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Flavonoids and antioxidant activity of Rosa agrestis leavesLeyla Bıtıs a; Sukran Kultur b; Gulay Melıkoglu c; Nurten Ozsoy d;Ayse Can d

a Department of Pharmacognosy, Faculty of Pharmacy, Marmara University, Istanbul, 34668Haydarpasa, Turkey b Department of Pharmaceutical Botany, Faculty of Pharmacy, IstanbulUniversity, Istanbul, 34116 Beyazıt, Turkey c Department of Pharmacognosy, Faculty of Pharmacy,Istanbul University, Istanbul, 34116 Beyazıt, Turkey d Department of Biochemistry, Faculty ofPharmacy, Istanbul University, Istanbul, Turkey

Online publication date: 13 April 2010

To cite this Article Bıtıs, Leyla , Kultur, Sukran , Melıkoglu, Gulay , Ozsoy, Nurten andCan, Ayse(2010) 'Flavonoids andantioxidant activity of Rosa agrestis leaves', Natural Product Research, 24: 6, 580 — 589To link to this Article: DOI: 10.1080/14786410903075507URL: http://dx.doi.org/10.1080/14786410903075507

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Natural Product ResearchVol. 24, No. 6, 10 April 2010, 580–589

Flavonoids and antioxidant activity of Rosa agrestis leaves

Leyla B|t|sa*, Sukran Kulturb, Gulay Mel|kogluc, Nurten Ozsoyd and Ayse Cand

aDepartment of Pharmacognosy, Faculty of Pharmacy, Marmara University, 34668Haydarpasa, Istanbul, Turkey; bDepartment of Pharmaceutical Botany, Faculty ofPharmacy, Istanbul University, 34116 Beyaz|t, Istanbul, Turkey; cDepartment ofPharmacognosy, Faculty of Pharmacy, Istanbul University, 34116 Beyaz|t, Istanbul, Turkey;dDepartment of Biochemistry, Faculty of Pharmacy, Istanbul University, 34116 Beyaz|t,Istanbul, Turkey

(Received 15 January 2009; final version received 25 June 2009)

In this work we report the isolation and characterisation of seven flavonoids,the levels of total phenolics, flavonoids and proanthocyanidins, and theantioxidant activity of the leaf extract of Rosa agrestis Savi (Rosaceae). Theresults showed that the R. agrestis leaf extract exhibited significantantioxidative activity as measured by 2,2-diphenyl-1-picrylhydrazyl(DPPH) (EC50¼ 47.4mgmL�1), inhibited both �-carotene bleaching anddeoxyribose degradation, quenched a chemically generated superoxideanion in vitro and showed high ferrous ion chelating activity. Reactivitytowards 2,20-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammo-nium salt (ABTS) radical cation and ferric-reducing antioxidant power(FRAP) values were equivalent to 2.30mML�1 Trolox, the water soluble�-tocopherol analogue, and 1.91mML�1 Fe2þ, respectively. The highantioxidant activity of the extract appeared to be attributed to its highcontent of total phenolics, flavonoids and proanthocyanidins. Theflavonoids isolated from R. agrestis leaves were diosmetin, kaempherol,quercetin, kaempherol 3-glucoside (astragalin), quercetin 3-rhamnoside(quercitrin), quercetin 3-xyloside and quercetin 3-galactoside (hyperoside).Diosmetin (5,7,30-trihydroxy-40-methoxyflavone) was isolated for the firsttime from Rosa species.

Keywords: antioxidant activity; flavonoids; polyphenol content; Rosaagrestis

1. Introduction

Rosa agrestis Savi (Rosaceae) is one of the 25 species of Rosa growing in Turkey(Kultur, 2004; Nilsson,1972). Rosa species are widely distributed in Turkey and someof them are used in folk medicine as diuretics, expectorants, stomachics, tonics andfor the treatment of dyspepsia, diarrhoea and nephritis (Kultur, 1998, 2007). Theyhave not yet been investigated for their flavonoids. This report is a part of a series ofinvestigations on the ethnobotanical survey, chemical composition and biologicalactivity of endemic Rosa species of Turkish flora.

*Corresponding author. Email: lbitis@yahoo.com

ISSN 1478–6419 print/ISSN 1029–2349 online

� 2010 Taylor & Francis

DOI: 10.1080/14786410903075507

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There is an increasing interest in natural antioxidants, namely polyphenols(e.g. flavonoids, tannins), present in medicinal and dietary plants that might helpprevent oxidative damage and therefore limit the risk of various diseases associatedto oxidative stress (Scalbert, Manach, Morand, Remesy, & Jimenez, 2005; Silva,Ferreres, Malva, & Dias, 2005). Antioxidants may be used to preserve food qualityfrom oxidative deterioration of lipids. Synthetic antioxidants, such as butylatedhydroxyanisole (BHA), butylated hydroxytoluen (BHT) and tert-butylhydroquinone(TBHQ) are widely used in the food industry, but BHA and BHT have beensuspected of being responsible for liver damage and carcinogenesis (Grice, 1986;Wichi, 1988). Therefore, the development and utilisation of more effectiveantioxidants of a natural origin are desired.

The only relatively recent publication on the flavonoids and antioxidant activityof the extract from R. agrestis leaves was by Nowak and Gawlik-Dziki (2007), whoreported quercetin and kaempherol content determined using HPLC and theantioxidant activity of the leaves of R. agrestis evaluated only by the DPPH method.Therefore, the aim of this study was to investigate the flavonoid content andantioxidant activity of the extract from R. agrestis leaves, including iron reductioncapacity, DPPH, ABTS cation, hydroxyl, superoxide anion radical scavenging andferrous ion chelating activities and the inhibition capacity in the �-carotene–linoleicacid model system. In addition, the results were compared with those of gallic acid,ascorbic acid or BHA. The levels of total phenolics, flavonoids and proanthocya-nidins were also determined.

2. Experimental

2.1. Chemicals

Gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH.), and phenazine methosulphate(PMS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA); �-carotene,linoleic acid, nitroblue tetrazolium (NBT), 2-deoxy-D-ribose, 2,20-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS.þ), 6-hydroxy-2,5,7,8,-tetramethylchroman-2-carboxylic acid (Trolox), and Tween 40 werepurchased from Fluka Chemical Co. (Bushs, Switzerland); L-ascorbic acid, 2,4,6-tripyridyl-S-triazine (TPTZ), thiobarbituric acid (TBA), BHA and ferrous chloridewere purchased fromMerck (Darmstadt, Germany). All other chemicals used were ofanalytical grade.

2.2. Plant material

Leaves of R. agrestis were collected in June 2000 from Istanbul (Turkey) andidentified by Dr S� ukran Kultur; voucher specimens have been deposited in theHerbarium of the Faculty of Pharmacy, University of Istanbul (ISTE 79592).

2.3. Extraction and isolation

The dried leaves (500 g) of R. agrestis were first extracted with petroleum etherand then with EtOH (95�) in a Soxhlet apparatus. The petroleum ether extractwas concentrated (A) and extracted with 60% ethanol. The ethanol extract was

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concentrated and extracted with chloroform (B). The concentrated EtOH extract wasdiluted with distilled water and extracted with benzene (C), chloroform (D) and ethylacetate (E) successively for fractionation.

For the purification of flavonoids, silica gel column chromatogrophy, preparativeTLC and paper chromatography (PC) were applied. The structures of the purecompounds were elucidated by Rf values, colour reactions and spectroscopicmethods in comparison with samples or their data (Mabry, Markham, & Thomas,1970). The E extract was dissolved in ethanol, and used for the assessment ofantioxidant activity.

2.4. Determination of total phenolic compounds, flavonoids and proanthocyanidins

Total phenolic compounds in the leaf extract of R. agrestis were determined withFolin–Ciocalteu reagent according to the method of Slinkard and Singleton (1977),with some modifications. Total flavonoid content was determined by using acolourimetric method (Sakanaka, Tachibana, & Okada, 2005). Proanthocyanidinswere determined by butanol–HCl assay (Hagerman, Harvey-Mueller, & Makkar,2000).

2.5. The b-carotene–linoleic acid model system

The antioxidant activity of the leaf extract of R. agrestis was evaluated using the�-carotene–linoleate model system as described by Sun and Ho (2005).

2.6. Total radical antioxidant potential (TRAP) assay

The total radical antioxidant potential of the extract was measured using the Troloxequivalent antioxidant coefficient (TEAC) assay, as described by Re, Pellegrini,Proteggente, Pannala, Yang and Rice-Evans (1999). The TEAC value is based on theability of the antioxidant molecules to quench the long-lived ABTS, a blue-greenchromophore with characteristic absorption at 734 nm, compared to that of Trolox,a water-soluble vitamin E analogue.

2.7. DPPH radical scavenging activity

The DPPH free radical scavenging activity of the leaf extract of R. agrestis wasmeasured according to the procedure described by Brand-Williams, Cuvelier, & Berset(1995).

2.8. Ferric reducing antioxidant power (FRAP) assay

The FRAP assay was carried out according to the procedure of Benzie and Strain(1996).

2.9. Superoxide radical scavenging activity

The effects of the leaf extract of R. agrestis, gallic acid or ascorbic acid on thegeneration of superoxide radicals were determined by the nitroblue tetrazoliumreduction method (Nishikimi, Rao, & Yagi, 1972).

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2.10. Hydroxyl radical scavenging activity

The effect of the extract on hydroxyl radicals was assayed using the deoxyribosemethod (Nagai, Myoda, & Nagashima, 2005).

2.11. Ferrous ion chelating activity

The chelating of ferrous ions by the leaf extract of R. agrestis was estimated by themethod of Dinis, Madeira and Almeida (1994).

3. Results and discussion

3.1. Flavonoid compounds

In this work, diosmetin (13.1mg; extract C), kaempherol (5.2mg; extract C),quercetin (12mg; extract C), astragalin (5.5mg; extract D), quercetin 3-xyloside(6.5mg; extract D, E), quercitrin (6.3mg; extract D, E) and hyperoside (7mg; extractD, E) were isolated (Table 1). Diosmetin (5,7,30-trihydroxy-40-methoxyflavone) wasisolated for the first time from Rosa species.

3.2. Total phenolic compounds, flavonoids and proanthocyanidins contents

The results revealed that the leaf extract of R. agrestis contained a significant amountof phenolic compounds (489.7mg g�1 extract) expressed in gallic acid equivalents(GAE) and flavonoids (265.0mg g�1 extract) (Table 2) expressed in catechinequivalents (CE). The antioxidative effectiveness in natural sources was reportedto be mostly due to phenolic compounds. It was observed that leaves contained107.4mg g�1 extract proanthocyanidins determined as leucoanthocyanidin equiva-lents (LE). It was reported that proanthocyanidins may be one of the secondaryplant metabolites to which antioxidant activity can be related (Rugna, Polo, Evelson,Gurni, Llesuy & Wagner, 2003).

3.3. The b-carotene–linoleic acid model system

We evaluated the antioxidant activity of the R. agrestis leaf extract by the�-carotene–linoleic acid model system. Figure 1 shows the decrease in absorbance of�-carotene in the presence of 0.5mgmL�1 of the extract or reference antioxidants(gallic acid and BHA). The addition of R. agrestis extract and BHA or gallic acid at0.5mgmL�1 was markedly effective in inhibiting the oxidation of linoleic acid andthe subsequent bleaching of �-carotene in comparison with control ( p50.05), whichcontained no antioxidant component. The addition of the extract prevented thebleaching of �-carotene by about 63.30� 2.27%. However, the extract was lesseffective in comparison to BHA (87.90� 2.26%), but showed more inhibitionactivity than the gallic acid (44.24� 2.64%) at the same concentration. The resultsindicated that the extract had acted as an effective antioxidant in the �-carotene–linoleic acid model system.

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Table

1.UV

data

forflavonoidsfrom

R.agrestis.

l max(nm)

Flavonoid

MeO

HNaOMe

AlCl 3

AlCl 3/H

Cl

NaOAc

NaOAc/H

3BO

3

Diosm

etin

342,276,254

390,300sh,

284,270

386,294,260

386,298,262

367,312sh,270

348,278

Kaem

pherol

367,322sh,294sh,

266,253sh

416(dec),316,

278

424,350,303sh,

268,260sh

424,348,303sh,

269,256sh

387,303,274

372,320sh,297sh,

267

Quercetin

370,301sh,269sh,

255

321(dec),247sh

458,333,

304sh,272

428,359,

301sh,265

390(dec),329,

274,257sh

388,303sh,261

Astragalin

351,296,266

402,327,275

398,351,304,275

397,348,304,279

383,304,272

354,300,267

Quercetin

3-xyloside

352,300sh,267sh,

256

395,327,271

433,334,

305sh,275

404,357,

303sh,273

376,322sh,275

370,300sh,263

Quercitrin

350,301sh,265sh,

256

393,326,270

430,333,

304sh,276

401,353,

303sh,272

372,322sh,272

367,300sh,260

Hyperoside

362,299sh

269sh,257

409,327,272

438,331sh,

305sh,275

405,366sh,

299sh,268

380,324,274

377,298sh,262

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3.4. TRAP assay

The total antioxidant capacity value in a sample was assessed as Trolox equivalentantioxidant capacity (TEAC); a value of 1 TEAC in a sample was defined asa concentration that is equivalent to 1mML�1 of Trolox. Based on the results ofthe TRAP assay, this extract contained the antioxidant equivalent of 2.30�0.016mML�1 of Trolox at a concentration of 0.5mgmL�1. Our results are in verygood agreement with TEAC values of many compounds and food samples (Huang,Ou, & Prior, 2005).

3.5. DPPH radical scavenging activity

The scavenging effect of the R. agrestis leaf extract on DPPH radicals sharplyincreased from 8 mgmL�1 (10.98� 2.26%) to 125mgmL�1 (90.68� 0.73%). Thescavenging effect of the leaf extract of R. agrestis was nearly equal to that of ascorbicacid (94.91� 1.42%) at a concentration of 125 mgmL�1. The maximum scavengingeffect of gallic acid (91.86� 1.09%) was reached with a smaller quantity(62.5 mgmL�1).

From the low value of EC50 (47.43� 0.33 mgmL�1) (the effective concentration atwhich the DPPH radicals were scavenged by 50%) (Table 2), it was also seen that theR. agrestis leaf extract had high DPPH radical scavenging activity. The EC50 value interms of scavenging ability of the extract on DPPH radicals was comparable withthat of ascorbic acid (42.96� 0.65 mgmL�1) but significantly different ( p50.05)from the EC50 value obtained for the gallic acid (22.20� 0.84mgmL�1). It wasevident that the extract from R. agrestis leaves showed the hydrogen donatingcapability to act as an antioxidant.

3.6. FRAP assay

The reducing capacity of the extract may serve as a significant indicator of itspotential antioxidant activity. In the FRAP assay, on the basis of the standard(Fe2þ), the FRAP value of the extract was 1.91� 0.03mML�1 at a concentration of

0

0.10.2

0.3

0.40.5

0.6

0.7

0.80.9

1

0 20 40 60 80 100 120Time (min)

Abs

orba

nce

at 4

70 n

m

Control

Extract

Gallic acid

BHA

Figure 1. Antioxidant activity of the R. agrestis leaf extract measured by the �-carotene–linoleic acid model system. Gallic acid and BHA were used as reference antioxidants. Valuesare means� SD (n¼ 3).

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Table

2.Polyphenolcontents

andsomeantioxidantactivitiesoftheextract

from

R.agrestisleaves.

Extract

Totalaphenolic

compounds(m

gg�1

plantextract)

(inGAE)

Flavonoidsb

(mgg�1plant

extract)(inCE)

Proanthocyanidinsc

(mgg�1extract)

(inLE)

Totalantioxidant

potentiald

(mM

TEAC)

FRAPvaluee

(mM

Fe2

+)

DPPH

EC50

(mgmL�1)

�-C

arotene–

linoleic

acid

model

system

inhibition(%

)

Rosa

leaves

489.76�22.33

265.01�30.33

107.46�7.24

2.30�0.016

1.91�0.03

47.43�0.33

63.30�2.27

Notes:Values

werethemeansofthreereplicates�standard

deviation.aExpressed

asmilligram

GAEper

gram

extract.bExpressed

asmilligram

CEper

gram

extract.cExpressed

asmilligram

LE

per

gram

extract.dExpressed

asmmolTroloxequivalents.eExpressed

asmmolferrousioneqivalents.

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0.5mgmL�1 (Table 2). It was comparable to that of ascorbic acid (1.96� 0.017mM)at the same concentration. Gallic acid showed high reducing power (1.84� 0.35) at alower concentration (0.125mgmL�1). Based on the FRAP value, the R. agrestis leafextract showed high reducing power in a dose-dependent manner, indicating highpotential antioxidant activity. Our results were in agreement with FRAP values ofdifferent phenolic extracts (Li et al., 2006).

The DPPH radical scavenging activity (r2¼ 0.9676) and TEAC value(r2¼ 0.9063) were well correlated with the ferric-reducing antioxidant power. Ourresults were in accordance with other investigators who have also reported thatantioxidant properties could be strongly correlated with the development of reducingpower (Kanatt, Chander, & Sharma, 2007).

3.7. Superoxide radical scavenging activity

The superoxide radical scavenging activity (97.09� 0.81%) of R. agrestis leaf extractat 2.5mgmL�1 compared well with that of the same dose of gallic acid(93.40� 0.54%), but was significantly higher (p50.05) than that exhibited byascorbic acid (44.38� 1.41%).

The superoxide radical scavenging activity of the extract, expressed in terms ofEC50 value (the effective concentration at which the superoxide radicals werescavenged by 50%), was significantly higher ( p50.05), with an EC50 value of0.870� 0.098mgmL�1, in comparison with ascorbic acid (2.51� 0.092mgmL�1)and gallic acid (1.13� 0.086mgmL�1). These results implied that the R. agrestis leafextract was a stronger superoxide radical scavenger than both ascorbic acid andgallic acid, and its capacity to scavenge superoxide radicals may contribute to theantioxidant activity of the extract.

3.8. Hydroxyl radical scavenging activity

The deoxyribose assay quantifies the extent to which a substance can inhibitdeoxyribose oxidation caused by hydroxyl radicals generated by Fenton reaction. TheRosa agrestis leaf extract scavenged hydroxyl radicals by 89.57� 0.06% at10mgmL�1, which is comparable to BHA (quenched 95.28� 0.37%) at1.25mgmL�1. The Rosa agrestis leaf extract was observed to possess stronger activitythan that of gallic acid. At 10mgmL�1, the hydroxyl radical scavenging activity ofgallic acid was significantly ( p50.05) lower (47.23� 1.06%).

Based on the EC50 values (the effective concentration at which the hydroxylradicals were scavenged by 50%), the extract (3.55� 0.081mgmL�1) showed lesshydroxyl radical scavenging activity than BHA (0.212� 0.017mg mL�1), but wasmuch more effective than gallic acid (13.33� 0.049mgmL�1).

3.9. Ferrous ion chelating activity

As metal chelation is an important antioxidant property, the leaf extract ofR. agrestis was assessed for its ability to compete with ferrozine for iron (II) ionsin free solution. The extract demonstrated an ability to chelate ferrous ions ina dose-dependent manner. The present study detected significant chelating activity

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(83.01� 0.98%) against Fe2þ at 2.5mgmL�1. EDTA, used as a positive control inthis assay system, showed an excellent chelating ability of 99.1� 0.05% at aconcentration as low as 0.01mgmL�1.

Although the chelating activity of the extract from R. agrestis leaves(0.921� 0.007mgmL�1), expressed in terms of EC50 value (the effective concentra-tion at which ferrous ions were chelated by 50%), was significantly lower (p50.05)than that of EDTA (5.23� 0.27 mgmL�1), it was evident that the extract couldinhibit free radical production by chelating the transition metal catalysts.

In conclusion, we have demonstrated that the extract from R. agrestis leavescontains high levels of total phenolic compounds, flavonoids and proanthocyanidinsand also shows reducing power and scavenging activities on free radicals. Theseproperties could, in part, explain the antioxidative activity of the extract. The extracthas an antioxidant activity comparable with or superior to gallic acid, ascorbic acidor BHA. The antioxidant activities measured using TRAP and DPPH radicalscavenging assays were highly correlated with phenolic (R2

¼ 0.9203 and 0.9063,respectively) content, indicating that phenolic compounds could be majorcontributors to antioxidant activity. According to the data presented, the extractof R. agrestis leaves may be a good source of natural antioxidants that could be usedas a possible supplement in the pharmaceutical industry.

4. Statistical analysis

Results were expressed as mean� the standard deviation of triplicate analyses.Statistical comparisons were performed with Student’s t-test. Differences wereconsidered significant at p50.05. The correlation coefficient (R2) between theparameters tested was established by regression analysis.

Acknowledgement

This work was supported by the Research Fund of the University of Istanbul, project number1660/30042001.

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