α-glucosidase inhibition and antioxidant properties of streptomyces sp.: in vitro

α-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 α-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 % α-glucosidase inhibition at the concentration of 860.50±2.68 μg/ml. Antioxidantproperties such as total phenolic content of EA-Loyola AR1 was 176.83±1.17 mg of catecholequivalents/g extracts. EA-Loyola AR1 showed significant scavenging activity on 2,2-diphenyl-picrylhydrazyl (50 % inhibition (IC50), 750.50±1.61 μg/ml), hydroxyl (IC50,690.20±2.38 μg/ml), nitric oxide (IC50, 850.50±1.77 μg/ml), and superoxide (IC50,880.08±1.80 μg/ml) radicals, as well as reducing power. EA-Loyola AR1 showedstrong suppressive effect on lipid peroxidation (IC50, 670.50±2.52 μg/ml). Antioxidants of β-carotene linoleate model system reveals significantly lower than butylated hydroxyanisole.

Keywords Soil sediment .α-Glucosidase inhibition . Antioxidant properties . Streptomyces sp.

Introduction

Actinomycetes are the most economically and biotechnologically worthful prokaryotes. Theyare responsible for the production of about half of the discovered bioactive secondarymetabolites, notably antibiotics [4], antitumor agents [7], immunosuppressive agents [23],

Appl Biochem Biotechnol (2014) 172:1687–1698DOI 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 protected]

and enzymes [29]. 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 [6]. Inthe course of screening for new metabolites, several studies were carried out in order to isolatenew Streptomyces species from different habitats.

α-Glucosidase is a very important enzyme responsible for the hydrolysis of dietarydisaccharides 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 [19]. There are many articlesrelated to antidiabetic compounds reported from Streptomyces hygroscopicus-limoneus [9] andStreptomyces calvus [22].

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 [35]. 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 [12]. 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 [31]. The present study evaluates in vitro α-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 13°06′36″ N latitude 80°10′12″ 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 10−5 and 0.1 ml of aliquots spread over actinomycetes isolation agar plates. Theplates were incubated at 28±2 °C for 7 days. Strains of actinomycetes were picked out andpurified by repeated streaking on yeast extract–malt extract agar (International StreptomycesProject, ISP2) and were preserved in slants at 4±2 °C.

1688 Appl Biochem Biotechnol (2014) 172:1687–1698

Identification of Isolate

The Streptomyces strain was characterized morphologically following methods given in theISP [32]. 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 28±2 °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 [27] with light microscopy.

DNA Preparation

The freshly cultured Loyola AR1 cells were pelleted by centrifuging for 2 min at 12,000 rpmto obtain 10–15 mg (wet weight). The cells were suspended thoroughly in 300 μl of lysissolution; 20 μl of RNase A solution was added, mixed, and incubated for 2 min at roomtemperature. About 20 μl 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 5–7 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 μl of lysis solution was added, mixed thoroughly by vortexing,and incubated at 55 °C for 10 min. To the lysate, 200 μl of ethanol (96–100 %) was added andmixed thoroughly by vortexing for 15 s. The lysate was transferred to new spin column and500 μl 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 μl of wash solutionand centrifuged at 10,000 rpm for 3 min. Of the elution buffer, 200 μl 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 (5′AGTTTGATCCTGGCTCAG 3′) and 1492R(5′ACGGCTACC 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.

Extraction

The selected antagonistic actinomycetes isolate was inoculated into MNG broth separately andincubated at 28±2 °C at 140 rpm for 7 days. After fermentation, broth was harvested and

Appl Biochem Biotechnol (2014) 172:1687–1698 1689

http://www.ncbi-nlm-nih.gov/

centrifuged to remove cell debris. Filtrate was collected and stored at 4 °C for further use [36].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 [2].

Determination of In Vitro α-Glucosidase Inhibition and Antioxidant Properties

In Vitro α-Glucosidase Inhibition

In order to investigate the inhibition activity of EA-Loyola AR1, an in vitro α-glucosidaseinhibition test was performed. α-Glucosidase from yeast is used extensively as a screeningmaterial for α-glucosidase inhibitors, but the results do not always agree with those obtained inmammals. Therefore, we used the mouse small-intestine homogenate as α-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 [8]. 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 α-glucosidase solution. The assaymixture consisted of 100 mM maleate buffer (pH 6.0), 2 % (w/v) sugar substrate solution(100 μl), and the EA-Loyola AR1 (200–1,000 μg/mL). It was preincubated for 5 min at 37 °C,and the reaction was initiated by adding the crude α-glucosidase solution (50 μl) 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 %ð Þ ¼hamount of glucose produced by the positive controlð Þ–ðamount of glucose produced by the addition of sampleÞ= amount of glucose produced by the positive controlð Þ

i� 100

Antioxidant Properties

Determination of Total Phenolic Content

Total phenolic content of EA-Loyola AR1 was assessed according to the Folin–Ciocalteaumethod [33] with some modifications. Briefly, 0.1 ml of extract (200–1,000 μg/ml), 1.9 ml ofdistilled water, and 1 ml of Folin–Ciocalteau 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 [30]. Different amounts of the extract (200–1,000 μg/ml) was suspended in distilled water


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 [13]. Themethanol DPPH solution (0.15 %) was mixed with serial dilutions (200–1,000 μg/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 %ð Þ ¼ A0−A1ð Þ=A0 � 100 ð1Þ

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 [10] with minor changes. Allsolutions were prepared freshly. One milliliter of the reaction mixture comprised with100 μl of 28 mM 2-deoxy-2-ribose (dissolved in phosphate buffer, pH 7.4), 500 μlsolution of various concentrations of EA-Loyola AR1 (200–1,000 μg/ml), 200 μl of200 μM FeCl3, 1.04 mM EDTA (1:1 v/v), 100 μl H2O2 (1 mM), and 100 μl 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 [11]. 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 (200–1,000 μg/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 measured at 540 nm. Vitamin C was used as positive control. Thescavenging activity was calculated using the formula (1).


Superoxide Anion Radical-Scavenging Activity

Superoxide anion radical-scavenging activity of EA-Loyola AR1 was determined by moni-toring the competition of those with NBT for the superoxide anion generated by the PMS–NADH system [21]. Superoxide radicals were generated in 1 ml of 20 mM Tris–HCl bufferpH 8.0 containing 0.05 mM NBT, 0.01 mM PMS, and different concentration of extracts(200–1,000 μg/ml) were preincubated for 2 min. The reaction was initiated by the addition of0.078 mM NADH. Blue chromogen, formed due to NBT reduction was read at 560 nm.Results were expressed as percentage of inhibition of superoxide radicals. Vitamin C was usedas a positive control. The scavenging activity was calculated using the formula (1).

Lipid Peroxidation Inhibition

The inhibition effect of EA-Loyola AR1 on lipid peroxidation was determined according to thethiobarbituric acid method. FeCl2–H2O2 was used to induce liver homogenate peroxidation[39]. In this method, 0.2 ml of different concentration of extract (200–1,000 μg/ml) was mixedwith 1 ml of 1 % liver homogenate (each 100 ml homogenate solution contains 1 g rat liver);then 50 μl of FeCl2 (0.5 mM) and H2O2 (0.5 mM). The mixture was incubated at 37 °C for60 min; then 1 ml of trichloroacetic acid (15 %) with thiobarbituric acid (0.67 %) was addedand the mixture was heated in boiling water for 15 min. The absorbance was recorded at532 nm. Vitamin C was used as positive control. The percentage of inhibition was calculatedusing the formula (1).

Antioxidant Activity of EA-Loyola AR1 Using β-Carotene Linoleate Model System

The antioxidant activity of EA-Loyola AR1 was evaluated by β-carotene linoleate model [26].A solution of β-carotene was prepared by dissolving 2 mg of β-carotene in 10 ml ofchloroform. Two milliliters of this solution were pipetted into a 100 ml round-bottom flask.After chloroform was removed under vacuum, 40 mg of purified linoleic acid, 400 mg ofTween 40 emulsifier, and 100 ml of aerated distilled water were added to the flask withvigorous shaking. Aliquots (4.8 ml) of this emulsion were transferred into different test tubescontaining different concentrations of the extracts (200–1,000 μg/ml). As soon as the emulsionwas added to each tube, the zero time absorbance was measured at 470 nm. The tubes werethen placed at 50 °C in a water bath. Butylated hydroxyanisole (BHA) was used as positivecontrol. Antioxidant activity (AA) was calculated using the following equation:

AA ¼ β−carotene content after 2 h of assay=initial β−carotene contentð Þ � 100

Statistical Analysis

The data for biochemical and physiological parameters were analyzed and expressedas mean±SD. The IC50 values were calculated from linear regression analysis. Resultswere processed by computer program, Microsoft Excel (2007).

Results

The morphological properties of Streptomyces strain showed subsequent changes insubstrate or vegetative mycelium, the nature of aerial mycelium, size, and shape


shown in (Fig. 1a, b; Table 1) and 16S rRNA partial sequences strongly proved thatLoyola AR1 belongs to the genus Streptomyces sp. A phylogenetic tree was con-structed based on neighbor-joining method (Fig. 2). In this, bioactive metabolitesrecovered from MNG broth and ethyl acetate was used for solvent extraction. Theresult for in vitro α-glucosidase inhibition assay for EA-Loyola AR1 and acarbosewere shown in (Table 2). Total phenolic content of EA-Loyola AR1 was found to be176.83±1.17 mg catechol equivalent/gram extract, respectively. Figure 3a shows thereductive capability of EA-Loyola AR1 that increased with the quantity of the sample.The extract could reduce the most Fe3+ ions, which had a lesser reductive activitythan the standard of BHT. EA-Loyola AR1 exhibited a significant dose-dependentinhibition of DPPH activity with a IC50 at a concentration of 750.50±1.61 μg/ml. TheIC50 value of vitamin C was 440.12±2.25 μg/ml (Fig. 3b). The concentrations for50 % hydroxyl radical scavenging inhibition were found to be 690.20±2.38 and290.10±2.55 μg/ml for the EA-Loyola AR1 and vitamin C shown in (Fig. 3c). Thescavenging of nitric oxide by EA-Loyola AR1 was increased in a dose-dependentmanner, 50 % of nitric oxide was scavenged at the concentration of 850.50±1.77 μg/ml. The IC50 value of vitamin C was 510.12±2.34 μg/ml (Fig. 3d). The superoxideanion derived from dissolved oxygen by phenazinemethosulphate/NADH couplingreaction reduces nitro blue tetrazolium. Superoxide anion radical scavenging activityof EA-Loyola AR1, vitamin C. The IC50 values, 880.08±1.80 and 470.30±2.73 μg/

Fig. 1 a Pure culture of Loyola AR1 strain. b Gram staining photograph of Loyola AR1 (40×)

Table 1 Morphological character-ization of Loyola AR1 strain Culture character Growth response

Growth under anaerobic condition −Gram staining +

Growth and shape Substrate, circular

Spores Pink color

Motility Nonmotile

Diffusible pigments −Optimum days for growth 7 days


ml, respectively, shown in (Fig. 3e). EA-Loyola AR1 showed inhibition of lipid peroxidationeffect with 50% of inhibition at 670.50±2.52 μg/ml. The IC50 value of vitamin C was 560.30±2.52 μg/ml, respectively, shown in (Fig. 3f). In the β-carotene linoleate system, β-caroteneundergoes rapid discoloration in the absence of antioxidants. The addition of extractsto this system prevents the bleaching of β-carotene at different degrees. EA-LoyolaAR1 of β-carotene bleaching on a dose-dependent manner. Based on 120 min reactiontime (Fig. 3g), the extract showed 50 % inhibition at 580.20±1.43 μg/ml and thevalue for BHA was 230.12±1.48 μg/ml.

Discussion

Actinomycetes are abundant in soils with high organic matter and low moisturecontents [20]. In the present study, Loyola AR1 strain was isolated from Ambattur

Fig. 2 Neighbor-joining phylogenetic tree analysis of Streptomyces sp. Loyola AR1

Table 2 α-Glucosidase inhibitionof EA-Loyola AR1 Sample Concentrations

(μg/ml)% of Inhibition IC50 (μg/ml)

EA-Loyola AR1 200 15.48±0.77 860.50±2.68

400 28.78±1.51

600 34.17±1.45

800 47.81±2.78

1,000 57.74±2.59

Acarbose 200 32.15±2.87 500.50±1.33

400 43.26±2.38

600 56.39±0.29

800 70.20±2.31

1,000 80.47±2.95


Lake soil sediments, Tamil Nadu, India. Streptomyces Loyola AR1 was identifiedaccording to Bergey’s Manual of Determinative Bacteriology [37]. Further, genomicDNA preparation and PCR amplification of Loyola AR1 strain resulted in 1,500 bp amplicon.16S rRNA sequence of the strain Streptomyces sp. Loyola AR1 was submitted to the GenBank(HM163569).

The drugs for lowering glucose are inhibitors of α-glucosidase enzyme. This enzymebreaks down the carbohydrates into absorbable monosaccharides [34]. Therefore, investi-gation on such agents from new, unexplored sources has become important. Also, there isa need to find new, safe, and effective therapeutic agents for the treatment of manydiseases and disorders associated with carbohydrate metabolism. For this purpose, weinvestigated the inhibitory effect of EA-Loyola AR1 extract from lake soil-derivedStreptomyces strain Loyola AR1 on α-glucosidase. There are reports on enzyme inhibitorproducing actinomycetes [15, 16].

Antioxidant properties of EA-Loyola AR1 were evaluated with varying parameters.Phenolic compounds were considered as a major group of compounds that contributedto the antioxidant activity [40]. The reducing power increased with increasing con-centration of the EA-Loyola AR1. The reducing capacity of a compound may serve asa significant indicator of its potential antioxidant activity [25]. DPPH test is usuallyused as the reduction of alcoholic DPPH solution in the presence of a hydrogen-donating antioxidant, due to the formation of the nonradical form DPPH-H by thereaction [5]. EA-Loyola AR1 has the ability to reduce the stable radical DPPH toyellow-colored diphenylpicrylhydrazine. The hydroxyl radical is an extremely reactivefree radical formed in biological systems and has been implicated as a highlydamaging species in free radical pathology, capable of damaging almost every mole-cule found in living cells [14]. Hydroxyl radical-scavenging capacity of an extract isdirectly related to its antioxidant activity [3]. EA-Loyola AR1 inhibited free radical-mediated deoxyribose damage remarkably. It has been reported that reducing power isassociated with antioxidant activity and may serve as a significant reflection of theantioxidant activity [28]. Compounds with reducing power indicate that they areelectron donors, and can reduce the oxidized intermediates of lipid peroxidationprocesses so that they can act as primary and secondary antioxidants [38]. For themeasurements of the reductive ability, we studied the Fe3+ to Fe2+ transformation inthe presence of EA-Loyola AR1. Nitric oxide radical inhibition study showed that theextract was a potent scavenger of nitric oxide. Scavengers of nitric oxide competedwith oxygen leading to reduced production of nitric oxide [24]. Superoxide, the one-electron reduced form of molecular oxygen, is a precursor of other ROS such ashydrogen peroxide, hydroxyl radical, and singlet oxygen that have the potential ofreacting with biological macromolecules and thereby inducing tissue damages [1].These results clearly indicated that EA-Loyola AR1 was a potent scavenger ofsuperoxide radicals in a dose-dependent manner. Lipid peroxidation is an oxidativealteration of polyunsaturated fatty acids in the cell membranes that generates anumber of degradation products. Malondialdehyde, one of the products of lipidperoxidation, has been studied widely as an index of lipid peroxidation and as amarker of oxidative stress [17]. β-Carotene in this model system undergoes rapiddiscoloration in the absence of an antioxidant. This is because of the coupledoxidation of β-carotene and linoleic acid, which generates free radicals [18]. In ourpresent study, the EA-Loyola AR1 extend β-carotene bleaching by neutralizing the linoleate-free radical and other free radicals formed in the system.


Conclusion

Our study suggests that Loyola AR1 strain belongs to Streptomyces sp. possessing α-glucosidase inhibition and oxidative stress mechanism in its system capability to reflect animbalance between the systemic manifestation of reactive oxygen species and a biologicalsystem’s ability to readily detoxify the reactive intermediates or to repair the resulting damage.It might be useful to prevent the progress of various oxidative stress-related diseases. Furtherinvestigation on the isolated component may lead the pharmaceutical industries for clinical use.

Acknowledgments Authors wish to thank the management of the Department of Plant Biology & PlantBiotechnology and M.Sc. Biotechnology Department, Loyola College, Chennai, Tamil Nadu, India, for provid-ing necessary facility to carry out this research work. Also, we thank to Sunil Christhudas (SRF, ICMR, NewDelhi), N. Murugan (SRF, Sankara Nethralaya, Chennai), and G. Ramesh Kumar (SRF, Aravind Eye Hospital,Tirunalveli) for their constant support throughout the research work.

References

1. Aruoma, O. I. (1998). Free radicals, oxidative stress and antioxidants in human health and disease. Journal ofthe American Oil Chemists Society, 75, 199–212.

2. Atta, H. M., Dabour, S. M., & Dsoukey, S. G. (2009). Sparsomycin antibiotic production by Streptomyces sp.AZ-NIOFD1: taxonomy, fermentation, purification and biological activities. An Eurasian Journal ofAgricultural and Environmental Sciences, 5(3), 368–377.

3. Babu, B. H., Shylesh, B. S., & Padikkala, J. (2001). Antioxidant and hepato protective effect of Alanthusicici focus. Fitoterapia, 72, 272–277.

4. Berdy, J. (2005). Bioactive microbial metabolites. Journal of Antibiotics, 58, 1–26.5. Brand-Williams, W., Cuvelier, M., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant

activity. Lebensm-Wiss Tech, 28, 25–30.6. Bull, A. T., & Stach, J. E. (2007). Marine Actinobacteria: new opportunities for natural product search and

discovery. Trends in Microbiology, 15, 491–499.7. Cragg, G. M., Kingston, D. G. I., & Newman, D. J. (Eds.). (2005). Anticancer agents from natural products

(pp. 475–497). Boca Raton, FL: Taylor & Francis.8. Dahlqvist, A. (1964). Method for assay of intestinal disaccharidases. Analytical Biochemistry, 7, 18–25.9. De Melo, E. B., Gomes, A. D. S., & Carvalho, I. (2006). α- and β-Glucosidase inhibitors: chemical structure

and biological activity. Tetrahedron, 62(44), 10277–10302.10. Elizabeth, K., & Rao, M. N. A. (1990). Oxygen radical scavenging activity of curcumin. International

Journal of Pharmaceutics, 58, 237–240.11. Garratt, D. C. (1964). The quantitative analysis of drugs (Vol. 3, pp. 456–458). Japan: Chapman and Hall.

Fig. 3 a Reducing power determination of different concentrations (200–1,000 μg/ml) of EA-Loyola AR1 and BHT.Each value presents the mean ± SEM of triplicate experiments. b DPPH radical scavenging activity of differentconcentrations (200–1,000μg/ml) of EA-LoyolaAR1 and vitaminC. Each value presents themean±SEMof triplicateexperiments. cHydroxyl radical scavenging activity of different concentrations (200–1,000μg/ml) of EA-Loyola AR1and vitamin C. Each value presents the mean ± SEM of triplicate experiments. dNitric oxide radical inhibition activityof different concentrations (200–1,000μg/ml) of EA-LoyolaAR1 and vitaminC. Each value presents themean ± SEMof triplicate experiments. e Superoxide anion radical-scavenging activity of different concentrations (200–1,000μg/ml)of EA-Loyola AR1 and vitamin C. Each value presents the mean ± SEMof triplicate experiments. fLipid peroxidationinhibition of different concentrations (200–1,000 μg/ml) of EA-Loyola AR1 and vitamin C. Each value presents themean ± SEM of triplicate experiments. gβ-Carotene bleaching effect in different concentrations (200–1,000 μg/ml) ofEA-Loyola AR1 and BHA. Each value presents the mean ± SEM of triplicate experiments

�


12. Gutteridge, J. M. (1993). Free radicals in disease processes: a compilation of cause and consequence. FreeRadical Research, 19, 141–158.

13. Hanato, T., Kagawa, H., Yasuhara, T., & Okuda, T. (1988). Two new flavonoids and other constituents inlicorice root: their relative astringency and radical scavenging effects. Chemical and PharmaceuticalBulletin, 36, 2090–2097.

14. Hochestein, P., & Atallah, A. S. (1988). The nature of oxidant and antioxidant systems in the inhibition ofmutation and cancer. Mutation Research, 202, 363–375.

15. Imada, C. (2005). Enzyme inhibitors and other bioactive compounds from marine actinomycetes. Antonievan Leeuwenhoek, 87, 59–63.

16. Imada, C., & Okami, Y. (1995). characterization of marine actinomycetes isolated from a deep-sea sedimentand production of β-glucosidase inhibitor. Journal of Marine Biotechnology, 2, 109–113.

17. Janero, D. R. (1990). Malondialdehyde and thiobarbituric acid reactivity as diagnostic indices of lipidperoxidation and peroxidative tissue injury. Free Radical Biology and Medicine, 9, 515–540.

18. Jayaprakasha, G. K., Singh, R. P., & Sakariah, K. K. (2001). Antioxidant activity of grape seed (Vitisvinifera) extracts on peroxidation models in vitro. Food Chemistry, 73, 285–290.

19. Jenkins, D. J., Taylor, R. H., Goff, D. F., Fielden, H., Misiewicz, J. J., Sarson, D. L., Bloom, S. R., & Alberti, K. G.(1981). Scope and specificity of acarbose in slowing carbohydrates absorption in man. Diabetes, 30, 951–954.

20. Lee, J. Y., & Hwang, B. K. (2002). Diversity of antifungal actinomycetes in various vegetative soils ofKorea. Canadian Journal of Microbiology, 48, 407–417.

21. Liu, F., Ooi, V. E. C., & Chang, S. T. (1997). Free radical scavenging activities of mushroom polysaccharideextracts. Life Sciences, 60(10), 763–771.

22. Mahmud, T. (2003). The C7N aminocyclitol family of natural products.Natural Product Reports, 20(1), 137–166.23. Mann, J. (2001). Natural products as immunosuppressive agents. Natural Product Reports, 18, 417–430.24. Marcocci, L., Packer, L., Droy-Lefai, M. T., Sekaki, A., & Gardes-Albert, M. (1994). Antioxidant action of

Ginkgo biloba extracts EGb 761. Methods in Enzymology, 234, 462–475.25. Meir, S., Kanner, J., Akiri, B., & Hadas, S. P. (1995). Determination and involvement of aqueous reducing

compounds in oxidative defense systems of various senescing leaves. Journal of Agricultural and FoodChemistry, 43, 1813–1817.

26. Miller, H. E. (1971). A simplified method for the evaluation of antioxidant. Journal of the American OilChemists Society, 18, 439–452.

27. Nolan, R., & Cross, T. (1988). Isolation and screening of actinomycetes. In M. Goodfellow, S. T. Williams,& M. Mordarski (Eds.), Actinomycetes in biotechnology (pp. 1–32). San Diego, CA: Academic.

28. Oktay, M., Gulcin, I., & Kufrevioglu, O. I. (2003). Determination of in vitro antioxidant activity of fennel(Foeniculum vulgare) seed extracts. Lebensmittel-Wissenschaft Und Technoogie, 36, 263–271.

29. Oldfield, C., Wood, N. T., Gilbert, S. C., Murray, F. D., & Faure, F. R. (1998). Desulphurisation ofbenzothiophene and dibenzothiophene by actinomycetes organisms belonging to the genus Rhodococcus,and related taxa. Antonie Van Leeuwenhoek, 74, 119–132.

30. Oyaizu, M. (1986). Studies on product of browning reaction prepared from glucose amine. Japanese Journalof Nutrition, 44, 307–315.

31. Rechner, A. R., Kuhnle, G., Bremmer, P., Hubbard, G. P., Moore, K. P., & Rice- Evans, C. A. (2002). Themetabolic fate of dietary polyphenols in humans. Free Radicals Biology and Medicine, 33, 220–235.

32. Shirling, E. B., & Gottlieb, D. (1966). Methods for characterization of Streptomyces species. InternationalJournal of Systematic Bacteriology, 16, 312–340.

33. Slinkard, K., & Singleton, V. L. (1977). Total phenol analyses: automation and comparison with manualmethods. American Journal of Enology and Viticulture, 8, 49–55.

34. Stuart, A. R., & Gulve, E. A. (2004). Chemistry and biochemistry of type 2 diabetes.Chemical Reviews, 104,1255–1282.

35. Schweder, T. (2005). Ulrike Lindequist and Michael Lalk. Advances in Biochemical Engineering/Biotechnology, 96, 1–48.

36. Umasankar, M. E., Kumar, G., Karthik, L., & Bhaskara Rao, K. V. (2010). Exploration of antagonisticactinobacteria from Amrithi forest. International Journal of Current Pharmaceutical Research, 2, 16–19.

37. Williams, S. T., Goodfellow, M., & Alderson, G. (1989). Genus Streptomyces Waksman and Henrici 1943,339AL. In S. T. Williams, M. E. Sharpe, & J. G. Holt (Eds.), Bergey’s manual of determinative bacteriology,vol. 4 (pp. 2453–2492). Baltimore: Williams & Wilkins.

38. Yen, G. C., & Chen, H. Y. (1995). Antioxidant activity of various tea extracts in relation to their anti-mutagenicity. Journal of Agricultural and Food Chemistry, 43, 27–32.

39. Yen, G. C., & Hsieh, C. L. (1998). Antioxidant activity of extracts from Du-zhong (Eucommia ulmoides)towards various peroxidation models in vitro. Journal of Agricultural and Food Chemistry, 46, 3952–3957.

40. Zielinski, H., & Kozłowska, H. (2000). Antioxidant activity and total phenolics in selected cereal grains andtheir different morphological fractions. Journal of Agricultural and Food Chemistry, 48, 2008–2016.


α-glucosidase inhibition and antioxidant properties of streptomyces sp.: in vitro

Documents