INTRODUCTION:

Natural products have always been a rich source of diverse molecules in drug discovery. Microbial diversity contribute an infinite resource for novel drug discovery. Identifying potential strains of microorganisms and isolating the bioactive molecules led to the discovery of many new drugs. Culturing microbes, identifying strains whose extracts have interesting biological activities, and purifying the molecules responsible for the activities, led to the discovery of many of the compounds Reactive oxygen species (ROS) are now recognized to participate in a growing number of disorders such as cancer, atherosclerosis and neurodegenerative diseases.¹

ROS production in excess of cellular anti-oxidant capacity may result in damage to lipids, proteins, and DNA. Oxidative damage to DNA leads to carcinogenesis, especially in the promotion stage.²In this context natural antioxidants are receiving increasing attention. Antioxidants, with free radical scavenging activities could have great importance as prophylactic and therapeutic agents in diseases in which oxidants or free radicals are implicated.³ Various research has shown that the microorganisms are rich sources of antioxidants. These antioxidants are not only useful as drugs but also find extensive applications in food industry. It suggested that because of the reducing action of microorganisms, the growth of a microbial flora on a fatty medium could lead to an inhibition of the oxidation of the fat by preventing the formation of peroxides and partially or completely destroying preformed peroxides.⁴ The antioxidant, 2-(hydroxy-2-metho xy-3,4-methylenedioxyphenyl)-benzofuran, isolated from baker's and brewer's yeast, was effective in protecting fats or food containing fats.^{5, 6} An oil soluble extract of Aspergillus oryzae contained a factor that prevented oxidative rancidity.⁷ Recent investigations have shown that a large number of microorganisms have the ability to decompose the peroxides present in fresh and rancid lard.⁸ This peroxide-decomposing activity of microorganisms appears to be enzymatic in nature. Therefore anticipating potent novel antioxidant molecules, extracellular as well as intracellular extracts of the microbial metabolites of 36 bacterial and 24 fungal isolates were subjected to antioxidant screening by diphenyl picryl hydrazyl (DPPH) radical scavenging method , ABTS [2, 2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging and superoxide radical scavenging by alkaline DMSO nitro blue tetrazolium (NBT) methods.

MATERIALS AND METHODS:

Sixty different microbial isolates, deposited at the Department of Pharmaceutical Biotechnology, MCOPS, Manipal, were used for the study. Of these, 36 were bacterial (Coded as B) and 24 were fungal isolates (Coded as F), isolated under unusual conditions such as high temperature, high Sodium Chloride and low glucose concentrations.⁹ Ascorbic acid used as standard and DMSO were purchased from SD Fine chemicals Pvt Ltd, India. DPPH, NBT and ABTS were purchased from Sigma Aldrich, Germany, and Potassium persulphate from Hi-media lab Pvt Ltd, Mumbai, India. All solvents used for extraction were purchased from Merck Ltd, Mumbai, India. Extracellular as well as intracellular extracts of the metabolites of these isolates were prepared as discussed below.

Inoculum development and production of metabolites

Nutrient broth was used for bacterial cultures whereas fungal broth containing 1% peptone and 2% dextrose was used for fungal cultures. In the case of bacterial cultures, 5 mL of inocula were developed in Nutrient broth by incubating at 37 °C for 24 h. Similarly, fungal inocula were developed in fungal broth by incubating at 27 °C for 72‑96 h. These were then used to inoculate the production media and incubated at 37 °C for 24 to 48 h in case of bacterial cultures, and 27 °C for 4-5 days in case of fungal cultures, at 150 rpm on a rotary shaker incubator. These broths were used for the extraction of extracellular and intracellular metabolites.

Extraction of bioactive principles:

The culture broth was centrifuged at 3000 rpm for 15 min to obtain a clear supernatant. Extracellular components were extracted successively with solvents such as Petroleum ether (P), Ethyl acetate (E) and chloroform (C) for extracellular extracts, followed by vacuum evaporation of these extracts to obtain the dry extracts. The cell sediment obtained on centrifugation of the inocula is used to prepare the intracellular extract. The cells were redispersed in 5 mL of methanol, probe sonicated to release the intracellular components and then centrifuged. The methanol in the supernatant was then evaporated off and the dried intracellular extract (M) was obtained on freeze drying.¹⁰ Each extract was tested in vitro for its antioxidant potential by DPPH, NBT and ABTS methods. The organic solvent extracts were dissolved in DMSO whereas methanolic extract was dissolved in sterile water to obtain the stock solution of test sample.

In vitro antioxidant studies:

The extracellular and intracellular extracts prepared were subjected to in vitro antioxidant screening by diphenyl picryl hydrazyl (DPPH) radical scavenging method (DPPH)¹¹, ABTS [2, 2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging¹² and superoxide radical scavenging by alkaline DMSO nitro blue tetrazolium (NBT) methods.¹³ All the experiments were performed thrice and results are averaged. Ascorbic acid was used as the standard for comparison.

DPPH radical scavenging activity:

The assay was carried in a 96 well microtitre plate. Briefly, 100µl of various concentrations of extract or standard in DMSO and 100µl of 200µM solution of DPPH was added to each well. The plates were incubated at 37̊C for 30 minutes in dark and absorbance was read using ELISA plate reader using 540 nm filter. DPPH radical-scavenging activity was calculated using the following equation:

% Inhibition = (A_x-A_y) / A_x × 100

Where A_x was the absorbance of the control (without extract) and A_y was the absorbance in the presence of the extract.

Scavenging of superoxide radical by alkaline DMSO NBT method:

0.1 ml of NBT (1 mg/ml) was added to the reaction mixture containing 1 ml of alkaline DMSO (1 ml of DMSO containing 5 mM NaOH in 0.1 ml of water) and 0.3 ml of extract/ fractions or standard (in DMSO), to give a final volume of 1.4 ml. The absorbance was measured at 560 nm.

Scavenging of ABTS radical:

To 50 ml of 2 mM ABTS in distilled water, 0.3 ml of 17 mM potassium persulphate was added. It was left overnight at room temperature in dark before usage. 1.0 ml of DMSO and 0.16 ml of ABTS solution were added to 0.2 ml of various concentrations of extracts /fractions or standard in DMSO, to make a final volume of 1.36 ml. Absorbance was measured after 20 min at 734 nm.

Preliminary chemical investigation:

Preliminary qualitative chemical analysis of most active extracts identified from each of the three methods (by DPPH, NBT and ABTS) were performed for the identification of various constituents of the extracts. All the analyses were carried out using 1-2 mL of extract solution. In the case of tests for carbohydrates, tests such as Molisch’s, Fehling’s, Benedict’s, and Barfoed’s tests were carried out. Tests for alkaloids (Dragendroff’s, Mayer’s, and Hager’s tests), test for sterols (Liebermann Burchard test and Salkowsky test), tests for the detection of phenolic compounds and tannins, tests for proteins and free amino acids (Biuret test and Ninhydrin test) and also tests for the detection of flavonoids, were performed.

Morphological studies:

Morphological studies of selected isolates were performed for partial characterization of the isolates and to test for any isolates present in repeats. In the case of bacterial isolates, Gram’s Staining and Motility Studies (Hanging Drop Method) were carried out, while Lactophenol cotton blue staining was used to visualize the morphology of the fungal isolates.^14,
15 (Figure 4)

RESULTS AND DISCUSSION:

In vitro antioxidant studies of bacterial and fungal isolates were done on various extracts prepared with Petroleum ether (P), Ethyl acetate (E) and Chloroform(C) for extracellular metabolites and Methanol (M) for intracellular metabolites by DPPH, Alkaline DMSO- NBT and ABTS methods. The results are expressed in terms of IC₅₀, as mean±SEM and shown in Tables 1-3. The graphical representation of IC₅₀ values of most active extracts are shown in Figures 1-3. The details of extracts with IC₅₀ values above 1000 μg/ml are not shown in the tables.

DPPH radical scavenging activity:

The extracellular and intracellular extracts of bacterial and fungal isolates such as B-13M, F-6E, F-12E, F-13M, F-14E, F-15E, F-19M, F-21E were found to have IC₅₀values between 125 and 500 μg/ml. Among these F-15E (VHS-4, Figure 4) showed the maximum antioxidant potential with IC₅₀ of 188.32±0.42 μg/ml. While bacterial and fungal extracts such as B-15M, F-8E, F-12M, F-13E, F-14M, F-15M, F-19E, F-21M showed an IC₅₀value between 500-1000 μg/ml. All the remaining extracts have IC₅₀above 1000 μg/ml. Results show that fungal extracts are more effective as antioxidants than bacterial extracts. It was also found that extracellular fungal metabolites are more active than intracellular metabolites (Table 1).

Scavenging of superoxide radical by alkaline DMSO NBT method:

Extracellular and intracellular extracts of bacterial and fungal isolates such as B-2P, B-2C, B-4P, B-4E, B-4C, B-13E, F-12E, F-14E, F-15E, F-19E, F-19M, F-21P, F-21E and F-21C showed an IC₅₀value between 125 and 500 μg/ml where as extracts like B-2E, F-12C, F-13P and F-13E showed an IC₅₀value between 500 and 1000 μg/ml. Maximum superoxide radical scavenging ability was shown by F-21E (VHSS-3, Figure 5) with an IC₅₀ value of 134.01±1.61 μg/ml. Unlike DPPH method bacterial extracts were showed better superoxide scavenging activity (Table 2).

Scavenging of ABTS radical:

In case of ABTS radical scavenging studies it is interesting to note that the extracellular ethyl acetate extract of F-12 (LS-2 Figure 6) showed an IC₅₀value of 10.57±0.14 μg/ml which is lower than the IC₅₀value of ascorbic acid (13.75±0.21 μg/ml) by ABTS method. The extracts which gave IC₅₀between 125 and 500 μg/ml were F-12C, F-13E, F-15E, and F-21E and those between 500 to 1000 μg/ml were B-2M, B-4P, B-4C, B-13M, F-6E, F-8E and F-19E. This shows that extracellular fungal principles were more efficient in scavenging ABTS monocation radical (Table 3).

Preliminary chemical investigation:

Among the extracellular and intracellular extracts of bacterial and fungal isolates only extracellular ethyl acetate fungal extracts showed maximum antioxidant potential (F-15E, F-21E & F12E). Preliminary qualitative chemical analysis of these most active extracts identified from each of the three methods (by DPPH, NBT and ABTS) were performed for the identification of various constituents of the extracts. All the three extracellular ethyl acetate fungal extracts (F-15E, F-21E & F12E) showed the presence of sterols. While the most active fungal extract F-12E additionally showed the presence of flavonoids (Table 4).

Table 1. Antioxidant activity of Intracellular and Extracellular Bacterial and Fungal Extracts by DPPH Method

Extract code	IC₅₀±SEM^a(μg/ml)
B13-M	474.12±0.37
B15-M	929.34±1.09
F6-E	260.55±0.74
F8-E	604.12±0.99
F12-E	271.47±0.78
F12-M	659.56±1.14
F13-E	602.72±1.34
F13-M	385.22±0.65
F14-E	383.75±0.79
F14-M	938.16±1.41
F15-E	188.32±0.42
F15-M	750.00±1.71
F19-E	607.06±0.88
F19-M	403.42±0.74
F21-E	248.00±0.49
F21-M	974.56±1.00
Ascorbic acid	5.12±0.12

^aAverage of three determinations

Table 2. Antioxidant activity of Intracellular and Extracellular Bacterial and Fungal Extracts by Alkaline DMSO - NBT Method

Extract code	IC₅₀±SEM^a(μg/ml)
B2-P	212.99±1.95
B2-E	526.84±2.31
B2-C	272.17±1.84
B4-P	144.67±1.01
B4-E	185.66±1.79
B4-C	240.01±1.37
B13-E	281.69±1.71
F8-E	675.66±3.12
F12-E	263.42±2.91
F12-C	847.72±3.97
F13-P	510.88±1.74
F13-E	576.54±2.17
F14-E	348.75±2.19
F14-M	666.84±1.97
F15-E	486.58±2.85
F15-M	633.38±2.31
F19-E	534.26±3.14
F19-C	500.00±2.97
F19-M	432.61±1.89
F21-P	474.89±2.84
F21-E	134.01±1.61
F21-C	179.00±1.72
Ascorbic acid	16.75±1.33

^aAverage of three determinations

Table 3. Antioxidant activity of Intracellular and Extracellular Bacterial and Fungal Extracts by ABTS Method

Extract code	IC₅₀±SEM^a(μg/ml)
B2-M	712.06±5.34
B4-P	586.62±3.67
B4-C	939.56±6.89
B13-M	932.28±7.73
F6-E	946.84±8.23
F8-E	569.12±4.11
F12-E	10.57±0.14
F12-C	307.17±3.44
F13-E	385.22±3.77
F15-E	277.28±1.95
F19-E	694.56±5.71
F21-E	293.31±2.73
Ascorbic acid	13.75±0.21

^aAverage of three determinations

Figure 3. Antioxidant activity of most active extracts in comparison with ascorbic acid by ABTS method

Figure 4. Lactophenol cotton blue staining to visualize the morphology of the most active fungal isolates

CONCLUSION:

Extracellular as well as intracellular extracts of the metabolites of thirty-six bacterial and twenty-four fungal isolates, grown under unusual conditions such as high temperature, high sodium chloride and low glucose concentrations, were in vitro tested for their antioxidant potential by DPPH, NBT and ABTS methods. Among the extracellular and intracellular extracts of bacterial and fungal isolates F-15E showed the maximum antioxidant potential with IC₅₀ of 188.32±0.42 μg/ml by DPPH method. While maximum superoxide radical scavenging ability was shown by F-21E with an IC₅₀ value of 134.01±1.61 μg/ml. In case of ABTS radical scavenging studies it is interesting to note that the extracellular ethyl acetate extract of F-12 (Fig.4) showed an IC₅₀value of 10.57±0.14 μg/ml which was better than standard ascorbic acid. Further studies can be carried out on bioassay-guided fractionation of the active extract for the isolation of active constituent, structural elucidation of the active constituent, systematic taxonomical studies for characterization of the isolates, optimization of production parameters, and on scaling up of the process.

ACKNOWLEDGEMENT:

The authors thank the Principal, Manipal College of Pharmaceutical Sciences, and Head, Dept. of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences, Manipal Academy of Higher Education for providing necessary research facilities.

CONﬂICT OF INTEREST:

Authors declare no financial/commercial conflicts of interest

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Received on 24.05.2019 Modified on 14.06.2019

Research J. Pharm. and Tech. 2019; 12(10):4916-4920.

DOI: 10.5958/0974-360X.2019.00852.7

Nature of Extracts	Alkaloids	Carbohydrates	sterols	Tannins & phenolic compounds	Proteins & Amino acids	Flavonoids
Ethyl acetate (F-15E)	-	-	+	-	-	-
Ethyl acetate F-21E	-	-	+	-	-	-
Ethyl acetate F12-E	-	-	+	-	-	+