research article influence of conventional and...

Research ArticleInfluence of Conventional and Ultrasonic-AssistedExtraction on Phenolic Contents Betacyanin Contentsand Antioxidant Capacity of Red Dragon Fruit(Hylocereus polyrhizus)

Nurul Shazini Ramli12 Patimah Ismail3 and Asmah Rahmat1

1 Department of Nutrition and Health Sciences Faculty of Medicine and Health Sciences Universiti Putra MalaysiaSerdang 43400 Selangor Malaysia

2 Department of Food Science Faculty of Food Science and Technology Universiti Putra Malaysia Serdang43400 Selangor Malaysia

3 Department of Biomedical Sciences Faculty of Medicine and Health Sciences Universiti Putra Malaysia Serdang43400 Selangor Malaysia

Correspondence should be addressed to Asmah Rahmat asmahupmedumy

Received 13 June 2014 Revised 22 August 2014 Accepted 22 August 2014 Published 14 October 2014

Academic Editor Dun Xian Tan

Copyright copy 2014 Nurul Shazini Ramli et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The aim of this study was to examine the effects of extraction methods on antioxidant capacities of red dragon fruit peel andflesh Antioxidant capacities were measured using ethylenebenzothiozoline-6-sulfonic acid (ABTS) radical cation assay and ferricreducing antioxidant power assay (FRAP) Total phenolic content (TPC) was determined using Folin-Ciocalteu reagent whilequantitative determination of total flavonoid content (TFC) was conducted using aluminium trichloride colorimetric methodBetacyanin content (BC) was measured by spectrophotometer Red dragon fruit was extracted using conventional (CV) andultrasonic-assisted extraction (UE) technique to determine the most efficient way of extracting its antioxidant components Resultsindicated that UE increased TFC reduced the extraction yield BC and TPC but exhibited the strongest scavenging activity for thepeel of red dragon fruit In contrast UE reduced BC TFC and scavenging activity but increased the yield for the flesh NonethelessUE slightly increases TPC in flesh Scavenging activity and reducing power were highly correlated with phenolic and flavonoidcompounds Conversely the scavenging activity and reducing power were weakly correlated with betacyanin content This workgives scientific evidences for the consideration of the type of extraction techniques for the peel and flesh of red dragon fruit inapplied research and food industry

1 Introduction

For decades evaluation of antioxidant properties of plantsfood has been part of the basis for screening their potentialin disease prevention and treatment [1] Many studies onantioxidant capacities have been conducted and publishedcontributing to the body of knowledge Even though in vitrostudies do not guarantee the effectiveness in vivo and inhuman studies [2] it does give ideas and support to possible

mechanismof action of the plant food in the biological system[3]

In order to maintain basic cell functions free radicals areproduced continuously in our body as a byproduct of energymetabolism and these free radicals are normally neutralizedby the endogenous antioxidants However environmentalexposure to poisons smoke and ionizing radiation can upsetthe balance of antioxidant system in the body hence resultingin oxidative stress [4] In retrospect oxidative stress has been

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 964731 7 pageshttpdxdoiorg1011552014964731

2 The Scientific World Journal

implicated in aging and the development of chronic diseaseslike cardiovascular diseases diabetes cancer atherosclerosisand Alzheimerrsquos disease [5 6]

Sample preparation and extraction techniques are verycrucial in antioxidant studies since it can influence therecovery of antioxidant compounds from the plant materialsand physiological function of the extracted substances Manyextraction techniques are available and have been usedextensively by researchers in determination of antioxidantcomponents and its capacity in plant food Nowadays novelextraction techniques have been developed to improve theextraction efficiency including microwave digestion enzy-matic extraction and ultrasound-assisted extraction (UE)[7ndash9] However the applications of new extraction tech-niques on specific food need solid scientific evidence basedon analysis of bioactive compounds and functional propertiesof the specific resulting food The comparison betweenconventional and ultrasonic-assisted extraction is made sinceconventional method is commonly used in research whileultrasound assisted extraction is cost effective to be usedcommercially

Red dragon fruit (Hylocereus polyrhizus) or sometimescalled red pitaya has been comprehensively researched forits antioxidant potential especially the polyphenols and beta-cyanin Betacyanin is a pigment that is responsible for the redpurple color of the fruit [10] A number of extraction tech-niques have been used for extracting bioactive compoundsfrom dragon fruit like enzyme-assisted extraction usingPectinexA Ultra SP-L microwave and ultrasonic extractionmethod [11ndash13] However to the best of our knowledge thecomparison between conventional and ultrasonic-assistedextraction has not yet been done Conventional extractionmethod using orbital shaker at high temperature involveslonger extraction hours and hence resulted in degradationof heat-sensitive bioactive compounds On the other handultrasonic-assisted extraction is very simple and inexpensive[14 15] and permits higher surface area contact betweensample and solvent [16]Thus the present study is conductedto investigate the influence of conventional and ultrasound-assisted extraction using sonicator on antioxidant capacitiesof peel and flesh of red dragon fruit

2 Materials and Methods

21 Materials All chemicals and reagents used for spec-trophotometric analysis were of analytical grade absoluteethanol Folin-Ciocalteu reagent and sodium carbonate 221015840-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)and potassium persulphate were purchased from Merck(Darmstadt Germany) aluminium chloride hexahydrate(AlCl3sdot6H20) was from Fischer Scientific (NH USA) gal-

lic acid acetate buffer 246-tripyridyl-s-triazine (TPTZ)iron(III) chloride hexahydrate (FeCl

3sdot6H20) and rutin were

from Sigma-Aldrich (MO USA)

22 Plant Material Dragon fruits were obtained from four-year-old plants fromMulti Rich farm inMantin Negeri Sem-bilan Malaysia The identification of the fruit was done by

a botanist from Biodiversity Unit Institute of BiosciencesUniversiti Putra Malaysia The voucher number is SK-244014 as previously described in Ramli et al [17] Dragonfruits were freshly harvested when reaching full ripeningstage 35ndash38 days after pollination and immediately trans-ported to theNutritional Laboratory Faculty ofMedicine andHealth sciences Universiti Putra Malaysia

23 Sample Preparation The fruits were cleaned and theresidual water was removed using paper towel before beingweighed The flesh and peel were cut into small cubes andstored in minus80∘C freezer (SonyUltra Low minus80∘C)The sampleswere spray-dried in a spray dryer (Niro AS GEA Germany)to reduce the moisture content for a more efficient extractionprocess Sample preparation was conducted in reduced lightcondition in order to minimize the pigment loss The spray-dried sample was then kept in airtight container beforefurther analysis The samples were extracted using eitherconventional method or ultrasonic-assisted extraction inorder to compare which extraction method is more efficientin extracting the antioxidant compounds of the samples

231 Conventional Extraction (CV) One g of the flesh washomogenized with 25mL of distilled water for a few min-utes at 125 (wv) The mixture was shaken using shakingincubator at 200 rpm for 120 minutes at 50∘C Mucilaginousmaterial was separated from the extract on a Buchner funnelthroughWhatmanNo 1 filter paper to give a colored solutionThe residue was reextracted with water for full pigmentrecovery The mixture was then centrifuged at 6000 rpmfor 15 minutes at room temperature and supernatant wassaved For the peel 55mL of distilled water was addedto 1 g of the sample at 155 (wv) The extraction for bothflesh and peel was conducted in triplicate (119899 = 3) Thenthe supernatants were used for the determination of totalphenolic and total flavonoid content betacyanin content andantioxidant activity using ferric reducing antioxidant power(FRAP) and ABTS+ radical scavenging assay

232 Ultrasonic-Assisted Extraction (UE) One g of the pulpwas homogenized with 25mL of distilled water for a fewminutes at 125 (wv) The mixture was then placed in ultra-sonic bath and sonicated at 50 kHz for 30 minutes at ambienttemperature (25∘C) Mucilaginous material was separatedfrom the extract on a Buchner funnel through WhatmanNo 1 filter paper to give a colored solution The residue wasreextracted with water for full pigment recoveryThemixturewas then centrifuged (Universal 32r Hettich ZentrifugenEngland) at 6000 rpm for 15 minutes at room temperatureand supernatant was saved For the peel 55mL of distilledwater was added to 1 g of the sample at 155 (wv) Theextraction for both flesh and peel was conducted in triplicateThen the supernatants were used for determination of totalphenolic and total flavonoid content betacyanin content anddetermination of antioxidant activity using ferric reducingantioxidant power (FRAP) and ABTS+ radical scavengingassay

The Scientific World Journal 3

24 Extraction Yield Extraction yield was measured basedon a method from Zhang et al [18] The resulting extractwas lyophilized in a freeze dryer (The Virtis CompanyInc Gardiner New York) Then the freeze dried extractwas weighed Extraction yield was calculated per 1 g of rawmaterial

25 Betacyanin Content Betacyanin content was quantifiedaccording to themethod byWybraniec andMizrahi [19] withslight modifications whereby the mean molar absorptivitywas set at 65000 for betanin instead of 60000 The beta-cyanin content of dry extracts was determined by the spec-trophotometricmethod and expressed as betanin equivalents(mg100 g of dry extracts) based on the formula below

Concentration of betacyanins (mg100 g dry extracts)

=

119860

538(MW) 119881 (DF) times 100120576119871119882

(1)

where 119860538= absorbance at 538 nm 119871 = path length 10

cm DF = dilution factor 119881 = pigment solutionvolume (mL) and119882 = dried pigment weight (g) For beta-nin 120576 (molar extinction coefficient) = 65000 and molecularweight (MW) = 550

26 Determination of Total Phenolic Content Determinationof total phenolic content was carried out using the Folin-Ciocalteursquos reagent as described by Singleton and Rossi [20]with slight modifications whereby the steps of heating at100∘C in a water bath are removed and the incubation time isincreased from 1 minute to 90 minutes The red dragon fruitextract (200120583L) was mixed with 15mL of Folin-Ciocalteureagent (previously diluted tenfold with distilled water) andallowed to stand at room temperature for 5min A 15mL ofsodium carbonate solution (60 gL)was added to themixtureThe tubes were vortexed covered with parafilm and allowedto stand for 90 minutes The absorbance was measured at750 nm using Secomamrsquos RS232 ultraviolet-visible (UV-vis)spectrophotometer (Cedex France) after a 90min incubationat room temperature The total phenolic content was calcu-lated using regression equations from standard curve of gallicacid (20ndash200120583gmL in water) The results were expressed asmg dry basis

27 Determination of Total Flavonoid Content The flavonoidcontent was estimated by using aluminium trichloride col-orimetric with slight modification based on the method byQuettier-Deleu et al [21] whereby 5mL of 2 AlCl

3 6H2O

has been reduced to 1mL In brief 1mL of extract solutionwas added to 1mL of 2 AlCl

3 6H2O The absorbance was

measured after 10min of incubation at 430 nm The resultwas expressed inmgdry basis by comparisonwith standardrutin treated in the same condition

28 Determination of Antioxidant Capacity

281 ABTS+ Scavenging Assay The ABTS+ radical scaveng-ing assay was done according to the method by Hurtado et al[22] The mixture of 7mM ABTS and 245mM potassiumpersulphate was kept in an amber bottle and incubatedin the dark at room temperature for 16 hours to produceABTS+ radical cation The solution was then diluted withdistilled water to get absorbance of 070 plusmn 01 at 734 nmmeasured using UV-Vis spectrophotometer (SECOMAMFrance) Then 20120583L of sample extract or standard (Trolox)to 10mL ABTS+ solution was added and mixed thoroughlyThe sample was diluted with the respective extraction mediato give 20ndash80 inhibition of the blank Trolox standard ata final concentration of 0004ndash024mM was prepared indistilled water and assayed under the same conditions TheTrolox equivalent antioxidant capacity (TEAC) of the reddragon fruit extracts was calculated based on the inhibitionexerted by the standard Trolox solution at 6min Considerthe following

of scavenging capacity = 1 minus (Absorbancesample

Absorbancecontrol) times 100

(2)

282 Ferric-Reducing Antioxidant Power Assay Determina-tion of Ferric-reducing power (FRAP) was carried out usinga method described by Benzie and Strain [23] with somemodifications whereby the incubation time was changedfrom 15 seconds to 15 minutes and samples were dilutedbefore it was mixed with FRAP reagent The FRAP reagentwas prepared by mixing 03M acetate buffer (pH 36) 001Mof TPTZ and 002M of FeCl

3sdot6H2O with a ratio of 10 1 1

at 37∘C Red dragon fruit extract (02mL) was mixed with3mL of FRAP reagent and themixture was incubated at 37∘CTriplicate measurements were conducted and the changes inabsorbance were measured at 593 nm after initial time andat 15min Trolox concentrations of 50ndash1000120583M were usedfor calibration of the FRAP assay and antioxidant power wasexpressed as mM of TEAC per 100 g dry weight

29 Statistical Analysis All data were analyzed using SPSSfor Windows version 190 Independent 119905-test is used to testwhether there are significant differences in total phenolictotal flavonoid betacyanin content and antioxidant capaci-ties between the two extraction methods of red dragon fruitpeel and flesh Pearson correlation coefficient was used totest the correlation between antioxidant capacities and totalphenolic total flavonoid and betacyanin content All dataare expressed as means plusmn standard error of mean Statisticalsignificance was set at 119875 = 005

3 Results and Discussion

31 Extraction Yield Table 1 summarizes the extraction yieldbetacyanin content and antioxidant capacities of red dragonfruit peel and fleshThe highest extraction yield was obtainedfrom the peel of red dragon fruit extracted using conventional


Table 1 Extraction yield betacyanin content scavenging activity and reducing power of peel and flesh from red pitaya of different extractionmethods

SamplesPeel

Extraction yield Betacyanin content Scavenging activity FRAP() (mg100 g dry extracts) () (120583mol Fe2+g dry extract)

UE 4707a 1764 plusmn 003b 606 plusmn 003a 255 plusmn 4994a

CV 9525b 1867 plusmn 050a 304 plusmn 036b 20083 plusmn 383a

FleshUE 9008a 7134 plusmn 100b 085 plusmn 055b 620 plusmn 5408a

CV 7327b 8279 plusmn 055a 573 plusmn 033a 60917 plusmn 5451a

UE ultrasonic-assisted extractionCV conventional extractionValues are expressed as mean plusmn SEM (119899 = 3)Means within a column followed by different letters within each fruit part were significantly different at 119875 lt 005

extractionmethod (9525)whereas the flesh extracted usingultrasonic-assisted extraction had the highest yield (9008)The peels of studied sample contained higher concentrationof pectin [24] and may require longer extraction time tocomplete the separation of compound for the compoundto be fully diffused into the solvent as applied in CV for 2hours compared to UE for only 30 minutes The flesh of reddragon fruit had the highest extraction yield using UE Itwas found that ultrasound-assisted extraction of polyphenolsfrom orange was able to reduce energy input from the higherextraction yield in shorter time [16] UE produces ultrasonicwaves that attack the integrity of plant cellular walls Thisresulted in increased permeability of cytoplasmicmembranesas evidenced by scanning electron microscopy [25] and moresolvent can enter into the plant cell while causing the releaseof more compounds into the solvent [26]

32 Betacyanin Content Results in Table 1 showed CV wasmore effective for extracting betacyanin from the peel andflesh Betacyanin is very sensitive to high temperature It wasfound that almost 90 of the pigment retention was reducedwith the increasing of temperature from 25∘C to 75∘C [27]Surprisingly the CV did not contribute to the decrease intotal betacyanin content in this present study In fact beta-cyanin content detected in this study was significantly highercompared to study by Tang and Norziah [27] whereby theyconducted the extraction in room temperature (25∘C) andfound 101 plusmn 06mg100 g dry weight and 67 plusmn 02mg100 gdry weight in flesh and peel respectively

33 Total Phenolic Content Figure 1 shows mean TPC ofspray-dried peel and flesh of red dragon fruit extracted usingconventional and ultrasonic-assisted extraction methodsThe results from this study found that TPC was significantlyhigher in peel extracted using CV (7384 plusmn 1594mg drybasis) compared to peel extracted by UE (6516 plusmn 152mgdry basis) On the other hand no difference between the twoextractionmethods for the flesh (12186plusmn489mg dry basisand 12830 plusmn 817mg dry basis extracted in CV and UEresp) It is possible that the use of CV increased the release

0

20

40

60

80

100

120

140

Peel Flesh

ConventionalUltrasonic

a

b

a a

mg

GA

E100

g dr

y ex

trac

t

Figure 1 Total phenolic contents extracted from red dragon fruitof different parts by using different methods (Mean with differentalphabets for the same plant parts are significantly different (119875 lt005))

of bound phenolic compound from the peel compared to UEmethod Some authors [28 29] have postulated that the loss ofTPC in dried samples at high temperature could be resultedfrom degradation of phenolic compounds due to thermaleffect andor reduced free phenolic compound Besides thereduction of TPC also contributed to the loss betacyaninas betacyanin also contained phenol structure in moleculeHowever the present study found higher total phenolic com-pounds which may be resulted from increased betacyanincontent when extracted using CV Previous research reportedthat 70 ethanolic extract of red dragon fruit contained 2816and 1972mg dry basis in peel and pulp respectively [30]Since the present study found more than 100 times of TPC inwater extract it seems that most of phenolic compounds inred dragon fruit are water soluble


0

100

200

300

400

500

600

Peel Flesh


ab

b

a

mg

rutin

100

g dr

y ex

trac

t

Figure 2 Total flavonoid contents extracted from red dragon fruitof different parts by using different methods (Mean with differentalphabets for the same plant parts are significantly different (119875 lt005))

34 Total Flavonoid Content Figure 2 illustrates mean TFCof red dragon fruit peel and flesh extracted using CV andUETFC in peel was higher when extracted using UE comparedto CV (19582 plusmn 550mg dry basis and 14588 plusmn 233mgdry basis extracted in CV and UE resp) Contrarily fleshextracted by using CV had higher TFC (51069 plusmn 754mgdry basis compared to UE (24531 plusmn 604mg dry basis)Flavonoid is a subset of phenolic compounds [31] howeverhigh flavonoid content does not lead to high total phenoliccontent in this present study

35 Antioxidant Capacity Assay

351 FRAP Assay Briefly the antioxidant power is equal toreducing capacity It can be seen from the results in Table 1that flesh exhibited FRAP values of 620 plusmn 5408 120583mol Fe2+gdry extract using UE and 60917 plusmn 5451 120583mol Fe2+g dryextract using CV It was noted that peel showed the lowestFRAP values of 20083plusmn6635 120583mol Fe2+g dry extract usingCV and 255 plusmn 8649 120583mol Fe2+g dry extracted using UEHowever the present study revealed no significant differencebetween the two extraction methods (119875 gt 005) for both peeland flesh in contrast with reports from a previous study [16]Besides Rebecca et al [32] reported that the reducing powerof red dragon fruit increased when the concentration of thesample was increased from 003125 g 00625 g 0125 g 025 gand 05 g

352 ABTS+ Scavenging Assay Determination of ABTS+scavenging assay was carried out based on the decoloriza-tion of the preformed radical monocation of 221015840-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) produced

Table 2 Correlation of the phytochemical contents with differentantioxidant indicators

Scavenging activity Reducing powerTotal phenolic content 0810 0990Total flavonoid content 0549 0890Betacyanin content 0382 0381Correlation is significant at the 005 level (1-tailed)

through the reaction between ABTS and potassium persul-fate This method has been reported to be relevant for bothlipophilic and hydrophilic antioxidants [31] As shown inTable 1 the scavenging effect of red dragon fruit peel rangedfrom 304 plusmn 036 to 606 plusmn 003 for spray dried sampleextracted by CV andUE respectively A significant differencewas found between the two extraction methods (119875 lt 005)Similarly the scavenging effect for flesh shows significantdifference between CV and UE For flesh the scavengingeffect was 573 plusmn 033 and 085 plusmn 055 when extractedusing CV and UE respectively In comparison with previousstudy the antioxidant assaymeasured byABTS for red dragonfruit flesh and peel was found to be 1029 and 81503 120583gTrolox equivalentsg FM [11]The scavenging effect of dragonfruit peel extracted using UE (606 plusmn 003) and dragonfruit flesh extracted using CV (573 plusmn 033) displayedhigher scavenging activity compared to Sapodilla fruit (430and 536 for the flesh and peel resp) [33] Findings fromprevious study pointed out the suitability of ultrasonic-assisted extraction in the preparation of antioxidant-richplant extracts especially in orange peel [16] While UEconducted at ambient temperature for 30 minutes resultedin about 50 increase in the scavenging activity of the peelthe CV of the flesh at 50∘C for 2 hours resulted in fourtimes increase in the scavenging activity Interestingly theeffects of time and temperature during sample preparationand extraction on antioxidant capacities are unique to thespecific plant parts

36 Correlation between PhenolicFlavonoid Compound andAntioxidant Activity The role of phenolic compounds asantioxidants has been recognized by means of their abil-ity to donate electron and formed a stable radical inter-mediates Esquivel et al [34] reported that betacyaninwas the major compound that contributed to the antiox-idant activity of red dragon fruit The present studyhowever found that scavenging activity and reducingpower were weakly correlated with betacyanin content(1199032 = 0382 and 1199032 = 0381 resp) (Table 2) Further-more the scavenging activity and reducing power werehighly correlated with phenolic (1199032 = 0810 and 1199032= 0990 resp) and flavonoid compounds (1199032 = 0549 and 1199032= 0890 resp) (Table 2) This finding was in agreement withprevious study whereby total phenolic and total flavonoidcontent of papaya extract were strongly correlated withantioxidant activity [30] It does show that flavonoid wasan important factor contributing to the radical scavengingactivity in this present study


4 Conclusions

The findings of this study suggest that ultrasonic-assistedextraction significantly reduces the extraction yield butincreases the flavonoid content and scavenging activity forthe peel of red dragon fruit Conversely ultrasonic-assistedextraction increases the extraction yield of the flesh butreduces total flavonoid content and scavenging activityHence it can be seen that the influence of extraction methoddepends upon the physical property of the sampleThis workgives scientific evidences for the consideration of the useof UE for the peel of red dragon fruit in applied researchand food industry Even though the extraction yield is animportant factor to consider for production of functionalfood in large scale manufacturing it is more crucial to takeinto consideration its biological effects Therefore the appli-cation of extraction methods for food or dietary supplementwarrant further research before being produced in large scalein order to identify the most efficient extraction techniquesfor extracting its antioxidants

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

Thiswork was supported by ResearchGrant from theUniver-siti PutraMalaysia under ResearchUniversityGrants (RUGS)(04-02-12-1759RU)

References

[1] M S Blois ldquoAntioxidant determinations by the use of a stablefree radicalrdquo Nature vol 181 no 4617 pp 1199ndash1200 1958

[2] B Halliwell ldquoFree radicals and antioxidants updating a per-sonal viewrdquo Nutrition Reviews vol 70 no 5 pp 257ndash265 2012

[3] R Tsao ldquoChemistry and biochemistry of dietary polyphenolsrdquoNutrients vol 2 no 12 pp 1231ndash1246 2010

[4] S Rao K Sireesha Y Aparna and M Sadanandam ldquoFreeradicals and tissue damage role of antioxidantsrdquo Free Radicalsand Antioxidant vol 1 no 4 pp 2ndash7 2011

[5] C Cencioni F Spallotta F Martelli et al ldquoOxidative stressand epigenetic regulation in ageing and age-related diseasesrdquoInternational Journal of Molecular Sciences vol 14 pp 17643ndash17663 2013

[6] S Reuter S C Gupta M M Chaturvedi and B B AggarwalldquoOxidative stress inflammation and cancer how are theylinkedrdquo Free Radical Biology amp Medicine vol 49 no 11 pp1603ndash1616 2010

[7] J Wu L Lin and F-T Chau ldquoUltrasound-assisted extractionof ginseng saponins from ginseng roots and cultured ginsengcellsrdquoUltrasonics Sonochemistry vol 8 no 4 pp 347ndash352 2001

[8] J G Wilkes E D Conte Y Kim M Holcomb J B Sutherlandand DWMiller ldquoSample preparation for the analysis of flavorsand off-flavors in foodsrdquo Journal of Chromatography vol 880no 1-2 pp 3ndash33 2000

[9] J F Parr V Dolic G Lancaster and W E Boyd ldquoA microwavedigestion method for the extraction of phytoliths from herbar-ium specimensrdquo Review of Palaeobotany and Palynology vol116 no 3-4 pp 203ndash212 2001

[10] S Wybraniec I Platzner S Geresh et al ldquoBetacyanins fromvine cactus hylocereus polyrhizusrdquo Phytochemistry vol 58 no8 pp 1209ndash1212 2001

[11] S Kunnika and A Pranee ldquoInfluence of enzyme treatment onbioactive compounds and colour stability of betacyanin in fleshand peel of red dragon fruit Hylocereus polyrhizus (Weber)Britton and Roserdquo International Food Research Journal vol 18no 4 pp 1437ndash1448 2011

[12] P Chaiwut A O-ki-la I Phuttisatien N Thitilertdecha andP Pintathong ldquoExtraction and stability of cosmetic bioactivecompounds fromdragon fruit peelrdquo inProceedings of the 1stMaeFah Luang University International Conference pp 1ndash11 2012

[13] A R Muhammad Shahairie Ultrasonic extraction of antioxi-dant compound from red pitaya [PhD thesis] Faculty of Chem-ical amp Natural Resources Engineering Universiti MalaysiaPahang 2009

[14] P V Hung and T L Duy ldquoEffects of drying methods onbioactive compounds of vegetables and correlation betweenbioactive compounds and their antioxidantsrdquo InternationalFood Research Journal vol 19 no 1 pp 327ndash332 2012

[15] J Wang B Sun Y Cao Y Tian and X Li ldquoOptimisation ofultrasound-assisted extraction of phenolic compounds fromwheat branrdquo Food Chemistry vol 106 no 2 pp 804ndash810 2008

[16] F Chemat Z-E Huma and M K Khan ldquoApplications ofultrasound in food technology processing preservation andextractionrdquo Ultrasonics Sonochemistry vol 18 no 4 pp 813ndash835 2011

[17] N S Ramli L Brown P Ismail and A Rahmat ldquoEffects ofred pitaya juice supplementation on cardiovascular and hepaticchanges in high-carbohydrate high-fat diet-induced metabolicsyndrome ratsrdquoBMCComplementary and AlternativeMedicinevol 14 article 189 2014

[18] S-Q Zhang H-M Bi and C-J Liu ldquoExtraction of bio-activecomponents from Rhodiola sachalinensis under ultrahighhydrostatic pressurerdquo Separation and Purification Technologyvol 57 no 2 pp 277ndash282 2007

[19] S Wybraniec and Y Mizrahi ldquoFruit flesh betacyanin pigmentsinHylocereus cactirdquo Journal of Agricultural and FoodChemistryvol 50 no 21 pp 6086ndash6089 2002

[20] V L Singleton and J A Rossi ldquoColorimetry of total oftotal phenolics with phosphomolybdemic-phosphotungsticacid reagentsrdquoTheAmerican Journal of Enology and Viticulturevol 16 pp 144ndash158 1965

[21] C Quettier-Deleu B Gressier J Vasseur et al ldquoPhenolic com-pounds and antioxidant activities of buckwheat (Fagopyrumesculentum Moench) hulls and flourrdquo Journal of Ethnopharma-cology vol 72 no 1-2 pp 35ndash42 2000

[22] N H Hurtado A L Morales M L Gonzalez-Miret M LEscudero-Gilete and F J Heredia ldquoColour pH stability andantioxidant activity of anthocyanin rutinosides isolated fromtamarillo fruit (Solanum betaceum Cav)rdquo Food Chemistry vol117 no 1 pp 88ndash93 2009

[23] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996


[24] K K Woo Y Y Chong S K li Hiong and P Y Tang ldquoPectinextraction and characterization from red dragon fruit (Hylo-cereus polyrhizus) a preliminary studyrdquo Journal of BiologicalSciences vol 10 no 7 pp 631ndash636 2010

[25] S Balachandran S E Kentish R Mawson and M Ashokku-mar ldquoUltrasonic enhancement of the supercritical extractionfrom gingerrdquo Ultrasonics Sonochemistry vol 13 no 6 pp 471ndash479 2006

[26] M Bimakr R A Rahman F Saleena Taip N M Adzahan ZIslam Sarker and A Ganjloo ldquoUltrasound-assisted extractionof valuable compounds from winter melon (Benincasa hispida)seedsrdquo International Food Research Journal vol 20 no 1 pp331ndash338 2013

[27] C S Tang and M Norziah ldquoBetacyanin stabilityrdquo IndonesianJournal of Chemistry vol 7 no 3 pp 327ndash331 2007

[28] J A Larrauri ldquoNew approaches in the preparation of highdietary fibre powders from fruit by-productsrdquo Trends in FoodScience and Technology vol 10 no 1 pp 3ndash8 1999

[29] A Omidizadeh R M Yusof A Ismail S Roohinejad LNateghi and M Z A Bakar ldquoCardioprotective compoundsof red pitaya (Hylocereus polyrhizus) fruitrdquo Journal of FoodAgriculture and Environment vol 9 no 3-4 pp 152ndash156 2011

[30] R Nurliyana S Zahir K Mustapha Suleiman M R AisyahandK Kamarul Rahim ldquoAntioxidant study of pulps and peels ofdragon fruits a comparative studyrdquo International Food ResearchJournal vol 375 pp 367ndash375 2010

[31] S R Nurul and R Asmah ldquoEvaluation of antioxidant propertiesin fresh and pickled papayardquo International Food ResearchJournal vol 19 no 3 pp 1117ndash1124 2012

[32] O P S Rebecca A N Boyce and S Chandran ldquoPigmentidentification and antioxidant properties of red dragon fruit(Hylocereus polyrhizus)rdquo African Journal of Biotechnology vol9 no 10 pp 1450ndash1454 2010

[33] P F Woo H S Yim H E Khoo C M Sia and Y K AngldquoEffects of extraction conditions on antioxidant properties ofsapodilla fruit (Manilkara zapota)rdquo International Food ResearchJournal vol 20 no 5 pp 2065ndash2072 2013

[34] P Esquivel F C Stintzing and R Carle ldquoPhenolic compoundprofiles and their corresponding antioxidant capacity of purplepitaya (Hylocereus sp) genotypesrdquo Journal of Biosciences vol 62no 9-10 pp 636ndash644 2007

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


implicated in aging and the development of chronic diseaseslike cardiovascular diseases diabetes cancer atherosclerosisand Alzheimerrsquos disease [5 6]

Sample preparation and extraction techniques are verycrucial in antioxidant studies since it can influence therecovery of antioxidant compounds from the plant materialsand physiological function of the extracted substances Manyextraction techniques are available and have been usedextensively by researchers in determination of antioxidantcomponents and its capacity in plant food Nowadays novelextraction techniques have been developed to improve theextraction efficiency including microwave digestion enzy-matic extraction and ultrasound-assisted extraction (UE)[7ndash9] However the applications of new extraction tech-niques on specific food need solid scientific evidence basedon analysis of bioactive compounds and functional propertiesof the specific resulting food The comparison betweenconventional and ultrasonic-assisted extraction is made sinceconventional method is commonly used in research whileultrasound assisted extraction is cost effective to be usedcommercially

Red dragon fruit (Hylocereus polyrhizus) or sometimescalled red pitaya has been comprehensively researched forits antioxidant potential especially the polyphenols and beta-cyanin Betacyanin is a pigment that is responsible for the redpurple color of the fruit [10] A number of extraction tech-niques have been used for extracting bioactive compoundsfrom dragon fruit like enzyme-assisted extraction usingPectinexA Ultra SP-L microwave and ultrasonic extractionmethod [11ndash13] However to the best of our knowledge thecomparison between conventional and ultrasonic-assistedextraction has not yet been done Conventional extractionmethod using orbital shaker at high temperature involveslonger extraction hours and hence resulted in degradationof heat-sensitive bioactive compounds On the other handultrasonic-assisted extraction is very simple and inexpensive[14 15] and permits higher surface area contact betweensample and solvent [16]Thus the present study is conductedto investigate the influence of conventional and ultrasound-assisted extraction using sonicator on antioxidant capacitiesof peel and flesh of red dragon fruit

2 Materials and Methods

21 Materials All chemicals and reagents used for spec-trophotometric analysis were of analytical grade absoluteethanol Folin-Ciocalteu reagent and sodium carbonate 221015840-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)and potassium persulphate were purchased from Merck(Darmstadt Germany) aluminium chloride hexahydrate(AlCl3sdot6H20) was from Fischer Scientific (NH USA) gal-

lic acid acetate buffer 246-tripyridyl-s-triazine (TPTZ)iron(III) chloride hexahydrate (FeCl

3sdot6H20) and rutin were

from Sigma-Aldrich (MO USA)

22 Plant Material Dragon fruits were obtained from four-year-old plants fromMulti Rich farm inMantin Negeri Sem-bilan Malaysia The identification of the fruit was done by

a botanist from Biodiversity Unit Institute of BiosciencesUniversiti Putra Malaysia The voucher number is SK-244014 as previously described in Ramli et al [17] Dragonfruits were freshly harvested when reaching full ripeningstage 35ndash38 days after pollination and immediately trans-ported to theNutritional Laboratory Faculty ofMedicine andHealth sciences Universiti Putra Malaysia

23 Sample Preparation The fruits were cleaned and theresidual water was removed using paper towel before beingweighed The flesh and peel were cut into small cubes andstored in minus80∘C freezer (SonyUltra Low minus80∘C)The sampleswere spray-dried in a spray dryer (Niro AS GEA Germany)to reduce the moisture content for a more efficient extractionprocess Sample preparation was conducted in reduced lightcondition in order to minimize the pigment loss The spray-dried sample was then kept in airtight container beforefurther analysis The samples were extracted using eitherconventional method or ultrasonic-assisted extraction inorder to compare which extraction method is more efficientin extracting the antioxidant compounds of the samples

231 Conventional Extraction (CV) One g of the flesh washomogenized with 25mL of distilled water for a few min-utes at 125 (wv) The mixture was shaken using shakingincubator at 200 rpm for 120 minutes at 50∘C Mucilaginousmaterial was separated from the extract on a Buchner funnelthroughWhatmanNo 1 filter paper to give a colored solutionThe residue was reextracted with water for full pigmentrecovery The mixture was then centrifuged at 6000 rpmfor 15 minutes at room temperature and supernatant wassaved For the peel 55mL of distilled water was addedto 1 g of the sample at 155 (wv) The extraction for bothflesh and peel was conducted in triplicate (119899 = 3) Thenthe supernatants were used for the determination of totalphenolic and total flavonoid content betacyanin content andantioxidant activity using ferric reducing antioxidant power(FRAP) and ABTS+ radical scavenging assay

232 Ultrasonic-Assisted Extraction (UE) One g of the pulpwas homogenized with 25mL of distilled water for a fewminutes at 125 (wv) The mixture was then placed in ultra-sonic bath and sonicated at 50 kHz for 30 minutes at ambienttemperature (25∘C) Mucilaginous material was separatedfrom the extract on a Buchner funnel through WhatmanNo 1 filter paper to give a colored solution The residue wasreextracted with water for full pigment recoveryThemixturewas then centrifuged (Universal 32r Hettich ZentrifugenEngland) at 6000 rpm for 15 minutes at room temperatureand supernatant was saved For the peel 55mL of distilledwater was added to 1 g of the sample at 155 (wv) Theextraction for both flesh and peel was conducted in triplicateThen the supernatants were used for determination of totalphenolic and total flavonoid content betacyanin content anddetermination of antioxidant activity using ferric reducingantioxidant power (FRAP) and ABTS+ radical scavengingassay





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OncologyJournal of





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