ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2013, том 49, № 6, с. 587-591
UDC 579.61
ANTIOXIDANT PROPERTIES OF FUNGAL METABOLITE NIGERLOXIN IN VITRO
© 2013 B. S. Suresha and K. Srinivasan
Department of Biochemistry and Nutrition, Central Food Technological Research Institute, CSIR, Mysore — 570 020, India
e-mail: ksri.cftri@gmail.com Received November 26, 2012
We have recently reported the beneficial influence of the fungal metabolite nigerloxin, a new aldose reductase inhibitor and a lipoxygenase inhibitor on oxidative stress in streptozotocin induced diabetic rats. In the present study we have investigated the antioxidant potential of nigerloxin in vitro as compared to one of the well known natural antioxidant, curcumin. The fungal metabolite nigerloxin was found to be an effective antioxidant in different in vitro assays including the phosphomolybdenum, 2,2-diphenyl-1-picrylhydrazyl (DPPH*), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS *+) and ferric reducing antioxidant power (FRAP) methods. The antioxidant potency of nigerloxin may be attributed to its electron donating nature. The ferric reducing potency of nigerloxin as demonstrated by FRAP assay method was even found to be superior to that of the natural antioxidant curcumin.
DOI: 10.7868/S0555109913060172
The role of free radicals (molecular species carring one or more unpaired electrons in their molecular orbitals) in the pathogenisis of many diseases is well documented [1]. In biological systems cellular metabolism is the most important source of production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and RNS include radicals such as
superoxide (O2-), hydroxyl (OH"), hydroperoxyl (HO2), alkoxyl (RO"), peroxyl (ROO"), nitric oxide
(NO"), nitrogen dioxide (NO2) and lipid peroxyl
(LOO") [2]. The deleterious influence of free radicals on biological system is termed oxidative and nitrosa-tive stresses [3—5]. These stresses may be due to overproduction of ROS/RNS or deficiency of enzymatic and non-enzymatic antioxidants in biological system. Some of other non radicals like hydrogen peroxide (H2O2), hypochlorous acid (HOCl), ozone (O3), per-oxynitrite (ONOO-), nitrous acid (HNO2), dinitro-gentrioxide (N2O3), lipid peroxide (LOOH) are also termed as oxidants and they are capable of leading to free radical reactions in living organisms.
The toxic influence of oxidative stress in many pathological conditions including cardiovascular disease, cancer, neurological disorders, diabetes, is-chemia/reperfusion, other diseases and ageing has been well studied [6-9]. Compounds capable of donating electrons or hydrogen atom are able to protect from deleterious influence of free radicals. Antioxidants have exhibited beneficial influence in many pathological conditions [10]. There are several novel approaches in the study of free radicals/antioxidants for the improvement of human health.
A new aldose reductase inhibitor molecule of [2-amido-3-hydroxy-6-methoxy-5-methyl-4-(prop-1'-enyl) benzoic acid] (nigerloxin) obtained from solidstate fermentation of Aspergillus niger CFR-W-105 has been reported to exhibit inhibitory activity on the partially purified aldose reductase and lipoxygenase in vitro [11]. Animal study have been recently carried out to verify if this fungal metabolite exhibits aldose reductase inhibitory activity in vivo and to examine the influence of nigerloxin on modulation of the renal and retinal lesions [12] associated with diabetes in experimental rats, by virtue of its aldose reductase inhibitory potential. The aim of the study was to assess antioxidant potential of nigerloxin in vitro as compared to one of the major natural antioxidant, cur-cumin.
MATERIALS AND METHODS
Chemicals. Nigerloxin was produced through solid state fermentation of A. niger CFR-W-105 (Central Food Technological Research Institute, Mysore, India) according to the procedure given by Rao et al. [13]. 2,2-Diphenyl-1 -picrylhydrazyl (DPPH); 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS); 2,4,6-tris-(2-pyridyl)-S-triazine (TPTZ); dimethyl sulfoxide (DMSO) and curcumin were obtained from Sigma-Aldrich (USA). All other chemicals and solvents used were of analytical grade obtained from Sisco Research Laboratories (India).
Antioxidant capacity of nigerloxin assessed by phosphomolybdenum method. Antioxidant potential of ni-gerloxin was evaluated by the protocol given by Prieto et al. [14] and compared with that of curcumin. Brief-
587
4*
0 20 40 60 80 100 120 Ascorbic acid,
Fig. 1. The linear regression curve of ascorbic acid.
ly, nigerloxin (2—10 ^g) or curcumin (2—10 ^g) taken in different concentrations in 10 ^L of DMSO was added to 1.0 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). Blank solution was constituted by adding 10 ^L of DMSO in place of sample solution with 1 mL of reagent. The tubes were capped and incubated in a boiling water bath for 90 min. After the samples had cooled to room temperature, the OD695 was measured against blank. Ascorbic was used as a standard and an-tioxidant capacities of nigerloxin and curcumin were expressed as ascorbic acid equivalents (^mol/mg of sample).
Free radical scavenging activity of nigerloxin assessed by DPPH assay. The DPPH radical scavenging potential of nigerloxin and curcumin were determined according to the method of Hatano et al. [15]. Briefly, nigerloxin (5—50 ^g) or curcumin (2—10 ^g) taken in different concentrations in 10 ^L of DMSO, was mixed with 1 mL of 0.2 mM DPpH in methanol. The mixture was vigorously shaken and incubated at 28°C for 30 min. The absorbance was measured at 517 nm. The free radical quenching ability of nigerloxin and curcumin were determined as percentage decrease in the absorbance with respect to control having appropriate concentration of solvent. The effective concentration (EC50) value was determined as the concentration of the sample required to reduce 50% of the absorbance with respect to control. Inhibition of free radical DPPH was calculated as follows:
Inhibition of DPPH radical % =
= (ODcontrol — ODsample) X 100/0Dcontrol.
ABTS radical cation decolorization assay. The
ABTS radical scavenging activity was determined according to the protocol given by Re et al. [16]. ABTS radical was generated by incubating a mixture of 2 mM ABTS and 2.45 mM potassium persulfate in water for 16 h in the dark at room temperature. The ABTS radical solution was diluted with ethanol to produce absor-bance of around 0.7 at 734 nm. Nigerloxin (5—50 ^g) or curcumin (2—10 ^g) taken in different concentrations in 10 ^L of DMSO was mixed with 1 mL of pre-diluted ABTS radical solution. The mixture was vigorously
shaken and incubated at room temperature for 30 min and OD734 was recorded. The percentage of radical scavenging was calculated for each concentration relative to a blank containing appropriate amount of solvent using the following equation and EC50 value was expressed in ^mol.
Inhibition of ABTS radical % =
= (ODcontrol — 0Dsample) X 100/0Dcontrol.
Ferric reducing antioxidant power (FRAP) assay.
The ferric reducing potential of nigerloxin and cur-cumin were estimated by the procedure given by Benzie and Strain [17]. The reagent was constituted by adding 2.5 mL of a 10 mM TPTZ solution in 40 mM HCl, 2.5 mL of 20 mM FeCl3 • 6H2O and 25 mL of 300 mM acetate buffer, pH 3.6. Reagent was freshly prepared and warmed at 37°C before used. From one to five ^g of nigerloxin or curcumin in several concentrations was prepared in 10 ^L of DMSO and added to 1 mL of FRAP reagent. Reaction mixture was mixed thoroughly and incubated at 37°C for 30 min and 0D593 was recorded. Ferrous sulphate was used as standard and FRAP value was expressed as mM of ferrous sulphate equivalents/mg sample.
RESULTS
A dose-dependent antioxidant influence of niger-loxin and curcumin on phosphomolybdenum complex is shown in Fig. 1 and 2. The regression curve of ascorbic acid was constructed by plotting absorbance against concentration and antioxidant potency of ni-gerloxin and curcumin were calculated using regression equation. The antioxidant potential of nigerloxin and curcumin by this method was found to be 3791 and 4684 ^M of ascorbic acid equivalents/mg, respectively. DPPH radical scavenging potential of nigerloxin and curcumin are presented in Fig. 3. Nigerloxin scavenged DPPH radical in a dose-dependent manner. Nigerloxin and curcumin exhibited EC50 value at the concentration of 68.56 and 22.57 ^M against DPPH radicals, respectively.
The dose-dependent inhibition of ABTS radical by nigerloxin and curcumin is shown in Fig. 4. Nigerloxin and curcumin potentially scavenged this radical and the EC50 value was found to be 8.97 ^M and 6.75 ^M, respectively. The ferric ion reducing potential of nigerloxin and curcumin is presented in Fig. 5 and 6. The linear regression curve, absorbance against concentration of ferrous sulphate was constructed and regression equation was used to calculate the reducing strength of nigerloxin and curcumin. Nigerloxin and curcumin reduced ferric complex in a dose depended manner. The FRAP value of nigerloxin and curcumin was found 14.44 and 8.20 mM ferrous sulphate equiva-lents/mg, respectively.
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ANTIOXIDANT PROPERTIES OF FUNGAL METABOLITE NIGERLOXIN IN VITRO
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Fig. 2. Antioxidant capacities of nigerloxi
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