α-Glucosidase Inhibition and Antioxidant Properties of Streptomyces sp.: In Vitro
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-Glucosidase Inhibition and Antioxidant Propertiesof Streptomyces sp.: In Vitro
P. Praveen Kumar & J. P. Preetam Raj &I. V. S. Nimal Christhudas & R. Sagaya Jansi &M. Narbert Raj & P. Agastian
Received: 6 February 2013 /Accepted: 8 November 2013 /Published online: 19 November 2013# Springer Science+Business Media New York 2013
Abstract Streptomyces strain isolated from the soil sediment was studied for its in vitro -glucosidase and antioxidant properties. Morphological characterization and 16S rRNA partialgene sequencing were carried out to confirm that the strain Loyola AR1 belongs to genusStreptomyces sp. Modified nutrient glucose broth was used as the basal medium for growthand metabolites production. Ethyl acetate extract of Loyola AR1 (EA-Loyola AR1) showed50 % -glucosidase inhibition at the concentration of 860.502.68 g/ml. Antioxidantproperties such as total phenolic content of EA-Loyola AR1 was 176.831.17 mg of catecholequivalents/g extracts. EA-Loyola AR1 showed significant scavenging activity on 2,2-diphenyl-picrylhydrazyl (50 % inhibition (IC50), 750.501.61 g/ml), hydroxyl (IC50,690.202.38 g/ml), nitric oxide (IC50, 850.501.77 g/ml), and superoxide (IC50,880.081.80 g/ml) radicals, as well as reducing power. EA-Loyola AR1 showedstrong suppressive effect on lipid peroxidation (IC50, 670.502.52 g/ml). Antioxidants of -carotene linoleate model system reveals significantly lower than butylated hydroxyanisole.
Keywords Soil sediment .-Glucosidase inhibition . Antioxidant properties . Streptomyces sp.
Actinomycetes are the most economically and biotechnologically worthful prokaryotes. Theyare responsible for the production of about half of the discovered bioactive secondarymetabolites, notably antibiotics , antitumor agents , immunosuppressive agents ,
Appl Biochem Biotechnol (2014) 172:16871698DOI 10.1007/s12010-013-0650-z
P. Praveen Kumar : J. P. Preetam Raj : I. V. S. Nimal Christhudas : R. Sagaya Jansi :M. Narbert Raj :P. AgastianDepartment of Plant Biology and Biotechnology, Loyola College, Chennai 600034, India
P. Agastian (*)Research Department of Plant Biology and Biotechnology, School of Life Science, Loyola College,Chennai 600034, Indiae-mail: firstname.lastname@example.org
and enzymes . Among the 140 described actinomycetes genera, only a few are responsiblefor the majority of 20,000 microbial natural products identified so far. In particular, the genusStreptomyces accounts for about 80 % of the actinomycetes natural products reported . Inthe course of screening for new metabolites, several studies were carried out in order to isolatenew Streptomyces species from different habitats.-Glucosidase is a very important enzyme responsible for the hydrolysis of dietary
disaccharides into absorbable monosaccharide in microbial system and in small intestine ofanimal digestive system. Glucosidase is not only essential for carbohydrate digestion but it isalso very important for processing of glycoproteins and glycolipids and are also involved invariety of metabolic disorders and other diseases such as diabetes . There are many articlesrelated to antidiabetic compounds reported from Streptomyces hygroscopicus-limoneus  andStreptomyces calvus .
Recent studies focusing on the response of antioxidant system of bacteria, fungi, andactinomycetes are important in terms of biotechnology, such as Streptomyces growth in variousoxidative stress conditions . Reactive oxygen species (ROS) are known to be implicated inmany cell disorders and in the development of many diseases including cardiovasculardiseases, atherosclerosis, chronic inflammation, and so on . Synthetic antioxidants arewidely used but their use is being restricted nowadays because of their toxic and carcinogeniceffects. Thus, interest in finding natural antioxidants, without any undesirable effect, hasincreased greatly . The present study evaluates in vitro -glucosidase inhibition andantioxidant properties of Streptomyces sp. Loyola AR1 isolated from the lake soil of AmbatturIndustrial estate, Tamil Nadu, India.
Materials and Methods
Chemicals and Reagents
All the chemicals used for preparation of different media and reagent were used from Himedia,Merck, Qualigens, etc., 1,1-diphenyl,2-picryl hydrazyl (DPPH), nitro blue tetrazolium (NBT),nicotinamide adenine dinucleotide phosphate reduced (NADH), phenazinemethosulphate(PMS), trichloro acetic acid (TCA), ferric chloride, and butylatedhydroxyltoluene (BHT) wereobtained from Sigma Chemical Co., USA. Ascorbic acid was obtained from SD Fine Chem.Ltd., Biosar, India.
Isolation of Actinomycetes
The Streptomyces strain used in this study was isolated from the Ambattur Lake soil sediments,Tamil Nadu, India with 130636 N latitude 801012 E longitude. Soils from differentplaces of lake were brought to the laboratory in aseptic condition. Actinomycetes from the soilwas isolated by pour plate technique on actinomycetes isolation agar (in gram per liter)containing 2 g sodium casinate, 0.1 g L-asparagines, 4 g sodium propionate, 0.5 g dipotassiumsulphate, 0.1 g magnesium sulphate, 0.001 g ferrous sulphate, 15 g agar, pH 8.1, and 1 Lsterile-distilled water, also supplemented with 20 mg/l actidione and 100 mg/l nalidixic acidwere added to minimize fungal and bacterial growth, respectively. Soil samples were seriallydiluted up to 105 and 0.1 ml of aliquots spread over actinomycetes isolation agar plates. Theplates were incubated at 282 C for 7 days. Strains of actinomycetes were picked out andpurified by repeated streaking on yeast extractmalt extract agar (International StreptomycesProject, ISP2) and were preserved in slants at 42 C.
1688 Appl Biochem Biotechnol (2014) 172:16871698
Identification of Isolate
The Streptomyces strain was characterized morphologically following methods given in theISP . The characters including colony morphology of the strains such as the color of aerialmycelium, reverse side color, size of the colony, and production of diffusible pigments wereobserved after incubation at 282 C for 7 days on actinomycetes isolation agar medium. Themicroscopic morphology of strains such as formation of aerial and substrate mycelium andspore management, which are highly characteristic and useful in the identification of actino-mycetes, were observed by cover slip technique  with light microscopy.
The freshly cultured Loyola AR1 cells were pelleted by centrifuging for 2 min at 12,000 rpmto obtain 1015 mg (wet weight). The cells were suspended thoroughly in 300 l of lysissolution; 20 l of RNase A solution was added, mixed, and incubated for 2 min at roomtemperature. About 20 l of the proteinase K solution (20 mg/ml) was added to the sample;mixed and resuspended cells were transferred to Hibead Tube and incubated for 30 min at55 C. The mixture was vortexed for 57 min and incubated for 10 min at 95 C followed bypulse vortexing. Supernatant was collected by centrifuging the tube at 10,000 rpm for 1 min atroom temperature. About 200 l of lysis solution was added, mixed thoroughly by vortexing,and incubated at 55 C for 10 min. To the lysate, 200 l of ethanol (96100 %) was added andmixed thoroughly by vortexing for 15 s. The lysate was transferred to new spin column and500 l of prewash solution was added to the spin column and centrifuged at 10,000 rpm for1 min and supernatant was discarded. The lysate was then washed in 500 l of wash solutionand centrifuged at 10,000 rpm for 3 min. Of the elution buffer, 200 l was pipetted out andadded directly into the column without spilling and incubated for 1 min at room temperature.Finally, the DNA was eluted by centrifuging the column at 10,000 rpm for 1 min (HipuraStreptomyces DNA spin kit-MB 527-20 pr from HiMedia, Mumbai, India).
PCR Amplification and Sequencing
The 16S ribosomal RNA was amplified by using the thermo cycler (Eppendorf ep. gradient)with Taq DNA polymerase and primers 27F (5AGTTTGATCCTGGCTCAG 3) and 1492R(5ACGGCTACC TTGTTACGACTT 3). The conditions for thermal cycling were as follows:denaturation of the target DNA at 94 C for 4 min followed by 30 cycles at 94 C for 1 min,primer annealing at 52 C for 1 min, and primer extension at 72 C for 1 min. At the end of thecycling, the reaction mixture was held at 72 C for 10 min and then cooled to 4 C. The PCRproduct obtained was sequenced by an automated sequencer (Genetic Analyzer 3130, AppliedBiosystems, USA). The same primers as above were used for sequencing. The sequence wascompared for similarity with the reference species of bacteria contained in genomic databasebanks using the National Center for Biotechnology Information (NCBI) BLAST available athttp://www.ncbi-nlm-nih.gov/. The DNA sequences were aligned and phylogenetic tree wasconstructed by using the Molecular Evolution Genetic Analysis (MEGA) software version 4.0.16S rRNA sequence was then submitted to the GenBank, NCBI, USA.
The selected antagonistic actinomycetes isolate was inoculated into MNG broth separately andincubated at 282 C at 140 rpm for 7 days. After fermentation, broth was harvested and
Appl Biochem Biotechnol (2014) 172:16871698 1689
centrifuged to remove cell debris. Filtrate was collected and stored at 4 C for further use .The bioactive metabolites were recovered from the harvested medium by solvent-extractionmethod. The filtrate was mixed with ethyl acetate (1:1 v/v) and shaken vigorously for 1 h in asolvent extraction funnel. Extraction was continued up to three times with the same solvent.The organic phase was concentrated and used for further studies .
Determination of In Vitro -Glucosidase Inhibition and Antioxidant Properties
In Vitro -Glucosidase Inhibition
In order to investigate the inhibition activit