Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2013 Article ID 385473 4 pageshttpdxdoiorg1011552013385473
Research ArticleQuantitative Determination of Flavonoids andChlorogenic Acid in the Leaves of Arbutus unedo L UsingThin Layer Chromatography
Celjan Maleš1 Darija ŠariT2 and Mirza BojiT1
1 University of Zagreb Faculty of Pharmacy and Biochemistry A Kovacica 1 10000 Zagreb Croatia2 Agency for Medicinal Products and Medical Devices of Croatia Ksaverska cesta 4 10000 Zagreb Croatia
Correspondence should be addressed to Mirza Bojic mirzabojicgmailcom
Received 26 May 2013 Accepted 11 July 2013
Academic Editor Shixin Deng
Copyright copy 2013 Zeljan Males et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
The plant species Arbutus unedo shows numerous beneficial pharmacological effects (antiseptic antidiabetic antidiarrheal astrin-gent depurative antioxidant antihypertensive antithrombotic and anti-inflammatory) For the medicinal use standardization ofextracts is a necessity as different compounds are responsible for different biological activities In this paper we analyze monthlychanges in the content of quercitrin isoquercitrin hyperoside and chlorogenic acid Methanolic extracts of the leaves are analyzedby HPTLC for the identification and quantification of individual polyphenol and DPPH test is used to determine antioxidantactivity Based on the results obtained the leaves should be collected in January to obtain the highest concentrations of hyperosideand quercitrin (035mgg and 194mgg resp) in June July andOctober for chlorogenic acid (145ndash146mgg) and for the fractionof quercitrin and isoquercitrin in November (198mgg and 033mgg resp) Optimal months for the collection of leaves with themaximum recovery of individual polyphenol suggested in this work could direct the pharmacological usage of the polyvalent herbaldrugs
1 Introduction
Arbutus unedo L (Ericaceae English strawberry tree) is anevergreen shrub or a small tree reaching up to 12m inheight It is found mainly in European Mediterranean regiongrowing in maquis evergreen scrub woodland marginsand on rocky slopes The leaves of A unedo are alternatesimple oblanceolate dark green leathery short-stalked andtoothed The flowers are bell shaped with recurved lobes8-9mm long white often tinged with pink or green andhoney scentedThe fruits are globose berries about 15ndash20mmin diameter ripening through yellow to scarlet and deepcrimson Since the fruits take about 12 months to ripen atree carries mature fruits and flowers at the same time andthe appearance of both during winter months also makes thisplant very popular for specimen plantings [1 2]
The leaves of A unedo are used as a urinary antisepticantidiabetic antidiarrheal astringent depurative antioxi-dant antihypertensive antithrombotic anti-inflammatory
agent [3ndash8] Chemical investigations of leaves and fruits showthe presence of essential oil flavonoids proanthocyanidinsiridoid glucosides sugars nonvolatile and phenolic acidsvitamins C and E and carotenoids [9ndash13]
As a pharmacological activity can rarely be attributed toa group of compounds as it is the case with polyphenols andantioxidant activity the identification and quantification ofindividual compounds responsible for a biological activityare of interest The objective of this paper was the identifi-cation and quantification of chlorogenic acid and flavonoidsquercitrin isoquercitrin and hyperoside using a simple thinlayer chromatography technique
2 Experimental
21 PlantMaterials Reagents Chemicals and Solutions Eachmonth in the year of 2003 the leaves of ten A unedo plantswere collected on five different locations on the island of Dugi
2 Journal of Analytical Methods in Chemistry
otok Bozava municipality (44∘ 81015840 3010158401015840N 14∘ 541015840 3010158401015840 E)Voucher specimens (no 99450-99461) were deposited at theDepartment of Pharmaceutical Botany Faculty of Pharmacyand Biochemistry University of Zagreb Solvents of theanalytical grade were obtained from Kemika (Croatia) andstandards (quercitrin isoquercitrin hyperoside and chloro-genic acid) were purchased from C Roth (Germany) 22-Diphenyl-1-picrylhydrazyl was supplied by Sigma-Aldrich(USA) and HPTLC silica gel 60 F254 by Merck (Germany)
22 Sample and Standard Preparation Extracts of A unedowere prepared by the reflux extraction of leaves powder inmethanol for 5 minutes final concentration being 01 gmLThe standards of polyphenols were prepared as 1mgmLsolutions in methanol
23Thin Layer Chromatography Thin layer chromatographywas performed on 10 times 20 cm HPTLC silica gel 60 F254plates (Merck Germany) Ethyl acetate-formic acid-aceticacid-water in volume ratio 100 11 11 26 was used as mobilephase [14] After development plates were air dried andrecorded at 254 and 366 nm identification and quantificationwere performed by TLC densitometry using CAMAG TLCScanner 3 andWinCATS software version 134 (Switzerland)Quantification was performed using calibration curves (peakarea of chromatogram versus mass of standard applied in theform of band) for individual standard in triplicate
24 DPPH Test Antioxidant activity was assessed using sta-ble free radical 22-diphenyl-1-picrylhydrazyl (DPPH)DPPHsolution was prepared by dissolving DPPH in ethanol toobtain the final concentration of 03mM Decolorization ofDPPH in the presence of extract (100 1 volume ration) wasmeasured on Varian Cary 50 Bio spectrophotometer (USA)Antioxidant activity (AA) was expressed as a percentage ofquenching of the stable free radical at 120582 = 518 nm as follows
AA =(1198600minus 119860)
1198600
times 100 (1)
where1198600represents the absorbance of blank (methanol) and
119860 absorbance of the extract measured 1 minute after mixing
3 Results and Discussion
Qualitative analysis of polyphenols in leaves and fruits ofA unedo [14] showed presence of nine bands in the methanolextracts Seven polyphenol standards were used and fourflavonoids and chlorogenic acid were identified out of whichwe were able to determine the content (quantify) of chloro-genic acid quercitrin isoquercitrin and hyperoside
Identification of each polyphenol was based on the colorof the band observed under 120582 = 366 nm 119877
119865value and
matching UV-Vis spectra in situ with the standard used(Table 1)
Volumes of extracts applied to the plate were adjusted tocorrespond to ranges of linearity for individual polyphenolthat were 02ndash16120583g per band for chlorogenic acid 10ndash50120583g per band for quercitrin 25ndash125120583g per band for
Table 1 Parameters of identification of the polyphenols analyzed
Polyphenol 119877119865
Color under 120582 = 366 nmQuercitrin 080 Yellow-greenIsoquercitrin 064 Yellow-greenHyperoside 057 Yellow-greenChlorogenic acid 049 Blue
30
35
40
45
50
55
60
Janu
ary
February
March
April
May
June July
August
Septem
ber
Octob
er
Novem
ber
Decem
ber
()
Figure 1 Variation of antioxidant activity expressed in percentagesduring the year
isoquercitrin and 02ndash06 120583g per band for hyperoside Theresults of HPTLC quantification of an individual polyphenolare presented in Table 2
Compared to the results of the total flavonoids of 130ndash200 g per 100 g of dried leaves from our previous study [14]quercitrin isoquercitrin and hyperoside (238mggmdashsum ofindividual flavonoids for June Table 2) can be accountedfor up to 12 of dry powder However it should be noticedthat methods for determination of total flavonoids usuallyuse hydrolyzed extractsmeaning that aglycones are analyzedwhereas all the analyzed polyphenols in this work presentflavonoid glycosides
As suggested by Oliveira et al [15] methanolic extractshave greater antioxidant activity compared to ethanolic andwater based The antioxidant activity values of methanolicextracts of A unedo were determined during a period of 12months (Figure 1)
The results of the antioxidant activity varywith themonthobserved and cannot be attributed to any of the analyzedpolyphenols individually (1199032 lt 025) rather it presents theoverall activity of the total extracted polyphenols includingphenolic acids and flavonoids other than analyzed chloro-genic acid quercitrin isoquercitrin and hyperoside Theseflavonoids are attributed up to only 12 of total flavonoids
Specific pharmacological activity is usually the result of aspecific substance for example the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase can be attributed toquercetin hyperoside rutin and chlorogenic acid fractionsof Crataegus pinnatifida Bge [16] Thus to achieve a specificaction of the extracts of A unedo leaves should be collectedduring the months when the concentration of individualsubstance responsible for specific action is the highestThis means based on the results obtained that the leaves
Journal of Analytical Methods in Chemistry 3
Table 2 Content of individual polyphenol during the year in the leaves of A unedo expressed in mg per g of dry sample
Results expressed as mean plusmn standard deviationnd not detected
should be collected in January to obtain the highest con-centrations of hyperoside and quercitrin (035mgg and194mgg resp) in June July and October for chlorogenicacid (145ndash146mgg) and for the fraction of quercitrin andisoquercitrin in November (198mgg and 033mgg resp)
4 Conclusion
Different studies have shown beneficial effects of A unedofor human health and suggested the usage of standardizedextracts inmedicinal products Although antioxidant activitywell correlates with the total content of polyphenols phenolicacids and flavonoids [15] a specific action for example anantiaggregatory [6] or an anti-inflammatory action [7] isprobably the consequence of specific compound(s) to whichextracts should be standardized For this purposes the thinlayer chromatography presents simple and readily availabletechnique
Conflict of Interest
The authors declare no conflict of interests
Acknowledgments
This work was supported by the Ministry of Science Educa-tion and Sports of the Republic of Croatia Grant nos 006-0061117-1237 and 006-0061117-1239
References
[1] RDomac Flora of CroatiaManual ForDetermination of PlantsSkolska knjiga Zagreb Croatia 2002
[2] C SilicAtlas of Trees and Shrubs Svjetlost Sarajevo Bosnia 3rdedition 1988
[3] S Afkir T B Nguelefack M Aziz et al ldquoArbutus unedoprevents cardiovascular and morphological alterations in L-NAME-induced hypertensive rats Part I cardiovascular and
renal hemodynamic effects of Arbutus unedo in L-NAME-induced hypertensive ratsrdquo Journal of Ethnopharmacology vol116 no 2 pp 288ndash295 2008
[4] D Andrade C Gil L Breitenfeld F Domingues and A PDuarte ldquoBioactive extracts from Cistus ladanifer and Arbutusunedo Lrdquo Industrial Crops and Products vol 30 no 1 pp 165ndash167 2009
[5] M Bnouham F Z Merhfour A Legssyer H Mekhfi SMaallem and A Ziyyat ldquoAntihyperglycemic activity ofArbutusunedo Ammoides pusilla and Thymelaea hirsutardquo Pharmazievol 62 no 8 pp 630ndash632 2007
[6] M El Haouari J J Lopez H Mekhfi J A Rosado and GM Salido ldquoAntiaggregant effects of Arbutus unedo extracts inhuman plateletsrdquo Journal of Ethnopharmacology vol 113 no 2pp 325ndash331 2007
[7] S Mariotto E Esposito R Di Paola et al ldquoProtective effectof Arbutus unedo aqueous extract in carrageenan-induced lunginflammation in micerdquo Pharmacological Research vol 57 no 2pp 110ndash124 2008
[8] A Pabuccuoglu B Kivcak M Bas and T Mert ldquoAntioxidantactivity of Arbutus unedo leavesrdquo Fitoterapia vol 74 no 6 pp597ndash599 2003
[9] L Barros A M Carvalho J S Morais and I C F R FerreiraldquoStrawberry-tree blackthorn and rose fruits detailed charac-terisation in nutrients and phytochemicals with antioxidantpropertiesrdquo Food Chemistry vol 120 no 1 pp 247ndash254 2010
[10] BKivcak TMert BDemirci andKHC Baser ldquoCompositionof the essential oil of Arbutus unedordquo Chemistry of NaturalCompounds vol 37 no 5 pp 445ndash446 2001
[11] P Lebreton and C Bayet ldquoThe physiological and biochemicalvariability of the strawberry tree Arbutus unedo L (Ericaceae)rdquoActa Pharmaceutica vol 52 no 2 pp 83ndash90 2002
[12] M M Ozcan and H Haciseferogullan ldquoThe strawberry (Arbu-tus unedo L) fruits chemical composition physical propertiesand mineral contentsrdquo Journal of Food Engineering vol 78 no3 pp 1022ndash1028 2007
[13] K Pallauf J C Rivas-Gonzalo M D del Castillo M P Canoand S de Pascual-Teresa ldquoCharacterization of the antioxidantcomposition of strawberry tree (Arbutus unedo L) fruitsrdquoJournal of Food Composition and Analysis vol 21 no 4 pp 273ndash281 2008
4 Journal of Analytical Methods in Chemistry
[14] Z Males M Plazibat V B Vundac and I Zuntar ldquoQualitativeand quantitative analysis of flavonoids of the strawberry treemdashArbutus unedo L (Ericaceae)rdquo Acta Pharmaceutica vol 56 no2 pp 245ndash250 2006
[15] I Oliveira V Coelho R Baltasar J A Pereira and P BaptistaldquoScavenging capacity of strawberry tree (Arbutus unedo L)leaves on free radicalsrdquo Food and Chemical Toxicology vol 47no 7 pp 1507ndash1511 2009
[16] X-L I Ye W-W Huang Z Chen et al ldquoSynergetic effect andstructure-activity relationship of 3-hydroxy-3-methylglutarylcoenzyme a reductase inhibitors from crataegus pinnatifidabgerdquo Journal of Agricultural and Food Chemistry vol 58 no 5pp 3132ndash3138 2010
otok Bozava municipality (44∘ 81015840 3010158401015840N 14∘ 541015840 3010158401015840 E)Voucher specimens (no 99450-99461) were deposited at theDepartment of Pharmaceutical Botany Faculty of Pharmacyand Biochemistry University of Zagreb Solvents of theanalytical grade were obtained from Kemika (Croatia) andstandards (quercitrin isoquercitrin hyperoside and chloro-genic acid) were purchased from C Roth (Germany) 22-Diphenyl-1-picrylhydrazyl was supplied by Sigma-Aldrich(USA) and HPTLC silica gel 60 F254 by Merck (Germany)
22 Sample and Standard Preparation Extracts of A unedowere prepared by the reflux extraction of leaves powder inmethanol for 5 minutes final concentration being 01 gmLThe standards of polyphenols were prepared as 1mgmLsolutions in methanol
23Thin Layer Chromatography Thin layer chromatographywas performed on 10 times 20 cm HPTLC silica gel 60 F254plates (Merck Germany) Ethyl acetate-formic acid-aceticacid-water in volume ratio 100 11 11 26 was used as mobilephase [14] After development plates were air dried andrecorded at 254 and 366 nm identification and quantificationwere performed by TLC densitometry using CAMAG TLCScanner 3 andWinCATS software version 134 (Switzerland)Quantification was performed using calibration curves (peakarea of chromatogram versus mass of standard applied in theform of band) for individual standard in triplicate
24 DPPH Test Antioxidant activity was assessed using sta-ble free radical 22-diphenyl-1-picrylhydrazyl (DPPH)DPPHsolution was prepared by dissolving DPPH in ethanol toobtain the final concentration of 03mM Decolorization ofDPPH in the presence of extract (100 1 volume ration) wasmeasured on Varian Cary 50 Bio spectrophotometer (USA)Antioxidant activity (AA) was expressed as a percentage ofquenching of the stable free radical at 120582 = 518 nm as follows
AA =(1198600minus 119860)
1198600
times 100 (1)
where1198600represents the absorbance of blank (methanol) and
119860 absorbance of the extract measured 1 minute after mixing
3 Results and Discussion
Qualitative analysis of polyphenols in leaves and fruits ofA unedo [14] showed presence of nine bands in the methanolextracts Seven polyphenol standards were used and fourflavonoids and chlorogenic acid were identified out of whichwe were able to determine the content (quantify) of chloro-genic acid quercitrin isoquercitrin and hyperoside
Identification of each polyphenol was based on the colorof the band observed under 120582 = 366 nm 119877
119865value and
matching UV-Vis spectra in situ with the standard used(Table 1)
Volumes of extracts applied to the plate were adjusted tocorrespond to ranges of linearity for individual polyphenolthat were 02ndash16120583g per band for chlorogenic acid 10ndash50120583g per band for quercitrin 25ndash125120583g per band for
Table 1 Parameters of identification of the polyphenols analyzed
Polyphenol 119877119865
Color under 120582 = 366 nmQuercitrin 080 Yellow-greenIsoquercitrin 064 Yellow-greenHyperoside 057 Yellow-greenChlorogenic acid 049 Blue
30
35
40
45
50
55
60
Janu
ary
February
March
April
May
June July
August
Septem
ber
Octob
er
Novem
ber
Decem
ber
()
Figure 1 Variation of antioxidant activity expressed in percentagesduring the year
isoquercitrin and 02ndash06 120583g per band for hyperoside Theresults of HPTLC quantification of an individual polyphenolare presented in Table 2
Compared to the results of the total flavonoids of 130ndash200 g per 100 g of dried leaves from our previous study [14]quercitrin isoquercitrin and hyperoside (238mggmdashsum ofindividual flavonoids for June Table 2) can be accountedfor up to 12 of dry powder However it should be noticedthat methods for determination of total flavonoids usuallyuse hydrolyzed extractsmeaning that aglycones are analyzedwhereas all the analyzed polyphenols in this work presentflavonoid glycosides
As suggested by Oliveira et al [15] methanolic extractshave greater antioxidant activity compared to ethanolic andwater based The antioxidant activity values of methanolicextracts of A unedo were determined during a period of 12months (Figure 1)
The results of the antioxidant activity varywith themonthobserved and cannot be attributed to any of the analyzedpolyphenols individually (1199032 lt 025) rather it presents theoverall activity of the total extracted polyphenols includingphenolic acids and flavonoids other than analyzed chloro-genic acid quercitrin isoquercitrin and hyperoside Theseflavonoids are attributed up to only 12 of total flavonoids
Specific pharmacological activity is usually the result of aspecific substance for example the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase can be attributed toquercetin hyperoside rutin and chlorogenic acid fractionsof Crataegus pinnatifida Bge [16] Thus to achieve a specificaction of the extracts of A unedo leaves should be collectedduring the months when the concentration of individualsubstance responsible for specific action is the highestThis means based on the results obtained that the leaves
Journal of Analytical Methods in Chemistry 3
Table 2 Content of individual polyphenol during the year in the leaves of A unedo expressed in mg per g of dry sample
Results expressed as mean plusmn standard deviationnd not detected
should be collected in January to obtain the highest con-centrations of hyperoside and quercitrin (035mgg and194mgg resp) in June July and October for chlorogenicacid (145ndash146mgg) and for the fraction of quercitrin andisoquercitrin in November (198mgg and 033mgg resp)
4 Conclusion
Different studies have shown beneficial effects of A unedofor human health and suggested the usage of standardizedextracts inmedicinal products Although antioxidant activitywell correlates with the total content of polyphenols phenolicacids and flavonoids [15] a specific action for example anantiaggregatory [6] or an anti-inflammatory action [7] isprobably the consequence of specific compound(s) to whichextracts should be standardized For this purposes the thinlayer chromatography presents simple and readily availabletechnique
Conflict of Interest
The authors declare no conflict of interests
Acknowledgments
This work was supported by the Ministry of Science Educa-tion and Sports of the Republic of Croatia Grant nos 006-0061117-1237 and 006-0061117-1239
References
[1] RDomac Flora of CroatiaManual ForDetermination of PlantsSkolska knjiga Zagreb Croatia 2002
[2] C SilicAtlas of Trees and Shrubs Svjetlost Sarajevo Bosnia 3rdedition 1988
[3] S Afkir T B Nguelefack M Aziz et al ldquoArbutus unedoprevents cardiovascular and morphological alterations in L-NAME-induced hypertensive rats Part I cardiovascular and
renal hemodynamic effects of Arbutus unedo in L-NAME-induced hypertensive ratsrdquo Journal of Ethnopharmacology vol116 no 2 pp 288ndash295 2008
[4] D Andrade C Gil L Breitenfeld F Domingues and A PDuarte ldquoBioactive extracts from Cistus ladanifer and Arbutusunedo Lrdquo Industrial Crops and Products vol 30 no 1 pp 165ndash167 2009
[5] M Bnouham F Z Merhfour A Legssyer H Mekhfi SMaallem and A Ziyyat ldquoAntihyperglycemic activity ofArbutusunedo Ammoides pusilla and Thymelaea hirsutardquo Pharmazievol 62 no 8 pp 630ndash632 2007
[6] M El Haouari J J Lopez H Mekhfi J A Rosado and GM Salido ldquoAntiaggregant effects of Arbutus unedo extracts inhuman plateletsrdquo Journal of Ethnopharmacology vol 113 no 2pp 325ndash331 2007
[7] S Mariotto E Esposito R Di Paola et al ldquoProtective effectof Arbutus unedo aqueous extract in carrageenan-induced lunginflammation in micerdquo Pharmacological Research vol 57 no 2pp 110ndash124 2008
[8] A Pabuccuoglu B Kivcak M Bas and T Mert ldquoAntioxidantactivity of Arbutus unedo leavesrdquo Fitoterapia vol 74 no 6 pp597ndash599 2003
[9] L Barros A M Carvalho J S Morais and I C F R FerreiraldquoStrawberry-tree blackthorn and rose fruits detailed charac-terisation in nutrients and phytochemicals with antioxidantpropertiesrdquo Food Chemistry vol 120 no 1 pp 247ndash254 2010
[10] BKivcak TMert BDemirci andKHC Baser ldquoCompositionof the essential oil of Arbutus unedordquo Chemistry of NaturalCompounds vol 37 no 5 pp 445ndash446 2001
[11] P Lebreton and C Bayet ldquoThe physiological and biochemicalvariability of the strawberry tree Arbutus unedo L (Ericaceae)rdquoActa Pharmaceutica vol 52 no 2 pp 83ndash90 2002
[12] M M Ozcan and H Haciseferogullan ldquoThe strawberry (Arbu-tus unedo L) fruits chemical composition physical propertiesand mineral contentsrdquo Journal of Food Engineering vol 78 no3 pp 1022ndash1028 2007
[13] K Pallauf J C Rivas-Gonzalo M D del Castillo M P Canoand S de Pascual-Teresa ldquoCharacterization of the antioxidantcomposition of strawberry tree (Arbutus unedo L) fruitsrdquoJournal of Food Composition and Analysis vol 21 no 4 pp 273ndash281 2008
4 Journal of Analytical Methods in Chemistry
[14] Z Males M Plazibat V B Vundac and I Zuntar ldquoQualitativeand quantitative analysis of flavonoids of the strawberry treemdashArbutus unedo L (Ericaceae)rdquo Acta Pharmaceutica vol 56 no2 pp 245ndash250 2006
[15] I Oliveira V Coelho R Baltasar J A Pereira and P BaptistaldquoScavenging capacity of strawberry tree (Arbutus unedo L)leaves on free radicalsrdquo Food and Chemical Toxicology vol 47no 7 pp 1507ndash1511 2009
[16] X-L I Ye W-W Huang Z Chen et al ldquoSynergetic effect andstructure-activity relationship of 3-hydroxy-3-methylglutarylcoenzyme a reductase inhibitors from crataegus pinnatifidabgerdquo Journal of Agricultural and Food Chemistry vol 58 no 5pp 3132ndash3138 2010
Results expressed as mean plusmn standard deviationnd not detected
should be collected in January to obtain the highest con-centrations of hyperoside and quercitrin (035mgg and194mgg resp) in June July and October for chlorogenicacid (145ndash146mgg) and for the fraction of quercitrin andisoquercitrin in November (198mgg and 033mgg resp)
4 Conclusion
Different studies have shown beneficial effects of A unedofor human health and suggested the usage of standardizedextracts inmedicinal products Although antioxidant activitywell correlates with the total content of polyphenols phenolicacids and flavonoids [15] a specific action for example anantiaggregatory [6] or an anti-inflammatory action [7] isprobably the consequence of specific compound(s) to whichextracts should be standardized For this purposes the thinlayer chromatography presents simple and readily availabletechnique
Conflict of Interest
The authors declare no conflict of interests
Acknowledgments
This work was supported by the Ministry of Science Educa-tion and Sports of the Republic of Croatia Grant nos 006-0061117-1237 and 006-0061117-1239
References
[1] RDomac Flora of CroatiaManual ForDetermination of PlantsSkolska knjiga Zagreb Croatia 2002
[2] C SilicAtlas of Trees and Shrubs Svjetlost Sarajevo Bosnia 3rdedition 1988
[3] S Afkir T B Nguelefack M Aziz et al ldquoArbutus unedoprevents cardiovascular and morphological alterations in L-NAME-induced hypertensive rats Part I cardiovascular and
renal hemodynamic effects of Arbutus unedo in L-NAME-induced hypertensive ratsrdquo Journal of Ethnopharmacology vol116 no 2 pp 288ndash295 2008
[4] D Andrade C Gil L Breitenfeld F Domingues and A PDuarte ldquoBioactive extracts from Cistus ladanifer and Arbutusunedo Lrdquo Industrial Crops and Products vol 30 no 1 pp 165ndash167 2009
[5] M Bnouham F Z Merhfour A Legssyer H Mekhfi SMaallem and A Ziyyat ldquoAntihyperglycemic activity ofArbutusunedo Ammoides pusilla and Thymelaea hirsutardquo Pharmazievol 62 no 8 pp 630ndash632 2007
[6] M El Haouari J J Lopez H Mekhfi J A Rosado and GM Salido ldquoAntiaggregant effects of Arbutus unedo extracts inhuman plateletsrdquo Journal of Ethnopharmacology vol 113 no 2pp 325ndash331 2007
[7] S Mariotto E Esposito R Di Paola et al ldquoProtective effectof Arbutus unedo aqueous extract in carrageenan-induced lunginflammation in micerdquo Pharmacological Research vol 57 no 2pp 110ndash124 2008
[8] A Pabuccuoglu B Kivcak M Bas and T Mert ldquoAntioxidantactivity of Arbutus unedo leavesrdquo Fitoterapia vol 74 no 6 pp597ndash599 2003
[9] L Barros A M Carvalho J S Morais and I C F R FerreiraldquoStrawberry-tree blackthorn and rose fruits detailed charac-terisation in nutrients and phytochemicals with antioxidantpropertiesrdquo Food Chemistry vol 120 no 1 pp 247ndash254 2010
[10] BKivcak TMert BDemirci andKHC Baser ldquoCompositionof the essential oil of Arbutus unedordquo Chemistry of NaturalCompounds vol 37 no 5 pp 445ndash446 2001
[11] P Lebreton and C Bayet ldquoThe physiological and biochemicalvariability of the strawberry tree Arbutus unedo L (Ericaceae)rdquoActa Pharmaceutica vol 52 no 2 pp 83ndash90 2002
[12] M M Ozcan and H Haciseferogullan ldquoThe strawberry (Arbu-tus unedo L) fruits chemical composition physical propertiesand mineral contentsrdquo Journal of Food Engineering vol 78 no3 pp 1022ndash1028 2007
[13] K Pallauf J C Rivas-Gonzalo M D del Castillo M P Canoand S de Pascual-Teresa ldquoCharacterization of the antioxidantcomposition of strawberry tree (Arbutus unedo L) fruitsrdquoJournal of Food Composition and Analysis vol 21 no 4 pp 273ndash281 2008
4 Journal of Analytical Methods in Chemistry
[14] Z Males M Plazibat V B Vundac and I Zuntar ldquoQualitativeand quantitative analysis of flavonoids of the strawberry treemdashArbutus unedo L (Ericaceae)rdquo Acta Pharmaceutica vol 56 no2 pp 245ndash250 2006
[15] I Oliveira V Coelho R Baltasar J A Pereira and P BaptistaldquoScavenging capacity of strawberry tree (Arbutus unedo L)leaves on free radicalsrdquo Food and Chemical Toxicology vol 47no 7 pp 1507ndash1511 2009
[16] X-L I Ye W-W Huang Z Chen et al ldquoSynergetic effect andstructure-activity relationship of 3-hydroxy-3-methylglutarylcoenzyme a reductase inhibitors from crataegus pinnatifidabgerdquo Journal of Agricultural and Food Chemistry vol 58 no 5pp 3132ndash3138 2010
[14] Z Males M Plazibat V B Vundac and I Zuntar ldquoQualitativeand quantitative analysis of flavonoids of the strawberry treemdashArbutus unedo L (Ericaceae)rdquo Acta Pharmaceutica vol 56 no2 pp 245ndash250 2006
[15] I Oliveira V Coelho R Baltasar J A Pereira and P BaptistaldquoScavenging capacity of strawberry tree (Arbutus unedo L)leaves on free radicalsrdquo Food and Chemical Toxicology vol 47no 7 pp 1507ndash1511 2009
[16] X-L I Ye W-W Huang Z Chen et al ldquoSynergetic effect andstructure-activity relationship of 3-hydroxy-3-methylglutarylcoenzyme a reductase inhibitors from crataegus pinnatifidabgerdquo Journal of Agricultural and Food Chemistry vol 58 no 5pp 3132ndash3138 2010
