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‘In vitro’ evaluation of the antioxidant activity in relation with structure and kinetic properties of scleraldehyde
Bioactive Carbohydrates and Dietary Fibre 2 (2013) 22 –29
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‘In vitro’ evaluation of the antioxidant activity in relation with structure and kinetic properties of scleraldehyde Nagarajan Usharani, Gladstone Christopher Jayakumar, Swarna V. Kanthn, Jonnalagadda Raghava Rao Council of Scientific and Industrial Research-Central Leather Research Institute, Adyar, Chennai 600 020, India
ar t ic l e in f o
abs tra ct
Article history:
Schizophyllum commune is the world′s most extensively disseminated mushroom except in
Received 20 May 2013
Antarctica. Schizophyllan is a polysaccharide fractions isolated from S. commune. The
Received in revised form
schizophyllan is oxidized to form scleraldehyde to introduce carbonyl group for additional
15 August 2013
functions. The relationship between structure, kinetic and antioxidant properties of
Accepted 17 August 2013
scleraldehyde was studied in order to establish the molecular characteristics related to its maximum radical scavenging activity. In our earlier paper we have reported that
Keywords:
scleraldehyde obtained from schizophyllan with the dialdehyde content of 28%. From the
Schizophyllan
absorption studies, it explains there is π–πn excitations involving the benzene groups.
Scleraldehyde
Moreover, the structural identification of scleraldehyde were characterized by bands at
Structure
1670 and 3600–3200 cm1, corresponding to the axial deformation of the C ¼O bond and the
Antioxidant activity
angular deformation of the C–H bond respectively. The IR spectrum confirms the identity
Kinetic property
of aldehyde. The scavenging assays of scleraldehyde was found to be reliant on their reactivity and concentration. Scavenging assays performed indicate that 100 μM of scleraldehyde showed the highest antioxidant activity compared to the ascorbic acid. Furthermore, The reducing ability of the scleraldehyde was 54.8170.92 μM of Fe(II)/g compared to the control (Ascorbic acid) respectively. The kinetic mechanism of scleraldehyde portrays a competitive inhibition against antioxidant enzymes on calculating for radical scavenging activity. By means of structure determination, kinetic assays and the antioxidant assays that hydroxyl group present in C2, C4 and C6 are the main reactive sites. The hypothesis converse that scleraldehyde can serve as scavenger of aqueous peroxyl radicals near the membrane surface of collagen. & 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
Schizophyllum commune is the world′s most widely distributed mushroom, produces number of enzymes such as bilirubin oxidase, fibrinolytic, acetylxylan transferase, extra cellular protease and a vital extracellular polysaccharide called
n
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2212-6198/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bcdf.2013.08.002
schizophyllan used both for medicinal and industrial applications (Maushmi, Shrikant, & Rekha, 2008). Schizophyllan, produced by S. commune is a neutral exopolysaccharide with a chemical structure consisting of 1,3 β-D-linked backbone of glucose residues and 1,6-β-D-glucosyl side chain at every third residue. Native schizophyllan with a typical molecular weight
Bioactive Carbohydrates and Dietary Fibre 2 (2013) 22 –29
of the order of 5.54 105 Da chemically two polysaccharides such as curdlan and scleroglucan produced by Agrobacterium sp and Sclerotium rotifis, respectively (Shrikant, Parag, & Rekha, 2006). In contrast to most polysaccharides, Schizophyllan exhibits a fairly high solubility in water forming the triple-helical conformation with a persistence length between 150 and 200 nm which is stable up to a temperature of 140 1C (David et al., 2007). Polysaccharides such as schizophyllan and scleroglucan contain periodate resistant β-1,3-linked D-glucan backbones with regularly distributed side chains consisting primarily of single β-1,6-linked D-glucose. Side chain oxidation of schizophyllan results in the formation of dialdehyde group called Scleraldehyde, and the production of formic acid. The mechanism of crosslinking of this scleraldehyde is explained by the formation of intermolecular hemiacetal linkages (Kristiansen, Potthast, & Christensen, 2010). Moreover the potential of scleraldehyde as an antimicrobial agent was also explored in our earlier study (Jayakumar, Swarna, Chandrasekaran, Rao, & Nair, 2010). The structural determinants of scleraldehyde molecule are incomprehensible, but the position and number of the hydroxylic groups have been suggested to play an important role in the antioxidant activity (Rice-Evans, Miller, & Paganga, 1996). The present study was extended by understanding the structural determinants relating to the activity of scleraldehyde, and in particular to establish the rate of inhibition using kinetic studies. The antioxidant activity was investigated in vitro by measuring the radical scavenging ability using the free radicals and antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase. Different chemical and physical principles was obtained from in-vitro activity values for antioxidant activity that vary according to the assays employed. Hence, the present investigation focussed on the structure-activity relationship of scleraldehyde.
2.
Materials and methods
2.1.
Materials
Media components such as glucose, sucrose, maltose, lactose, starch, magnesium sulfate, yeast extract, peptone were purchased from Hi-Media Limited, Mumbai, India. All other chemicals were purchased from S.D. Fine Chemicals Ltd, Mumbai, India. Strains of Schizophyllum commune (S. commune) MTCC 1096 were procured from Microbial Type Culture (MTCC), Chandigarh.
2.2.
Cultivation of Schizophyllum commune
S. commune was maintained on solid medium containing 20 g of malt extract/L, 1 g of peptone/L, 20 g of dextrose/L, and 20 g of agar/L. A liquid culture was started by transferring approximately 7 mm of a 7–10 day-old colony into 250 mL of the same medium without agar in a 500-mL fluted Erlenmeyer flask with three 10-mm glass beads. This culture was grown for 4 days at 30 1C and 240 rpm. Suspensions (0.5 mL) were used to inoculate 300-mL flasks containing 50 mL (w/v) of
23
corn fiber pretreated as previously described with alkaline H2O2 in a basal medium containing 0.67% yeast nitrogen base (Difco), 0.2% asparagine, and 0.5% KH2PO4. Cultures containing treated corn fiber were grown for 7 days at 30 1C and 240 rpm (Jayakumar et al., 2010).
2.3.
Isolation of schizophyllan
Mycelial growth was removed from cultures by centrifugation in 50 mL conical tubes at 3220g for 30 min at 4 1C. Polysaccharides were recovered from culture supernatants using 95% ethanol diluted to 50% (v/v) and 67% (v/v). Polysaccharide pellets were resuspended in distilled H2O, transferred to 15 mL conical tubes, and dried under vacuum at 50 1C for analysis.
2.4.
Extraction of schizophyllan from pellet
S. commune was cultivated in 1000 mL yeast malt broth for seven days. The seven days culture was centrifuged for 20 min at 2500 rpm at 4 1C and the pellet was obtained. 300 mL of deionized water was then added to the whole pellet and incubated for 3 h at 100 1C. After 3 h, 300 mL of 80% ethanol was added to the above sample and incubated at 100 1C for 3 h and 300 mL of 1% ammonium oxalate solution is added to the sample and incubated for 6 h at 100 1C. 150 mL of 5% NaOH was then added to the above sample and incubated for 6 h at 80 1C. Final residue (Schizophyllan) obtained after 6 h was weighed (Jayakumar et al. 2012).
2.5.
Periodate oxidation of schizophyllan
Schizophyllan (10 g) was hydrolyzed in 5 N hydrochloric acid (10 h, 85 1C). Hydrolyzed schizophyllan was suspended in demineralized water and subsequently cooled in an ice bath. 12 g of sodium periodate was added to the sample while stirring with a magnetic stirrer. The pH of the solution was maintained around 4 during the reaction. The reaction was performed in the dark at room temperature and stopped after 48 h to obtain scleraldehyde of 99% oxidation. The product obtained was centrifuged with t-butyl alcohol (1:3 sample: solvent). The product was resuspended in the same volume of demineralized water and the centrifugation carried out several times. The product was dried at room temperature. The oxidation of schizophyllan was determined by the release of formic acid using procedures reported earlier.
2.6.
Structural determinations
2.6.1.
UV–visible spectroscopy
The UV–visible absorption spectra were recorded on a Cary 100 UV–VIS–NIR spectrometer with a spectral resolution of 2 nm in the wavelength range 200–600 nm (Xu, Pamela, Noel, & Jeffrey, 1993).
2.6.2.
Fourier transform—Infrared spectroscopy
Infrared spectra of the sample was obtained from KBr pellets at room temperature using a Jasco Spectrometer with a resolution of 4 cm1 averaging 50 scans in the 4000– 400 cm1 wavenumber range (Ross, 1953).
24
2.6.3.
Bioactive Carbohydrates and Dietary Fibre 2 (2013) 22 –29
GC–MS analysis
Vanden Heuvel and Horning have used the hydrazone products for gas chromatographic analyses, relying solely on retention times for characterization (Vandenheuvl & Horning, 1963). Gas chromatographic analyses were performed on a JEOL GC Mate gas chromatograph with a FID detector. The column employed was a 1.8-m 2-ram ID glass column packed with 3% SP-2100 on 100/120 mesh. Electron impact spectra were generated with a source potential of 70 eV; chemical ionization spectra were generated using ultrapure methane (Airco, Incorporated) as both the carrier gas and the ionizing gas. The internal source pressure was 0.5 Torr, with a source potential of 200 eV.
2.7.
Antioxidant activity of scleraldehyde
2.7.1.
Radical scavenging activity (DPPH model system)
Scleraldehyde (10 μg/mL) and positive control ascorbic acid (10 μg/mL) in methanol (0.1 mL) was added to 3.9 mL of a 6 105 M DPPH solution in methanol (Chen & Ho, 1997). The exact initial DPPH concentration in the reaction medium was calculated from a calibration curve. Decrease in absorbance was determined at 515 nm for 1 h for every 5 min, and every 60 min until the reaction reached a plateau (about 6 h). Antiradical activity was expressed as the EC50, i.e. antioxidant concentration required to decrease the initial amount of DPPH by 50% (Blois, 1958).
2.7.2.
ABTS radical scavenging assay
ABTS radical scavenging activities of scleraldehyde (0–100 μM) and positive control ascorbic acid (100 μM) are determined by the method described by Re et al. (1999). with slight modification. The stock solution of ABTS radicals is made by mixing 5.0 mL of 7 mM ABTS solution with 88 μL of 140 mM potassium persulfate, and kept in the dark for 12–16 h at 37 1C. An aliquot of stock solution is diluted with phosphate buffer, PB (5 mM, pH 7.4) containing 0.15 M NaCl, to prepare the working solution of ABTS radicals to an absorbance of 0.7070.02 at 734 nm. Samples are mixed 1:4 (v/v) with diluted ABTS, or only buffer (control), incubated for 10 min at 37 1C in the dark, and then absorbance is measured at 734 nm. The percent reduction of ABTSþ to ABTS is calculated according to the following equation: ð%Þ ¼ ½ðAbscontrol Abssample Þ=ðAbscontrol Þ 100 Abscontrol is the absorbance of ABTS radical and methanol; Abssample is the absorbance of ABTS radical and sample extract/standard.
dark condition. Readings of the colored product (ferrous tripyridyltriazine complex) were taken at 593 nm. The standard curve was linear between 50 and 1000 μM FeSO4. Results are expressed in μM Fe(II)/g dry mass and compared with that of ascorbic acid (100 μM) (Benzie & Strain, 1996).
2.8.
The enzyme inhibition was monitored continuously for 20–60 min by adding the enzyme such as superoxide dismutase, catalase and glutathione peroxidase to a solution of buffer with inhibitor (scleraldehyde). The appropriate substrate used for the enzymes were 6-hydroxy dopamine, hydrogen peroxide and phospholipid hydroperoxide respectively. The concentrations of ethanol extract of scleraldehyde were used and compared with the kinetic behavior of ascorbic acid as a control. Enzyme concentration and total volume of reaction mixture was kept constant but the concentration of substrate such as used varied in the range of 0.1–1 mM. The results were analyzed in terms of Michaelis–Menten treatment. Kinetic data were analyzed by double reciprocal plots and computer-fitted to appropriate rate equations by means of non-linear regression analysis program (EZFIT) (Knight, 1995). The nomenclature used in this study is that of Cleland. From the Lineweaver–Burk plots of v1 vs [S]1, the kinetic parameters such as Vmax, maximum velocity and Km, the Michaelis–Menten constant of the enzyme were calculated. Dixon plots of v1 vs [I] were used to determine the inhibition constant (Ki) for competitive and non-competitive inhibition. Ki for mixed inhibition was determined using secondary plots, by replotting slopes and intercepts of Lineweaver–Burk plots vs. [I]. Cornish–Bowden plots of [S]/v vs. [I] were used to calculate Ki for uncompetitive inhibition. In all the cases, the intercept on the [I] axis gives Ki.
3.
Results and discussions
A novel antioxidant which is eligible for standardization requires an attention to some structural requirements of antioxidant potency (Liu et al., 2009). The present investigation involves the characterization of scleraldehyde as possible antioxidant agent. This has been examined by understanding the structural parameters of scleraldehyde relating to the antioxidant activity. Radical scavenging action of scleraldehyde is found to be dependent on both reactivity and concentration of the antioxidant.
3.1. 2.7.3.
Kinetic of investigations
Preparation and characterization of scleraldehyde
Reducing ability (FRAP assay)
The determination of the total antioxidant activity (FRAP assay) of the synthesized compounds was evaluated by modified method of Benzie and Strain (1996). The stock solutions included 300 mM acetate buffer, pH 3.6, 10 mM TPTZ (2,4,6-tripyridyl-S-triazine) solution in 40 mM HCl and 20 mM FeCl3 6H2O solution. Working solution was prepared by mixing acetate buffer (25 mL), TPTZ (2.5 mL) and FeCl3 6H2O (2.5 mL). The temperature of the solution was raised to 37 1C before use. Scleraldehyde (100 μM) was allowed to react with 1500 μL of the FRAP solution for 30 min in the
Scleraldehyde was obtained by oxidizing schizophyllan by sodium metaperiodate. Periodate oxidation specifically cleaves the vicinal glycols in schizophyllan to form their dialdehyde derivatives called scleraldehyde. It is observed that scleraldehyde consumes up to 2 mol of periodate per mole of repeating unit with simultaneous release of up to 1 mol of formic acid as reported in our earlier paper. The dialdehyde content of scleraldehyde was found to be 28%. The yield of schizophyllan and scleraldehyde from pellet and supernatant is given Table 1. Higher amount of scleraldehyde
25
Bioactive Carbohydrates and Dietary Fibre 2 (2013) 22 –29
is obtained from pellet when compared with preparation of scleraldehyde from supernatant. The antioxidant activities of scleraldehyde is compared with the standard control, ascorbic acid.
3.2.
Structural characterization of scleraldehyde
3.2.1.
UV–visible Spectroscopic determination
Intermediate intensity peaking around 240 nm (1.2 eV, ε (max)E1000 M (1) cm (1)) is dominated by ππn excitations within the arene function. Finally, strong absorptions (ε(max) E10,000 M(1) cm(1)) is observed around 210 nm (4.0 eV) which is ascribe to ππn excitations involving the benzene groups.
3.2.2.
IR spectrum of scleraldehyde
Source of extraction
Amount of schizophyllan
Amount of scleraldehyde
The IR spectra (Fig. 2) of scleraldehyde are characterized by bands at 1670 and 3600–3200 cm1, corresponding to the axial deformation of the C¼O bond and the angular deformation of the C–H bond respectively. The IR spectrum confirms the identity of aldehyde. Moreover, the scleraldehyde spectrum can be observed with intense band at 1618 cm1, probably related to the asymmetric stretching vibration of the –C¼O group. The acidification of the derivative produces the disappearance of this peak and the appearance of a new peak at 1734 cm1, probably due to the stretching vibration of the C¼O in the undissociated acid.
Pellet Supernatant
9.66 g/30 g of pellet 5.98 g/1000 mL of supernatant
6.54 g 3.79 g
3.2.3.
The UV–visible absorption spectrum of scleraldehyde is shown in Fig. 1. The degree of conjugation of the molecule, and some evidence of identity through spectral matching was observed. The spectra of scleraldehyde isomers are characterized by weak (ε(max)E100 M (1) cm(1)) transitions around 290 nm (2.6 eV), arising from nπn absorptions.
Absorbance
Table 1 – Production profile of scleraldehyde.
GC–MS analysis
0.12
The mass spectra of scleraldehyde shows molecular ion (M)þ at m/z 225 for scleraldehyde. In addition, scleraldehyde derivative gives a base fragment peak at m/z 181 and a fragment peak at m/z 44.
0.10
3.3.
Antioxidant activity of scleraldehyde
0.08
3.3.1.
DPPH scavenging activity
0.06
The comparison of antioxidant activity of scleraldehyde and the positive control ascorbic acid, as estimated in in vitro tests, is reported in Table 2. The results are expressed as the
0.04
Table 2 – Antioxidant activities of scleraldehyde each value is the mean of at least three independent experiments7SD.
0.02 0.00 200
250
300
350
Wavelength (nm)
Fig. 1 – UV spectroscopic analysis of scleraldehyde.
400
Compound
DPPH EC50 (μg/mL)
Scleraldehyde Ascorbic acid
80.9870.21 78.1270.01
Fig. 2 – IR spectra for scleraldehyde.
26
Bioactive Carbohydrates and Dietary Fibre 2 (2013) 22 –29
EC50 for each sample used. One parameter that is introduced recently for the interpretation of the results from the DPPH method, is the “efficient concentration” or EC50 value (BrandWilliams, Cuvelier, & Berset, 1995). This is defined as the concentration of substrate that causes 50% loss of the DPPH activity. Stronger the antioxidant activity, EC50 value is minimum. In all samples, presence of 100 μM scleraldehyde shows the highest antioxidant activity compared to the ascorbic acid. Increasing values of EC50 are observed for derivatives with minimum concentration of scleraldehyde (o100 μM) reaching values about 5 and 3 times higher (p, 0.01) than control, respectively.
3.3.2.
ABTS assay
Similarly, from ABTS assay it is observed that the antioxidant behavior of scleraldehyde at 100 μM concentration shows to have similarly higher activity with ABTS when compared ascorbic acid (100 μM). ABTS scavenging activity of scleraldehyde at different concentration (0–100 μM) is shown in Fig. 3. From the figure, the percentage of scavenging activity is increased as the concentration of scleraldehyde increases. The presence of 100 μM concentration of scleraldehyde induces 70% scavenging activity against the control ascorbic acid with 89% activity (Re et al., 1999).
3.3.3.
Total antioxidant activity
The ferric reducing/antioxidant power (FRAP assay) is widely used in the evaluation of the antioxidant component in organic compounds (Luximon-Ramma, Bahorun, Soobrattee, & Aruoma, 2002). The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. The antioxidant potential of scleraldehyde is estimated from their ability to reduce TPRZ-Fe(III) complex to TPTZ-Fe(II). The reducing ability of the scleraldehyde is 54.8170.92 μM of Fe(II)/g while that of control is (72.0770.06 μM), respectively.
3.4.
Kinetics of inhibition
In order to establish the rate of inhibition of antioxidant enzymes by scleraldehyde, hydrolysis of the appropriate substrates at different concentrations of scleraldehyde is investigated. The
ABTS scavenging activity (%)
80
60
40
20
Scleraldehyde Ascorbic acid
0 0
20
40 60 Concentration (uM)
80
100
Fig. 3 – ABTS scavenging activity of scleraldehyde at different concentration such as 20, 40, 60, 80 and 100 μM.
Michaelis–Menten constant obtained for superoxide dismutase is Km ¼0.58 mM for the 6-hydroxydopamine substrate at 25 1C, pH 8 in 0.2 M Tris HCl. Similarly, for Michaelis–Menten constant for catalase is Km ¼ 0.32 mM for the hydrogen peroxide substrate and for glutathione peroxidase is Km ¼0.26 mM for the phospholipid hydroperoxide substrate. The kinetics of inhibition and mechanism of action of inhibitor scleraldehyde is performed by analyzing the nature of inhibition observed by varying concentrations of substrates. The mode of inhibition by scleraldehyde is determined by plotting double reciprocal Lineweaver–Burk plots (v1 against [S]1) to study the effect of inhibitor on antioxidant activity. The kinetic parameters Km, Vmax and Ki are listed in Table 3. The Lineweaver–Burk plots (Fig. 4) obtained for respective substrates by incubated in the presence of different concentrations of scleraldehyde (20, 40, 60, 80 and 100 mM) reveal that there is no appreciable change in Km values. Addition of scleraldehyde at concentrations of 20, 40, 60, 80 and 100 mM to superoxide dismutase caused a decrease in the velocity to 0.61, 0.422, 0.314, 0.237 and 0.219 mmol s1, respectively, without appreciable change in the Km value as shown in Fig. 4a. The double reciprocal plot suggests that scleraldehyde apparently acts as a non-competitive inhibitor of superoxide dismutase during inhibition against substrate. A Dixon plot of v1 vs [I] at different substrate concentrations yields an inhibition constant (Ki) of 34 mM. Similarly at different concentration of scleraldehyde, the presence of catalase cause a decrease in velocity such as 0.59, 0.40, 0.29, 0.20 and 0.19 mmol s1, respectively and yields an inhibition constant Ki of 31 as shown in Fig. 4b. and glutathione dismutase also results in decrease in velocity such as 0.55, 0.36, 0.25, 0.18 and 0.17 mmol s1, respectively, and yields an inhibition constant Ki of 29 mM as shown in Fig. 4c. The activity of scleraldehyde towards superoxide dismutase is prominent compared to that of other enzymes. The study on the influence of scleraldehyde on the activity of antioxidant enzymes illustrates the function of scleraldehyde as an potent antioxidant agent which can be used in various biomedical applications (Li, Liu, Fan, Ai, & Shan, 2011).
3.5.
Mechanistic inhibition action of scleraldehyde
According to kinetic studies of radical formation and decomposition reactions, the antioxidant activity of scleraldehyde closely related to its chemical structure. The structural requirements are important for high antioxidant activity of an aldehyde. The 2,3-double bond, in conjugation with the 4-oxo function, enhancing electron-transfer and radical scavenging actions through electron-delocalization. The presence of both 3- and 5OH groups enabled the formation of stable quinonic structures upon oxidation. Substitution of the 3-OH resulted in increase in the tortion angle, loss of coplanarity structure and reduced antioxidant activity. The scleraldehyde as an inhibitor undergoes non-competitive inhibition towards antioxidant enzymes. In which the inhibitor would have no effect on substrate binding and vice versa. The inhibitor and substrate unite reversibly, arbitrarily, and independently at different sites and do not bind to the active site of the enzymes as shown in Fig. 5. The substrate binds to enzymes (for e.g. Superoxide dismutase (PDB ID: 2CW2) and Enzyme-Inhibitor complex (Superoxide dismutase-Scleraldehyde complex) and Inhibitor (Scleraldehyde) binds to Enzyme and
27
Bioactive Carbohydrates and Dietary Fibre 2 (2013) 22 –29
Table 3 – Michaelis–Menten parameters for antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GTP) oxidation of appropriate substrate at 25 1C, pH 8 in the presence of varying concentrations of scleraldehyde. Inihibtor
Enzyme
Concentration [μM]
Km (mM)
Vmax (mmol s1)
Ki [μM]
Type of inhibition
Control (only enzyme)
SOD CAT GTP SOD
– – – 20 40 60 80 100 20 40 60 80 100 20 40 60 80 100
0.5870.06 0.3270.02 0.2670.01 0.92570.005 0.90370.002 0.86270.003 0.81970.008 0.71470.004 0.90570.005 0.87370.001 0.82270.002 0.79470.007 0.70470.001 0.89570.002 0.84370.005 0.76270.009 0.74970.003 0.66470.004
0.24470.02 0.22470.04 0.18470.06 0.61370.04 0.42270.02 0.31470.01 0.23770.03 0.21970.01 0.59370.01 0.40270.06 0.29470.02 0.20770.04 0.19970.05 0.55370.01 0.36270.02 0.25470.04 0.18770.08 0.17970.01
– – – 3470.6
– – – Non-competitive
3170.7
Non-competitive
2970.8
Non-competitive
Scleraldehyde
CAT
GTP
40
100µM Scl
Superoxide dismutase
35 80µM Scl
1/v (m mol-1.s)
30 25
60µM Scl
20
40µM Scl 20µM Scl
15
0µM Scl
10 5 0
-2
-5
0
2
4
6
1/[S]
(mM-1)
8
100µM Scl 28
80µM Scl
24
60µM Scl
20
40µM Scl
16
20µM Scl
12
0µM Scl
8
24
80µM Scl
20
60µM Scl
16
40µM Scl
12
20µM Scl
8
0µM Scl
0
0 -4
Glutathione peroxidase
4
4 -2
12
100µM Scl
28
Catalase
1/v (m mol-1.s)
1/v (m mol-1.s)
32
10
0
2
4
6
8
10
12
-1)
1/[S] (mM
0
2
4 1/[S]
6
8
10
(mM-1)
Fig. 4 – Line weaver burk plot of scleraldehyde against antioxidant enzymes such as (a) superoxide dismutase (b) catalase and (c) glutathione dismutase.
Enzyme-Substrate (Superoxide dismutase-6 hydroxy dopamine). There appears to be a reduced amount of active enzyme at any given concentration of the scleraldehyde possibly because of the formation of a non-productive Enzyme-Substrate-Inhibitor
complex (Superoxide dismutase-6-hydroxy dopamine-Scleraldehyde). Similarly, Scleraldehyde on catalase and glutathione peroxidase undergo non-competitive inhibition as shown in Fig. 5.
28
Bioactive Carbohydrates and Dietary Fibre 2 (2013) 22 –29
Fig. 5 – Kinetic mechanism of action of scleraldehyde as an inhibitor (I) on superoxide dismutase (E) along with 6 hydroxydopamine as substrate (S).
4.
Conclusions
One important function of antioxidants toward free radicals is to suppress free radical mediated oxidation by inhibiting the formation of free radicals and/or by scavenging radicals. The present study indicates that the more polar compounds possess reactive oxygen species in the aqueous phase. The free radicals were readily scavenged by the polar antioxidants such as scleraldehyde in in vitro system. Therefore, oxidation property will relatively decrease. In conclusion, the hydroxyl group present in C2, C4 and C6 of scleraldehyde are the main reactive sites for the antioxidant enzymes and increases its antioxidant ability in the biological system. A mechanism indicated the fact that aldehyde and hydroxyl group addition increases the polarity of the molecule and thus the polar antioxidants can retard peroxidation by scavenging water soluble oxygen species more effectively.
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Ross, J. H. (1953). Infrared spectra for analysis of aldehyde and ketone 2,4-dinitrophenylhydrazones. Analytical Biochemistry, 25 (9), 1288–1303. Shrikant, A. S., Parag, S. S., & Rekha, S. S. (2006). Production of scleroglucan from Sclerotium rolfsii MTCC 2156. Bioresource Technology, 97, 989–993. Vandenheuvl, W. J. A., & Horning, E. C. (1963). Gaschromatographic characterization of steroid ketones as N,Ndimethylhydrazones. Biochimica et Biophysica Acta, 74, 560–563.
Xu, H., Pamela, J. W., Noel, W. H., & Jeffrey, A. J. (1993). Temperature dependent near-UV molar absorptivities of aliphatic aldehydes and ketones in aqueous solution. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 49, 1171–1178.
29
Available online at www.sciencedirect.com
www.elsevier.com/locate/bcdf
‘In vitro’ evaluation of the antioxidant activity in relation with structure and kinetic properties of scleraldehyde Nagarajan Usharani, Gladstone Christopher Jayakumar, Swarna V. Kanthn, Jonnalagadda Raghava Rao Council of Scientific and Industrial Research-Central Leather Research Institute, Adyar, Chennai 600 020, India
ar t ic l e in f o
abs tra ct
Article history:
Schizophyllum commune is the world′s most extensively disseminated mushroom except in
Received 20 May 2013
Antarctica. Schizophyllan is a polysaccharide fractions isolated from S. commune. The
Received in revised form
schizophyllan is oxidized to form scleraldehyde to introduce carbonyl group for additional
15 August 2013
functions. The relationship between structure, kinetic and antioxidant properties of
Accepted 17 August 2013
scleraldehyde was studied in order to establish the molecular characteristics related to its maximum radical scavenging activity. In our earlier paper we have reported that
Keywords:
scleraldehyde obtained from schizophyllan with the dialdehyde content of 28%. From the
Schizophyllan
absorption studies, it explains there is π–πn excitations involving the benzene groups.
Scleraldehyde
Moreover, the structural identification of scleraldehyde were characterized by bands at
Structure
1670 and 3600–3200 cm1, corresponding to the axial deformation of the C ¼O bond and the
Antioxidant activity
angular deformation of the C–H bond respectively. The IR spectrum confirms the identity
Kinetic property
of aldehyde. The scavenging assays of scleraldehyde was found to be reliant on their reactivity and concentration. Scavenging assays performed indicate that 100 μM of scleraldehyde showed the highest antioxidant activity compared to the ascorbic acid. Furthermore, The reducing ability of the scleraldehyde was 54.8170.92 μM of Fe(II)/g compared to the control (Ascorbic acid) respectively. The kinetic mechanism of scleraldehyde portrays a competitive inhibition against antioxidant enzymes on calculating for radical scavenging activity. By means of structure determination, kinetic assays and the antioxidant assays that hydroxyl group present in C2, C4 and C6 are the main reactive sites. The hypothesis converse that scleraldehyde can serve as scavenger of aqueous peroxyl radicals near the membrane surface of collagen. & 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
Schizophyllum commune is the world′s most widely distributed mushroom, produces number of enzymes such as bilirubin oxidase, fibrinolytic, acetylxylan transferase, extra cellular protease and a vital extracellular polysaccharide called
n
Corresponding author. Tel.: þ91 44 2491 1386; fax: þ91 44 2491 1589. E-mail address: [email protected] (S.V. Kanth).
2212-6198/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bcdf.2013.08.002
schizophyllan used both for medicinal and industrial applications (Maushmi, Shrikant, & Rekha, 2008). Schizophyllan, produced by S. commune is a neutral exopolysaccharide with a chemical structure consisting of 1,3 β-D-linked backbone of glucose residues and 1,6-β-D-glucosyl side chain at every third residue. Native schizophyllan with a typical molecular weight
Bioactive Carbohydrates and Dietary Fibre 2 (2013) 22 –29
of the order of 5.54 105 Da chemically two polysaccharides such as curdlan and scleroglucan produced by Agrobacterium sp and Sclerotium rotifis, respectively (Shrikant, Parag, & Rekha, 2006). In contrast to most polysaccharides, Schizophyllan exhibits a fairly high solubility in water forming the triple-helical conformation with a persistence length between 150 and 200 nm which is stable up to a temperature of 140 1C (David et al., 2007). Polysaccharides such as schizophyllan and scleroglucan contain periodate resistant β-1,3-linked D-glucan backbones with regularly distributed side chains consisting primarily of single β-1,6-linked D-glucose. Side chain oxidation of schizophyllan results in the formation of dialdehyde group called Scleraldehyde, and the production of formic acid. The mechanism of crosslinking of this scleraldehyde is explained by the formation of intermolecular hemiacetal linkages (Kristiansen, Potthast, & Christensen, 2010). Moreover the potential of scleraldehyde as an antimicrobial agent was also explored in our earlier study (Jayakumar, Swarna, Chandrasekaran, Rao, & Nair, 2010). The structural determinants of scleraldehyde molecule are incomprehensible, but the position and number of the hydroxylic groups have been suggested to play an important role in the antioxidant activity (Rice-Evans, Miller, & Paganga, 1996). The present study was extended by understanding the structural determinants relating to the activity of scleraldehyde, and in particular to establish the rate of inhibition using kinetic studies. The antioxidant activity was investigated in vitro by measuring the radical scavenging ability using the free radicals and antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase. Different chemical and physical principles was obtained from in-vitro activity values for antioxidant activity that vary according to the assays employed. Hence, the present investigation focussed on the structure-activity relationship of scleraldehyde.
2.
Materials and methods
2.1.
Materials
Media components such as glucose, sucrose, maltose, lactose, starch, magnesium sulfate, yeast extract, peptone were purchased from Hi-Media Limited, Mumbai, India. All other chemicals were purchased from S.D. Fine Chemicals Ltd, Mumbai, India. Strains of Schizophyllum commune (S. commune) MTCC 1096 were procured from Microbial Type Culture (MTCC), Chandigarh.
2.2.
Cultivation of Schizophyllum commune
S. commune was maintained on solid medium containing 20 g of malt extract/L, 1 g of peptone/L, 20 g of dextrose/L, and 20 g of agar/L. A liquid culture was started by transferring approximately 7 mm of a 7–10 day-old colony into 250 mL of the same medium without agar in a 500-mL fluted Erlenmeyer flask with three 10-mm glass beads. This culture was grown for 4 days at 30 1C and 240 rpm. Suspensions (0.5 mL) were used to inoculate 300-mL flasks containing 50 mL (w/v) of
23
corn fiber pretreated as previously described with alkaline H2O2 in a basal medium containing 0.67% yeast nitrogen base (Difco), 0.2% asparagine, and 0.5% KH2PO4. Cultures containing treated corn fiber were grown for 7 days at 30 1C and 240 rpm (Jayakumar et al., 2010).
2.3.
Isolation of schizophyllan
Mycelial growth was removed from cultures by centrifugation in 50 mL conical tubes at 3220g for 30 min at 4 1C. Polysaccharides were recovered from culture supernatants using 95% ethanol diluted to 50% (v/v) and 67% (v/v). Polysaccharide pellets were resuspended in distilled H2O, transferred to 15 mL conical tubes, and dried under vacuum at 50 1C for analysis.
2.4.
Extraction of schizophyllan from pellet
S. commune was cultivated in 1000 mL yeast malt broth for seven days. The seven days culture was centrifuged for 20 min at 2500 rpm at 4 1C and the pellet was obtained. 300 mL of deionized water was then added to the whole pellet and incubated for 3 h at 100 1C. After 3 h, 300 mL of 80% ethanol was added to the above sample and incubated at 100 1C for 3 h and 300 mL of 1% ammonium oxalate solution is added to the sample and incubated for 6 h at 100 1C. 150 mL of 5% NaOH was then added to the above sample and incubated for 6 h at 80 1C. Final residue (Schizophyllan) obtained after 6 h was weighed (Jayakumar et al. 2012).
2.5.
Periodate oxidation of schizophyllan
Schizophyllan (10 g) was hydrolyzed in 5 N hydrochloric acid (10 h, 85 1C). Hydrolyzed schizophyllan was suspended in demineralized water and subsequently cooled in an ice bath. 12 g of sodium periodate was added to the sample while stirring with a magnetic stirrer. The pH of the solution was maintained around 4 during the reaction. The reaction was performed in the dark at room temperature and stopped after 48 h to obtain scleraldehyde of 99% oxidation. The product obtained was centrifuged with t-butyl alcohol (1:3 sample: solvent). The product was resuspended in the same volume of demineralized water and the centrifugation carried out several times. The product was dried at room temperature. The oxidation of schizophyllan was determined by the release of formic acid using procedures reported earlier.
2.6.
Structural determinations
2.6.1.
UV–visible spectroscopy
The UV–visible absorption spectra were recorded on a Cary 100 UV–VIS–NIR spectrometer with a spectral resolution of 2 nm in the wavelength range 200–600 nm (Xu, Pamela, Noel, & Jeffrey, 1993).
2.6.2.
Fourier transform—Infrared spectroscopy
Infrared spectra of the sample was obtained from KBr pellets at room temperature using a Jasco Spectrometer with a resolution of 4 cm1 averaging 50 scans in the 4000– 400 cm1 wavenumber range (Ross, 1953).
24
2.6.3.
Bioactive Carbohydrates and Dietary Fibre 2 (2013) 22 –29
GC–MS analysis
Vanden Heuvel and Horning have used the hydrazone products for gas chromatographic analyses, relying solely on retention times for characterization (Vandenheuvl & Horning, 1963). Gas chromatographic analyses were performed on a JEOL GC Mate gas chromatograph with a FID detector. The column employed was a 1.8-m 2-ram ID glass column packed with 3% SP-2100 on 100/120 mesh. Electron impact spectra were generated with a source potential of 70 eV; chemical ionization spectra were generated using ultrapure methane (Airco, Incorporated) as both the carrier gas and the ionizing gas. The internal source pressure was 0.5 Torr, with a source potential of 200 eV.
2.7.
Antioxidant activity of scleraldehyde
2.7.1.
Radical scavenging activity (DPPH model system)
Scleraldehyde (10 μg/mL) and positive control ascorbic acid (10 μg/mL) in methanol (0.1 mL) was added to 3.9 mL of a 6 105 M DPPH solution in methanol (Chen & Ho, 1997). The exact initial DPPH concentration in the reaction medium was calculated from a calibration curve. Decrease in absorbance was determined at 515 nm for 1 h for every 5 min, and every 60 min until the reaction reached a plateau (about 6 h). Antiradical activity was expressed as the EC50, i.e. antioxidant concentration required to decrease the initial amount of DPPH by 50% (Blois, 1958).
2.7.2.
ABTS radical scavenging assay
ABTS radical scavenging activities of scleraldehyde (0–100 μM) and positive control ascorbic acid (100 μM) are determined by the method described by Re et al. (1999). with slight modification. The stock solution of ABTS radicals is made by mixing 5.0 mL of 7 mM ABTS solution with 88 μL of 140 mM potassium persulfate, and kept in the dark for 12–16 h at 37 1C. An aliquot of stock solution is diluted with phosphate buffer, PB (5 mM, pH 7.4) containing 0.15 M NaCl, to prepare the working solution of ABTS radicals to an absorbance of 0.7070.02 at 734 nm. Samples are mixed 1:4 (v/v) with diluted ABTS, or only buffer (control), incubated for 10 min at 37 1C in the dark, and then absorbance is measured at 734 nm. The percent reduction of ABTSþ to ABTS is calculated according to the following equation: ð%Þ ¼ ½ðAbscontrol Abssample Þ=ðAbscontrol Þ 100 Abscontrol is the absorbance of ABTS radical and methanol; Abssample is the absorbance of ABTS radical and sample extract/standard.
dark condition. Readings of the colored product (ferrous tripyridyltriazine complex) were taken at 593 nm. The standard curve was linear between 50 and 1000 μM FeSO4. Results are expressed in μM Fe(II)/g dry mass and compared with that of ascorbic acid (100 μM) (Benzie & Strain, 1996).
2.8.
The enzyme inhibition was monitored continuously for 20–60 min by adding the enzyme such as superoxide dismutase, catalase and glutathione peroxidase to a solution of buffer with inhibitor (scleraldehyde). The appropriate substrate used for the enzymes were 6-hydroxy dopamine, hydrogen peroxide and phospholipid hydroperoxide respectively. The concentrations of ethanol extract of scleraldehyde were used and compared with the kinetic behavior of ascorbic acid as a control. Enzyme concentration and total volume of reaction mixture was kept constant but the concentration of substrate such as used varied in the range of 0.1–1 mM. The results were analyzed in terms of Michaelis–Menten treatment. Kinetic data were analyzed by double reciprocal plots and computer-fitted to appropriate rate equations by means of non-linear regression analysis program (EZFIT) (Knight, 1995). The nomenclature used in this study is that of Cleland. From the Lineweaver–Burk plots of v1 vs [S]1, the kinetic parameters such as Vmax, maximum velocity and Km, the Michaelis–Menten constant of the enzyme were calculated. Dixon plots of v1 vs [I] were used to determine the inhibition constant (Ki) for competitive and non-competitive inhibition. Ki for mixed inhibition was determined using secondary plots, by replotting slopes and intercepts of Lineweaver–Burk plots vs. [I]. Cornish–Bowden plots of [S]/v vs. [I] were used to calculate Ki for uncompetitive inhibition. In all the cases, the intercept on the [I] axis gives Ki.
3.
Results and discussions
A novel antioxidant which is eligible for standardization requires an attention to some structural requirements of antioxidant potency (Liu et al., 2009). The present investigation involves the characterization of scleraldehyde as possible antioxidant agent. This has been examined by understanding the structural parameters of scleraldehyde relating to the antioxidant activity. Radical scavenging action of scleraldehyde is found to be dependent on both reactivity and concentration of the antioxidant.
3.1. 2.7.3.
Kinetic of investigations
Preparation and characterization of scleraldehyde
Reducing ability (FRAP assay)
The determination of the total antioxidant activity (FRAP assay) of the synthesized compounds was evaluated by modified method of Benzie and Strain (1996). The stock solutions included 300 mM acetate buffer, pH 3.6, 10 mM TPTZ (2,4,6-tripyridyl-S-triazine) solution in 40 mM HCl and 20 mM FeCl3 6H2O solution. Working solution was prepared by mixing acetate buffer (25 mL), TPTZ (2.5 mL) and FeCl3 6H2O (2.5 mL). The temperature of the solution was raised to 37 1C before use. Scleraldehyde (100 μM) was allowed to react with 1500 μL of the FRAP solution for 30 min in the
Scleraldehyde was obtained by oxidizing schizophyllan by sodium metaperiodate. Periodate oxidation specifically cleaves the vicinal glycols in schizophyllan to form their dialdehyde derivatives called scleraldehyde. It is observed that scleraldehyde consumes up to 2 mol of periodate per mole of repeating unit with simultaneous release of up to 1 mol of formic acid as reported in our earlier paper. The dialdehyde content of scleraldehyde was found to be 28%. The yield of schizophyllan and scleraldehyde from pellet and supernatant is given Table 1. Higher amount of scleraldehyde
25
Bioactive Carbohydrates and Dietary Fibre 2 (2013) 22 –29
is obtained from pellet when compared with preparation of scleraldehyde from supernatant. The antioxidant activities of scleraldehyde is compared with the standard control, ascorbic acid.
3.2.
Structural characterization of scleraldehyde
3.2.1.
UV–visible Spectroscopic determination
Intermediate intensity peaking around 240 nm (1.2 eV, ε (max)E1000 M (1) cm (1)) is dominated by ππn excitations within the arene function. Finally, strong absorptions (ε(max) E10,000 M(1) cm(1)) is observed around 210 nm (4.0 eV) which is ascribe to ππn excitations involving the benzene groups.
3.2.2.
IR spectrum of scleraldehyde
Source of extraction
Amount of schizophyllan
Amount of scleraldehyde
The IR spectra (Fig. 2) of scleraldehyde are characterized by bands at 1670 and 3600–3200 cm1, corresponding to the axial deformation of the C¼O bond and the angular deformation of the C–H bond respectively. The IR spectrum confirms the identity of aldehyde. Moreover, the scleraldehyde spectrum can be observed with intense band at 1618 cm1, probably related to the asymmetric stretching vibration of the –C¼O group. The acidification of the derivative produces the disappearance of this peak and the appearance of a new peak at 1734 cm1, probably due to the stretching vibration of the C¼O in the undissociated acid.
Pellet Supernatant
9.66 g/30 g of pellet 5.98 g/1000 mL of supernatant
6.54 g 3.79 g
3.2.3.
The UV–visible absorption spectrum of scleraldehyde is shown in Fig. 1. The degree of conjugation of the molecule, and some evidence of identity through spectral matching was observed. The spectra of scleraldehyde isomers are characterized by weak (ε(max)E100 M (1) cm(1)) transitions around 290 nm (2.6 eV), arising from nπn absorptions.
Absorbance
Table 1 – Production profile of scleraldehyde.
GC–MS analysis
0.12
The mass spectra of scleraldehyde shows molecular ion (M)þ at m/z 225 for scleraldehyde. In addition, scleraldehyde derivative gives a base fragment peak at m/z 181 and a fragment peak at m/z 44.
0.10
3.3.
Antioxidant activity of scleraldehyde
0.08
3.3.1.
DPPH scavenging activity
0.06
The comparison of antioxidant activity of scleraldehyde and the positive control ascorbic acid, as estimated in in vitro tests, is reported in Table 2. The results are expressed as the
0.04
Table 2 – Antioxidant activities of scleraldehyde each value is the mean of at least three independent experiments7SD.
0.02 0.00 200
250
300
350
Wavelength (nm)
Fig. 1 – UV spectroscopic analysis of scleraldehyde.
400
Compound
DPPH EC50 (μg/mL)
Scleraldehyde Ascorbic acid
80.9870.21 78.1270.01
Fig. 2 – IR spectra for scleraldehyde.
26
Bioactive Carbohydrates and Dietary Fibre 2 (2013) 22 –29
EC50 for each sample used. One parameter that is introduced recently for the interpretation of the results from the DPPH method, is the “efficient concentration” or EC50 value (BrandWilliams, Cuvelier, & Berset, 1995). This is defined as the concentration of substrate that causes 50% loss of the DPPH activity. Stronger the antioxidant activity, EC50 value is minimum. In all samples, presence of 100 μM scleraldehyde shows the highest antioxidant activity compared to the ascorbic acid. Increasing values of EC50 are observed for derivatives with minimum concentration of scleraldehyde (o100 μM) reaching values about 5 and 3 times higher (p, 0.01) than control, respectively.
3.3.2.
ABTS assay
Similarly, from ABTS assay it is observed that the antioxidant behavior of scleraldehyde at 100 μM concentration shows to have similarly higher activity with ABTS when compared ascorbic acid (100 μM). ABTS scavenging activity of scleraldehyde at different concentration (0–100 μM) is shown in Fig. 3. From the figure, the percentage of scavenging activity is increased as the concentration of scleraldehyde increases. The presence of 100 μM concentration of scleraldehyde induces 70% scavenging activity against the control ascorbic acid with 89% activity (Re et al., 1999).
3.3.3.
Total antioxidant activity
The ferric reducing/antioxidant power (FRAP assay) is widely used in the evaluation of the antioxidant component in organic compounds (Luximon-Ramma, Bahorun, Soobrattee, & Aruoma, 2002). The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. The antioxidant potential of scleraldehyde is estimated from their ability to reduce TPRZ-Fe(III) complex to TPTZ-Fe(II). The reducing ability of the scleraldehyde is 54.8170.92 μM of Fe(II)/g while that of control is (72.0770.06 μM), respectively.
3.4.
Kinetics of inhibition
In order to establish the rate of inhibition of antioxidant enzymes by scleraldehyde, hydrolysis of the appropriate substrates at different concentrations of scleraldehyde is investigated. The
ABTS scavenging activity (%)
80
60
40
20
Scleraldehyde Ascorbic acid
0 0
20
40 60 Concentration (uM)
80
100
Fig. 3 – ABTS scavenging activity of scleraldehyde at different concentration such as 20, 40, 60, 80 and 100 μM.
Michaelis–Menten constant obtained for superoxide dismutase is Km ¼0.58 mM for the 6-hydroxydopamine substrate at 25 1C, pH 8 in 0.2 M Tris HCl. Similarly, for Michaelis–Menten constant for catalase is Km ¼ 0.32 mM for the hydrogen peroxide substrate and for glutathione peroxidase is Km ¼0.26 mM for the phospholipid hydroperoxide substrate. The kinetics of inhibition and mechanism of action of inhibitor scleraldehyde is performed by analyzing the nature of inhibition observed by varying concentrations of substrates. The mode of inhibition by scleraldehyde is determined by plotting double reciprocal Lineweaver–Burk plots (v1 against [S]1) to study the effect of inhibitor on antioxidant activity. The kinetic parameters Km, Vmax and Ki are listed in Table 3. The Lineweaver–Burk plots (Fig. 4) obtained for respective substrates by incubated in the presence of different concentrations of scleraldehyde (20, 40, 60, 80 and 100 mM) reveal that there is no appreciable change in Km values. Addition of scleraldehyde at concentrations of 20, 40, 60, 80 and 100 mM to superoxide dismutase caused a decrease in the velocity to 0.61, 0.422, 0.314, 0.237 and 0.219 mmol s1, respectively, without appreciable change in the Km value as shown in Fig. 4a. The double reciprocal plot suggests that scleraldehyde apparently acts as a non-competitive inhibitor of superoxide dismutase during inhibition against substrate. A Dixon plot of v1 vs [I] at different substrate concentrations yields an inhibition constant (Ki) of 34 mM. Similarly at different concentration of scleraldehyde, the presence of catalase cause a decrease in velocity such as 0.59, 0.40, 0.29, 0.20 and 0.19 mmol s1, respectively and yields an inhibition constant Ki of 31 as shown in Fig. 4b. and glutathione dismutase also results in decrease in velocity such as 0.55, 0.36, 0.25, 0.18 and 0.17 mmol s1, respectively, and yields an inhibition constant Ki of 29 mM as shown in Fig. 4c. The activity of scleraldehyde towards superoxide dismutase is prominent compared to that of other enzymes. The study on the influence of scleraldehyde on the activity of antioxidant enzymes illustrates the function of scleraldehyde as an potent antioxidant agent which can be used in various biomedical applications (Li, Liu, Fan, Ai, & Shan, 2011).
3.5.
Mechanistic inhibition action of scleraldehyde
According to kinetic studies of radical formation and decomposition reactions, the antioxidant activity of scleraldehyde closely related to its chemical structure. The structural requirements are important for high antioxidant activity of an aldehyde. The 2,3-double bond, in conjugation with the 4-oxo function, enhancing electron-transfer and radical scavenging actions through electron-delocalization. The presence of both 3- and 5OH groups enabled the formation of stable quinonic structures upon oxidation. Substitution of the 3-OH resulted in increase in the tortion angle, loss of coplanarity structure and reduced antioxidant activity. The scleraldehyde as an inhibitor undergoes non-competitive inhibition towards antioxidant enzymes. In which the inhibitor would have no effect on substrate binding and vice versa. The inhibitor and substrate unite reversibly, arbitrarily, and independently at different sites and do not bind to the active site of the enzymes as shown in Fig. 5. The substrate binds to enzymes (for e.g. Superoxide dismutase (PDB ID: 2CW2) and Enzyme-Inhibitor complex (Superoxide dismutase-Scleraldehyde complex) and Inhibitor (Scleraldehyde) binds to Enzyme and
27
Bioactive Carbohydrates and Dietary Fibre 2 (2013) 22 –29
Table 3 – Michaelis–Menten parameters for antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GTP) oxidation of appropriate substrate at 25 1C, pH 8 in the presence of varying concentrations of scleraldehyde. Inihibtor
Enzyme
Concentration [μM]
Km (mM)
Vmax (mmol s1)
Ki [μM]
Type of inhibition
Control (only enzyme)
SOD CAT GTP SOD
– – – 20 40 60 80 100 20 40 60 80 100 20 40 60 80 100
0.5870.06 0.3270.02 0.2670.01 0.92570.005 0.90370.002 0.86270.003 0.81970.008 0.71470.004 0.90570.005 0.87370.001 0.82270.002 0.79470.007 0.70470.001 0.89570.002 0.84370.005 0.76270.009 0.74970.003 0.66470.004
0.24470.02 0.22470.04 0.18470.06 0.61370.04 0.42270.02 0.31470.01 0.23770.03 0.21970.01 0.59370.01 0.40270.06 0.29470.02 0.20770.04 0.19970.05 0.55370.01 0.36270.02 0.25470.04 0.18770.08 0.17970.01
– – – 3470.6
– – – Non-competitive
3170.7
Non-competitive
2970.8
Non-competitive
Scleraldehyde
CAT
GTP
40
100µM Scl
Superoxide dismutase
35 80µM Scl
1/v (m mol-1.s)
30 25
60µM Scl
20
40µM Scl 20µM Scl
15
0µM Scl
10 5 0
-2
-5
0
2
4
6
1/[S]
(mM-1)
8
100µM Scl 28
80µM Scl
24
60µM Scl
20
40µM Scl
16
20µM Scl
12
0µM Scl
8
24
80µM Scl
20
60µM Scl
16
40µM Scl
12
20µM Scl
8
0µM Scl
0
0 -4
Glutathione peroxidase
4
4 -2
12
100µM Scl
28
Catalase
1/v (m mol-1.s)
1/v (m mol-1.s)
32
10
0
2
4
6
8
10
12
-1)
1/[S] (mM
0
2
4 1/[S]
6
8
10
(mM-1)
Fig. 4 – Line weaver burk plot of scleraldehyde against antioxidant enzymes such as (a) superoxide dismutase (b) catalase and (c) glutathione dismutase.
Enzyme-Substrate (Superoxide dismutase-6 hydroxy dopamine). There appears to be a reduced amount of active enzyme at any given concentration of the scleraldehyde possibly because of the formation of a non-productive Enzyme-Substrate-Inhibitor
complex (Superoxide dismutase-6-hydroxy dopamine-Scleraldehyde). Similarly, Scleraldehyde on catalase and glutathione peroxidase undergo non-competitive inhibition as shown in Fig. 5.
28
Bioactive Carbohydrates and Dietary Fibre 2 (2013) 22 –29
Fig. 5 – Kinetic mechanism of action of scleraldehyde as an inhibitor (I) on superoxide dismutase (E) along with 6 hydroxydopamine as substrate (S).
4.
Conclusions
One important function of antioxidants toward free radicals is to suppress free radical mediated oxidation by inhibiting the formation of free radicals and/or by scavenging radicals. The present study indicates that the more polar compounds possess reactive oxygen species in the aqueous phase. The free radicals were readily scavenged by the polar antioxidants such as scleraldehyde in in vitro system. Therefore, oxidation property will relatively decrease. In conclusion, the hydroxyl group present in C2, C4 and C6 of scleraldehyde are the main reactive sites for the antioxidant enzymes and increases its antioxidant ability in the biological system. A mechanism indicated the fact that aldehyde and hydroxyl group addition increases the polarity of the molecule and thus the polar antioxidants can retard peroxidation by scavenging water soluble oxygen species more effectively.
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