DPPH Radical-Scavenging Activity and Kinetics of Antioxidant AgentHesperidin in Pure Aqueous Micellar Solutions
Morteza Jabbari* and Azam Jabbari
School of Chemistry, Damghan University, 36716-41167 Damghan, Iran
E-mail: [email protected]
Received: March 19, 2016; Accepted: May 6, 2016; Web Released: August 15, 2016
Morteza JabbariMorteza Jabbari received B.Sc. degree from Ferdowsi University of Mashhad in 2004. He received his M.Sc.(2006) and Ph.D. (2011) degrees in physical chemistry from Shahid Beheshti University of Tehran undersupervision of Prof. Farrokh Gharib. Since 2012, he worked at Damghan University as an assistantprofessor. His research interests focus on the kinetics of antioxidant reactions and thermodynamics ofsolutions.
AbstractThe antioxidant ability of bioactive agent hesperidin was
assessed in terms of radical-scavenging activity (RSA) againstthe 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical in aqueouscolloidal media containing micelle using UVvis spectropho-tometry. The DPPH assay was carried out at 25.0 0.1 C andcationic surfactant CTAB and anionic surfactant SDS at variousconcentrations above the critical micelle concentration (CMC).The rates of the antioxidant reaction (Rs) of hesperidin werealso measured in the micelle systems. The activity and rateof the DPPH radical scavenging by hesperidin were found todepend on concentration and nature of the surfactants used, sothat both RSA and Rs values increase with increasing concen-tration of micelles CTAB and SDS. Finally, the micelle effectson the antioxidant efficiency were explained based on possi-ble interaction modes between hesperidin and the micellarsurfaces.
Hesperidin (C28H34O15), (s)-7-[[6-o-(6-deoxy--L-manno-pyranosyl)--D-glucopyranosyl]oxy]-2,3-dihydro-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, is abioflavonoid diglycoside abundantly found in citrus fruits likeorange, lemon, petitgrain, etc., such that the peel and mem-branous parts of lemons and oranges have the highest hes-peridin concentrations. This compound possesses a wide rangeof potential beneficial effects for human health such as anti-oxidant, anti-inflammatory, anticancer, hypolipidemic, anticar-cinogenic, and antimicrobial properties.13 Besides the protec-tive effects (most notable in the heart and brain, but extendto every organ), hesperidin may be able to reduce a lack ofappetite and have minor antiallergic properties. Hesperidin is awhite to yellow crystalline solid with poor aqueous solubility.
This limits its dissolution rate in water, which finally results inpoor in vivo bioavailability. Hesperidin has also been reportedas insoluble in most of the physiologically safe organic sol-vents useful in pharmaceutical dosage form development.1,4,5 Asketch of the molecular structure of hesperidin is shown inScheme 1.
Flavonoids or bioflavonoids are polyphenolic phytochemi-cals that are frequently found in nature and easily extractedfrom different parts of the plants, such as the roots, stems,leaves, flowers, fruits, or seeds. They are extremely safe andwith low toxicity, which makes them excellent disease-preventing dietary supplements and cancer-preventive agents.6
Scheme 1. Chemical structure of bioflavonoid hesperidin,gallic acid, and DPPH radical.
Bull. Chem. Soc. Jpn. 2016, 89, 869875 | doi:10.1246/bcsj.20160095 2016 The Chemical Society of Japan | 869
Many of the pharmacological effects are usually ascribed to theability of flavonoids to trap free radicals, an ability common toall phenols that is generally equated to antioxidant activity.This property of flavonoids is a biological function, importantin keeping the oxidative stress levels below a critical point inthe body and can thus help preserve neuronal health. Radicalscavenging by flavonoids occurs by electron or hydrogen dona-tion from the free hydroxyl groups on the flavonoid nucleus.This leads to the formation of a less reactive flavonoid aroxylradical that is stabilized by resonance and therefore plays amoderator role in the propagation of radical-induced damage inbiological systems.7,8 Numerous research groups have focusedon the free radical-scavenging effectiveness evaluation offlavonoids until now. Some of them have confirmed that thefree radical-scavenging activity of flavonoids depends not onlyon the environmental parameters like pH, temperature, natureof the solvent, etc.,7,911 but also on a number of other factorssuch as their partitioning between the different regions of thesystem.12,13 A general methodology for modeling this factoris to employ a microheterogeneous environment such as amicellar system. Surfactant unimers in aqueous solution self-assemble into micelles at a specific concentration called thecritical micelle concentration (CMC). The CMC is an importantcharacteristic, specific to each individual surfactant. Surfactantmicelles are dynamic entities and can have different shapes,such as spherical, spheroid, oblate, and prolate. Most micellesare spherical and contain between 60 and 100 surfactant mole-cules.14,15 The information obtained in the aqueous micellarsystems can play a crucial role in understanding antioxidantactivity of water-insoluble bioactive compounds.
Various methods have been designed to measure antioxidantpower of bioactive compounds so far. These assays can givedifferent results depending on the specific free radical beingused as a reactant. Among them, the one which is the mostwidely used for in vitro testing is based on employing the stablefree radical 2,2-diphenyl-1-picryhydrazyl (DPPH, Scheme 1)which has an unpaired valence electron at one atom of thenitrogen bridge.16 The DPPH assay has often been performedin pure organic or mixed aquo-organic solvents due to theinsolubility of species used in water. Therefore, the purpose ofthe present investigation was to develop a DPPH assay forevaluation of the radical-scavenging activity (RSA) of naturalpolyphenol hesperidin in pure aqueous solution using surfactantmicelles of CTAB and SDS without need of an organic co-solvent. The effects of the type of surfactant, concentration ofthe surfactant and the flavonoid as factors that can affect theantioxidant capacity are examined. Additionally, the reactionrates of DPPH radical with hesperidin are evaluated in pureaqueous micellar media to study antioxidant behavior of thisflavonoid kinetically.
2.1 Chemicals. All chemicals used were of analytical gradepurity. Gallic acid (Scheme 1), hesperidin and the stable DPPHfree radical were supplied from Fluka and used without furtherpurification. The anionic surfactant SDS and cationic surfac-tant CTAB (Scheme 2) of the highest quality available werepurchased from Sigma-Aldrich. The deionized doubly distilledwater (1.2 0.11 cm1 conductivity) was used throughout.
2.2 DPPH Radical-Scavenging Assay (RSA). The relativestability of radical DPPH, sensitivity and the technical simplic-ity of DPPH assay execution makes this colorimetric methodpopular for measuring antioxidant capacity of food products orplant extracts. This method is based on the decay of the mainabsorption band in the visible spectrum of DPPH radical. Inthe presence of an electron- or hydrogen-donating compoundlike flavonoids, the DPPH radical would be scavenged throughelectron or hydrogen donation, and its absorbance is decreased.The decreased extent of absorbance is taken as a measure forthe antioxidant activity.17 The DPPH assay was carried out inpresence of the cationic CTAB and anionic SDS micelles asthe following procedure. The aqueous stock solutions of DPPH(0.030mM) and hesperidin (0.025mM) were daily prepared inthe same surfactant concentration and then stored in a refrig-erator until needed. To a known volume of aqueous micellesolution taken in a 5mL volumetric flask, 2mL DPPH solutionwas pipetted together with a volume of deionized water. Thereaction was started by adding an aliquot (0.160.60mL) ofhesperidin to the mixture to make a final volume of 3mL. Thereaction mixture was then vigorously shaken by hand andallowed to stand in the darkness for 5min at ambient tem-perature for completion of the reaction. The same procedurewas repeated for at least five different concentrations of thesurfactants tested in a range 6.508.50mM of CTAB and15.0025.00mM of SDS. In each concentration of micelle, sixdifferent concentrations of hesperidin were used. The DPPH inaqueous micelle solution without an antioxidant was used ascontrol of this experiment. After 5min of starting the reaction,the samples were analyzed using a Perkin-Elmer (Lambda 25)UVvis spectrophotometer in the wavelength range of 450 to650 nm. The aqueous micelle solutions were used as blank. Theradical-scavenging potential in each surfactant micelle solutionwas derived based on the value of the DPPH visible absorbanceat the maximum according to the following expression:
%RSA Abscontrol AbssampleAbscontrol
where Abscontrol and Abssample are the absorbance at maximawavelength of the control and the sample, respectively. Allassays were performed in three independent runs and the RSAvalues in each micelle system were calculated as a mean ofthese measurements. The RSAvalues were usually compared toa reference antioxidant of which its antiradical capacity wasmeasured under the same conditions. In this work, gallic acidwas used as a reference standard compound.
Scheme 2. Sketch of molecular str