Antioxidative activities of white rose flowerextract and pharmaceutical advantages of itshexane fraction via free radical scavengingeffects

Dongsun Park, Jeong Hee Jeon, Sang-Chul Kwon, Sunhee Shin, Ja Young Jang,Heon Sang Jeong, Do Ik Lee, Yun-Bae Kim, and Seong Soo Joo

Abstract: In this study, we determined the antioxidant activities of two different solvent fractions(butanol and hexane) ob-tained from white Rosa rugosa flowers by employing various assays such as 2,2-diphenyl-1-picrylhydrazyl hydrate(DPPH), 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, and nitric oxide (NO)scavenging and inhibition activity in S-nitroso-N-acetylpenicillamine (SNAP) in the RAW264.7 model. In addition, moreadvanced antioxidant assays were conducted, including lipid peroxidation, hydroxyl radical-mediated oxidation, DNA frag-mentation, apoptosis, and cell growth. The results revealed that the hexane fraction, which contained a significant amountof polyphenols and volatile components, had excellent antioxidant potency and could scavenge free radicals of DPPH andABTS. Interestingly, the hexane fraction inhibited lipid peroxidation to almost the same degree as a chemical antioxidant.In the NO assay, the hexane fraction effectively scavenged free radicals at all dose ranges and is expected to inhibit NOproduction in mammalian cells. The hexane fraction effectively prevented oxidative damage, which was induced by Cu2+/H2O2, to target proteins at lower concentrations (>1 mg�mL–1). The DNA fragmentation and the cell-level assays suggestthat the hexane fraction may play a crucial role in inhibiting peroxynitrite and H2O2 attack. Based on the findings de-scribed in this study, the hexane fraction holds promise for use as a novel pharmaceutical antioxidant.

Key words: antioxidant activity, radical scavenging, Rosa rugosa flowers, reactive oxygen species, lipid peroxidation.

Resume : Dans cette etude, nous avons caracterise les activites antioxydantes de deux fractions organiques differentes(butanol/hexane) obtenues des fleurs de Rosa rugosa blanc a l’aide de differents tests, notamment la mesure de l’activitede desactivation (scavenger) des radicaux de l’hydrate de 2,2-diphenyl-1-picrylhydrazyle (DPPH) et du 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), de l’activite de desactivation de l’oxyde nitrique (NO) et de l’activite inhibitricede la S-nitroso-N-acetylpenicillamine (SNAP) dans le modele des cellules RAW264,7. De plus, des mesures plus avanceesdu potentiel antioxydant ont ete realisees, notamment la peroxydation des lipides, l’oxydation par les radicaux hydroxyle,la fragmentation d’ADN, l’apoptose et la croissance cellulaire. Les resultats ont revele que la fraction hexane, qui conte-nait une quantite significative de polyphenols et de composes volatils, possedait un excellent potentiel antioxydant et pou-vait desactiver les radicaux libres du DPPH et de l’ABTS. Fait interessant, la fraction hexane inhibait la peroxydationlipidique de facon presque aussi efficace que les antioxydants chimiques. Lors du test de desactivation du NO, la fractionhexane desactivait de facon efficace les radicaux libres a toutes les doses et ce faisant, devrait pouvoir inhiber la produc-tion de NO dans les cellules mammiferes. La fraction hexane prevenait aussi efficacement le dommage oxydatif induit parle Cu+2/H2O2 pour cibler les proteines a faibles concentrations (>1 mg�mL–1). Les tests de fragmentation d’ADN et decroissance cellulaire suggerent que la fraction hexane joue un role crucial en inhibant l’attaque par les peroxynitrites et leH2O2. Selon les resultats decrits dans cette etude, la fraction hexane constitue un nouvel antioxydant pharmaceutique pro-metteur.

Mots-cles : activite antioxydante, desactivation des radicaux, fleurs de Rosa rugosa, especes d’oxygene reactives, peroxy-dation des lipides.

[Traduit par la Redaction]

Received 8 May 2009. Revision received 10 July 2009. Accepted 4 August 2009. Published on the NRC Research Press Web site atbcb.nrc.ca on 6 November 2009.

D. Park, J.H. Jeon, S. Shin, J.Y. Jang, Y.-B. Kim, and S.S. Joo.1 College of Veterinary Medicine and Research Institute of VeterinaryMedicine, Chungbuk National University, 410 Seongbongro (Gaeshin-dong), Cheongju, Chungbuk 361-763, Republic of Korea.S.-C. Kwon. Chamsunjin Total Food Co. Ltd., 373-18 Sangsin-ri, Jincheon-eup, Jincheon-gun, Chungbuk 365-801, Republic of Korea.H.S. Jeong. College of Agriculture, Life & Environment Sciences, Chungbuk National University, 410 Seongbongro (Gaeshin-dong),Cheongju, Chungbuk 361-763, Republic of Korea.D.I. Lee. College of Pharmacy, Chung-Ang University, 221, Huksuk-dong, Dongjak-ku, Seoul 156-756, Republic of Korea.

1Corresponding author (e-mail: larryjoo@hanmail.net).

943

Biochem. Cell Biol. 87: 943–952 (2009) doi:10.1139/O09-065 Published by NRC Research Press

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Introduction

Plants have provided a rich and vital source of com-pounds for the development of medicines and researchersfrom many different countries have continually identifiedplants yielding beneficial health effects. Over the past deca-des, many scientists have been interested in seeking and de-veloping synthetic or semi-synthetic analogs orphytochemicals from plant compounds that can be used indrugs, therapeutic adjuvant, or foods. Plants can produce aremarkably diverse range of low-molecular-weight naturalproducts (e.g., ascorbic acid, glutathione, uric acid, toco-pherol, carotinoids, polyphenols), known as secondary meta-bolic products, which protect against reactive oxygenspecies (ROS) (Vranova et al. 2002; Argolo et al. 2004).Although these products, which accumulate in plants thathave been subjected to stresses including various elicitors orsignal molecules, may have a variety of functions in plants,it is likely that their ecological function may provide uniqueresources for pharmaceuticals and may also possess benefi-cial medicinal properties in humans (Zhao et al. 2005).Among the several small molecules retained in plants, poly-phenols (hydrolysable tannins, phenylpropanoids) are themost abundant antioxidants in our diet and have been shownto play a possible role in the prevention of several chronicdiseases (Hotta et al. 2002). Polyphenol antioxidants, suchas resveratrol, inhibit the occurrence and (or) growth ofmammalian tumors (Jang et al. 1997), and a wide variety ofphenolic substances derived from plants have been reportedto retain marked antioxidant and anti-inflammatory activitiesthat contribute to their chemopreventive potential (Surh etal. 2001). Recent studies have shown that the antioxidantproperties of plants can be correlated with defense againstoxidative stress and different human diseases associatedwith the aging process (Stajner et al. 1998; Sanchez-Morenoet al. 1999; Malencic et al. 2000). When ROS productionexceeds cellular antioxidant capacity, cellular proteins, lip-ids, and DNA will become modified, which can lead to celldeath or to acceleration in aging and age-related diseases(Duffy et al. 1996). To fully elucidate the entire profile ofthe antioxidant capacity of natural sources, it is necessary toexamine the entire spectrum of potential ROS/reactive nitro-gen species such as O2

�–, HO�, and ONOO– (Prior et al.2005).

White Rosa rugosa is a species of rose that is native tolarge parts of eastern Asia. It has been primarily used as aningredient in tea, a source of rose oil, and for producing jamfrom its fruits (Kamijo et al. 2008). The roots of R. rugosahave been used in traditional medicines to treat diabetesmellitus, pain, and chronic inflammatory diseases in Korea(Kyohaksa Company 2003). Recently, it was reported thatthe petals of R. rugosa possess antioxidant activity throughthe condensed hydrolysable tannins, which may have strongantioxidant and anti-inflammatory properties (Cho et al.2003a, 2003b; Ng et al. 2005). Moreover, volatile compo-nents of plants have been reported to exhibit varying antiox-idant activities, which may support or directly act as aneffective antioxidant (Jang et al. 2008).

However, there have been no studies on the white roseflower extract (WRFE) or its solvent fractions in regards totheir antioxidant properties. Therefore, in the present study,

we examined if the WRFE fractions (water, butanol, hexane)possessed antioxidant properties that would make them ame-nable to use in pharmaceutical medicines.

Materials and methods

Plant materialFresh white R. rugosa flowers were collected at Jincheon,

Chungbuk, Korea, in May 2006, and completely dried undersunlight to obtain dried petals. Dried petals were thenground in a rotor speed mill (Laval Lab Inc., Laval, Que.),and the pulverized flowers were sterilized using a 70% etha-nol spray, followed by drying at 80 8C for 24 h and storageat 4 8C before use.

ChemicalsL-ascorbic acid, dimethyl sulfoxide (DMSO), hydroperox-

ide (H2O2), S-nitroso-N-acetylpenicillamine (SNAP), 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH), 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), gallicacid, butylated hydroxyanisole (BHA), bovine serum albu-min (BSA), and Folin–Ciocalteu’s phenol reagent werepurchased from Sigma (St. Louis, Mo.). Peroxynitrite(ONOO–) was purchased from Calbiochem (San Diego,Calif.).

Separation of extractFinely ground flower powders were extracted with metha-

nol at room temperature for 24 h. The ratio of flower pow-der to solvent was 1:20 (w/v). The resulting slurries werefiltered through Whatman no. 1 filter paper. This procedurewas repeated twice for the residue, and the filtrates werecombined. All of the filtrates were collected and concen-trated under vacuum at 50 8C and freeze-dried. Then, thefreeze-dried methanol extract (1 g) was redissolved in meth-anol (1.5 mL), and 20 mL of water was added (methanol–water, 1.5:20, v/v). This mixture was then sequentially parti-tioned with hexane and butanol (same solvent ratio aswater). Three resulting fractions (H2O, butanol, and hexanelayer) were obtained, evaporated to dryness under vacuum,and used directly in the antioxidant tests. Each fraction wasdissolved in 10% DMSO, and stock solutions were preparedat high concentrations so that the DMSO concentration wasnot higher than 0.1% during all experiments.

AnimalsMale Sprague–Dawley rats weighing 300–350 g were ob-

tained from Samtako, Inc. (Kyung Gi, Korea) and accli-mated for at least 1 week prior to experimental use.Animals were individually housed with access to food andwater, and maintained under a normal light:dark cycle. Thepresent study was approved by the Institutional Animal Careand Use Committee of the Laboratory Animal ResearchCenter at Chungbuk National University, Korea.

Cell cultureRaw 264.7 (mouse leukaemic monocyte macrophage cell

line) cells were grown in Dulbecco’s modified Eagle’s me-dium (DMEM) (Hyclone, Utah) supplemented with 10% fe-tal bovine serum (FBS), 4 mmol�L–1 L-glutamate(Invitrogen, Calif.), 100 U�mL–1 penicillin and 100 mg�mL–1

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streptomycin (Invitrogen). Cultures were maintained under5% CO2 at 37 8C in tissue culture flasks. For all experi-ments, the cells were grown to greater than 90% confluencyand subjected to no more than 20 cell passages.

Determination of total phenolic compoundsThe total amount of phenolic compounds in each fraction

was determined and expressed as milligrams of gallic acidequivalents per gram of fraction (Hsieh et al. 2008) withsome modifications using a 96-well microplate reader. Inbrief, 10 mL of each fraction was transferred into a 96-wellmicroplate containing 160 mL of distilled water and 10 mLof 1 mol�L–1 Folin–Ciocalteu’s phenol reagent (Sigma).Twenty microlitres of 20% aqueous sodium carbonate solu-tion was then added. The mixture was allowed to stand atambient temperature for 20 min. Absorbance of the devel-oped dark blue – purple color was measured using a spectro-photometer at 735 nm (Spectra Max 340, Molecular Devices,Calif.). The total number of phenolic compounds was calcu-lated using a calibration curve established with a gallicstandard solution that ranged from 25 to 800 mg�mL–1.

Gas chromatography – mass spectometry (GC-MS)analysis

The volatile compounds of the hexane fraction of WRFEwere determined using an Agilent 6890 gas chromatograph/5973N mass selective detector (Palo Alto, Calif.), and sepa-rated on an HP-FFAP (HP-free fatty acid phase) capillarycolumn (30 m length � 0.25 mm internal diameter,0.25 mm film thickness). Helium was used as the carriergas at a constant flow rate of 1 mL�min–1, and the oven tem-perature was set at 50 8C for 5 min, raised to 230 8C in in-crements of 4 8C�min–1, and held for 20 min. The detectorand injector temperatures were maintained at 250 8C. Theionizing energy of the mass selective detector was set at70 eV, with a scanning mass range of m/z 35–500. Mostpeaks were identified using the computer library (Wiley275L program). Chromatographic peaks were checked fortheir homogeneity with the aid of mass chromatograms forthe characteristic fragment ions.

High-performance liquid chromatography (HPLC)analysis

The hexane fraction of WRFE was analyzed by HPLC us-ing a Younglin-ACME 9000 system (Younglin, Anyang,South Korea) with a YL9160 PDA detector. Spectral datawere recorded from 190 to 900 nm during the whole run.For a separation, a Mightysil RP-18 GP column (4.6 �250 mm, 5 mm, Kanto Chemical, Japan) was used, and thesolvent flow rate was held constant at 0.6 mL�min–1. Themobile phase used for the separation consisted of solvent A(acetonitrile) and solvent B (10 mmol�L–1 phosphoric acid,pH 2.5). A gradient elution procedure was used as follows:0 min 10% A, 0–10 min 5% A, 10–40 min 50% A, 40–41 min 100% A, 41–51 min 100% A, 51–61 min 10% A.The injection volume was 20 mL for analysis and the gallicacid standard was prepared in HPLC-grade methanol. Con-centration of gallic acid was determined by standard curvesprepared by injecting different concentrations of gallic acidstandards. The gallic acid was identified and quantified by

comparing their retention time and UV – visible spectraldata to known previously injected standards.

Measurement of DPPH and ABTS radical scavengingactivity

The DPPH radical is one of the few stable organic nitro-gen radicals, which bears a deep-purple color. To evaluatethe free radical scavenging activity, fractions were allowedto react with the DPPH solution (Espın et al. 2000). Eachlyophilized fraction was dissolved in DMSO as a stock solu-tion (100 mg�mL–1) and each fraction was reacted with0.3 mmol�L–1 DPPH in methanol. Various concentrations ofeach fraction (1–32 mg�mL–1) were reacted with the DPPHradical solution for 30 min at room temperature. The absorb-ance was then measured at 517 nm. The DPPH free-radicalscavenging activity was calculated using the following equa-tion:

½1� DPPH scavenging activity ¼ ½Ac � ðA� AsÞ�Ac

� 100%

where Ac is the absorbance of the control DPPH solution, Ais the absorbance of the sample with the DPPH solution,and As is the absorbance of the sample alone. ABTS formsa relatively stable free radical, which is colorless in its nonradical form. ABTS�+ was prepared by combining2 mmol�L–1 ABTS in H2O with 2.5 mmol�L–1 potassiumpersulfate (K2S2O8), and storing in the dark at room tem-perature for 4 h. Then, 100 mL of the pre-formed ABTS�+solution was reacted with each fraction in a 96-well micro-plate. After a pre-determined period of time, the remainingABTS�+ was quantified spectrophotometrically at 734 nm.

Measurement of lipid peroxidationLipid peroxidation was measured by determining the for-

mation of malondialdehyde on the basis of the presence ofthiobarbituric acid reactive substances in the brain (Ohkawaet al. 1979; Callaway et al. 1998). In brief, rat brains werehomogenized in 10 volumes of buffer after perfusion. Thehomogenate was centrifuged at 4000 r�min–1 to obtain super-natant. To induce lipid peroxidation, the brain homogenate(450 mL) was mixed with 50 mmol�L–1 ferric chloride(25 mL) in the presence or absence of the fractions (25 mL)and incubated for 30 min at 37 8C. The reaction was thenstopped by the addition of sodium dodecyl sulphate(500 mL of 8.1% w/v solution) and 1 mL of 20% acetic acid(adjusted to pH 3.5). Aliquots (500 mL) of the clear superna-tants were mixed with an equal volume of the thiobarbituricacid solution (0.8% w/v) and heated in a glass tube cappedwith aluminum foil at 95 8C for 30 min. Samples werecooled on ice and 100 mL of each sample was pipetted into96 well plates and the absorbance was read at 532 nm (Mo-lecular Devices). Butylated hydroxyanisole (BHA) was usedas a control chemical antioxidant.

Nitric oxide scavenging assayTo determine whether the hexane fraction possessed nitric

oxide (NO) scavenging activity, a modified non-cell NOscavenging assay (SNAP assay) was performed. In brief,solutions of 100 mmol�L–1 SNAP (NO donor), 10% DMSO,and varying concentrations of hexane fractions (0.1 to100 mg�mL–1) were prepared. The final volume of the reac-

Park et al. 945

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tion was set to 150 mL and incubated at 37 8C for 3 h. Aftercompletion of the reaction, each sample (50 mL) was mixedwith the Griess reagent according to the manufacturer’s di-rections (Promega, Madison, Wis.) and the absorbance wasmeasured at 540 nm.

Nitric oxide production in RWA264.7A mammalian cell line, RAW264.7, was used to acquire a

more advanced understanding of the antioxidant propertiesof the hexane fractions. In these experiments, RAW264.7cells (1 � 106 cells�mL–1) were seeded on a 24-well tissueculture plate and pre-incubated at 37 8C for 12 h to achievestable attachment. Next, the wells were washed with phos-phate buffered saline (PBS) after pre-incubation, refreshedwith 1% FBS DMEM containing the samples (lipopolysac-charide (LPS), ascorbic acid (Asc), and hexane fractions(0.1 to 25 mg�mL–1)), and incubated for 24 h. NO productionwas then monitored by measuring nitrite levels in the culturemedia using the Griess reagent (Promega).

Hydroxyl radical mediated oxidation assayHydroxyl radical mediated oxidation experiments were

performed using a metal-catalysed reaction as described pre-viously (Mayo et al. 2003), with some modifications. Thetarget protein, BSA, was dissolved in a 150 mmol�L–1 phos-phate buffer (pH 7.3) at a final concentration of 0.5 mg�mL–1.The BSA solution was incubated both with and without100 mmol�L–1 copper (Cu2+) and 2.5 mmol�L–1 H2O2 in thepresence and absence of the fractions. The control antioxi-dant was 50 mmol�L–1 of ascorbate, which was directly dis-solved in PBS. The reactions were carried out in open tubesand placed in a shaking water bath that was maintained at37 8C. After the reaction was complete, each mixture wasseparated on a 10% sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS–PAGE), and stained with 0.1%Coomassie blue solution.

DNA fragmentation and determination of apoptotic cellsRaw 264.7 cells were grown on 60 cm2 culture dishes

(BD Bioscience, Calif.), starved with FBS-free DMEM for6 h. After the indicated treatment, cells were harvested byscraping, washed twice with ice-cold PBS, and lysed in lysisbuffer (10 mmol�L–1 Tris–HCl (pH 8.0), 10 mmol�L–1

EDTA, and 0.2% Triton X-100) on ice for 30 min. The cellswere then centrifuged at 13 500 r�min–1 and 4 8C for 10 minand the supernatant was transferred to a new tube. To re-move RNA contamination, 10 mg�mL–1 RNase A (Promega)was added and incubated at 37 8C for 30 min. SolubleDNAs were isolated by phenol – chloroform – isoamylalco-hol extraction and ethanol precipitation. Air-dried DNA pel-lets were dissolved in ionized distilled water and resolved on1.6% agarose gel for 1 h. DNA fragments were visualizedby staining with ethidium bromide. To determine the pres-ence of apoptotic cells, HaCaT (human keratinocyte cellline) cells were treated with 0.5 mmol�L–1 SNAP (NO do-nor) in the presence or absence of hexane fractions (0.1–50 mg�mL–1), and incubated for 6 h. Cells were then washed3 times with ice-cold PBS, fixed with 4% paraformaldehydefor 15 min, and stained with Annexin V-FITC and propio-dium iodide (1:2.5 ratio) for 1 h on ice in the dark. Apop-totic cells were determined using an Olympus IX71 inverted

microscope (Olympus, Tokyo, Japan) and morphologicchanges were compared by merging figures captured at thesame sites.

Antioxidant analysis in Escherichia coliCells were directly stressed with 1 mmol�L–1 H2O2 after

pre-treatment with the hexane fraction at various concen-trations (1–1000 mg�mL–1), and cultured in liquid Luria–Bertani (LB) media for 2 h at 37 8C, 250 r�min–1. Onedrop from each culture tube was then inoculated on anLB agar plate in duplicate, and incubated overnight at37 8C. Cell growth was determined by comparison with co-horts exposed to stress without being treated with ascorbicacid (0.5 mmol�L–1) or fractions.

Statistical analysisThe results obtained were expressed as mean ± SD, and

one-way ANOVA (SPSS 12.0 version), followed by Tukey’stest, was used to statistically compare the groups. Resultswith p value < 0.05 were considered statistically significant.

Results

Determination of total phenolic compoundsThe crude extracts from dried flowers of white rose (R.

rugosa) were prepared by fractionating with two differentsolvents (hexane and butanol) and water. The total amountsof phenolic components in these different fractions werethen determined to elucidate the nature of these compounds.The total amounts of polyphenols in the 3 fractions ofWRFE are shown in Fig. 1. The hexane fraction containedthe highest abundance of total polyphenols (330.3 ±20.1 milligrams of gallic acid equivalent per gram drymass), which was approximately 2-fold higher than that ofthe butanol fraction (177.3 ± 7.1 mg) and 2.6-fold higherthan that of the water fraction (127.1 ± 10.8 mg). These re-sults suggest that the hexane fraction may have a morepotent antioxidant activity, and was thus selected for morein-depth analysis.

GC-MS and HPLC analysisIn GC-MS analysis, we found that the hexane fraction

contained different volatile components, which may havevarying antioxidant activities (Jang et al. 2008). The majoraroma constituents of the hexane fraction from WRFE weredodecyl acrylate (41.17%) and cyclododecane (23.83%)(Table 1). Moreover, higher amounts of gallic acid (>10%)in the hexane fraction were identified by HPLC (Fig. 1B).The presence of gallic acid was confirmed by UV spectrumanalysis of gallic acid in the hexane fraction and the stand-ard compound (data not shown).

DPPH and ABTS radical scavenging activityDPPH is usually used as a reagent to evaluate the free-

radical scavenging activity of various antioxidant substan-ces. DPPH is a stable free radical and accepts an electronor hydrogen radical to become a stable diamagnetic mole-cule. ABTS also forms a relatively stable free radical, whichdecolorizes in its non-radical form. Thus, these radicals wereused in radical scavenging assays to initially assess the anti-oxidant potency of the different fractions. In the DPPH as-

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say, the hexane fraction plateued at 2 mg�mL–1, whereas thebutanol fraction reached a plateau at 4 mg�mL–1 in 20 min.However, the water fraction did not effectively scavengefree radicals compared with the hexane and butanol fractions(Fig. 2A). Moreover, the hexane fraction was more activeand more rapid in scavenging free radicals than the butanolfraction (Fig. 2B). In addition, the ABTS radical scavengingactivity assay also indicated that the hexane fraction hadmore antioxidant potency than the butanol or water fractions(Fig. 2C).

Inhibition of lipid peroxidationThe lipid peroxidation assay, which utilizes thiobarbituric

acid reacting substances (TBARS), is a widely adopted andsensitive method for assessing antioxidant activity. The po-

Fig. 1. Determination of the total amounts of phenolic compounds and chromatogram analysis. (A) The total amounts of phenolic com-pounds (milligrams of gallic acid equivalent) were determined in water, butanol, and hexane fractions of WRFE as described in Materialsand methods. Data are expressed as mean ± SD on per gram dry mass basis from 3 different experiments. **, p < 0.01 compared with waterfraction; {{{, p < 0.001 compared with butanol fraction. (B) Chromatogram of a hexane fraction of WRFE. Peak 4 (arrow) indicates a gallicacid existing in a high proportion.

Table 1. Volatile components identified by GC-MS inhexane fraction of WRFE.

CompoundRetention time(min) Content (%)

1-Butanol 7.17 7.52Dodecane 9.10 2.14Tetradecane 17.37 1.71Benzene 18.36 1.25Hexadecane 25.45 0.87Cyclododecane 38.54 23.83Dodecyl acrylate 39.32 41.17Ethanone 49.56 2.61

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tency of the two solvent fractions (hexane and butanol) fromWRFE in inhibiting the formation of MDA was examined at532 nm (Fig. 3). A concentration-dependent decrease in ab-sorbance (TBARS formation) was found with the addition ofBHA and the hexane fraction. Surprisingly, the hexane frac-tion showed a similar inhibitive effect at 31.6 mg�mL–1 asthe positive control, BHA. However, the butanol fractionwas not that effective in inhibiting lipid peroxidation. Thesecombined results suggest that the hexane fraction may have

a pharmacological benefit that is equal to chemical antioxi-dants.

Nitric oxide scavenging and inhibition activityTo assess the antioxidant, scavenging, and inhibitory

properties of the hexane fraction in a more comprehensiveway, NO assays were carried out in both non-cell and in vi-tro cell lines using the Griess reagent method, as describedin the Materials and methods. In the non-cell NO scaveng-ing assay, 100 mmol�L–1 SNAP was mixed with hexanefractions and the absorbance was measured in a time- anddose-dependent manner for up to 3 h. In these experiments,the hexane fraction displayed remarkable NO scavengingproperties at all hexane concentrations tested (0.1–100 mg�mL–1) (Fig. 4A), and the scavenging patterns fol-lowed the ascorbic acid pattern throughout the indicatedreaction time (0 to 3 h). The IC50 of the hexane fraction forNO was 0.9 mg�mL–1. In addition, inhibition of NO produc-tion was examined in RAW264.7 cells. To stimulate NOproduction, 5 mg�mL–1 LPS was added and varying concen-trations of the cells were co-treated with the hexane fraction(0.1 to 25 mg�mL–1) for up to 36 h. As shown in Fig. 4C, thehexane fraction significantly inhibited NO production at10 mg�mL–1, which was comparable with 50 mmol�L–1 ascor-bic acid. More interestingly, a longer incubation (36 h) re-vealed that the hexane fraction was more effective thanascorbic acid in controlling NO production (Fig. 4D). Thesetwo results suggest that the hexane fraction may continu-

Fig. 2. Radical scavenging activity at different times and concentrations. (A) DPPH free radical scavenging activity in water, butanol, andhexane fractions at different concentrations (1–32 mg�mL–1) was determined for a fixed time (20 min). (B) The DPPH free radical scaven-ging activity of the butanol and hexane fractions over a different time scale (1 to 30 min) was determined at 2 mg�mL–1. (C) ABTS radicalscavenging activity in water, butanol, and hexane fractions at different concentrations (0.1–1000 mg�mL–1) was determined for a fixed time(30 min). Data are expressed as mean ± SD.

Fig. 3. Measurement of lipid peroxidation. The inhibition ofTBARS formation in rat brain homogenates was measured at var-ious concentrations of the butanol and hexane fraction (10, 31.6,and 100 mg�mL–1) at 532 nm as described in the Materials andmethods. BHA was used as a positive control. NC, normal control.

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ously control the NO concentration at the cellular level byboth NO scavenging and inhibiting NO production.

Hydroxyl radical-mediated oxidation assayTo determine whether the hexane fraction had protein-

level antioxidant properties, degradation of BSA by Cu2+

and H2O2 was monitored in the presence of 0.1 to10 mg�mL–1 hexane fraction, as described in the Materialsand methods. As shown in Fig. 5, the hexane fraction pro-tected against the breakdown of BSA as did 0.5 mmol�L–1

ascorbic acid. The hexane fraction efficiently inhibited thedegradation of BSA by free radicals in a dose-dependantmanner with a maximum effect at a concentration of10 mg�mL–1. No noise effect from the vehicle control(DMSO) was detected during the degradation of BSA.

Cell damage and growth inhibition assayNuclear DNA fragmentation, which is the biochemical

hallmark of apoptosis, was examined in RAW264.7 cellsco-treated with the hexane fractions and peroxynitrite

Fig. 4. Nitric oxide scavenging and inhibition. Two separate experiments were carried out for non-cell based NO scavenging, and cell basedNO inhibition. (A) NO scavenging was determined using SNAP at different concentrations of the hexane fraction (0.1–100 mg�mL–1). As-corbic acid (50 mmol�L–1) was used as a positive control and 10% DMSO was used to determine the noise of the vehicle. (B) Time depen-dency of NO scavenging by the hexane fraction was examined at different time scales (0–3 h) in the presence of the hexane fraction(10 mg�mL–1) or ascorbic acid (50 mmol�L–1) with 100 mmol�L–1 SNAP. (C and D) RAW264.7 cells were seeded on a 96-well culture plate,treated with the hexane fractions (0.1–25 mg�mL–1), and incubated for 24 or 36 h. Ascorbic acid (50 mmol�L–1) was used as a positive con-trol, and LPS (5 mg�mL–1) was used as a cell stimulator. The presence of NO was then detected using the Griess reagent and measured at540 nm. The experiments were performed in triplicate and the results are expressed as the mean ± SD. *, p < 0.05; **, p < 0.01; ***, p <0.001 compared with positive control, LPS.

Fig. 5. PAGE profiles of the BSA protein with Cu2+/H2O2 in thepresence of the hexane fraction. The gels show the protein obtainedwithout treatment, with Cu2+/H2O2, and at different concentrationsof the hexane fractions (0.1–10 mg�mL–1). Ascorbic acid(0.5 mmol�L–1) and 10% DMSO were used as positive and vehiclecontrols, respectively. The final steps include the incubation of allreactants, including BSA, for 2 h and electrophoresis in 10% SDS–PAGE.

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(ONOO–). As shown in Fig. 6A, DNA ladders were ob-served in DNA extracted from cells treated with low con-centrations of the hexane fraction; however, higherconcentrations of the hexane fraction remarkably reducedDNA fragmentation in a dose-dependent manner. These re-sults may suggest that the hexane fraction effectively scav-enged ONOO–, which can be generated in highconcentrations in the presence of increased levels of NO,and thus prevented cells from DNA damage. In addition,Fig. 6B shows that the hexane fraction effectively protectedcells from NO-induced apoptosis at higher concentrations(>50 mg�mL–1). These scavenging results were also con-firmed by the cell growth of E. coli when co-cultured with1 mmol�L–1 H2O2. Interestingly, in the cell growth analysis,we found that the hexane fraction effectively scavenged

H2O2 at lower concentrations (~ 1 mg�mL–1), whereas higherconcentrations of the hexane fraction (>100 mg�mL–1) werebactericidal (Fig. 6B), suggesting that the hexane fraction canact as either an ROS scavenger or bactericidal agent in a dose-selective manner.

Discussion

During the past decades, ROS have aroused significant in-terest among scientists because the mechanisms of ROS maybe important in the pathogenesis of certain diseases andaging. Recently, many reports have supported the use ofantioxidant supplementation in reducing the level of oxida-tive stress and in slowing or preventing the development ofcomplications associated with diseases (Ross et al. 1982).

Fig. 6. Cytoprotection and cell growth analysis. (A) Agarose gel electrophoresis of DNA extracted from RAW264.7 cells. RAW264.7 cellswere co-treated with various concentrations of the hexane fraction (0.1–50 mg�mL–1) and ONOO– (0.5 mmol�L–1), and DNA fragmentationwas analyzed as described in the Materials and methods. Ascorbic acid (0.1 mmol�L–1) was used as a positive control. M, 100 bp DNAmarker. (B) Captures of apoptotic cells. HaCaT cells were treated with and hexane fractions (0.1–50 mg�mL–1) in the presence or absence of0.5 mmol�L–1 SNAP for 6 h, and Annexin V–FITC + propidium iodide (PI) were added as described in the Materials and methods. PI-stained condensed cells represent an apoptosis. (C) Comparison of the cell growth of E. coli. Cells were co-treated with various concentra-tions of the hexane fraction (1–1000 mg�mL–1) and 1 mmol�L–1 H2O2, and analyzed on LB solid agar plates as described in the Materialsand methods. 1Ascorbic acid (0.5 mmol�L–1) and 10% DMSO were used as the positive control and vehicle, respectively. The fading circlerepresents the inhibitory activity of the hexane fraction.

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Experimentally, many synthetic antioxidant componentshave been shown to be toxic and (or) possess mutagenic ef-fects, which have caused researchers to focus on naturallyoccurring free radical scavengers or antioxidants (Aruomaand Cuppett 1997). Polyphenols are a large and diverse classof compounds, many of which occur naturally in a widerange of food and plants. Importantly, the antioxidant prop-erties of numerous plant sources have been attributed totheir phenolic nature (Toda et al. 1988). In addition, volatile(aromatic) plant components were also proven to have anti-oxidant activity. During the characterization of the fractionsby GC-MS, we identified different volatile components inthe hexane fraction of WRFE, which may play an importantrole in antioxidant activity in combination with phenoliccompounds (i.e., gallic acid).

In the present study, we demonstrated that the solventfractions (hexane and butanol) from WRFE are potent anti-oxidants. The antioxidant potential of polyphenols has beencorrelated to their capacity to donate hydrogen radicals. Thenumber and configuration of H-donating hydroxyl groupsare both important structural features influencing the antiox-idant capacity of phenolic compounds (Soobrattee et al.2005). The antioxidant properties of both fractions were ini-tially assessed by analyzing their ability to scavenge DPPHand ABTS, a stable free radical. Since DPPH picks up oneelectron in the presence of a free radical scavenger, the in-crease in percent scavenging by both fractions was propor-tional to the resulting discoloration, which isstochiometrically related to the number of electrons gained(Silva et al. 2005). Both fractions also decreased the ABTSabsorbance in a dose-dependent manner, suggesting thatthese fractions possess effective radical cation scavengingactivity. Interestingly, both experiments indicated that thehexane fraction was a more rapid and effective radical scav-enger than the butanol fraction. The hexane fraction drasti-cally inhibited lipid peroxidation by almost as much asBHA, whereas the butanol fraction was not effective in in-hibiting lipid peroxidation. These findings indicate that thehexane fraction, which contains more active polyphenols (2-fold higher than the butanol fraction), holds promise for useas a highly effective antioxidant candidate in pharmaceuticalapplications. NO, which can form peroxynitrite, was effi-ciently scavenged by the hexane fraction in the non-cellSNAP assay. These results were also confirmed by the NOinhibition assay in RAW264.7 cells. These combined resultssuggest that the hexane fraction scavenges NO in its originalform and also inhibits NO production. Oxidative damage toproteins, lipids, or DNA may all be seriously deleterious andmay occur concomitantly. However, proteins are possiblythe most immediate vehicle for inflicting oxidative damageon cells (Dalle-Donne et al. 2003). Using PAGE, we foundthat the hexane fraction dose-dependently inhibited Cu2+/H2O2 damage, which is specific degradation of BSA. How-ever, the hexane fraction was effective in protecting againstDNA damage when co-treated with ONOO– in RAW264.7cells, suggesting that the hexane fraction may get rid of un-wanted ONOO–, a powerful oxidizing and nitrating agent inall major classes of biological molecules including proteins,lipids, cellular thiols, and nucleic acids that results from theoverproduction of nitric oxide and superoxide in cells (Kimet al. 2005). In cell metabolism, ONOO– is thought to be in-

volved in both cell death and increased cancer risk and hasbeen shown to induce single-strand breaks and base damagein DNA (Spencer et al. 1996). Interestingly, the hexanefraction effectively protected keratinocyte cells from NO-induced apoptosis, suggesting that the fraction may also bea beneficial therapeutic candidate for oxidative stresses.Moreover, we found that hexane sufficiently protected cellgrowth from H2O2 attack at lower concentrations, andacted as a non-antibiotic antimicrobial agent at higher con-centrations. These dual properties of antioxidant and anti-microbial activity also indicate that the hexane fractionmay have an excellent pharmaceutical advantage in regardsto drug development. Notably, the non-antibiotic antimicro-bial activity of the hexane fraction will be important to de-veloping novel therapeutic approaches that cover a broadspectrum of microbial infections.

In conclusion, the hexane fraction of WRFE was found topossess outstanding antioxidant activity in non-cell and cellmodels, and was clearly shown to inhibit cellular damagesby reducing intracellular lipid and protein oxidation levelsand acting as ROS scavengers, which may be helpful in pre-venting or slowing the progress of various oxidative stress-related diseases due to the combination of phenolic and vol-atile components in the fraction. Thus, the hexane fractionfrom WRFE may be a potential source of antioxidants or inpart non-antibiotic antimicrobial agents, which is advanta-geous for pharmaceutical applications.

AcknowledgementsThis work was supported by the Korea Research Founda-

tion Grant funded by the Korean Government (KRF-2008-005-J02801).

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