Research Article The Content of Secondary Metabolites and...

Research ArticleThe Content of Secondary Metabolites and Antioxidant Activityof Wild Strawberry Fruit (Fragaria vesca L)

Magdalena Dyduch-SiemiNska1 Agnieszka Najda2 Jan Dyduch2

Magdalena Gantner3 and Kamila Klimek4

1Department of Genetics and Horticultural Plant Breeding University of Life Sciences in Lublin Akademicka Street 1520-068 Lublin Poland2Department of Vegetable and Medicinal Plants University of Life Sciences in Lublin K Leszczynskiego Street 5820-068 Lublin Poland3Division of Engineering in Nutrition Warsaw University of Life Sciences Nowoursynowska Street 159 02-776 Warsaw Poland4Department of Applied Mathematics and Informatics University of Life Sciences in Lublin Głęboka Street 28 20-612 Lublin Poland

Correspondence should be addressed to Agnieszka Najda agnieszkanajdauplublinpl

Received 1 March 2015 Accepted 20 May 2015

Academic Editor Constantinos Georgiou

Copyright copy 2015 Magdalena Dyduch-Sieminska et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Chemical analyses carried out in 2011ndash2013 aimed at evaluating the contents of flavonoids free phenolic acids tanninsanthocyanins and antioxidant activity () by means of DPPH radical neutralization ability in fresh and air-dried fruits of threewild strawberry cultivars Examinations revealed differences in contents of biologically active substances determined in raw versusdried material depending on the cultivar Mean concentrations of flavonoids and tannins were highest in raw fruits of ldquoBaron vonSolemacherrdquo cv which amounted to 1244mgsdotgminus1 and 609 respectively Fresh fruits of ldquoReginardquo cv were characterized by thehighest average content of phenolic acids and anthocyanins 4987mgsdotgminus1 and 0636mgsdot100 gminus1 The pattern of mean contents ofbiologically active substances analyzed in air-dried fruits was similar Significant differences in abilities to neutralize the DPPHradical to diphenylpicrylhydrazine by extracts made of examined wild strawberry fruits were also indicated

1 Introduction

Fragaria vesca L is a well-known and valuable plant specieshowever its cultivation is still much poorly spread [1]Currently grown cultivars of Fragaria vesca are derived froma wild species which can be found in woods and grass-lands in Europe Western Asia North America and tem-perate areas in Chile [2] Fragaria vesca ssp vesca havebeen cultivated for centuries in European gardens Theirwidespread temperate growing range self-compatibility andlong history of cultivation coupled with selection for favor-able recessive traits such as day neutrality nonrunneringand yellow-fruited forms offer extensive genotypic diversity[3] Despite high price wild strawberries fruits are a productthat is highly appreciated by consumers [1] Their natural

aspect color nutritional values and high natural antioxidantcompounds content are their most attractive characteristicsA high percentage of these fruits are sold as a frozen productwhich is used in the manufacturing of cakes ice creamsor milk desserts [1 4] There are an increasing demand forfresh berries and consequently a need to increase theirdistribution ratio and shelf life [1] Fragaria spp is unusual inwhat is called the fruit actually originates from the expansionof the flower base (the receptacle) as a pseudocarp with thereal one seeded fruits (achenes) on the epidermal layer [5ndash7]Wild strawberry fruits ripen during 3-4 weeks after flowersdevelop although the period greatly depends on the weatherconditions Many epidemiological studies have shown thata higher consumption of fruit and vegetables is associatedwith the prevention of chronic diseases such as diabetes heart

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2015 Article ID 831238 8 pageshttpdxdoiorg1011552015831238

2 Journal of Analytical Methods in Chemistry

disease and certain cancers Apart from essential nutrientsfruit and vegetables also contain a variety of different phy-tochemicals that can act as antioxidants prevent oxidationand also exhibit other bioactive physiological propertiesDifferent antioxidants such as flavonoids phenolic acidscarotenoids and ascorbic acid have been proposed to actanticarcinogenically [8ndash13] Red spherical sweet taste fruits(Fragariae fructus) with unforgettable flavor are one of theraw materials achieved from wild strawberry [13] Accordingtomuchof the research donewild strawberry fruits have highantioxidant activity which has been linked to their content ofphenolic compounds [4 14]

Small fruit breeding programs are currently used toacquire new cultivars improved for specific agronomic (yieldand size) qualitative (firmness sugars content and acidity)and sensorial (colour and aroma) characteristics all com-bined to increased disease resistance and plant adaptabilityNowadays besides all these parameters it is necessary tolook for the specific bioactive components well known fortheir effect on human health This aspect is now highlyrequested by the consumer [15] Flavonoids and phenolicacids are the most common phenolic compounds in smallfruits with strong antioxidant capacity [13 16ndash18] Takingthis into account the following study was carried out tomake chemical analyses aim at evaluating the contentsof flavonoids free phenolic acids tannins anthocyaninsand antioxidant activity () by means of DPPH radicalneutralization ability in fresh and air-dried fruits of threewild strawberry cultivars The aim of these studies was toidentify cultivars which were characterized by the highestcontent of investigated biologically active substances andthe highest ability to free radicals elimination Fruits ofthese cultivars will be characterized by the highest prohealthproperties The results from the performed experiments willalso have practical application during the breeding programpreparation in order to obtain new cultivars of this species

2 Experiment

The study material consisted of fruits collected from threecultivated wild strawberry cultivars ldquoBaron von SolemacherrdquoldquoYellow Wonderrdquo and ldquoReginardquo originating from agrotech-nical experiments carried out at Department of Vegetableand Medicinal Plants University of Life Sciences Lublin(Poland 51∘231015840 N 22∘561015840 E) Seeds of tested cultivar weresown manually on March 5th 2010 into boxes filled with asubstrate (peat substrate) and covered with thin sand layerAfter emergence and forming 2-3 true leaves the seedlingswere transferred into boxes at 5 times 35 cm spacing Plants wereset into their permanent place on June 20th 2010 in plots of75m2 area (20 times 375m) at 40 times 25 cm spacing (14 plants perrow ie 93 plantssdotmminus2) in three replicatesThe agrotechnicalexperiment was carried out on dusty soil characterized bygood abundance in nutrients and neutral reaction Soilunder wild strawberry cultivation was prepared accordingto commonly accepted recommendations applying manure(40 kgsdotha) for the forecrop (onion) Phosphorus potassiumandmagnesium fertilizers were used before seedling planting

Table 1 Mean monthly air temperatures amount and total hoursof precipitation at ES Felin in the years 2011ndash2013

Month 2011 2012 2013 Mean for 1951ndash2000Temperature ∘C

IV 88 87 93 75V 149 150 128 130VI 181 181 177 165VII 191 192 183 179

Amount of precipitation mmIV 174 174 558 406V 805 815 1016 583VI 878 878 259 658VII 870 870 771 780

Total hours in sunshine hrsIV 2388 1396 2915 1566V 2686 1830 2746 2809VI 2722 3166 2003 2287VII 2367 2423 2796 1580

at the following amounts 80 kgsdothamdashP2

O5

100 kgsdothamdashK2

OStarter rate (Nmdash30 kgsdotha) was applied when the seedling wastaken the rootsThe wild strawberry plantation was regularlymanually deweeded First harvest was in second growingyears The fruit harvest was carried out at the full of fruitingstage in years 2011ndash2013 Fruits were harvested once in theearly morning in June every year

Weather conditions during growth and studies uponFragaria vesca are presented in Table 1

Directly after the harvest part of material was subjectto laboratory analyses as raw while another part was driedout The drying process was performed in a drying facilityat 40∘C till the moment of a constant air-dried fruit weightachievement Raw material was subject to determinations ofdry matter () by means of drier method [19] weight lossafter drying-moisture () [20] content flavonoids (mgsdotgminus1)[20] sum of phenolic acids (mgsdotgminus1) [21] tannins () [20]anthocyanins (mgsdot100 gminus1) [22] and antioxidant capacity ()as an ability to neutralize the DPPH radicals [23] Biochem-ical analysis was performed in Laboratory for Vegetable andHerbal Material Quality at the Department of Vegetable andMedicinal Plants University of Life Sciences Lublin

21 Dry Matter Dry matter determination was carried outby means of drier method according to Charłampowicz [19]Aliquots of about 1 g (00001 g accuracy) of raw and groundfruits were weighed Samples were placed in a drier anddried at 105∘C for 6 hours The drying process was repeatedtill the constant weight of samples (difference between twosubsequent weighings should not be greater than 05mg)Thedifference of weights before and after drying was the waterloss and then the result was recalculated onto the percentageof dry matter Determinations were made in three replicates

22Weight Loss after Drying-Moisture Content The loss afterdrying was determined by means of gravimetric method

Journal of Analytical Methods in Chemistry 3

according to Polish Pharmacopoeia VII [20] Samples of1 g of three cultivars of ground wild strawberry fruits wereweighed in vessels Samples were then placed in a drier at105∘C and dried for 2 hours after that they were cooled toambient temperature in desiccator over silica gel andweighedagain The drying was repeated until the constant weight(difference between two subsequent weighings should notbe greater than 05mg) The difference of weights beforeand after drying was the water loss (moisture content) alldeterminations were made in 3 replicates

23 Total Flavonoids Estimation Total flavonoids were esti-mated according to the spectrophotometricmethod of Christand Muller [20] after their extraction as recommended bythe European Pharmacopoeia For this purpose 20 g ofcrushed fruit was added to a round-bottomed flask 20mL ofacetone 2mL of HCl (281 gsdotLminus1) and 1mL of methenamine(5 gsdotLminus1) were then added and the mixture was maintainedfor 30min under reflux on a water bath The hydrolysate wasfiltered through cottonwool into a volumetric flask of 100mLthen placed in a flask together with the cotton pellet and20mL of acetone and refluxed for 10min Next 20mL ofsolution was dispensed into a separatory funnel with 20mLof water and extracted with ethyl acetate in 15mL portions 3times with 10mL The combined organic layers were washedtwice with 40mL of water filtered into a volumetric flask of50mL and supplemented with ethyl acetate To determineflavonoid content 2 samples were prepared to 10mL ofthe stock solution 2mL of a solution of aluminum chloride(20 gsdotLminus1) was added supplemented with a mixture (1 19) ofacetic acid (102 kgsdotLminus1) and methanol (25mL) To preparethe comparative solution stockwas supplementedwith 10mLof a mixture (1 19) of acetic acid (102 kgsdotLminus1) and methanol(25mL) After 45min the absorbance of the solutions wasread at 120582 = 425 nm onHITACHI U-2900 spectrophotometerusing the reference solution for comparison Samples wereanalyzed in 3 replicates The total content of flavonoids(mgsdotgminus1) was expressed as quercetin QE equivalent accordingto the following formula

119883 =

119896 times 119860

119898

(1)

where 119883 are total flavonoids (mgsdotgminus1) 119860 is the absorbanceof the solution being studied 119896 is the convection factor forquercetin and equal to 8750 119898 is the sample with the rawmaterial (g) which was the amount of fresh and dry material

24 Total Phenolic Acids Estimation Total phenolic acidsestimation was carried out according to Arnov method [21]which corresponds to the recommendations of the EuropeanPharmacopoeia To 50 g of homogenized rawmaterial placedin a round-bottomed flask 20mL of methanol was addedand the mixture was heated for 30min at 70∘C in a waterbath at reflux The hydrolysate was filtered through a hardfilter paper into an Erlenmeyer flask of 100mL The filteredmedium was returned to the round-bottomed flask with20mL of methanol and heated at reflux for 30min Thisprocess was repeated 3 times The combined filtrates were

taken to the tube with 1mL of blueberry extract 1mL of 05Nhydrochloric acid 1mL of Arnov reagent and 1mL of 1Nsodium hydroxide made up to 10mL with distilled waterThe absorbance was measured at 120582 = 490 nm The total phe-nolic acid content expressed as acid equivalent weight ofcaffeic acid (CAE) in the fruit was calculated from theequation obtained from the calibration curve of caffeic acid(119910 = 17321119909 + 00227 1198772 = 09992) Samples were analyzedin triplicate

25 Tannin Estimation Theamount of tannin estimationwasdetermined using Pharmacopoeia procedure [20] The con-tent of tannins was expressed as fresh and dry weight per-centage

26 Anthocyanins Estimation by means of Colorimetry Sam-ples of raw material (10 g) were extracted with 50mL HCl(1molsdotdm3) and heated in water bath for 1 hourThe obtainedextract was hydrolyzed with 20mL n-butanol and then twoportions of 10mL n-butanol were added as a solution Antho-cyanin extract was rinsed in 50mL flask with n-butanolThe absorbance was measured immediately at 533 nm [22]The percentage of anthocyanins as delphinidin chloride wascalculated from the expression

119875 =

119860 times 119881 times 119865

119898

(2)

where 119875 are total anthocyanins (mgsdot100 gminus1)119860 is absorbanceat 533 nm119881 is value of butanol phase (50mL)119865 is coefficientfor delphinidin chloride (26) 119898 is mass of sample to beexamined (mg)

27 Antioxidant Activity Antioxidant activity () was evalu-ated on a base of the ability to neutralize the DPPH radicalsby means of spectrophotometry according to Chen and Ho[23] to do this water extracts were prepared from fruitsextracts were then evaporated till dried and lyophilizedAnalyses were performed for 20 120583gsdotmLminus1 concentration Theabsorbance measurements were made at 120582 = 517 nm wave-length using spectrophotometer HITACHI U-2900

28 Chemicals All reagents and solvents were of analyticalgrade chemicals fromMerck (DarmstadtGermany) or SigmaChemical Co (St Louis MO USA) and POCH (GliwicePoland)

29 Statistical Analysis Achieved results from laboratoryexperiments were statistically processed bymeans of varianceanalysis method and Tukeyrsquos confidence intervals at 5 con-fidence level

3 Results

31 Principal Physicochemical Parameters of Studied MaterialDeterminations related to the chemical composition of threewild strawberry cultivars fruits here presented were precededwith the evaluation of general physicochemical parameters


Table 2 Dry matter () content in raw and dried fruits as well as moisture content in dried fruits in successive years of the study

CultivarsRaw material

Fresh material Dry material2011 2012 2013 Mean 2011 2012 2013 Mean

ldquoBaron von Solemacherrdquo 3520cA 3619cA 3773cA 3637 1136aA 1133aA 1124aA 1131ldquoYellowWonderrdquo 2847aA 2795aA 2823aA 2822 1126aA 1105aA 1101aA 1111ldquoReginardquo 3112bA 3098bA 3117bA 3109 1135aA 1049aA 1081aA 1088Mean 3160 3171 3238 3189 1132 1096 1102 1110Explanatory notes different letters a b c and A B C in the same column and line indicate statistically significant differences (119875 lt 005) In each columnand for each cultivar different letters mean significant differences (119875 lt 005)

Table 3 Content of flavonoids TFL (as quercetin) mgsdotgminus1 phenolic acids TPC (as caffeic acid) mgsdotgminus1 tannins () and anthocyanins ACN(as delphinidin mgsdot100 gminus1) in raw material


Fresh material Dry materialTFL TPC TAN ACN TFL TPC TAN ACN

ldquoBaron von Solemacherrdquo 0593c 2454b 340c 11400a 1245b 4858b 609b 30000bldquoYellowWonderrdquo 0471a 1648a 219a 9000a 1178a 4483a 483a 21461aldquoReginardquo 0524b 2840b 270b 16050b 1210ab 4987b 500a 44425cMean 0530 2314 276 12150 1211 4776 531 31962Explanatory notes see Table 2

that is dry matter content in raw fruits and weight loss afterdrying-moisture content in dried fruits Data presented inTable 2 indicate that dry matter content in raw fruits rangedfrom 2795 to 3773 Among compared cultivars ldquoBaronvon Solemacherrdquo fruits were characterized by the highestconcentration of the component (3637 on average) whileldquoYellowWonderrdquo contained the lowest level of the parameter(2822) Significant differences between cultivars in subse-quent years of study were observed Considering the watercontent in dried material fruits of studied wild strawberrycultivars slightly differed from each other and statisticalanalysis did not reveal any significant differences Regardlessof the cultivar moisture content of air-dried fruits oscillatedaround 1110 on average (Table 2)

Among compared cultivars ldquoBaron von Solemacherrdquofruits were characterized by the highest concentration of thecomponent (3637 on average) while ldquoYellow Wonderrdquocontained the lowest level of the parameter (2822) Sig-nificant differences between cultivars in subsequent years ofstudy were observed Considering the water content in driedmaterial fruits of studied wild strawberry cultivars slightlydiffered from each other and statistical analysis did notreveal any significant differences Regardless of the cultivarmoisture content of air-dried fruits oscillated around 1110on average (Table 2)

32 Flavonoids Contents Different levels of flavonoids inraw and dried fruits of three wild strawberry cultivars arepresented in Figure 1 and Table 3The highest concentrationsof flavonoids was found in fruits of Baron von Solemachercv 0593mgsdotgminus1 (rawmaterial) and 1245mgsdotgminus1 (driedmate-rial) On the other hand the lowest quantities of analyzedcompounds were recorded in fruits of Yellow Wonder cv

0 02 04 06 08 1 12 14

2011

2012

2013

2011

2012

2013

Fres

h m

ater

ial

Dry

mat

eria

l

(mgmiddotgminus1)

Baron von SolemacherYellow WonderRegina

Figure 1 Content of flavonoids (as quercetin) in raw material

(0471 and 1178mgsdotgminus1 resp for raw and dried material)Significant differences in flavonoids contents over the yearsof study for both analyzed types of material were observedThe dried fruits of all studied genotypes were characterizedby over twice as high flavonoids amounts as compared to rawfruits

33 Total Phenolic Acids Contents Analysis of phenolic acidsconcentration revealed that raw and dried fruits of Reginacv were the best sources of these compounds 2840mgsdotgminus1 inraw and 4987mgsdotgminus1 in driedmaterial (Table 3 and Figure 2)

Slightly less phenolic acids were found in fruits of Baronvon Solemacher cv 2454mgsdotgminus1 and 4858mgsdotgminus1 in raw anddried fruits respectively Fresh fruits of Yellow Wonder cv


0

1

2

3

4

5

6

2011 2012 2013 2011 2012 2013Fresh material Dry material

Phen

olic

acid

s (m

gmiddotgminus

1)

Baron von SolemacherYellow Wonder Regina

Figure 2 Content of phenolic acids (as caffeic acid) mgsdotgminus1 in rawmaterial in successive years of the study

contained almost twice as low phenolic acids as Regina cvfruits However mean values recorded for dried fruits of allanalyzed genotypes slightly differed and statistical analysisconfirmed that the differences were significant

34 Total Tannins Contents Changes in tannins contents forall studied wild strawberry fruits were similar as those forflavonoids Raw fruits contained 276of tannins on averagewhile dried ones contained 531 (Table 3 and Figure 3) Sig-nificant influence of all examined factors on tannins contentsin studied materials was found on a base of determinationsperformed

35 Total Anthocyanins Contents Anthocyanins were an-other group of substances analyzed in wild strawberryfruits (Table 3 and Figure 4) Fruits of Regina cv were thebest source of anthocyanins as similar as phenolic acidsFrom 14412mgsdot100 gminus1 to 17707mgsdot100 gminus1 were recordedin raw fruits of this cultivar whereas dried fruits con-tained from 39854mgsdot100 gminus1 to 46040mgsdot100 gminus1 Amongcompared cultivars the Yellow Wonder cv appeared to bethe worst since it contained only from 8097mgsdot100 gminus1 to9900mgsdot100 gminus1 (raw material) and from 19252mgsdot100 gminus1to 23690mgsdot100 gminus1 (dried material) of anthocyanins Thelowest levels of analyzed substances for all studied genotypeswere recorded in 2012

36 Antioxidant Activity Significant differences in the abilityto neutralize the free DPPH (diphenylpicrylhydrazyl) radicalby extracts made of examined wild strawberry fruits wererecorded (Table 4)

Extracts prepared from dried fruits revealed definitelyhighest ability (2393) as compared to rawmaterial (1311)When comparing studied cultivars the highest neutralizingcapacity was shown by extracts made of Regina cv fruits1427 (raw material) and 2460 (dried fruits) The free

0 1 2 3 4 5 6 7

2011

2012

2013

2011

2012

2013

Fres

h m

ater

ial

Dry

mat

eria

l

Tannins ()


Figure 3 Content of tannins () in rawmaterial in successive yearsof the study

0

100

200

300

400

500


Baron von SolemacherYellow Wonder

Regina

Ant

hocy

anin

s (m

gmiddot100

gminus1)

Figure 4 Content of anthocyanins (mgsdot100 gminus1) in raw material

radicals were worse reduced by extracts prepared from fruitsof YellowWonder cv and Baron von Solemacher cv

4 Discussion

Fragaria vesca has traditionally been a popular deliciousfruit for its flavortaste fresh use freezing and processingMorphological biometric and agronomical characteristicshave been widely used to describe wild strawberry culti-vars In recent years cultivated berries have become veryattractive for consumers because of potentially beneficialphytochemicals contained in these fruits The importance offlavonoids and other phenolics has been suggested to playa preventive role in the development of cancer and heartdisease [24] Considerable data suggests that higher contentof flavonoids phenolic acids tannins and anthocyanins inberry fruits contributes to their higher antioxidant activity[17 25] Following substances should be counted as themost important bioactive components of wild strawberry


Table 4 Antioxidant activity () expressed as the ability to neutralize the DPPH radical in water extracts made of studied materials insuccessive years of the study



ldquoBaron von Solemacherrdquo 1265a 1134a 1402b 1267 2384a 2151b 2605b 2380ldquoYellowWonderrdquo 1242a 1116a 1363a 1240 2344a 2105a 2570a 2340ldquoReginardquo 1430b 1321b 1530c 1427 2466b 2215c 2698c 2460Mean 1312 1190 1431 1311 2398 2157 2624 2393Explanatory notes see Table 2

phenolic acids (ellagic p-coumaric gallic acids) flavonoids(flavonols quercetin and kaempferol) proanthocyanidinsand anthocyanins (pelargonidin cyanidin) [26ndash28] Due tothese compounds the fruits have anticarcinogenic antioxi-dant anticoagulant immunomodulating anti-inflammatoryblood pressure and cholesterol regulating features [29 30]Measuring such parameters is wildly used to evaluate thepotential health benefits of breeding material or variousagronomic factors [9] However little is known on thephenolic profiles and antioxidant potential of wild straw-berry in important local cultivars In this paper we usethe contents of secondary metabolites for cultivar identifi-cation The differences in the concentrations of secondarycompounds determine the nutritional importance of theanalyzed cultivars Furthermore they provide informationon the marketing potential of the 3 cultivars and present animportant chemical insight into the popular cultivars grownin this region

Secondary metabolites content in berry fruits variesamong species and cultivars but it can also be affectedby growth conditions including environmental factors andcultivation techniques [10 11 31ndash34] It has been shownthat higher growing temperatures (day and night) increasethe flavonols and anthocyanins contents in strawberries[26] Authors of the present study on a base of performedresearch proved differentiated contents of biologically activesubstances flavonoids phenolic acids tannins and antho-cyanins in raw and dried fruits depending onwild strawberrycultivar Furthermore they analyzed the antioxidant ability ofextracts made of studied materials by means of neutralizingthe free DPPH radical Mean content of flavonoids andtannins in fresh fruits of ldquoBaron von Solemacherrdquo cv wasthe highest and amounted to 1245mgsdotgminus1 and 609 respec-tively Raw fruits of ldquoReginardquo cv were characterized by thehighest average concentrations of phenolic acids and antho-cyanins 4987mgsdotgminus1 and 44425mgsdot100 gminus1 respectivelyAccording to Antal et al [35] the quantity of anthocyaninsin raw berries ranges from 30mgsdot100 gminus1 (Fragaria moschata)to 165mgsdot100 gminus1 (Vaccinium myrtillus) Huang et al [14]found higher contents of anthocyanins in berry fruits whichcorrespond with values achieved in the present study for rawfruits of examined wild strawberry genotypes In opinion ofOlsson et al [11] the amount of phenolic substances as well asantioxidant activity was different within Fragaria x ananassaDuch genus Antal et al [35] evaluated fresh fruits and found

that strawberries had total phenolics of 3680mgsdotkg freshweight As it follows from performed analyses mean contentof phenolic acids for fresh wild strawberry fruits was at thelevel of 2314mgsdotgminus1 The differences in polyphenol contentof strawberry fruit from the literature may be due to thedifferent conditions during the growth of plants (climaticconditions temperature precipitation and soil conditions)the length of the growing season and harvest date Theymay also be caused by using various analytical proceduresor methods identifying the active ingredients by scientists invarious centers Identification of chromatographic methodsusually has a lower content of active substances because itstask is to determine theminimumcontent and separation andidentification of individual compounds in the raw materialCommonly used spectrophotometric procedures are basedon similar assumptions so after taking into account differ-ences methods are comparable to those applied in this paperDuring the three years of the research the highest contents ofanalyzed biologically active substances were recorded in 2013while the lowest were recorded in 2012

In a previous investigation we found that fresh wildstrawberry fruits possess high amounts of bioactive com-pounds However fresh fruits are not available all yearround being harvested in Poland only in JunendashSeptemberTherefore it is important to find a proper substitute that couldbe used when fresh berries are not available It was decidedto prepare dried fruits to determine the contents of someimportant bioactive compounds and their antioxidant poten-tial therein and to compare them with the same parametersin fresh fruits Dried fruits have a greater nutrient densitygreater fiber content increased shelf life and significantlygreater phenol antioxidant content compared to fresh fruitsThe quality of the antioxidants in the processed dried fruitis the same as in the corresponding fresh fruit Phenols indried fruit may be important antioxidants [24] Data pre-sented in Tables 2ndash4 indicate diverse contents of biologicallyactive compounds in raw and dried materials depending onthe cultivar The highest mean concentrations of analyzedsubstances in air-dried fruits were similar as for fresh onesDried fruits of analyzed genotypes were characterized by overtwice as high quantities of biologically active substances andantioxidant activity as fresh fruits Results from the presentstudy are a confirmation of the results achieved by Vinson etal [24] related to the secondarymetabolites in fresh and driedfruits


5 Conclusion

As a conclusion our results clearly demonstrate that con-siderable variation exists in the phenolic compounds amongwild strawberry genotypes The obtained results allowed theidentification of cultivars which were characterized by thehighest content of investigated biologically active substancesand the highest ability to free radicals elimination Fruits ofthese cultivars were characterized by the highest prohealthproperties The results from the performed experimentsalso have practical application during the breeding programpreparation in order to obtain new cultivars of this speciesTherefore dried fruits are good source of important bioactivecompounds andmore dried fruits should be recommended tobe added to the diet by dieticians and nutritionists

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] E Almenar P Hernandez-Munoz J M Lagaron R Catala andR Gavara ldquoControlled atmosphere storage of wild strawberryfruit (Fragaria vesca L)rdquo Journal of Agricultural and FoodChemistry vol 54 no 1 pp 86ndash91 2006

[2] Alpine strawberry Fragaria vesca 2008 httpwwwsuperb-herbsnetalpinestrawberryhtm

[3] V Shulaev D J Sargent R N Crowhurst et al ldquoThe genomeof woodland strawberry (Fragaria vesca)rdquo Nature Genetics vol43 no 2 pp 109ndash116 2011

[4] B R Cordenunsi M I Genovese J R O Nascimento N M AHassimoto R J Santos and FM Lajolo ldquoEffects of temperatureon the chemical composition and antioxidant activity of threestrawberry cultivarsrdquo Food Chemistry vol 91 no 1 pp 113ndash1212005

[5] P Perkins-Veazie ldquoGrowth and ripening of strawberry fruitrdquoHorticultural Review vol 17 pp 267ndash297 1995

[6] A Aharoni ldquoFunctional genomics in strawberryrdquo Acta Horti-culturae vol 649 pp 29ndash33 2004

[7] A Bombarely C Merchante F Csukasi et al ldquoGeneration andanalysis of ESTs from strawberry (Fragaria xananassa) fruitsand evaluation of their utility in genetic and molecular studiesrdquoBMC Genomics vol 11 article 503 2010

[8] T Jurikova O Rop J Mlcek et al ldquoPhenolic profile of edi-ble honeysuckle berries (genus Lonicera) and their biologicaleffectsrdquoMolecules vol 17 no 1 pp 61ndash79 2012

[9] M J Anttonen and R O Karjalainen ldquoEnvironmental andgenetic variation of phenolic compounds in red raspberryrdquoJournal of Food Composition and Analysis vol 18 no 8 pp 759ndash769 2005

[10] J Scalzo A Politi N Pellegrini B Mezzetti and M BattinoldquoPlant genotype affects total antioxidant capacity and phenoliccontents in fruitrdquo Nutrition vol 21 no 2 pp 207ndash213 2005

[11] M E Olsson C S Andersson S Oredsson R H Berglund andK-E Gustavsson ldquoAntioxidant levels and inhibition of cancercell proliferation in vitro by extracts from organically and con-ventionally cultivated strawberriesrdquo Journal of Agricultural andFood Chemistry vol 54 no 4 pp 1248ndash1255 2006

[12] S Tulipani BMezzetti F Capocasa et al ldquoAntioxidants pheno-lic compounds and nutritional quality of different strawberrygenotypesrdquo Journal of Agricultural and Food Chemistry vol 56no 3 pp 696ndash704 2008

[13] J Milivojevic V Maksimovic M Nikolic J Bogdanovic RMaletic and DMilatovic ldquochemical and antioxidant propertiesof cultivated and wild fragaria and rubus berriesrdquo Journal ofFood Quality vol 34 no 1 pp 1ndash9 2011

[14] W-Y Huang H-C Zhang W-X Liu and C-Y Li ldquoSurveyof antioxidant capacity and phenolic composition of blueberryblackberry and strawberry in Nanjingrdquo Journal of ZhejiangUniversity Science B (Biomedicine amp Biotechnology) vol 13 no2 pp 94ndash102 2012

[15] F Capocasa J Diamanti B Mezzetti S Tulipani and MBattino ldquoBreeding strawberry (Fragaria X ananassa Duch) toincrease fruit nutritional qualityrdquo BioFactors vol 34 no 1 pp67ndash72 2008

[16] S Y Wang and H-S Lin ldquoAntioxidant activity in fruits andleaves of blackberry raspberry and strawberry varies withcultivar and developmental stagerdquo Journal of Agricultural andFood Chemistry vol 48 no 2 pp 140ndash146 2000

[17] J Oszmianski and A Wojdyło ldquoComparative study of phenoliccontent and antioxidant activity of strawberry puree clear andcloudy juicesrdquoEuropean FoodResearch andTechnology vol 228no 4 pp 623ndash631 2009

[18] M Ding R Feng S Y Wang et al ldquoCyanidin-3-glucoside anatural product derived from blackberry exhibits chemopre-ventive and chemotherapeutic activityrdquoThe Journal of BiologicalChemistry vol 281 no 25 pp 17359ndash17368 2006

[19] Z Charłampowicz Analyses of Fruit Vegetable and MushroomProducts WPLS Warsaw Poland 1966

[20] PT Farm Polish Pharmacopoeia VI PT Farm WarszawaPoland 2002

[21] Polish Pharmacopoeia VII PTFarm Warsaw Poland 2006[22] K Miłkowska and H Strzelecka ldquoFlos Hibiscimdashidentification

methods and evaluating the materialrdquo Herba Polonica vol 41no 1 pp 11ndash16 1995

[23] J H Chen and C-T Ho ldquoAntioxidant activities of caffeic acidand its related hydroxycinnaminic acid compoundsrdquo Journal ofAgricultural and Food Chemistry vol 45 no 7 pp 2374ndash23781997

[24] J A Vinson L Zubik P Bose N Samman and J Proch ldquoDriedfruits excellent in vitro and in vivo antioxidantsrdquo Journal of theAmerican College of Nutrition vol 24 no 1 pp 44ndash50 2005

[25] A Bunea DO Rugina AM Pintea Z Sconta C I Bunea andC Socaciu ldquoComparative polyphenolic content and antioxidantactivities of some wild and cultivated blueberries from roma-niardquoNotulae Botanicae Horti Agrobotanici Cluj-Napoca vol 39no 2 pp 70ndash76 2011

[26] S Y Wang and W Zheng ldquoEffect of plant growth temperatureon antioxidant capacity in strawberryrdquo Journal of Agriculturaland Food Chemistry vol 49 no 10 pp 4977ndash4982 2001

[27] J Milivojevic V Maksimovic M Nikolic J Bogdanovic RMaletic and D Milatovic ldquoChemical and antioxidant proper-ties of cultivated and wild Fragaria and Rubus berriesrdquo Journalof Food Quality vol 34 no 1 pp 1ndash9 2011

[28] J Oszmianski AWojdyło and PMatuszewski ldquoIn polyphenolscompounds changes in the industrial production process ofconcentrated strawberry juicerdquo Food Science Technology Qual-ity vol 1 no 50 pp 94ndash104 2007


[29] E Jabłonska-Rys M Zalewska-Korona and J KalbarczykldquoAntioxidant capacity ascorbic acid and phenolics contentinwild edible fruitsrdquo Journal of Fruit and Ornamental PlantResearch vol 17 no 2 pp 115ndash120 2009

[30] S M Hannum ldquoPotential impact of strawberries on humanhealth a review of the sciencerdquo Critical Reviews in Food Scienceand Nutrition vol 44 no 1 pp 1ndash17 2004

[31] N Deighton R Brennan C Finn and H V Davies ldquoAntiox-idant properties of domesticated and wild Rubus speciesrdquoJournal of the Science of Food and Agriculture vol 80 no 9 pp1307ndash1313 2000

[32] R Moyer K Hummer R E Wrolstad and C Finn ldquoAntioxi-dant compounds in diverse ribes and rubus germplasmrdquo ActaHorticulturae vol 585 pp 501ndash505 2002

[33] E Kruger H Dietrich E Schopplein S Rasim and P KurbelldquoCultivar storage conditions and ripening effects on physicaland chemical qualities of red raspberry fruitrdquo PostharvestBiology and Technology vol 60 no 1 pp 31ndash37 2011

[34] Y J Shin J-A Ryu R H Liu J F Nock and C B WatkinsldquoHarvest maturity storage temperature and relative humidityaffect fruit quality antioxidant contents and activity and inhibi-tion of cell proliferation of strawberry fruitrdquo Postharvest Biologyand Technology vol 49 no 2 pp 201ndash209 2008

[35] D S Antal G Garban and Z Garban ldquoThe anthocyansbiologically-active substances of food and pharmaceutic inter-estrdquo inAnnals of the University Dunarea de Jos of Galati FascicleVI Food Technology pp 106ndash115 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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disease and certain cancers Apart from essential nutrientsfruit and vegetables also contain a variety of different phy-tochemicals that can act as antioxidants prevent oxidationand also exhibit other bioactive physiological propertiesDifferent antioxidants such as flavonoids phenolic acidscarotenoids and ascorbic acid have been proposed to actanticarcinogenically [8ndash13] Red spherical sweet taste fruits(Fragariae fructus) with unforgettable flavor are one of theraw materials achieved from wild strawberry [13] Accordingtomuchof the research donewild strawberry fruits have highantioxidant activity which has been linked to their content ofphenolic compounds [4 14]

Small fruit breeding programs are currently used toacquire new cultivars improved for specific agronomic (yieldand size) qualitative (firmness sugars content and acidity)and sensorial (colour and aroma) characteristics all com-bined to increased disease resistance and plant adaptabilityNowadays besides all these parameters it is necessary tolook for the specific bioactive components well known fortheir effect on human health This aspect is now highlyrequested by the consumer [15] Flavonoids and phenolicacids are the most common phenolic compounds in smallfruits with strong antioxidant capacity [13 16ndash18] Takingthis into account the following study was carried out tomake chemical analyses aim at evaluating the contentsof flavonoids free phenolic acids tannins anthocyaninsand antioxidant activity () by means of DPPH radicalneutralization ability in fresh and air-dried fruits of threewild strawberry cultivars The aim of these studies was toidentify cultivars which were characterized by the highestcontent of investigated biologically active substances andthe highest ability to free radicals elimination Fruits ofthese cultivars will be characterized by the highest prohealthproperties The results from the performed experiments willalso have practical application during the breeding programpreparation in order to obtain new cultivars of this species

2 Experiment

The study material consisted of fruits collected from threecultivated wild strawberry cultivars ldquoBaron von SolemacherrdquoldquoYellow Wonderrdquo and ldquoReginardquo originating from agrotech-nical experiments carried out at Department of Vegetableand Medicinal Plants University of Life Sciences Lublin(Poland 51∘231015840 N 22∘561015840 E) Seeds of tested cultivar weresown manually on March 5th 2010 into boxes filled with asubstrate (peat substrate) and covered with thin sand layerAfter emergence and forming 2-3 true leaves the seedlingswere transferred into boxes at 5 times 35 cm spacing Plants wereset into their permanent place on June 20th 2010 in plots of75m2 area (20 times 375m) at 40 times 25 cm spacing (14 plants perrow ie 93 plantssdotmminus2) in three replicatesThe agrotechnicalexperiment was carried out on dusty soil characterized bygood abundance in nutrients and neutral reaction Soilunder wild strawberry cultivation was prepared accordingto commonly accepted recommendations applying manure(40 kgsdotha) for the forecrop (onion) Phosphorus potassiumandmagnesium fertilizers were used before seedling planting

Table 1 Mean monthly air temperatures amount and total hoursof precipitation at ES Felin in the years 2011ndash2013

Month 2011 2012 2013 Mean for 1951ndash2000Temperature ∘C

IV 88 87 93 75V 149 150 128 130VI 181 181 177 165VII 191 192 183 179

Amount of precipitation mmIV 174 174 558 406V 805 815 1016 583VI 878 878 259 658VII 870 870 771 780

Total hours in sunshine hrsIV 2388 1396 2915 1566V 2686 1830 2746 2809VI 2722 3166 2003 2287VII 2367 2423 2796 1580

at the following amounts 80 kgsdothamdashP2

O5

100 kgsdothamdashK2

OStarter rate (Nmdash30 kgsdotha) was applied when the seedling wastaken the rootsThe wild strawberry plantation was regularlymanually deweeded First harvest was in second growingyears The fruit harvest was carried out at the full of fruitingstage in years 2011ndash2013 Fruits were harvested once in theearly morning in June every year

Weather conditions during growth and studies uponFragaria vesca are presented in Table 1

Directly after the harvest part of material was subjectto laboratory analyses as raw while another part was driedout The drying process was performed in a drying facilityat 40∘C till the moment of a constant air-dried fruit weightachievement Raw material was subject to determinations ofdry matter () by means of drier method [19] weight lossafter drying-moisture () [20] content flavonoids (mgsdotgminus1)[20] sum of phenolic acids (mgsdotgminus1) [21] tannins () [20]anthocyanins (mgsdot100 gminus1) [22] and antioxidant capacity ()as an ability to neutralize the DPPH radicals [23] Biochem-ical analysis was performed in Laboratory for Vegetable andHerbal Material Quality at the Department of Vegetable andMedicinal Plants University of Life Sciences Lublin

21 Dry Matter Dry matter determination was carried outby means of drier method according to Charłampowicz [19]Aliquots of about 1 g (00001 g accuracy) of raw and groundfruits were weighed Samples were placed in a drier anddried at 105∘C for 6 hours The drying process was repeatedtill the constant weight of samples (difference between twosubsequent weighings should not be greater than 05mg)Thedifference of weights before and after drying was the waterloss and then the result was recalculated onto the percentageof dry matter Determinations were made in three replicates

22Weight Loss after Drying-Moisture Content The loss afterdrying was determined by means of gravimetric method




119883 =

119896 times 119860

119898

(1)






119875 =

119860 times 119881 times 119865

119898

(2)





3 Results














0 02 04 06 08 1 12 14

2011

2012

2013

2011

2012

2013

Fres

h m

ater

ial

Dry

mat

eria

l

(mgmiddotgminus1)







0

1

2

3

4

5

6


Phen

olic

acid

s (m

gmiddotgminus

1)








0 1 2 3 4 5 6 7

2011

2012

2013

2011

2012

2013

Fres

h m

ater

ial

Dry

mat

eria

l

Tannins ()



0

100

200

300

400

500



Regina

Ant

hocy

anin

s (m

gmiddot100

gminus1)



4 Discussion












5 Conclusion




References














































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




119883 =

119896 times 119860

119898

(1)






119875 =

119860 times 119881 times 119865

119898

(2)





3 Results














0 02 04 06 08 1 12 14

2011

2012

2013

2011

2012

2013

Fres

h m

ater

ial

Dry

mat

eria

l

(mgmiddotgminus1)







0

1

2

3

4

5

6


Phen

olic

acid

s (m

gmiddotgminus

1)








0 1 2 3 4 5 6 7

2011

2012

2013

2011

2012

2013

Fres

h m

ater

ial

Dry

mat

eria

l

Tannins ()



0

100

200

300

400

500



Regina

Ant

hocy

anin

s (m

gmiddot100

gminus1)



4 Discussion












5 Conclusion




References














































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of













0 02 04 06 08 1 12 14

2011

2012

2013

2011

2012

2013

Fres

h m

ater

ial

Dry

mat

eria

l

(mgmiddotgminus1)







0

1

2

3

4

5

6


Phen

olic

acid

s (m

gmiddotgminus

1)








0 1 2 3 4 5 6 7

2011

2012

2013

2011

2012

2013

Fres

h m

ater

ial

Dry

mat

eria

l

Tannins ()



0

100

200

300

400

500



Regina

Ant

hocy

anin

s (m

gmiddot100

gminus1)



4 Discussion












5 Conclusion




References














































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0

1

2

3

4

5

6


Phen

olic

acid

s (m

gmiddotgminus

1)








0 1 2 3 4 5 6 7

2011

2012

2013

2011

2012

2013

Fres

h m

ater

ial

Dry

mat

eria

l

Tannins ()



0

100

200

300

400

500



Regina

Ant

hocy

anin

s (m

gmiddot100

gminus1)



4 Discussion












5 Conclusion




References














































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of











5 Conclusion




References














































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Research Article The Content of Secondary Metabolites and...

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Transcript of Research Article The Content of Secondary Metabolites and...