ResearchArticle Castanea sativa Mill. Flowers amongst the ... 202.pdf · m/z 915, 631, 451 and 301) of peak 8 were in agreement with those attributed to castalagin or vescalagin isomers

Research ArticleCastanea sativa Mill Flowers amongstthe Most Powerful Antioxidant Matrices A PhytochemicalApproach in Decoctions and Infusions

Maacutercio Carocho12 Lillian Barros1 Albino Bento1 Celestino Santos-Buelga3

Patricia Morales2 and Isabel C F R Ferreira1

1 Mountain Research Center (CIMO) ESA Polytechnic Institute of Braganca Campus de Santa Apolonia 11725301-855 Braganca Portugal

2 Department of Nutrition and Bromatology II Faculty of Pharmacy Complutense University of Madrid Pza Ramon y Cajalsn 28040 Madrid Spain

3 Grupo de Investigacion en Polifenoles (GIP) Faculty of Pharmacy University of Salamanca Campus Miguel de Unamuno37007 Salamanca Spain

Correspondence should be addressed to Isabel C F R Ferreira iferreiraipbpt

Received 20 February 2014 Accepted 28 March 2014 Published 14 April 2014

Academic Editor P J Oefner

Copyright copy 2014 Marcio Carocho et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Infusions and decoction of chestnut tree flowers have been used for different medical purposes but their phytochemical profileand antioxidant activity are still mostly unknown Herein decoctions and infusions of flowers from the two most appreciatedchestnut cultivars (longal and judia) in Tras-os-Montes Portugal were prepared and characterized with regard to their content infree sugars organic acids and phenolic compounds such as flavonoids and hydrolyzable tannins and their antioxidant activityOverall the decoction of the cultivar judiawas the sample with both the highest quantity of flavonoids and antioxidant activityThephenolic compound with the highest abundance in all samples was trigalloyl-HHDP-glucoside followed by pentagalloyl glucosideThe sample with the highest quantity of total phenolic compounds was judia infusion closely followed by longal decoction whichalso gave the highest quantities of ellagitannins Regarding sugars and organic acids the profiles were more similar These resultscorroborate ancestral claims of the health benefits of infusions and decoctions of chestnut flowers

1 Introduction

In the Tras-os-Montes region of Portugal and across a goodpart of the Mediterranean countries chestnut trees are aconsiderable part of the landscape These trees and theirrespective nuts have been important in the past and are stilla source of income for those regions In Portugal chestnutsare almost totally exported translating into a revenue of 32million euros in 2012 [1 2] The chestnut tree has a variety ofapplications the nuts are used for human and animal feedbeing widely appreciated and even transformed into manytypical dishes and desserts The wood is used for high classfurniture The leaves are used in many ethnobotanic formu-lations against colds coughs diarrhea and even high bloodcholesterol [3] Furthermore some patents indicate the use of

chestnut flowers in beverages like teas and refreshments [4ndash6]

The nutritional and bioactive properties of the fruits andflowers of chestnuts have been reported [7ndash9] Barros et al[8 9] described the high antioxidant potential and phenoliccompounds profile of methanolic extracts obtained from theflowers In fact some of the most antioxidant molecules arenot always found in fruits but in richer polyphenolic matri-ces like flowers Other antioxidant molecules include someorganic acids that among many other beneficial activities areknown to act against free radicals [8 10]

The characterization of antioxidant molecules present inflowers is fundamental to draw conclusions concerning theirantioxidant potential which could be interesting for the foodindustry by adding antioxidant extracts to foodstuffs or using

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 232956 7 pageshttpdxdoiorg1011552014232956

2 BioMed Research International

them in coatings in order to extend their shelf-life and reduceconsumption of chemical additives [11]

In this report the decoctions and infusions of flowersfrom the two most appreciated chestnut cultivars (longal andjudia) from Tras-os-Montes Portugal were characterizedregarding their content of hydrophilic antioxidant molecules(free sugars organic acids and phenolic compounds) reduc-ing power free radicals scavenging activity and lipid peroxi-dation inhibition

2 Materials and Methods

21 Standards and Reagents Acetonitrile 999 of high per-formance liquid chromatography (HPLC) grade andsulphuric acid were acquired from Fisher Scientific(Lisbon Portugal) Formic acid was acquired from Panreac(Barcelona Spain) Sugar standards [D(minus)-fructose D(+)-glucose anhydrous andD(+)-sucrose] organic acid standards(malic acid shikinic acid oxalic acid and quinic acid) andtrolox (6-hydroxy-2578-tetramethylchroman-2-carboxylicacid) were acquired from Sigma Chemical Co (Saint LouisMO USA) Phenolic compound standards (catechin gallicacid isorhamnetin 3-O-glucoside kaempferol 3-O-glucosidekaempferol 3-O-rutinoside myricetin quercetin 3-O-gluco-side and quercetin 3-O-rutinoside) were purchased fromExtrasynthese (Genay France) 22-Diphenyl-1-picrylh-ydrazyl (DPPH) was obtained from Alfa Aesar (Ward HillMA USA) Water was treated by means of a Milli-Q waterpurification system (TGI Pure Water Systems GreenvilleSC USA)

22 Flower Samples Castanea sativaMill flowers of the cul-tivars judia and longal were collected in June 2013 in OleirosBraganca (north-eastern Portugal) (41∘5110158400210158401015840N 6∘4910158405410158401015840W)The specimens were lyophilized (FreeZone 45 LabconcoKansas USA) milled down to a fine powder and finallystored at minus5∘C until analysis

23 Preparation of the Decoctions and Infusions For the infu-sions preparation the lyophilized flowers (1 g) were added to200mL of boiling distilled water left to stand for 5min andfinally filtered through a Whatman filter paper The obtainedinfusions were frozen and lyophilized

For the decoctions preparation the lyophilized flowers(1 g) were added to 200mL of boiling distilled water boiledfor 5min and then left to stand at room temperature for5 more minutes After filtration through a Whatman filterpaper the obtained decoctions were frozen and lyophilized

24 Analysis of Free Sugars Free sugars were determined byHPLCcoupled to a refraction index (RI) detector as describedpreviously [8] The equipment consisted of a pump (KnauerSmartline System 1000 Berlin Germany) a degasser (Smart-line Manager 5000) an autosampler (AS-2057 Jasco EastonMD USA) and a RI detector (Knauer Smartline 2300) Theidentification was achieved by comparing the relative reten-tion times of sample peakswith standardsQuantificationwasmade by the internal standard method and the results areexpressed in mg per g of lyophilized decoction or infusion

25 Analysis of Organic Acids Organic acids were deter-mined following a procedure previously optimized anddescribed by the authors [10] Analysis was performedon a Shimadzu 20A series ultra-fast liquid chromatograph(UFLC Shimadzu Cooperation Kyoto Japan) coupled tophotodiode array detector (PDA Shimadzu) using 215 nmand 245 nm as the preferred wavelengths Separation wasachieved on a SphereClone (Phenomenex Torrance CAUSA) reverse phase C18 column (5 120583m 250mm times 46mmid) thermostatted at 35∘C Analytes were eluted with 36mMsulphuric acid at a flow-rate of 08mLminThe organic acidsfound were quantified by comparison of the area of theirpeaks recorded at 215 nm or 245 nm (for ascorbic acid) withcalibration curves obtained from commercial standards ofeach compound oxalic acid (119910 = 1119909107119909 + 96178 1198772 =0999) quinic acid (119910 = 601768119909 + 88532 1198772 = 1) malicacid (119910 = 952269119909 + 17803 1198772 = 1) shikimic acid (119910 =8119909107+55079 1198772 = 0999)The results were expressed inmgper g of lyophilized decoction or infusion

26 Analysis of Phenolic Compounds Phenolic compoundswere determined by HPLC (Hewlett-Packard 1100 AgilentTechnologies Santa Clara CA USA) as previously described[9] Double online detection was carried out with a PDAusing 280 nm and 370 nm as the preferred wavelengths anda mass spectrometer (MS) connected to the HPLC systemvia the PDA cell outlet Mass spectrometric detection wasperformed by means of an API 3200 (Applied BiosystemsDarmstadt Germany) triple quadrupole-ion trap analyserequipped with an ESI source Spectra were recorded innegative ion mode between mz 100 and 1700 The phenoliccompounds were characterized according to their UV massspectra retention times and comparison with authenticstandards when available For the quantitative analysis ofphenolic compounds a baseline to valley integration withbaseline projection mode was used to calculate peak areasFor quantification calibration curves were generated byinjection of known concentration (25ndash100 120583gmL) of stan-dard compounds catechin (119910 = 13276119909 minus 59658 1198772 = 1)gallic acid (119910 = 55694119909minus73837 1198772 = 0999) isorhamnetin-3-O-glucoside (119910 = 26231119909minus98958 1198772 = 1) kaempferol-3-O-glucoside (119910 = 19075119909 minus 36158 1198772 = 1) kaempferol-3-O-rutinoside (119910 = 17502119909 minus 43877 1198772 = 0999) myricetin(119910 = 778119909 minus 14543 1198772 = 0999) quercetin-3-O-glucoside(119910 = 31648119909 minus 29142 1198772 = 1000) and quercetin-3-O-rutinoside (119910 = 22279119909 minus 24311 1198772 = 0999) Theresults were expressed in mg per g of lyophilized decoctionor infusion

27 Evaluation of Antioxidant Activity The in vitro antioxi-dant activity assays were performed following the previouslydescribed methodology of Barros et al [8] The lyophilizedinfusions and decoctions were dissolved in water (finalconcentration 10mgmL) the final solution was furtherdiluted to different concentrations to be used in the followingassays DPPH radical-scavenging activity was evaluated usingan ELX800 microplate Reader (Bio-Tek Instruments IncWinooski VT USA) and calculated as a percentage of DPPH

BioMed Research International 3

0 5 10 15 20 25 30

(mAU

)

020406080

100120140160

Time (min)

11

12

10

13 14

15

1617

1918 20212223

25 26 27

(a)

Time (min)0 5 10 15 20 25

(mAU

)

0250500750

10001250150017502000

1

23 4

5

6

7 8

10

913 20

24

(b)

Figure 1 Phenolic profile of the infusion of Castanea sativa flowers (judia cultivar) recorded at 370 nm (a) and 280 nm (b)

discolouration after 1 hour of incubation with the antioxidantextract using the formula [(119860DPPH minus 119860119878)119860DPPH] times 100where 119860

119878is the absorbance of the solution containing the

sample at 515 nm and 119860DPPH is the absorbance of the DPPHsolution Reducing power was evaluated by the capacity toreduce Fe3+ into Fe2+ measuring the absorbance at 690 nmin the microplate reader mentioned Inhibition of 120573-carotenebleaching was evaluated through the 120573-carotenelinoleateassay the neutralization of linoleate free radicals avoids 120573-carotene bleaching which is measured by the formula (120573-carotene absorbance after 2 h of assayinitial absorbance) times100 Lipid peroxidation inhibition in porcine (Sus scrofa)brain homogenates was evaluated by the decrease in thiobar-bituric acid reactive substances (TBARS) the colour intensityof the malondialdehyde-thiobarbituric acid (MDA-TBA) wasmeasured by its absorbance at 532 nm the inhibition ratio() was calculated using the following formula [(119860minus119861)119860]times100 where 119860 and 119861 were the absorbance of the controland the sample solution respectively The results of theantioxidant activity were expressed in EC

50value (sample

concentration providing 50 of antioxidant activity or 05 ofabsorbance in the reducing power assay)

28 Statistical Analysis All the assays were carried out intriplicate for both decoctions and infusions of each culti-var The results are expressed as mean values plusmn standarddeviations (SD) The statistical differences represented byletters were obtained through one-way analysis of variance(ANOVA) followed by Tukeyrsquos honestly significant differencepost hoc test with 120572 = 005 Statistical analyses were carriedout using the SPSS v 180 program

3 Results and Discussion

31 Analysis of Free Sugars and Organic Acids Three freesugars namely fructose glucose and sucrose were detectedin all the samples (Table 1) Sucrose was the least abundantsugar while fructose and glucose were the most abundant inthe cultivars judia and longal respectivelyThis had been alsoreported for methanolic extracts of C sativa flowers [9] Glu-cose and fructose were present at higher levels in decoctionsthan infusions probably due to the longer extraction timealbeit significant differenceswere only found in the case of thecultivar longal By contrast higher concentrations of sucrosewere extracted in the infusions although the increase was

only statistically significant in the case of judiaNo significantdifferences were observed among samples

Oxalic quinic malic and shikimic acids were quantifiedin all the samples with quinic acid being the most abundantacid (Table 1) Decoctions generally yielded higher quantitiesof oxalic acid in comparison to infusions and statisticaldifferences for this compound were only noted for the infu-sion of longal The longal cultivar did not display statisticaldifferences for quinic acid in both extraction methods butfor judia statistical differences were found between extractionmethods Malic acid and shikimic acid displayed similar con-centrations in all the samples whereas some differences werefound among samples regarding quinic acid The decoctionof judia displayed the highest concentrations of total organicacids of the samples analysed

32 Analysis of Phenolic Compounds An exemplary phenolicprofile of the judia cultivar of C sativa recorded at 280 nmand 370 nm is shown in Figure 1 Twenty-seven phenoliccompounds were identified in both judia and longal cultivars(infusion and decoction preparations) Peak characteristicsand tentative identities are presented in Table 2

Peaks 1ndash4 6ndash10 13 20 24 and 25 showed UV spectracoherent with galloyl and hexahydroxydiphenoyl (HHDP)derivatives [12ndash14] According to the literature the maincharacteristic in the mass spectra of these compounds isthe deprotonated molecule [M-H]minus and the loss of oneor more ellagic acid (302mu) gallic acid (170mu) andorgalloyl groups (152mu) [12 15 16] Peaks 1 and 2 presented asingly charged pseudomolecular ion [M-H]minus at mz 783 anddaughter ions at mz 481 and 301 which together with theirearly elution allowed their identification as pedunculagin(ie bis-HHDP-glucose) isomers [17] Mass characteristicsof peak 3 ([M-H]minus at mz 633 fragment ions at mz 463and 301) coincided with a galloyl-HHDP-glucose isomer[16 17] whereas peaks 4 10 and 13 ([M-H]minus at mz 937fragment ions at mz 767 637 467 and 301) were coherentwith trigalloyl-HHDP-glucose isomers [13] already reportedin C sativa heartwood [18] and flowers [9] Peak 6 presenteda pseudomolecular ion [M-H]minus at mz 939 yielding MS2fragment ions atmz 631 [M-2galloyl-3H]minus 469 [M-2galloyl-3H-glu]minus being identified as pentagalloylglucose Peak 7witha pseudomolecular ion [M-H]minus at mz 935 and MS2 productions at mz 633 and 301 likely due to the loss of HHDPand galloyl-glucose moieties was consistent with a galloyl-


Table 1 Content of free sugars and organic acids in mgg of lyophilized decoctioninfusion of the flowers of two Castanea sativa cultivars

Decoction judia Decoction longal Infusion judia Infusion longalSugars

Fructose 16041 plusmn 001a 15208 plusmn 652a 14894 plusmn 460a 12358 plusmn 176b

Glucose 14909 plusmn 004b 19191 plusmn 735a 14571 plusmn 563b 16407 plusmn 231b

Sucrose 2701 plusmn 045b 2569 plusmn 099b 3568 plusmn 145a 2667 plusmn 050b

Total 33651 plusmn 048a 36969 plusmn 1485a 33032 plusmn 1169a 31432 plusmn 457a

Organic AcidsOxalic acid 7291 plusmn 182a 7185 plusmn 052ab 4342 plusmn 075c 5584 plusmn 182bc

Quinic acid 8461 plusmn 064a 5259 plusmn 314b 6904 plusmn 281ab 6163 plusmn 440b

Malic acid 2717 plusmn 136a 2294 plusmn 010a 2503 plusmn 052a 2082 plusmn 070a

Shikimic acid 183 plusmn 002a 126 plusmn 008a 135 plusmn 002a 135 plusmn 007a

Total 18652 plusmn 384a 14865 plusmn 279ab 13883 plusmn 261b 13964 plusmn 699b

In each row different letters mean significant differences with a 119875 lt 005 Letters refer to Tukeyrsquos post hoc test therefore significant different values wereclassified using letters in alphabetic order

Table 2 Retention time (Rt) wavelength of maximum absorption (120582max) mass spectral data and tentative identification of phenoliccompounds in decoctions and infusions of Castanea sativa flowers

Peak Rt (min) 120582max(nm)

Pseudomolecularion [M-H]minus (mz)

MS2 (mz)( of base peak) Tentative identification

1 496 276 783 481 (10) 301 (41) Pedunculagin isomer (bis-HHDP-glucose)2 579 268 783 481 (6) 301 (45) Pedunculagin isomer (bis-HHDP-glucose)3 706 272 633 463 (17) 301 (100) Galloyl-HHDP-glucose4 767 274 937 637 (15) 467 (2) 301 (4) Trigalloyl-HHDP-glucose5 802 278 289 245 (91) 203 (60) 137 (38) (+)-Catechin6 1377 276 939 631 (31) 469 (66) 169 (100) Pentagalloyl glucose7 1492 276 935 633 (15) 301 (18) Galloyl-bis-HHDP-glucose

8 1525 274 933 915 (5) 633 (8) 451 (24)301 (7) Castalaginvescalagin

9 1546 278 907 767 (3) 607 (24) 467 (35)169 (5) Galloyl-HHDP derivative

10 1613 274 937 767 (2) 637 (8) 467 (68)301 (10) Trigalloyl-HHDP-glucoside

11 1665 358 493 317 (100) Myricetin O-glucuronide12 1707 350 479 317 (100) Myricetin 3-O-glucoside


14 1989 356 609 301 (100) Quecetin 3-O-rutinoside15 2064 356 477 301 (100) Quercetin 3-O-glucuronide16 2086 356 463 301 (100) Quercetin 3-O-glucoside17 2130 356 463 301 (100) Quercetin O-hexoside18 2354 350 593 285 (100) Kaempferol 3-O-rutinoside19 2405 354 433 301 (100) Quercetin O-pentoside


21 2511 348 477 285 (100) Kaempferol 3-O-glucoside22 2600 354 477 315 (100) Isorhamnetin O-hexoside23 2625 354 491 315 (100) Isorhamnetin O-glucuronide


25 2987 250368 343 328 (97) 313 (100) 298 (36) Tri-O-methylellagic acid26 3238 358 609 463 (76) 301 (40) Quercetin O-rhamnosyl hexoside27 3313 356 519 477 (5) 315 (77) Isorhamnetin O-acetylhexoside


bis-HHDP-glucose isomer [15 17 19] Pseudomolecular ion([M-H]minus at mz 933) and fragmentation pattern (ions atmz 915 631 451 and 301) of peak 8 were in agreementwith those attributed to castalagin or vescalagin isomers[17 20] already reported in C sativa heartwood [18] Peaks9 20 and 24 are most likely galloyl-HHDP derivativeswith an unusual parent ion [M-H]minus at mz 907 but withcharacteristic fragments of this type of compounds (mz at767 467 and 169) No structure could be assigned to thesepeaks that remain unidentified A fragment ion with mz at907was reported as released from the cleavage of a di(HHDP-galloylglucose)-pentose found in pomegranate juice [21] andfrom unknown ellagitannins present in blackberries [17]Peak 25 presented an ellagic acid-like UV spectrum (120582maxaround 250 and 368 nm) and a pseudo molecular ion [M-H]minus at mz 343 releasing three fragments at mz 328313 and 298mu corresponding to the successive losses ofthree methyl groups (minus15mu) which allowed its tentativeidentification as a tri-O-methylellagic acid

Regarding flavonoids flavonol derivatives were the maincompounds found in the analyzed samples (Table 2) Cate-chin (peak 5) myricetin 3-O-glucoside (peak 12) quercetin3-O-rutinoside (peak 14) quercetin 3-O-glucoside (peak 16)kaempferol 3-O-rutinoside (peak 18) and kaempferol 3-O-glucoside (peak 21) were positively identified according totheir retention mass and UV-vis characteristics by compar-ison with commercial standards Peak 11 was assigned to amyricetin O-glucuronide according to the pseudomolecularion [M-H]minus atmz 497 andMS2 fragment released atmz 317([M-H-176]minus loss of glucuronyl moiety) Peaks 15 17 19 and26 presented UV spectra with 120582max around 350 nm and anMS2 product ion at mz 301 indicating that they correspondto quercetin derivatives According to their pseudomolecularions they were identified as quercetin 3-O-glucuronide (peak15 [M-H]minus atmz 477) which was confirmed by comparisonwith a standard obtained in our laboratory [22] quercetinO-hexoside (peak 17 [M-H]minus at mz 463) quercetin O-pentoside (peak 19 [M-H]minus at mz 433) and quercetin O-rhamnosyl hexoside (peak 26 [M-H]minus at mz 609) Simi-larly reasoning also allowed assigning peaks 22 and 23 asisorhamnetin O-hexoside and isorhamnetin O-glucuroniderespectively Peak 27 should correspond to isorhamnetin O-acetylhexoside according to the pseudomolecular ion [M-H]minus at mz 519 and MS2 fragment released at mz 315 ([M-H-42-162]minus loss of an acetylhexosidemoiety)The individualpolyphenol with the highest concentration in all samples wasa trigalloyl-HHDP-glucoside (peak 10) followed by penta-galloyl glucose (peak 6) whereas quercetin 3-O-glucuronide(peak 15) and a quercetin hexoside (peak 17) were the mostabundant flavonoids in judia and longal cultivars respectively(Table 3) The sample with the highest concentration of totalpolyphenols was the infusion of judia closely followed by thedecoction of longalThe preparations of the cultivar judia pre-sented higher flavonoid levels while those of longal displayedhigher concentrations of hydrolyzable tannins (Table 3) Thecompounds present in the samples were to some extentdifferent from those found in a C sativa hydromethanolicextract [9] Nevertheless the main ellagitannins found in thisstudy were in accordance with the mentioned study

33 Antioxidant Activity The antioxidant activity of theC sativa samples was determined through various assaysnamely DPPH scavenging activity reducing power throughPrussian-blue assay inhibition of 120573-carotene bleachingand finally inhibition of TBARS formation in brain cellhomogenates (Table 4) Decoctions showed greater antioxi-dant activity than infusions this might be explained by thelonger time at boiling point that decoctions were subjected toduring extraction In terms of scavenging of DPPH radicalsstatistically significant differenceswere foundbetweendecoc-tions and infusions Reducing power 120573-carotene bleachinginhibition and TBARS assays gave statistically significantdifferent results among samples but the lowest EC

50values

were always obtained in the TBARS assay this could be dueto its specificity sensibility and low quantity of interferences

This assay was the only one that reported lower EC50

values for infusions which can be related to the low heatresistance of antioxidants that inhibit lipid peroxidation liketocopherols and other vitamins [23]

Among the four samples the decoction of judia provedto be the most antioxidant among the samples analysed Thiscould be explained by antioxidant variability of cultivarstranslating into a higher antioxidant potential of judia whencompared to longal Further research should be carriedout to determine what antioxidants apart from phenoliccompounds are present in these flowers that could help clarifythis antioxidant variability between cultivars Nevertheless ingeneral all the studied samples proved to be powerful antiox-idants when compared to other herbal matrices previouslystudied by the authors [8 9]

4 Conclusion

The ancestral claims of health benefits of chestnuts flowerswere partially corroborated in this paper The organic acidscould be responsible for some antioxidant potential butfurther research can be carried out in terms on tocopherolsand other vitamins and minerals that could provide moreinsight into the total amount of antioxidants Further researchshould be carried out on the potentiality of these flowersin the pharmaceutical and food industries among othersPharmaceutical industries could use the flowers as excipientsfor dietary supplements benefiting from their natural contentin polyphenols for health purposes The food industrywhich has recently started to intensify its search for naturalconservatives and additives to add to transformed foodstuffsmeeting the consumers habits can use the high antioxidantpower andnatural high abundance of tannins in the flowers topreserve food and inhibit lipid deterioration andmicroorgan-ism development Although quite beneficial the flowers usedfor these purposes should not be picked from the tree butrather from the ground to avoid interference of pollinationMethanolic extractions have also been performed for chest-nut flowers and yielded good results [8 9] although the usageof methanol is not recommended in the food industry buton the other hand extractions with this solvent could carryhydrophobic antioxidant compounds


Table 3 Quantification of phenolic compounds in infusions and decoctions of Castanea sativa flowers expressed in mgg of lyophilizeddecoctioninfusion

Compounds Decoction judia Decoction longal Infusion judia Infusion longalPedunculagin isomer (bis-HHDP-glucose) 521 plusmn 011 848 plusmn 051 598 plusmn 016 768 plusmn 011Pedunculagin isomer (bis-HHDP-glucose) 112 plusmn 009 144 plusmn 019 092 plusmn 092 121 plusmn 004Galloyl-HHDP-glucose 307 plusmn 014 318 plusmn 000 318 plusmn 007 305 plusmn 009Trigalloyl-HHDP-glucose 281 plusmn 025 551 plusmn 006 260 plusmn 006 430 plusmn 047(+)-Catechin 114 plusmn 012 158 plusmn 014 111 plusmn 003 110 plusmn 005Pentagalloyl glucose 561 plusmn 008 604 plusmn 015 573 plusmn 014 647 plusmn 015Galloyl-bis-HHDP-glucose 045 plusmn 004 029 plusmn 007 066 plusmn 001 030 plusmn 001Castalaginvescalagin 075 plusmn 006 073 plusmn 011 106 plusmn 009 161 plusmn 004Galloyl-HHDP derivative 038 plusmn 002 055 plusmn 007 360 plusmn 016 372 plusmn 001Trigalloyl-HHDP-glucoside 2672 plusmn 015 2816 plusmn 011 3070 plusmn 040 2873 plusmn 134Myricetin O-glucuronide 113 plusmn 002 009 plusmn 000 096 plusmn 001 008 plusmn 000Myricetin 3-O-glucoside 066 plusmn 001 009 plusmn 001 060 plusmn 001 009 plusmn 000Trigalloyl-HHDP-glucoside 133 plusmn 000 054 plusmn 001 161 plusmn 005 070 plusmn 004Quecetin 3-O-rutinoside 157 plusmn 001 052 plusmn 003 131 plusmn 010 048 plusmn 005Quercetin 3-O-glucuronide 355 plusmn 010 156 plusmn 004 338 plusmn 001 149 plusmn 007Quercetin 3-O-glucosidelowast 180 plusmn 001 089 plusmn 002 160 plusmn 004 091 plusmn 010Quercetin O-hexoside 221 plusmn 007 182 plusmn 005 211 plusmn 002 185 plusmn 006Kaempferol 3-O-rutinoside 021 plusmn 001 021 plusmn 003 018 plusmn 001 021 plusmn 001Quercetin O-pentoside 043 plusmn 002 020 plusmn 001 034 plusmn 000 022 plusmn 002Galloyl-HHDP derivative 418 plusmn 010 454 plusmn 014 241 plusmn 014 196 plusmn 012Kaempferol 3-O-glucoside 053 plusmn 000 049 plusmn 001 041 plusmn 000 046 plusmn 004Isorhamnetin O-hexoside 033 plusmn 004 027 plusmn 001 027 plusmn 000 030 plusmn 001Isorhamnetin O-glucuronide 035 plusmn 003 050 plusmn 002 033 plusmn 001 046 plusmn 004Galloyl-HHDP derivative 204 plusmn 006 202 plusmn 003 072 plusmn 001 059 plusmn 001Tri-O-methylellagic acid 010 plusmn 001 tr 008 plusmn 001 trQuercetin O-rhamnosyl hexoside 023 plusmn 000 010 plusmn 001 023 plusmn 003 009 plusmn 000Isorhamnetin O-acetylhexoside 011 plusmn 000 008 plusmn 001 012 plusmn 001 004 plusmn 000Total flavonoids 1426 plusmn 014a 838 plusmn 001c 1294 plusmn 017b 779 plusmn 035dTotal hydrolyzable tannins 5378 plusmn 031c 6149 plusmn 080a 5925 plusmn 003b 6032 plusmn 187ab

Total phenolic compounds 6804 plusmn 018c 6988 plusmn 081bc 7220 plusmn 014a 6810 plusmn 152c

In each row different letters mean significant differences with a119875 lt 005They refer to Tukeyrsquos post hoc test therefore significant different values were classifiedusing letters in alphabetic order tr compound detected in trace amount lowastEstimation due to its low resolution

Table 4 Antioxidant activity of decoctions and infusions of the flowers of two Castanea sativa cultivars

Antioxidant activity(EC50 values 120583gmL) Decoction judia Decoction longal Infusion judia Infusion longal

DPPH scavengingactivity 9947 plusmn 0006b 10004 plusmn 001b 12661 plusmn 0005a 13356 plusmn 0005a

Reducing power 6851 plusmn 0001d 7607 plusmn 0001c 9065 plusmn 0001b 9879 plusmn 0001a

120573-carotene bleachinginhibition 4789 plusmn 0002d 18492 plusmn 0001b 17723 plusmn 0004c 19510 plusmn 001a

TBARS inhibition 3873 plusmn 0001b 4863 plusmn 0000a 1524 plusmn 0002d 1979 plusmn 0003c

EC50 values correspond to the sample concentration achieving 50 of antioxidant activity or 05 of absorbance in the reducing power assay In each rowdifferent letters mean significant differences with a 119875 lt 005 They refer to Tukeyrsquos post hoc test therefore significant different values were classified usingletters in alphabetic order

Conflict of Interests

The authors declare that there is no conflict of interests regar-ding the publication of this paper

Acknowledgments

The authors are grateful to Project PRODER n∘ 46577-Plant-Lact and to Foundation for Science and Technology (FCT

Portugal) for financial support to the research centre CIMO(PEst-OEAGRUI06902011) and for L Barrosrsquo contract(Compromisso para a Ciencia 2008)

References

[1] O P Borges J Soeiro Carvalho P Reis Correia and A PaulaSilva ldquoLipid and fatty acid profiles of Castanea sativaMill Che-stnuts of 17 native Portuguese cultivarsrdquo Journal of Food Com-position and Analysis vol 20 no 2 pp 80ndash89 2007


[2] INE Instituto Nacional de Estatıstica Chestnuts nationalstatistics 2012 httpwwwineptxportalxmainxpid=INEampxpgid=ine publicacoesampPUBLICACOESpub boui=153380933ampPUBLICACOESmodo=2

[3] T K Lim Edible Medicinal and Non-Medicinal Plants vol 1 ofFruits Springer 2012

[4] Patent CN102524895 B httpswwwgooglecompatentsCN102524895Bcl=enampdq=chestnut+floweramphl=enampsa=Xampei=pmGTUvuJBJKI7AbTqIHABQampved= 0CEAQ6AEwAQ

[5] Patent CN102334573 B httpswwwgooglecompatentsCN102334573Bcl=enampdq=chestnut+floweramphl=enampsa=Xampei=pmGTUvuJBJKI7AbTqIHABQampved= 0CH8Q6AEwCA

[6] PatentCN102972838A httpshttpwwwgooglecompatentsCN102972838Acl=enampdq=chestnut+floweramphl=enampsa=Xampei=pmGTUvuJBJKI7AbTqIHABQampved= 0CHYQ6AEwBw

[7] M C B M de Vasconcelos R N Bennett E A S Rosaand J V Ferreira-Cardoso ldquoComposition of European chestnut(Castanea sativaMill) and association with health effects freshand processed productsrdquo Journal of the Science of Food andAgriculture vol 90 no 10 pp 1578ndash1589 2010

[8] L Barros S Oliveira AM Carvalho and I C F R Ferreira ldquoInvitro antioxidant properties and characterization in nutrientsand phytochemicals of sixmedicinal plants from the Portuguesefolk medicinerdquo Industrial Crops and Products vol 32 no 3 pp572ndash579 2010

[9] L Barros C T Alves M Duenas et al ldquoCharacterization ofphenolic compounds in wild medicinal flowers from Portugalby HPLC-DAD-ESIMS and evaluation of antifungal proper-tiesrdquo Industrial Crops and Products vol 44 pp 104ndash110 2013

[10] C Pereira L Barros A M Carvalho and I C F R FerreiraldquoUse of UFLC-PDA for the analysis of organic acids in thirty-five species of food and medicinal plantsrdquo Food AnalyticalMethods vol 6 pp 1337ndash1344 2013

[11] L Day R B Seymour K F Pitts I Konczak and L LundinldquoIncorporation of functional ingredients into foodsrdquo Trends inFood Science and Technology vol 20 no 9 pp 388ndash395 2009

[12] KM BarryNWDavies andC LMohammed ldquoIdentificationof hydrolysable tannins in the reaction zone of Eucalyptusnitens wood by high performance liquid chromatography-electrospray ionisation mass spectrometryrdquo PhytochemicalAnalysis vol 12 no 2 pp 120ndash127 2001

[13] E Cantos J C Espın C Lopez-Bote L De la Hoz J AOrdonez and F A Tomas-Barberan ldquoPhenolic compounds andfatty acids from acorns (Quercus spp) the main dietary con-stituent of free-ranged Iberian pigsrdquo Journal of Agricultural andFood Chemistry vol 51 no 21 pp 6248ndash6255 2003

[14] A Romani M Campo and P Pinelli ldquoHPLCDADESI-MSanalyses and anti-radical activity of hydrolyzable tannins fromdifferent vegetal speciesrdquoFoodChemistry vol 130 no 1 pp 214ndash221 2012

[15] J-P Salminen V Ossipov J Loponen E Haukioja and K Pih-laja ldquoCharacterisation of hydrolysable tannins from leaves ofBetula pubescens by high-performance liquid chromatography-mass spectrometryrdquo Journal of Chromatography A vol 864 no2 pp 283ndash291 1999

[16] B Zywicki T Reemtsma and M Jekel ldquoAnalysis of commer-cial vegetable tanning agents by reversed-phase liquid chr-omatography-electrospray ionization-tandem mass spectrom-etry and its application to wastewaterrdquo Journal of Chromatogra-phy A vol 970 no 1-2 pp 191ndash200 2002

[17] T J Hager L R Howard R Liyanage J O Lay and R L PriorldquoEllagitannin composition of blackberry as determined by

HPLC-ESI-MS and MALDI-TOF-MSrdquo Journal of Agriculturaland Food Chemistry vol 56 no 3 pp 661ndash669 2008

[18] M Sanz E Cadahıa E Esteruelas et al ldquoPhenolic compoundsin chestnut (Castanea sativaMill) heartwood Effect of toastingat cooperagerdquo Journal of Agricultural and Food Chemistry vol58 no 17 pp 9631ndash9640 2010

[19] K R Maatta-Riihinen A Kamal-Eldin and A R TorronenldquoIdentification and quantification of phenolic compounds inberries of Fragaria and Rubus species (family Rosaceae)rdquo Jour-nal of Agricultural and Food Chemistry vol 52 no 20 pp 6178ndash6187 2004

[20] M D Bubba L Checchini U Chiuminatto S Doumett andD F E Giordani ldquoLiquid chromatographicelectrospray ion-ization tandemmass spectrometric study of polyphenolic com-position of four cultivars of Fragaria vesca L berries and theircomparative evaluationrdquo Journal of Mass Spectrometry vol 47pp 1207ndash1220 2012

[21] PMena L Calani C DallrsquoAsta et al ldquoRapid and comprehensiveevaluation of (poly)phenolic compounds in pomegranate (Pun-ica granatum L) juice by UHPLC-MS119899rdquo Molecules vol 17 pp14821ndash14840 2012

[22] M Duenas H Mingo-Chornet J J Perez-Alonso R Di Paola-Naranjo A M Gonzalez-Paramas and C Santos-Buelga ldquoPre-paration of quercetin glucuronides and characterization byHPLC-DAD-ESIMSrdquo European Food Research and Technologyvol 227 no 4 pp 1069ndash1076 2008

[23] D R Janero ldquoMalondialdehyde and thiobarbituric acid-reacti-vity as diagnostic indices of lipid peroxidation and peroxidativetissue injuryrdquo Free Radical Biology and Medicine vol 9 no 6pp 515ndash540 1990

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

Hindawi Publishing Corporationhttpwwwhindawicom

GenomicsInternational Journal of

Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y


Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


them in coatings in order to extend their shelf-life and reduceconsumption of chemical additives [11]

In this report the decoctions and infusions of flowersfrom the two most appreciated chestnut cultivars (longal andjudia) from Tras-os-Montes Portugal were characterizedregarding their content of hydrophilic antioxidant molecules(free sugars organic acids and phenolic compounds) reduc-ing power free radicals scavenging activity and lipid peroxi-dation inhibition

2 Materials and Methods

21 Standards and Reagents Acetonitrile 999 of high per-formance liquid chromatography (HPLC) grade andsulphuric acid were acquired from Fisher Scientific(Lisbon Portugal) Formic acid was acquired from Panreac(Barcelona Spain) Sugar standards [D(minus)-fructose D(+)-glucose anhydrous andD(+)-sucrose] organic acid standards(malic acid shikinic acid oxalic acid and quinic acid) andtrolox (6-hydroxy-2578-tetramethylchroman-2-carboxylicacid) were acquired from Sigma Chemical Co (Saint LouisMO USA) Phenolic compound standards (catechin gallicacid isorhamnetin 3-O-glucoside kaempferol 3-O-glucosidekaempferol 3-O-rutinoside myricetin quercetin 3-O-gluco-side and quercetin 3-O-rutinoside) were purchased fromExtrasynthese (Genay France) 22-Diphenyl-1-picrylh-ydrazyl (DPPH) was obtained from Alfa Aesar (Ward HillMA USA) Water was treated by means of a Milli-Q waterpurification system (TGI Pure Water Systems GreenvilleSC USA)

22 Flower Samples Castanea sativaMill flowers of the cul-tivars judia and longal were collected in June 2013 in OleirosBraganca (north-eastern Portugal) (41∘5110158400210158401015840N 6∘4910158405410158401015840W)The specimens were lyophilized (FreeZone 45 LabconcoKansas USA) milled down to a fine powder and finallystored at minus5∘C until analysis

23 Preparation of the Decoctions and Infusions For the infu-sions preparation the lyophilized flowers (1 g) were added to200mL of boiling distilled water left to stand for 5min andfinally filtered through a Whatman filter paper The obtainedinfusions were frozen and lyophilized

For the decoctions preparation the lyophilized flowers(1 g) were added to 200mL of boiling distilled water boiledfor 5min and then left to stand at room temperature for5 more minutes After filtration through a Whatman filterpaper the obtained decoctions were frozen and lyophilized

24 Analysis of Free Sugars Free sugars were determined byHPLCcoupled to a refraction index (RI) detector as describedpreviously [8] The equipment consisted of a pump (KnauerSmartline System 1000 Berlin Germany) a degasser (Smart-line Manager 5000) an autosampler (AS-2057 Jasco EastonMD USA) and a RI detector (Knauer Smartline 2300) Theidentification was achieved by comparing the relative reten-tion times of sample peakswith standardsQuantificationwasmade by the internal standard method and the results areexpressed in mg per g of lyophilized decoction or infusion

25 Analysis of Organic Acids Organic acids were deter-mined following a procedure previously optimized anddescribed by the authors [10] Analysis was performedon a Shimadzu 20A series ultra-fast liquid chromatograph(UFLC Shimadzu Cooperation Kyoto Japan) coupled tophotodiode array detector (PDA Shimadzu) using 215 nmand 245 nm as the preferred wavelengths Separation wasachieved on a SphereClone (Phenomenex Torrance CAUSA) reverse phase C18 column (5 120583m 250mm times 46mmid) thermostatted at 35∘C Analytes were eluted with 36mMsulphuric acid at a flow-rate of 08mLminThe organic acidsfound were quantified by comparison of the area of theirpeaks recorded at 215 nm or 245 nm (for ascorbic acid) withcalibration curves obtained from commercial standards ofeach compound oxalic acid (119910 = 1119909107119909 + 96178 1198772 =0999) quinic acid (119910 = 601768119909 + 88532 1198772 = 1) malicacid (119910 = 952269119909 + 17803 1198772 = 1) shikimic acid (119910 =8119909107+55079 1198772 = 0999)The results were expressed inmgper g of lyophilized decoction or infusion

26 Analysis of Phenolic Compounds Phenolic compoundswere determined by HPLC (Hewlett-Packard 1100 AgilentTechnologies Santa Clara CA USA) as previously described[9] Double online detection was carried out with a PDAusing 280 nm and 370 nm as the preferred wavelengths anda mass spectrometer (MS) connected to the HPLC systemvia the PDA cell outlet Mass spectrometric detection wasperformed by means of an API 3200 (Applied BiosystemsDarmstadt Germany) triple quadrupole-ion trap analyserequipped with an ESI source Spectra were recorded innegative ion mode between mz 100 and 1700 The phenoliccompounds were characterized according to their UV massspectra retention times and comparison with authenticstandards when available For the quantitative analysis ofphenolic compounds a baseline to valley integration withbaseline projection mode was used to calculate peak areasFor quantification calibration curves were generated byinjection of known concentration (25ndash100 120583gmL) of stan-dard compounds catechin (119910 = 13276119909 minus 59658 1198772 = 1)gallic acid (119910 = 55694119909minus73837 1198772 = 0999) isorhamnetin-3-O-glucoside (119910 = 26231119909minus98958 1198772 = 1) kaempferol-3-O-glucoside (119910 = 19075119909 minus 36158 1198772 = 1) kaempferol-3-O-rutinoside (119910 = 17502119909 minus 43877 1198772 = 0999) myricetin(119910 = 778119909 minus 14543 1198772 = 0999) quercetin-3-O-glucoside(119910 = 31648119909 minus 29142 1198772 = 1000) and quercetin-3-O-rutinoside (119910 = 22279119909 minus 24311 1198772 = 0999) Theresults were expressed in mg per g of lyophilized decoctionor infusion

27 Evaluation of Antioxidant Activity The in vitro antioxi-dant activity assays were performed following the previouslydescribed methodology of Barros et al [8] The lyophilizedinfusions and decoctions were dissolved in water (finalconcentration 10mgmL) the final solution was furtherdiluted to different concentrations to be used in the followingassays DPPH radical-scavenging activity was evaluated usingan ELX800 microplate Reader (Bio-Tek Instruments IncWinooski VT USA) and calculated as a percentage of DPPH


0 5 10 15 20 25 30

(mAU

)

020406080

100120140160

Time (min)

11

12

10

13 14

15

1617

1918 20212223

25 26 27

(a)

Time (min)0 5 10 15 20 25

(mAU

)

0250500750

10001250150017502000

1

23 4

5

6

7 8

10

913 20

24

(b)





50value (sample









































50values





4 Conclusion
















Acknowledgments



References

































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 5 10 15 20 25 30

(mAU

)

020406080

100120140160

Time (min)

11

12

10

13 14

15

1617

1918 20212223

25 26 27

(a)

Time (min)0 5 10 15 20 25

(mAU

)

0250500750

10001250150017502000

1

23 4

5

6

7 8

10

913 20

24

(b)





50value (sample









































50values





4 Conclusion
















Acknowledgments



References

































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

































50values





4 Conclusion
















Acknowledgments



References

































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





50values





4 Conclusion
















Acknowledgments



References

































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















Acknowledgments



References

































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology





Volume 2014


















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

ResearchArticle Castanea sativa Mill. Flowers amongst the ... 202.pdf · m/z 915, 631, 451 and 301) of peak 8 were in agreement with those attributed to castalagin or vescalagin isomers