrazine Derivatives

Novel Tetra hydro imidazo[1, 2-a] pyrazine derivatives have been synthesized by Treatment of pyrazine-2carboxylic acid with thionyl chloride in methanol yielded methyl pyrazine-2-carboxylate. This up on treatment with alcoholic ammonia gave pyrazinamide, further it was undergone Hoffman's degradation, gave 2-amino pyrazine after that it was refluxed for 26 hrs with chloro acetaldehyde, sodium bicarbonate gave imidazo[1, 2-a] pyrazine and hydrogen gas was passed on it in the presence of palladium on carbon yielded tetra hydro imidazo[1, 2-a] pyrazine, which was treated with different substituent's to get the novel derivatives. Synthesized compounds were identified and characterized by Melting point, TLC, FT-IR, 1H and 13C NMR, MASS and percent purity done by HPLC. Based on characterization and percent purity novel derivatives were subjected for molecular docking, QSAR studies and pharmacological evaluation. Selected compounds were docked on Anti oxidant activity using “Thioredoxin peroxidase B” as a target site. QSAR parameters like partition coefficient was studied and showed polar surface area below 140 A0 and passed ADME parameters. Analysis of docking and QSAR studies suggested that remarkable inhibitory activity was exhibited by the derivatives. Hydrogen bond interactions were mapped to confirm their potencies. They were evaluated for In-Vitro Anti-oxidant activity by DPPH method and In-Vitro Anti-diabetic activity by alpha-amylase inhibition assay by DNSA method. Results suggested that electron withdrawing groups like nitro, bromo having compounds shown potent activity. Rest of compounds showed mild to moderate activity.

Production of Cellulase and Xylanase by &amp;lt;i&amp;gt;Aspergillus terreus&amp;lt;/i&amp;gt; KJ829487 Using Cassava Peels as Subtrates

Cassava (Manihot esculenta, Crantz) is one of the most important food plants in West Africa. Its peels are made up of cellulose, hemicellulose and lignin. This lignocellulolytic biomass can be converted using microbial enzymes to fermentable sugars which have wide range of biotechnological relevance in many fermentation processes. The aim of this study is to screen filamentous fungi from decaying cassava peels that are good producers of xylanases and cellulases. Decaying parts of cassava peels were obtained and brought to the laboratory for further work. Fungi were isolated, identified and screened for cellulase and xylanase production. Isolate with highest frequency of occurrence and enzyme production was identified using phenotypic and molecular method. Optimisation of growth conditions for enzymes production was monitored using the DNSA method, also saccharification of cassava peel were carried out using the enzymes obtained from the isolate. Aspergillus terreus KJ829487 was the predominant fungus. It produces cellulases and xylanases optimally at 40°C, pH 6 and 8, utilising carboxymethylcellulose (CMC) or xylose and yeast extracts as its carbon and nitrogen sources respectively. Saccharification of the peels yielded 584 mg/L glucose, 78 mg/L xylose and 66 mg/L rhamnose. Aspergillus terreus KJ829487 obtained from cassava peels have the ability to produce high concentration cellulases and xylanases which effectively hydrolysed the lignocelluloses’ biomass to fermentable sugars.

Anti-diabetic potential of flavonoids and other crude extracts of stem bark of Mangifera indica Linn: A comparative study

One important therapeutic approach for the treatment of Type 2 Diabetes Mellitus is by decreasing the postprandial increase of glucose. This is possible by inhibiting carbohydrate hydrolyzing enzymes like alpha-amylase. In the present study, the percentage inhibitory effects of flavonoids and crude extracts isolated from stem bark of Mangifera indica Linn. collected from Jaipur and Bharatpur districts of Rajasthan were studied with salivary alpha amylase and starch as substrate using chromogenic DNSA method &amp; starch iodine method. All experiments were performed in 3 different sets each in triplicates. The data are expressed as mean ± SEM (standard error of the mean). The results clearly indicated that highest inhibition (57.08% and 58.24%) was obtained at the concentration of 1.5 mg/ml of flavonoids extracted from plants in both Jaipur and Bharatpur districts respectively, with an IC50 value of 0.009 mg/ml and 0.021 mg/ml respectively. All crude extracts have shown less than 50% inhibition of alpha amylase activity. Thus the results are clearly indicating that the flavonoids might be more effective than other crude extracts in lowering postprandial hyperglycemia.

Effect of different C: N sources on the activity of alkaline α-amylase from Bacillus licheniformis

Amylases are among the most important enzymes and are of great significance in present day biotechnology. Alkaline amylases are used in the starch, textile industries and as an ingredient in detergents. Water samples are collected from Lonar lake of Buldana district (M.S.). The collected water samples are analyzed for isolation of bacteria on the nutrient agar which directly prepared in Lonar lake water. Bacterial isolate is identified on the basis of cultural, morphological and biochemical characterization. The results are found that the Bacillus licheniformis is produced efficient zone of starch hydrolysis on the starch agar by producing the enzyme alkaline α -amylase. The optimum α - amylase was produced by using different carbon and nitrogen sources. Then assay of the activity of alkaline α amylase performed by DNSA method. The optimum alkaline amylase production was observed in the 1% starch as a carbon source and 0.5% peptone and 0.5% meat extracts as a nitrogen source .

Reducing values: dinitrosalicylate gives over-oxidation and invalid results whereas copper bicinchoninate gives no over-oxidation and valid results

A comparative study was made between two carbohydrate reducing value methods, a relatively old, highly alkaline, 3,5-dinitrosalicylic acid (DNSA) method and a relatively newer, low alkaline (pH 10.5), copper bicinchoninate (CuBic) method. Reducing values for a series of equimolar amounts of maltose−maltohexaose, isomaltose−isomaltohexaose, and cellobiose−cellohexaose were compared by the two methods. The DNSA method gave over-oxidation for equimolar amounts of all three of the oligosaccharide series. The amount of oxidation increased as the sizes of the oligosaccharides increased, giving inflated, inaccurate reducing values. The CuBic method gave constant reducing values, for equimolar amounts of the oligosaccharides, indicating that there was no over-oxidation, as the sizes of the oligosaccharides were increased. The two methods were used to determine the number average molecular weights (MWn) for six polysaccharides. The DNSA method was not able to determine the MWn for any of the polysaccharides tested due to the low sensitivity of the method, compared with the CuBic method that did not give over-oxidation and gave reasonable MWn values for all six of the polysaccharides tested.

Comparative study of alpha amylase inhibitory activity of flavonoids of Vitex negundo Linn. and Andrographis paniculata Nees

Background: One important therapeutic approach for the treatment of Type 2 Diabetes Mellitus is by decreasing the postprandial increase of glucose. This is possible by inhibiting certain carbohydrate hydrolyzing enzymes like alpha‑amylase. Aim: The objective of the present study was to evaluate the alpha amylase inhibitory activity of flavonoids extracts of different parts of Vitex negundo Linn and Andrographis paniculata Nees. Materials and Methods: In the present study, the percentage inhibitory effect of flavonoids isolated from different parts of Vitex negundo and Andrographis paniculata were studied with salivary alpha amylase and starch as substrate using chromogenic DNSA method and starch iodine method for qualitative estimation. Statistical Analysis: All experiments were performed in 3 different sets with each set in triplicates. The data are expressed as mean ± SEM (standard error of the mean). Statistical difference and linear regression analysis were performed using Graph pad prism 5 statistical software. Statistical analysis was performed for ANOVA (analysis of variance) using Statistical Package for Social Sciences (SPSS) version 11.5. Values of P which were ≤0.05 were considered as significant. Results and Conclusion: The results clearly indicated that highest inhibition (62.22%) was obtained at the concentration of 1.5 mg/ml of flavonoids extracted from the leaves of A. paniculata, with an IC50 value of 0.004 mg/ml. Except Vitex leaf flavonoids extract, all other tested flavonoids of different parts of both the plants have shown more than 50% inhibition of alpha amylase activity. Thus the results are clearly indicating that the flavonoids of V. negundo and A. paniculata might be effective in lowering post prandial hyperglycemia. Key words: Alpha amylase inhibition, Andrographis paniculata, flavonoids, post‑prandial hyperglycemia, Vitex negundo

Cellulase Activity and Kinetics in Rice Grasshopper Hieroglyphus banian (Orthoptera: acrididae)

Grasshopper ( Hieroglyphus banian ), an orthopteran species is considered to be a serious pest of rice plant in India. To study the activity and kinetics of cellulase in grasshopper this experiment is undertaken and accordingly foregut and midgut homogenates were prepared. The cellulase enzyme activity and kinetics was measured by DNSA method of Miller,(1959) by taking different concentrations of crystalline cellulose substrate in spectrophotometer at 540nm. Cellulase activity in foregut and midgut were found to be 0.482 u/mg of tissue and 0.687u/mg of tissue respectively. K m values were determined separately both in foregut and midgut and were found to be 3 mg/dl and 2 mg/dl respectively. Experimental data indicates the presence of high cellulolytic activity in the midgut which may suggest that cellulases of endogenous origin are present in this organism. It can be expected that in future, detailed study of these efficient lignocellulolytic systems will help in identification of novel enzymes possessing features that optimize biotechnological applications for the biofuel industry. Moreover, identification of crucial insect cellulases may help in the development of insecticidal technologies aimed at inhibiting their vital digestive role. Considering that this grasshopper species is a pest of paddy and grass, characterization of insect cellulolytic systems may aid in reducing the grasshopper pest attack in Indian subcontinent.