Vol. 1 Issue 1, pp: (1-16), March 2016. Available online at: http://www.prudentjournals.org/IRJC

Full Length Research Paper RETRACTED

Isolation, characterisation and in vitro evaluation of the antioxidant properties of α-lipoic acid from

de-oiled rice bran.

Surashree Sen Gupta1* and Mahua Ghosh1

1Department of Chemical Technology, University of Calcutta, Kolkata

West Bengal 700 009, India

*Corresponding author: surashree_sg@yahoo.co.in, surashree.sengupta@gmail.com

Received 20 January, 2016; Accepted 22 February, 2016.

ABSTRACT

α-lipoic acid was isolated from de-oiled rice bran by solvent extraction, followed by purification. α-lipoic

acid was isolated using ten different solvents/solvent mixtures. A 1:10 proportion of de-oiled bran and

solvents/solvent mixture was maintained. The mixtures were intensely blended followed by

ultrasonication. Finally, they were purified by protein removal and treatment with alkali. The most

effective solvent system for maximum yield of α-lipoic acid was water:ethanol (2:1) which yielded an

amount of (0.43+0.02)% α-lipoic acid. Identity of the isolated and purified product was confirmed by

HPLC, FTIR, NMR and GC analysis methods. HPLC retention time at 2.96 minutes and GC retention

time at 19.80 minutes were obtained which were similar to those of the procured standard α-lipoic acid.

FTIR and NMR techniques confirmed the atypical S–S linkage of α-lipoic acid, its carboxylic acid group

and the ring structure. Finally varying concentrations of α-lipoic acid were prepared in water and their

antioxidant efficacy were analysed by virtue of seven in vitro assay entities. A positive outcome was

observed with α-lipoic acid in response to all the antioxidant assays performed. A concentration of 0.1%

α-lipoic acid indicated to be the most effective one in terms of providing maximum antioxidant potency.

Key words: α-lipoic acid; antioxidant; isolation; purification; rice bran; solvent extraction.

International Research Journal of Chemistry Article Number: PRJA45923613 Copyright ©2016 Author(s) retain the copyright of this article

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons

Attribution 4.0 International License.

INTRODUCTION

Alpha-lipoic acid (ALA) or thioctic acid or 1,2-dithiolane-3-pentanoic acid, a natural antioxidant, prevents massive impairment of body cells by scavenging the sole genesis, ‘free radicals’. In fact, ALA is well-known for the management of several age-related endemics [1]. A distinctive advantage of ALA is that it is both fat- and water-soluble. Hence it contrives both in body membranes and in aqueous mediums. ALA is changed into dihydrolipoic acid in cells of the body [1]. It is produced mostly in micro-organisms and present in trace amounts in some fruits, vegetables and body cells [1-3]. ALA acts as an important diet supplement that promotes healthy living, especially in adults beyond 40 years, due to its therapeutic effects [1, 4]. Rice bran is an unconventional yet valuable vegetable source of this unique food supplement [5] which has not been utilized for the extraction of ALA till date. In fact lipoic acid is known to be a popular antioxidant, especially due to its dual solubility [1]. A protective action of alpha lipoic acid was observed against high cholesterol related and oxidation related problems when tested on rabbits [6]. Similar results were obtained from another study involving rats [7]. Alpha lipoic acid was also observed to minimise the toxicity generated by a common insecticide Diazinon [8]. A study conducted on horses indicated that alpha lipoic acid was able to prevent stress due to oxidation [9].

Rice is an extremely valuable cereal crop world-wide. In India, one of the principal centres of rice cultivation, it serves as a staple diet. In annexation, the de-oiled bran is mostly used as

a low-cost feed for livestock. In reality the bran is a reservoir of several precious nutrients, a predominant one being ALA [5, 10, 11]. Successful isolation of ALA would ensure the recovery of ‘WEALTH’ (‘ALA’) from ‘WASTE’ (the ‘rice bran’), an unconventional source. Studies performed so far have reported that crystallized ALA has only been isolated from liver residue by application of benzene, which was followed by acid-hydrolysis with 6N sulphuric acid. Thereafter the product was purified to obtain almost 2.5% of ALA [12]. Rice bran has remained an unexplored by-product of the rice oil industries, which can be applied for extrication of such priceless nutraceutical, viz., ALA.

In the present work, a novel technology for the isolation of ALA from de-oiled rice bran was developed. Isolation of ALA was carried out by solvent extraction with application of ultrasonication, followed by step-wise purification process. The developed modus operandi would initiate the utilization of a neglected product. Furthermore, such a valuable product isolated from a vegetable source is undoubtedly more lucrative than others extracted from an animal source or produced synthetically, especially in terms of avoiding untoward side-effects. The isolated ALA was later identified by various physico-chemical techniques. Finally, several antioxidant assays were performed on five different aqueous concentrations of ALA to determine its most potent formulation which would serve as a therapeutic dose.

MATERIALS AND METHODS

Materials

Authentic rice bran was procured from a local

rice bran oil dealer of West Bengal, India. They

were crushed into fine particles. The crushed

bran was de-oiled using a Soxhlet apparatus

with hexane as the extracting solvent. The de-

oiled hull was dried and then stored at 4°C for

further use. Standard α-lipoic acid was

purchased from Sigma–Aldrich Chemie Gmbh

(Munich, Germany). All other reagents were of

analytical grade and procured from Merck Ltd.,

Mumbai, India.

Selection of extraction solvents for isolation of α-lipoic acid

Solubility of standard α-lipoic acid was checked

in different solvents. Previously ALA was

extracted from liver residue using benzene as

the extraction solvent [12]. However today

isolation of ALA from natural resources is quite

rare as there is a scarcity of the yield of ALA

from such sources. So these days ALA is mostly

2 Int. Res. J. Chem.

synthesized by some chemical process. Hence

the present study is one of its kinds as here an

attempt has been made to isolate ALA from a

natural product using different solvent systems.

Ten different solvents/solvent-systems showing

complete solubility of α-lipoic acid were

shortlisted for extracting ALA from the de-oiled

rice bran. The solvents/solvent-systems were i.

water, ii. ethanol, iii. acetonitrile, iv.

water:ethanol (1:1), v. water:ethanol (2:1), vi.

water:acetonitrile (1:1), vii. water:acetonitrile

(2:1), viii. ethanol:acetonitrile (1:1), ix.

ethanol:acetonitrile (2:1) and x.

water:ethanol:acetonitrile (1:1:1). The de-oiled

bran and the solvents were mixed in a 1:10

proportion (bran:extraction solvent). The final

blends were vigorously agitated using high

speed disperser (OMNI International GLH;

Model: GLH-220) at 20,000 rpm for 3 hours. The

mixtures were further homogenized at 10,000

rpm with 4 re-circulations using homogenizer

(Universal Motors; Type: RQ-127A, Remi

Motors Ltd., Mumbai-53, India) at 1.1 Amp and

220 V. The high pressure and intense mixing

during the processes encouraged diffusion of

ALA in to the solvent medium. Finally, sound-

wave assisted cell rupture was induced by

ultrasonication (Ultrasonicator, Vibronics, 3172

Meco-G) of the mixtures for 1 hour at 200 Volt

for all the solvents/solvent mixtures, while

maintaining a temperature of 10°C throughout

by means of an ice bath.

Purification of extracted products

The extracts were centrifuged at 10°C and 15,000 rpm. The supernatants were collected in volumetric flasks. They were tested for the presence of protein residue by the ninhydrin test [13]. The supernatants of all extracts were dried by a rotary evaporator. As the undesired protein residues of rice bran were insoluble in ethanol, the dried products from extraction systems ‘i’, ‘iii’, ‘vi’ and ‘vii’ were dissolved in fresh ethanol. Complete absence of protein from the ethanol extracts was confirmed by ninhydrin test. All the extracts (i to x) were completely dried by rotary evaporation.

The dried products were dissolved in de-ionised water. Rice bran is a potent source of heavy metals, especially that of inorganic arsenic [14], which were co-extracted along with ALA. To

remove these metals, the extracts were treated with 0.1N aqueous solution of sodium hydroxide. The base was gradually added to the extracts with continuous stirring until alkaline to litmus. The mixtures were then vigorously agitated for 3 hours at a temperature of 40°C. After the reactions were over the metals precipitated as heavy metal oxides/hydroxides. The reaction mixtures were centrifuged at 10,000 rpm and 30°C. The supernatants were concentrated using rotary evaporation technique and then they were collected in proper vials. The concentrated products were then acidified with 0.1N hydrochloric acid until distinctly acidic to litmus paper, in order to release free ALA, which was previously converted to its sodium salt during alkaline treatment. The final ALA product was freeze-dried to get a mild yellow coloured powder-like substance. It was collected and preserved at -20°C for subsequent analysis.

Analysis using High Performance Liquid Chromatography (HPLC)

Analyses were performed using a Waters HPLC (Milford, Massachusetts, US) instrument equipped with a Waters 1525 consisting of binary HPLC pump with a vacuum degasser and a Waters 2487 dual λ absorbance detector. Normal phase separation with Betasil Diol100 column was used. HPLC analyses of isolated and standard α-lipoic acid samples were carried out at a wavelength of 215 nm. The mobile phase consisted of an isocratic system of solution A (HPLC grade hexane) and solution B (0.5% trifluroacetic acid in HPLC grade isopropanol) mixed in the ratio of 95:5. Flow rate was 1ml/minute, injection volume of 30μl, run time 30 minutes and a normal room temperature [15].

Analysis using Fourier-transformed Infrared spectroscopy (FTIR)

FTIR of ALA was recorded on a JASCO FT/IR-6300 type A (Serial no. A014461024), ATR PRO 450-S spectrophotometer, Version – 2.07.01 [Build 1] in the range 650–4000 cm−1. A very thin film of the samples were applied to demountable liquid cell with luer filling, CaF2

0.05 mm. Each spectrum was Fourier-transformed, phase-corrected, and integrated using Sadtler Database – Bio-Rad Laboratories. Resolution was 4 cm−1, aperture 7.1 mm, scanning speed 2 mm/sec and filter 10000 Hz.

Surashree and Mahua 3

Analysis using Nuclear magnetic resonance spectroscopy (NMR)

To elucidate the structure of α-lipoic acid it was analysed by NMR technique (1H NMR). The experiments were performed on a Bruker AV 300 MHz Supercon NMR System Dual Probe. Proton magnetic resonance spectroscopy was done in CD3OD solvent.

Analysis by Gas chromatography (GC)

The α-lipoic acid sample and standard were converted into methyl esters [16] and analyzed by gas chromatography (AGILENT; Model: 6890 N; FID detector; DB Wax column). The retention times of the sample and standard were compared.

Antioxidant assays

The antioxidant activities of α-lipoic acid were examined by seven different in vitro assay systems by preparing different concentrations of 0.001%, 0.005%, 0.01%, 0.05% and 0.1% (w/v) in water.

Assay of DPPH radical-scavenging activity

The scavenging activity of ALA for the stable DPPH free radical was studied. The method was described by Katerere and Eloff (2005) [17]. Absorbance at the λmax value of 517 nm was measured. The activity was expressed as the inhibition percentage. It was calculated according to the formulae, Radical scavenging activity (%) = [(A0 – A1)/A0] x 100, where A0 is the absorbance of the control and A1 is the absorbance of the sample.

Assay of reductive potential

The reductive potential of ALA was determined according to the method of Dorman and Hiltunen (2004) [18]. Different concentrations of ALA were treated with phosphate buffer and potassium ferricyanide. The absorbance was measured at 700 nm after addition of trichloroacetic acid and FeCl3. Higher absorbance of the reaction mixture indicated greater reductive potential.

Assay of metal chelating activity

The Fe2+ chelating ability of ALA was estimated by the method of Dinis, Madeira and Almeida (1994) [19]. Absorbance was thereby measured spectrophotometrically at 562 nm. Percentage of inhibition of ferrozine-Fe2+ complex formation was calculated as follows: % inhibition = [(A0 – A1)/A0] x 100, where A0 is the absorbance of the control and A1 is the absorbance of the sample.

ABTS.+ radical scavenging activity

ABTS.+ radical scavenging activity of ALA were determined after a few modifications by following the method of Re et al. (1999) [20] and Zhao et al.

(2005) [21]. 7mM ABTS.+ (5 ml) was mixed with 140 mM K2S2O8 (88 µl) and kept overnight. The following day the mixture was diluted with 50% ethanol and 20µl ALA solution was added, mixed and the absorbance was measured at 734 nm at 1 second intervals for 5 minutes.

Lipid oxidation in a linoleic acid emulsion model system

Inhibition of lipid peroxidation by ALA was studied by following the procedure described by Gulcin et al. (2004) [22]. A linoleic acid pre-emulsion was prepared by mixing 0.28 g of linoleic acid with 0.28 g of Tween 20 in 50 mL of 0.05 µ phosphate buffer (pH 7.4). About 0.2 ml of ALA solution was mixed with 2.5 ml linoleic acid emulsion and 2.3 ml phosphate buffer (0.2 µ) by vortexing and then incubated at 37°C overnight. Aliquots of 100 μL were withdrawn from the mixture at 1, 24, 48, 72, 96 and 120 hours of incubation and 5 mL of ethanol (75%), 0.1 mL of NH4SCN (30% w/v), and 0.1 mL of FeCl2 (0.1% w/v) were added, mixed well and analysed by measurement of absorbance. The absorbance was measured at 500 nm against ethanol. Prevention of peroxidation (%) = [(A0 – A1)/A0] x 100, where A0 is the absorbance of the control and A1 is the absorbance of the sample.

Superoxide anion scavenging assay

Superoxide scavenging activity of ALA was determined by following the procedure of Zhao et al. (2003) [23] and Wang et al. (2008) [24]. 4.5 ml Tris-HCl buffer (50 mmol/L, pH 8.2) and 1.0 ml of ALA solutions were mixed in test tubes with caps. The mixtures were incubated for 20 minutes in the water bath at 25°C. Then 0.4 ml of 25 mmol/L pyrogallol which was preheated at 25°C was added immediately to the mixture. After 4 minutes, the reaction was terminated by 0.1 ml HCl solution (8 mol/L) and the mixture was centrifuged at 4000 rpm for 15 minutes. The absorbance of the supernatants of both sample and control were determined by UV spectrophotometer at 325 nm. Using the equation, superoxide anion scavenging activity (%) = (A0-As)/A0 ×100, where A0 is the absorbance of control, and As is absorbance of sample, scavenging activity was calculated.

Assay of in vitro anti-denaturation effects

Determination of anti-denaturation activity of ALA was carried out based on the method described by Williams et al. (2008) [25]. About 2.5 ml 1% BSA was mixed with 2.5 ml tris acetate buffer (0.05 M) and 2.5 ml of the ALA solutions. The mixtures were heated at 69°C for 4 minutes, then cooled and the absorbance of the turbidities was measured at 660 nm. Inhibition of denaturation (%) = (A0-As)/A0 ×100, where A0 is the absorbance of control and As is absorbance of the samples.

4 Int. Res. J. Chem.

Statistical analysis

Statistical analysis was performed with one-way analysis of variance (ANOVA). When differences were observed between mean values, they were compared using Tukey’s test.

For statistical studies OriginLab software (Northampton, UK) was used. All analyses were carried out in triplicate. Statistical significance was expressed as P<0.05. Studies are performed in triplicate and the values are expressed as Mean ± S.E.M.

RESULTS AND DISCUSSION

Isolation of α-lipoic acid

ALA extracts sans ethanol (viz. i, iii, vi and vii solvent systems) showed positive response to ninhydrin tests. This unique behaviour can be explained by the fact that isolations carried out using ethanol prevented the abstraction of enzymes and protein residues from bran. Rice bran contains almost (15 – 18)% proteins [26]. However, such enzymes and proteins in rice bran were notably soluble in water and hence co-extracted with ALA when water or water-rich solvents were used for extraction. As proteinaceous compounds were insoluble in ethanol, noteworthy isolation of singularly ALA, was observed in such prodigious cases.

Yield of ALA was noted to be (0.43+0.02)% with respect to rice bran, when water:ethanol (2:1) solvent system was used. This solvent system yielded the highest amount of ALA in comparison to the other systems (Table 1). This was followed by water and then ethanol:water (1:1) solvent systems The complete solubility of ALA in water and ethanol resulted in the increased recovery of ALA.

Some undesired nuisances co-extracted with ALA are heavy metals like arsenic and silica, which are present in sufficient quantities in rice

bran [14]. Hence an important step during the purification process is removal of such metals. Augmentation of alkali for treating ALA extracts displaced the heavy metals to be removed, with low molecular weight sodium. The sodium salt of lipoic acid remained soluble in the medium, while the heavy metals precipitated and were removed by filtration. A high temperature was applied for the precipitation of silica, which remains water-soluble under cold conditions. Finally, the reaction medium containing sodium salt of lipoic acid was collected and the free acid was liberated by acidification.

HPLC and GC analysis

ALA eluted as a single peak in the HPLC chromatogram as observed in Fig. 1A. The retention time for ALA was 2.96 minutes. The identity of the peak was confirmed by comparison with standard lipoic acid run under similar conditions as the isolated ALA.

Pure ALA appeared as a single peak even in the gas chromatographic analysis at a retention time of 19.80 minutes. The sample peak was correlated with the gas chromatogram of the standard ALA. The gas chromatogram of the isolated sample is shown in Fig. 1B.

Surashree and Mahua 5

Fig. 1: Characterisation of α-lipoic acid (A) HPLC chromatogram of isolated α-lipoic acid; (B) Gas chromatographic analysis of α-lipoic acid; (C) FTIR spectrum of α-lipoic acid; (D) PMR spectrum of α-lipoic acid.

Identification of functional groups of the isolated product with FTIR spectrophotometry

The main band regions observed in the FTIR spectra and their assignments to functional groups are discussed. The band at 3846 – 3000 cm-1 region indicates the O-H stretching originating from the -OH group of the carboxylic acid (Fig. 1C).

Due to intermolecular hydrogen bonding a weakening of the O-H bond was observed which resulted in the broadening and shifting of the bands to lower frequency range of 3600 – 2900 cm-1. The peak at 2350 cm-1 clearly indicates the presence of a carboxylic acid group as observed

in Fig. 1C. This data is supported by the minor absorptions arising in the region between 1300 and 1100 cm-1, due to the C–O stretching mode. The significant pair of bands near 2000 cm-1

indicates CH2-CH bond stretching and the smaller one indicating coupled C–O stretching and O–H deformation. The two extended region of bands covering 1500 cm-1 to 1300 cm-1 and 1200 cm-1 to 1000 cm-1 show the appearance of two strong bands originating due to S–S bonding of α-lipoic acid due to asymmetric and symmetric stretchings respectively. Furthermore, the presence of sulphur group is confirmed by the broad O–H stretching absorption centred at ~3000 cm-1.

Identification of α-lipoic acid using NMR Spectroscopy

The proton magnetic resonance spectrum of ALA is shown in Fig. 1D and the assignments to

functional groups are discussed. The 1H NMR spectrum of α-lipoic acid showed the presence of a typical carbonyl group at δ 2.025 – 2.098 ppm associated with the carboxylic acid group of ALA (Fig. 1D). The absorption frequency

6 Int. Res. J. Chem.

displayed greater de-shielding effect of the carbonyl group relative to that of the aromatic ring (Table 2).

The signals at δ 0.76 – 0.80 ppm referred to the methyl group (-CH3) and that appearing at δ 1.18 – 1.53 ppm indicated the methylene group (-CH2-). The methylene proton signals (-CH2-) were shifted up-field to δ 1.53 ppm. Another

distinctive feature was the hydroxyl proton (-OH) of the carboxylic acid group which yielded a minor signal at δ 4.36 ppm. Chemical shift of –CH– groups moved downfield due to the de-shielding effect of hydroxyl groups. A sharp signal at δ 7.19 ppm was due to the characteristic aromatic hydrogen unit of the sulphated ring substrate of ALA.

TABLE 1: YIELDS (%) OF Α-LIPOIC ACID FROM RICE BRAN USING DIFFERENT SOLVENTS/SOLVENT SYSTEMS

Extraction Solvents or Solvent Systems used

Yields of α-lipoic acid with respect to the de-oiled rice bran (%)

Water 0.31+0.06a

Ethanol 0.062+0.01b

Acetonitrile 0.0002+0.001c

Water:Ethanol (1:1) 0.29+0.03a

Water:Ethanol (2:1) 0.43+0.02d

Water:Acetonitrile (1:1) 0.15+0.06e

Water:Acetonitrile (2:1) 0.19+0.007f

Ethanol:Acetonitrile (1:1) 0.041+0.004g

Ethanol:Acetonitrile (2:1) 0.061+0.05b

Water:Ethanol:Acetonitrile (1:1:1) 0.25+0.08a

Values are expressed as mean ± S.E.M, n=3. Means were compared using Tukey’s test. Rows not sharing a common superscript are statistically significant (p<0.05).

TABLE 2: PMR SIGNALS FOR Α-LIPOIC ACID

δ (ppm) Assignment

0.76―0.80 ―CH3

1.18―1.53 ―CH2

2.02―2.09 ―C=O

4.36 ―OH

7.19 aromatic H group

Antioxidant assay of α-lipoic acid

Five different concentrations of α-lipoic acid (0.001%, 0.005%, 0.01%, 0.05%, 0.1%) were prepared in water and subjected to seven different in vitro antioxidant assays.

Assay of DPPH radical-scavenging activity

The DPPH radical scavenging activity of ALA increased with its increasing concentration (Fig. 2). Highest activity was at 0.1% concentration and the lowest at 0.001% concentration. This was possibly

Surashree and Mahua 7

due to the extensive water solubility of ALA. With increasing ALA concentration its capacity to scavenge the free radicals effectively increased. ALA was observed to reduce and decolourise 1,1-diphenyl-2-picrylhydrazyl (DPPH) effectively due to the lone pair electron donation capability of the sulphur atoms of ALA. As the concentration of ALA increased, rapid reaction of the lone pair of electrons

of ALA with DPPH radicals were more favoured as evident from the dose–response curve. Till date studies have shown that ALA scavenges hydroxyl radicals, hypochlorous acid, and singlet oxygen and undergoes metal chelation, thereby acting as a biological antioxidant. It was proved also that ALA effectively prevented and even treated diabetic conditions [27].

Fig. 2: DPPH radical scavenging activity of α-lipoic acid at different concentrations. Values are Mean+SEM of 3 observations. At the 0.05 level, the difference in superscripts indicates significant difference in means for 0.005% (a), 0.01% (b), 0.05% (c) and 0.1% (d).

8 Int. Res. J. Chem.

Fig. 3: Reducing activity of α-lipoic acid at different concentrations. Values are Mean+SEM of 3 observations. At the 0.05 level, the difference in superscripts indicates significant difference in means for 0.001% (a), 0.005% (b), 0.01% (c), 0.05% (d) and 0.1% (e).

Fig. 4: Metal-chelation activity of α-lipoic acid at different concentrations. Values are Mean+SEM of 3 observations. At the 0.05 level, the difference in superscripts indicates significant difference in means for 0.001% (a), 0.005% (b), 0.01% (c), 0.05% (d) and 0.1% (e).

Surashree and Mahua 9

Assay of reductive potential

Reducing activity showed a similar pattern like the DPPH radical scavenging activity. Reducing activity increased with increasing concentration of ALA, with maximum reducing activity at 0.1% concentration (Fig. 3). Oxidised ALA (hydroxyl lipoic acid) also acts as an antioxidant with abilities to scavenge electrons, which is further beneficial. The stability of the cation formed on oxidation of ALA is increased due to the ring structure attached to it [28]. Higher concentrations of ALA probably induce further development of such stable radicals leading to its greater reducing activities. The reducing nature of ALA is evident from its biological activities as demonstrated in previous studies [7, 8].

Assay of metal chelating activity

In contrary to the previous assays metal chelation activity showed a different trend (Fig. 4). The maximum activity of ALA was at 0.001%

concentration. Thereafter the metal chelation activity decreased with increasing concentration up to 0.05% ALA concentration. Beyond this point the activity was found to increase rapidly at 0.1% concentration. In this case with increase in concentration of ALA, higher pro-oxidant effect influenced the activity of the antioxidant. This effect was absent at lower concentrations. After 0.05% ALA concentration the pro-oxidant effect exceeded its antioxidant effect. Hence reverse behaviour was observed from this point. Ferrozine [disodium salt of 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine] forms a magenta-coloured complex of Fe(II) with λmax at 562 nm. In presence of a reducing agent like ALA the complex formation process is de-activated and limited amount of coloured products are produced. The metal chelation activity of ALA is already well proved in biological systems as well as in minimizing the effect of mercury induced toxicity in humans [27, 29].

Fig. 5: ABTS.+ radical scavenging activity of α-lipoic acid at different concentrations. Values are Mean+SEM of 3 observations. At the 0.05 level, the difference in superscripts indicates significant difference in means, where at 0.001% concentration superscripts used are (a, b, c, d, e, f); at 0.005% concentration superscripts used are (g, h, i, j, k, k); at 0.01% concentration superscripts used are (l, m, n, o, p, q); at 0.05% concentration superscripts used are (r, s, t, t, u, v); at 0.1% concentration superscripts used are (w, x, x, y, z, z).

10 Int. Res. J. Chem.

Assay of ABTS.+ radical scavenging activity

At concentrations of 0.005%, 0.01% and 0.05% ALA did not display any ABTS.+ radical scavenging activity. However, at 0.001% concentration ALA showed a lowering of absorbance after 180 seconds, indicating increase in the scavenging activity of lipoic acid. The best results were displayed by ALA at a concentration of 0.1% (Fig. 5), where a steadily decreasing absorbance value was noted, indicating

that the time-dependant ABTS.+ radical scavenging activity was dominant beyond this point. Just like the DPPH scavenging and reducing activities, ALA showed the maximum potency as a radical scavenging agent at the highest concentration. The activity of ALA at 0.001% concentration after 180 seconds still needs to be justified. The ABTS.+ radical scavenging activity of the antioxidant was also proved on a cellular level previously [30].

Fig. 6: Prevention of lipid peroxidation at different concentrations of α-lipoic acid. Values are Mean+SEM of 3 observations. At the 0.05 level, the difference in superscripts indicates significant difference in means, where at 0.001% concentration superscripts used are (a, b); at 0.005% concentration superscripts used are (c, d, e, f); at 0.01% concentration superscripts used are (g, h, i, h); at 0.05% concentration superscripts used are (j, k, l, m, n); at 0.1% concentration superscripts used are (x, y, x).

Lipid oxidation in a linoleic acid emulsion model system

At a concentration of 0.001%, ALA did not show any prevention of lipid peroxidation till 96 hours. At 120 hours however the lipid peroxidation activity reduced. In case of 0.005% and 0.01% concentrations the activities were observed after 48 hours and in case of 0.1% concentration the activity was observed after 72 hours (Fig. 6). However, the best results were observed for 0.05% ALA concentration where activity increased after 24 hours.

In all the cases initially an increasing value of lipid peroxidation prevention was observed followed by a lowering after 72 hours beyond which once again an increment occurred after 96 hours. Finally, continuous lowering was noted after 120 hours, indicating that the optimum time for attaining highest prevention was achieved. Lipid hydroperoxides are formed due to the peroxidation process. The observed prevention of peroxidation by ALA was probably due to its ability to scavenge free radicals which are produced. The activity of ALA in preventing lipid peroxidation, and thus inhibition of diabetes is a well proved fact [31].

Surashree and Mahua 11

Fig. 7: Superoxide scavenging activity of α-lipoic acid at different concentrations. Values are Mean+SEM of 3 observations. At the 0.05 level, the difference in superscripts indicates significant difference in means for 0.001% (a), 0.005% (b), 0.01% (c), 0.05% (d) and 0.1% (e).

Superoxide anion scavenging assay

Pyrogallol is a powerful free radical initiator which acts by absorbing oxygen from the air and forms superoxide anions. Superoxide is an anion which contains oxygen at its central position. It is reactive in nature that damages cells and tissues [32].

2 O2¯ + 2H+ → H2O2 + O2

During this reaction ALA effectively scavenges the peroxide molecules formed in-situ thus preventing the superoxide-mediated free radical generation.

A gradual lowering of superoxide scavenging activity of ALA was observed with increasing concentration. Hence maximum activity was displayed by the concentration of 0.001% ALA (Fig. 7) and minimum by the concentration of 0.1% ALA. Once again this can be attributed to the pro-oxidant effect of ALA beyond certain strength. The superoxide scavenging activities of dihydrolipoic acid and other compounds akin to it are already proved by the chemiluminescence method [33].

12 Int. Res. J. Chem.

Fig. 8: Inhibition of denaturation by α-lipoic acid at different concentrations. Values are Mean+SEM of 3 observations. At the 0.05 level, the difference in superscripts indicates significant difference in means for 0.001% (a), 0.005% (b), 0.01% (c), 0.05% ( c) and 0.1% ( d).

Assay of in vitro anti-denaturation effects

Anti-denaturation activity of ALA showed a similar pattern like DPPH radical scavenging and reducing activities. Activity increased with increasing concentration of ALA and the highest activity was displayed at a concentration of 0.1% aqueous solution of ALA (Fig. 8). Unlike many popular bioactive components available today, in case of ALA, the pro-oxidant effect did not appear to be a dominant factor and did not affect antioxidant activities at high concentrations. When BSA protein is heated, it undergoes denaturation which can be

associated to different inflammatory diseases. ALA stabilizes the denaturation process efficiently thus preventing such abnormalities [25]. Anti-inflammatory drugs stabilize heat treated BSA at pH (6.2 – 6.5), which fact has been exploited here. In fact ALA in combination with superoxide dismutase has been used in the treatment of severe pain in the lower back and the results were outstanding [34]. However till date study on the anti-denaturation effects of lipoic acid have not been carried out. Hence the present study will open new avenues in terms of preventing inflammation related diseases.

Surashree and Mahua 13

CONCLUSIONS

The successful isolation and purification of a universal antioxidant like ALA from de-oiled rice bran, a potential waste product, is undoubtedly a profitable and lucrative achievement. The protective action of ALA will definitely attract food industries to adopt the developed methodology for its isolation. ALA will safeguard the body against uncontrolled production of free radicals which are responsible for the advent of several degenerative diseases. From the present study it was observed that ALA

displayed different antioxidant activities, being bereft of any nocuous effects. Furthermore, it showed pronounced lipid and water solubility which will serve as a contributing factor in its enhanced biological activities in multiple organs of the body. A concentration of 0.1% ALA in water will prove to be efficacious for gaining vital antioxidant potency. Hence subsequently this system can be successfully utilized in dosage formulations for several nutraceutical supplements.

ACKNOWLEDGEMENT

The authors would like to acknowledge the ‘Council of Scientific and Industrial Research’ (CSIR) for their financial support. Furthermore the Centre for Research in Nanoscience and

Nanotechnology (CRNN), University of Calcutta, is acknowledged for extending their FTIR facilities.

CONFLICT OF INTEREST

The authors have declared no conflict of interest.

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