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: email@example.com
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.
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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 activity of EA-Loyola AR1, an in vitro -glucosidaseinhibition test was performed. -Glucosidase from yeast is used extensively as a screeningmaterial for -glucosidase inhibitors, but the results do not always agree with those obtained inmammals. Therefore, we used the mouse small-intestine homogenate as -glucosidase solu-tion because we speculated that it would better reflect the in vivo state. The inhibitory effectwas measured using the method slightly modified from . After fasting for 20 h, the smallintestine was incised, rinsed with ice-cold saline, and homogenized with 12 ml of maleatebuffer (100 mM, pH 6.0). The homogenate was used as the -glucosidase solution. The assaymixture consisted of 100 mM maleate buffer (pH 6.0), 2 % (w/v) sugar substrate solution(100 l), and the EA-Loyola AR1 (2001,000 g/mL). It was preincubated for 5 min at 37 C,and the reaction was initiated by adding the crude -glucosidase solution (50 l) to it followedby incubation for 10 min at 37 C. The rate of carbohydrate decomposition was calculated asthe percentage ratio to the amount of glucose obtained when the carbohydrate was completelydigested. The rate of inhibition was calculated by the following formula:
Inhibition % hamount of glucose produced by the positive control
amount of glucose produced by the addition of sample= amount of glucose produced by the positive control
Determination of Total Phenolic Content
Total phenolic content of EA-Loyola AR1 was assessed according to the FolinCiocalteaumethod  with some modifications. Briefly, 0.1 ml of extract (2001,000 g/ml), 1.9 ml ofdistilled water, and 1 ml of FolinCiocalteau reagent were seeded in a tube, and then 1 ml of100 g/l Na2CO3 was added. The reaction mixture was incubated at 25 C for 2 h and theabsorbance of the mixture was read at 765 nm. The sample was tested in triplicate and acalibration curve with six data points for catechol was obtained. The results were compared toa catechol calibration curve and the total phenolic content of EA-Loyola AR1 extract wasexpressed as milligram of catechol equivalents per gram of extract.
Determination of Reducing Power
Determination of reducing power in EA-Loyola AR1 was evaluated according to the methodof . Different amounts of the extract (2001,000 g/ml) was suspended in distilled water
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and mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1 % K3Fe(CN)6. Themixture was incubated at 50 C for 20 min; 2.5 ml of 10 % TCAwas added to the mixture andcentrifuged at 3,000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed withdistilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1 %), and the absorbance was measured at700 nm. BHT was used as standard.
DPPH Radical-Scavenging Activity
DPPH radical-scavenging activity of EA-Loyola AR1 was measured according to . Themethanol DPPH solution (0.15 %) was mixed with serial dilutions (2001,000 g/ml) ofthe extract and after 10 min, the absorbance was read at 515 nm. Vitamin C was used asstandard. The antiradical activity was expressed as 50 % inhibition (IC50; in microgram permilligram). The ability to scavenge the DPPH radical was calculated using the followingequation:
DPPH scavenging effect % A0A1 =A0 100 1
Where A0 is the absorbance of the control at 30 min and A1 is the absorbance of the sampleat 30 min. All samples were analyzed in triplicate.
Hydroxyl Radical-Scavenging Activity
The assay was performed as described by the method of  with minor changes. Allsolutions were prepared freshly. One milliliter of the reaction mixture comprised with100 l of 28 mM 2-deoxy-2-ribose (dissolved in phosphate buffer, pH 7.4), 500 lsolution of various concentrations of EA-Loyola AR1 (2001,000 g/ml), 200 l of200 M FeCl3, 1.04 mM EDTA (1:1 v/v), 100 l H2O2 (1 mM), and 100 l ascorbicacid (1 mM). After an incubation period of 1 h at 37 C, the extent of deoxyribosedegradation was measured by thiobarbituric acid reaction. The absorbance was read at532 nm against the blank solution. Vitamin C was used as a positive control. Thescavenging activity was calculated using formula (1).
Nitric Oxide Radical Inhibition Activity
Sodium nitroprusside in an aqueous solution at physiological pH spontaneously generatesnitric oxide; it interacts with oxygen to produce nitrite ions, which can be estimated bythe use of GriessIllosvoy reaction . In the present investigation, GriessIllosvoy reagentwas modified using naphthylethylenediamine dihydrochloride (0.1 % w/v) instead of 1-naphthylamine (5 %). The reaction mixture (3 ml) containing sodium nitroprusside(10 mM, 2 ml), phosphate buffer saline (0.5 ml), and different concentrations of EA-Loyola AR1 (2001,000 g/ml) or standard solution (0.5 ml) was incubated at 25 C for150 min. After incubation, 0.5 ml of the reaction mixture containing nitrite was pipettedand mixed with 1 ml of sulphanilic acid reagent (0.33 in 20 % glacial acetic acid) andallowed to stand for 5 min for completing diazotization. Then, 1 ml ofnaphthylethylenediamine dihydrochloride (1 %) was added, mixed, and allowed to standfor 30 min. A pink-colored chromophore was formed in diffused light. The absorbance ofthese solutions was...