research article binary solvent extraction system and extraction...

Research ArticleBinary Solvent Extraction System and ExtractionTime Effects on Phenolic Antioxidants from Kenaf Seeds(Hibiscus cannabinus L) Extracted by a PulsedUltrasonic-Assisted Extraction

Yu Hua Wong1 Hwee Wen Lau1 Chin Ping Tan2 Kamariah Long3 and Kar Lin Nyam1

1 Department of Food Science and Nutrition Faculty of Applied Sciences UCSI University No 1 Jalan Menara Gading UCSI Heights56000 Cheras Kuala Lumpur Malaysia

2 Department of Food Technology Faculty of Food Science and Technology Universiti Putra Malaysia43400 Serdang Selangor Malaysia

3Malaysian Agricultural Research amp Development Institute (MARDI) 43400 Serdang Selangor Malaysia

Correspondence should be addressed to Kar Lin Nyam nyamklucsiuniversityedumy

Received 28 August 2013 Accepted 12 November 2013 Published 23 January 2014

Academic Editors D Benke and A A Iglesias

Copyright copy 2014 Yu Hua Wong et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aimof this studywas to determine the best parameter for extracting phenolic-enriched kenaf (Hibiscus cannabinusL) seeds by apulsed ultrasonic-assisted extractionThe antioxidant activities of ultrasonic-assisted kenaf seed extracts (KSE) were determined bya 22-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity assay 221015840-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)(ABTS) radical scavenging assay 120573-carotene bleaching inhibition assay and ferric reducing antioxidant power (FRAP) assay Totalphenolic content (TPC) and total flavonoid content (TFC) evaluations were carried out to determine the phenolic and flavonoidcontents in KSE The KSE from the best extraction parameter was then subjected to high performance liquid chromatography(HPLC) to quantify the phenolic compounds The optimised extraction condition employed 80 ethanol for 15min with thehighest values determined for the DPPH ABTS and FRAP assay KSE contained mainly tannic acid (230220mg100 g extract)and sinapic acid (119822mg100 g extract) which can be used as alternative antioxidants in the food industry

1 Introduction

Bioactive constituents with antioxidant activities have beenfound at high concentrations in plants [1] Because of thepotential health risks and toxicity some synthetic antioxi-dants must be replaced with natural antioxidants [2] There-fore there is great interest in the replacement of syntheticantioxidants with natural sources especially from plantmaterials [3] Kenaf (Hibiscus cannabinus L) is well knownin Asia and Africa for its multipurpose use and it hasbeen widely cultivated in some Mediterranean areas [4]According to Cheng et al [5] the traditional use of thisplant is focused on fibre production such as making ropessacks canvases and carpets Keshk et al [6] showed thatnew kenaf applications have been developed for the pulpand paper industry oil adsorption and potting media boardmaking filtration media and animal feed The kenaf seeds

are considered a waste product and they are used mainlyfor animal feeds in the cattle sheep camel and poultryindustries Using this waste is very important for kenafcultivation and for increasing grower income In additionthis plant was also composed of various active componentswhich have long been prescribed in traditional folk medicinefromAfrica and India [7] According to Nyam et al [8] therewere seven main phenolic compounds identified in the kenafseed oil namely gallic acid p-hydroxybenzoic acid caffeicacid vanillic acid syringic acid p-coumaric and ferulic acidIncreasing interest in replacing synthetic antioxidants has ledto investigations of natural antioxidant sources especially inplant materials

Ultrasound-assisted extraction provides a mechanicaleffect allowing greater solvent penetration into the samplematrix increasing the contact surface area between the solidand liquid phase and as a result the solute quickly diffuses

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 789346 7 pageshttpdxdoiorg1011552014789346

2 The Scientific World Journal

from the solid phase to the solvent [9 10] Kenaf seed oilhas normally been extracted by Soxhlet extraction [11] andsupercritical carbon dioxide fluid extraction [12] Howeverthere have been limited studies on the extraction of kenaf seedextract Yusri et al [13] have extracted phenolic compoundsfrom kenaf seed by normal solvent extraction There is noinformation regarding the ultrasonic-assisted extraction ofkenaf seeds Therefore pulse ultrasound-assisted extraction(PUAE) was chosen to extract kenaf seeds in this studyPUAE required lower electrical energy consumption highextraction time reduction higher antioxidant yield andincreased antioxidant activity

The objectives of this study were to determine thebest parameters (ethanol concentration and extractiontime) for extracting phenolic-enriched kenaf seeds bypulsed ultrasonic-assisted extraction (PUAE) to compare theantioxidant properties among different extraction parame-ters and to determine the phenolic compounds in kenaf seedextract which were extracted by using the best parameters

2 Materials and Methods

21 Sample Dried kenaf (Hibiscus cannabinus L) seed wasobtained from the Malaysian Agricultural Research andDevelopment Institute (MARDI Serdang Malaysia) and wasground into powder with a grinder (SHARP Japan) Thekenaf seed powder particle size was 1mm

22 Sample Extraction

221 Pulsed Ultrasound-Assisted Extraction (PUAE) Nine500mL Scott bottles were prepared Each set of three bottleswas filled with 60 80 and 100 ethanol Fifty gramsof ground kenaf seed was added to the solvent to undergoultrasonic extraction (Ultrasonic Homogeniser Labsonic P400W Sartorius AG) with a 5min pulse duration period and5min pulse interval period A 5min pulse duration periodand 5min pulse interval period were considered as 1 extrac-tion cycle Extractions were carried out with 1 extractioncycle 2 extraction cycles and 3 extraction cycles for eachethanol concentration The resulting kenaf seed extract wascentrifuged at 3500 rpm for 10min The supernatant of thekenaf seed extract was collected and underwent filtration andthe pellet was discarded The filtered extract was evaporatedby a rotary evaporator (Rotavapor R-200 Buchi Switzerland)and left in the centrifuge tube which was wrapped withaluminium foil

23 Antioxidant Activities

231 22-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scav-enging Capacity Assay The 22-diphenyl-1-picrylhydrazyl(DPPH) radical scavenging capacity assay for all nine kenafseed extracts was determined according to Liu et al [14] withslight modifications The kenaf seed extract (200120583L) withconcentration of 1mgmLwas added to 28mL of ethanol and1mL of 0004 of DPPH solution After 30min of incuba-tion the absorbance was measured against a blank reagent

(ethanol) at 517 nm by using a UV-Vis spectrophotometer(Model XTD 5 Secomam Domont France) All DPPHradical scavenging activities of the KSE were expressed aspercentage inhibition Inhibition percentage was calculatedby using the formula below

Inhibition percentage (IP) = (blank minus KSE)blank

times 100 (1)

where blank and KSEwere the absorbance values of the blankand the KSE

232 221015840-Azino-bis(3-ethylbenzothiazoline-6-sulphonicAcid) (ABTS) Radical Scavenging Assay ABTS was carriedout according to Floegel et al [15] with slight modificationsABTS (7mM) and 245mM of potassium persulphate(K2O8S2) were mixed and kept in the dark at room

temperature for 12ndash16 h for activation The activated mixturewas then adjusted with ethanol at 734 nm to 07 AU (plusmn002)Fifty milliliters of the sample was added to 1950120583L of ABTSand allowed to react for 6min The absorbance at 734 nmwas measured by using a UV-Vis spectrophotometer (ModelXTD 5 Secomam Domont France) against a blank reagent

All ABTS radical scavenging activities of the KSE wereexpressed as percentage inhibition Inhibition percentagewascalculated by using the formula below

Inhibition Percentage (IP) = (blank minus KSE)blank

times 100 (2)

where blank and KSE are the absorbance values of the blankand the KSE

233 120573-Carotene Bleaching (BCB) The 120573-carotene bleachingactivity of the extracts was evaluated by the 120573-carotenelinoleate model system [16] A solution of 120573-carotene wasprepared by dissolving 120573-carotene (5mg) in chloroform(50mL) Three milliliters of this solution was pipetted intoa round-bottom flask Then the chloroform was removedat 40∘C under vacuum The linoleic acid (40mg) Tween 20emulsifier (400mg) and distilled water (100mL) were addedto the flask with vigorous shaking An aliquot (3mL) of thisemulsion was transferred into different test tubes containingdifferent concentrations of the extracts (01mL) The tubeswere shaken and incubated at 50∘C for 30min in a waterbath As soon as the emulsion was added to each tube thezero time absorbance was measured at 470 nm using a UV-Vis spectrophotometer (Model XTD 5 Secomam DomontFrance) The BCB antioxidant activity was expressed as inhi-bition percentage with reference to the control after 30minstandardized incubation time with the following formula

AA = 100 times(DRcontrol minus DRsample)

DRcontrol (3)

where AA is the antioxidant activity DRcontrol is the degra-dation rate of the control = [(119886119887)30] DRsample is thedegradation rate in the presence of the sample = [(119886119887)30]where 119886 is the absorbance at zero time (first result recorded)while 119887 is the absorbance at 30min (second result recorded)

The Scientific World Journal 3

234 Ferric Reducing Antioxidant Power (FRAP) FRAP wascarried out according toWootton-Beard et al [17] with slightmodifications The FRAP reagent was prepared by mixing25mL of acetate buffer with 25mL of 246-tripyridyl-s-triazine (TPTZ) and 25mL of FeCl

3sdot6H2OThe mixture was

maintained in a 37∘C water bath (Lab Companion Korea)Fifty milliliters of the sample was added to 950 120583L of FRAPreagent in a test tube The mixture was allowed to react for30min in the dark at room temperature The absorbancewas measured at 593 nm by using UV-Vis spectrophotometer(Model XTD 5 Secomam Domont France) against a blankreagent after 30min The calibration equation for Troloxwas 119910 = 15535119909 + 04198 The results were expressed inmilligrams of Trolox100 g of kenaf seed extract

235 Total Phenolic Content (TPC) The total phenolic con-tent of the kenaf seed extract was determined using Folin-Ciocalteu assay based on the method described by Limet al [18] with slight modifications A kenaf seed extract(300 120583L) of 10mgmL was added to 15mL of Folin-Ciocalteureagent (FCR) and mixed well The mixture was allowedto stand at room temperature for 5min Then 12mL ofsodium carbonate (75 wv) solution was added and mixedthoroughly by using a vortex mixer and allowed to stand for30min After 30min the absorbance was measured against ablank reagent (ultrapure water mixed with FCR and sodiumcarbonate) at 765 nm using a UV-Vis spectrophotometer(Model XTD 5 Secomam France) The calibration equationfor gallic acid was 119910 = 8525119909 + 00331 (1199032 = 09989) Thetotal phenolic content was expressed in milligrams of gallicacid100 g of kenaf seed extractThe results were expressed inmilligrams of gallic100 g of kenaf seed extract

236 Total Flavonoid Content (TFC) The total flavonoidconcentration of the kenaf seed extract was measured byusing a colorimetric assay in accordance with the method ofVerzelloni et al [19] with slight modifications A kenaf seedextract (250 120583L) of 10mgmL was transferred into a test tubeOne millilitre of deionised water and 150 120583L of 150mgmLsodium nitrite were added to the test tube and allowed tostand for 6min Then 75120583L aluminium chloride (AlCl

3)

(100mgmL) was addedThe test tubes were allowed to standfor 5min then 1mL of 40mgmL sodium hydroxide (NaOH)was added Finally 25 120583L of deionised water was added fora final volume of 25mL The absorbance was measured at510 nm using a UV-Vis spectrophotometer (Model XTD 5Secomam France) against a blank reagent The calibrationequation for catechin was 119910 = 2895119909 minus 00185 (1199032 = 09956)The results were expressed in milligrams of catechin100 g ofkenaf seed extract

24 Phenolic Compound Quantification The phenolic com-pounds in the kenaf seed extract were quantified by HPLCas described by Baydar et al [20] with a slight modifica-tion The HPLC system was equipped with a diode arraydetector (DAD) Phenolic compound separation was carriedout using a reversed-phase HPLC column (Purospher star5 120583m times 250mm times 46mm Merck Germany) The column

temperature and detection wavelength were set at 30∘C and210 nm respectively A gradient elution system of solvent A(water with 01 phosphoric acid) and solvent B (methanolwith 01 phosphoric acid) was used (5 B (0min) 50B (5min) 55 B (65min) and 5 B (70min)) The flowrate was 1mLmin and the injected volume was 20 120583L Thephenolic compound chromatographic peaks were confirmedby comparing their retention timeswith those of the referencestandards Gallic acid tannic acid catechin hydrate 4-hydroxybenzaldehyde 4-hydroxybenzoic acid syringic acidsinapic acid ferulic acid naringin and protocatechuic acidwere used as phenolic compound standards

25 Statistical Analysis All experiments andor measure-ments were duplicated Analysis of variance (ANOVA) wascarried out and the average values were compared withFisherrsquos multiple comparison test All statistical analyses wereperformed using Minitab 13 for Windows (PennsylvaniaUnited States)

3 Results and Discussion

31 Extraction Solvent Evaluation In the present study anextraction using a binary ethanol and water solvent wasadopted after considering health and safety in handling [21]This experimental result was in accordance with previousreports suggesting that a binary solvent system was superiorto a monosolvent system (water or pure ethanol) in theextraction of phenolic compounds with regard to their rel-ative polarity [22 23] The solubility of phenolic compoundsdepends on the chemical nature of the plant tissue and thepolarity of the solvent system [24] Ethanol (60) was chosenas the lowest solvent concentration in this study This solventwas chosen because previous studies showed that antioxidantcompounds from plant sources were best extracted by 70ndash80 ethanol concentrations [25ndash27] In this study the kenafseeds extracted by 80 ethanol had better 22-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity assayresults than the 100 ethanol kenaf seed extract (Table 1)This circumstance may be explained by the inability of100 ethanol to extract phenolic compounds which aremore water-soluble (hydrophilic) The presence of water inthe extraction eases the release of hydrophilic antioxidantsTrabelsi et al [28] showed that the addition of 20 water tomethanol acetone or ethanol can enhance the extraction ofantioxidants from Limoniastrum monopetalum leaves

DPPH radical scavenging capacity assay is frequentlyused to determine the free radical scavenging ability ofvarious food components [29 30] In addition the 221015840-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)radical scavenging assay is also used to study the free radicalscavenging ability of a sample by using a moderately stablenitrogen-centred radical species [31] DPPH and ABTS bothyielded consistent results in the measurement of antioxidantactivity [32] Therefore DPPH and ABTS were chosen todetermine the best extraction parameters In this studythe 80 ethanol and 15min condition were the optimisedmethod for kenaf seed extraction In addition ferric reducing


Table 1 22-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity 221015840-azino-bis(3 ethylbenzothiazoline-6-sulphonic acid) (ABTS)radical scavenging assay total phenolic content (TPC) total flavonoid content (TFC) ferric reducing antioxidant power (FRAP) and 120573-carotene bleaching (BCB) assays of kenaf seed extract extracted by different ethanol concentrations and extraction times

Extraction parameterDPPH () ABTS () TPC

(mgGAE100 g)TFC

(mgCE100 g)FRAP

(mgTEAC100 g) BCB ()Ethanolconcentration

Time(min)

605 2871 plusmn 055i 1286 plusmn 034e 157994 plusmn 5623cd 59240 plusmn 489h 101191 plusmn 3446i 2893 plusmn 068c

10 3504 plusmn 062g 2354 plusmn 033d 175765 plusmn 2780b 66667 plusmn 446g 113743 plusmn 3735h 3107 plusmn 294abc

15 4282 plusmn 033e 4702 plusmn 099c 191308 plusmn 214a 84974 plusmn 446f 129997 plusmn 2127g 3356 plusmn 070a

805 5289 plusmn 070c 4533 plusmn 149c 140721 plusmn 1325f 167098 plusmn 1069e 355455 plusmn 8652c 2887 plusmn 058c

10 6035 plusmn 035b 5544 plusmn 145b 150692 plusmn 738e 239465 plusmn 435d 442195 plusmn 3946b 3422 plusmn 054a

15 6668 plusmn 038a 6978 plusmn 066a 153859 plusmn 1325d 270553 plusmn 490c 566109 plusmn 2991a 3346 plusmn 110ab

1005 3207 plusmn 087h 872 plusmn 032g 123331 plusmn 951h 408204 plusmn 331a 324718 plusmn 2127d 3196 plusmn 040b

10 3968 plusmn 076f 1146 plusmn 039f 159431 plusmn 832c 283679 plusmn 590b 157033 plusmn 1609e 1835 plusmn 076d

15 4694 plusmn 057d 1262 plusmn 020e 127877 plusmn 1663g 239378 plusmn 446d 140296 plusmn 2849f 1586 plusmn 051e

Value are presented in means plusmn standard deviation (119899 = 4) mean values at the same column with different superscripts are significantly different (119875 lt 005)GAE gallic acid equivalentsCE catechin equivalentsTEAC Trolox equivalent

antioxidant power (FRAP) and 120573-carotene bleaching assays(BCB) were also in accordance with this observation inwhich the highest value in each analysis was identified How-ever the antioxidant activities indicated by the 120573-carotenebleaching assay were relatively lower than those of the DPPHABTS and FRAP assays The 120573-carotene bleaching test wascarried out in a state of emulsionThe result was based on thefading of the characteristic yellow 120573-carotene colour whichwas caused by its reaction with radicals formed from theoxidation of linoleic acid Therefore it can be deduced thatthe hydrophilic antioxidant content is higher compared tohydrophobic antioxidants in kenaf seed extract

In this study the optimised condition (80 ethanolfor 15min) that produced the highest antioxidant contentfor kenaf seed extract did not give the optimised resultsfor the total phenolic content (TPC) and total flavonoidcontent (TFC) assays Based on the present experimentalresults it was predicted that kenaf seed contained diversephenolic compounds with different polaritiesThus no singleethanol concentrationwas able to recover all of the individualphenolic compounds from the samples Durling et al [33]suggested that the TPC which had its highest value at lowethanol concentrations contained a higher proportion ofhydrophilic compoundsThe TFCs recovered at high ethanolconcentrations were lipophilic compounds Additionally ahigh individual phenolic compound yield will not necessarilybe associated with a high antioxidant capacity which indeedis dependent on the synergistic effects of the extractedphenolics Therefore DPPH and ABTS analyses were moresuitable for choosing the optimised parameter

32 Extraction Time Evaluation The DPPH and ABTSassays showed increasing scavenging activity trends whenthe extraction time increased at all ethanol concentrations(Table 1) However the total phenolic content (TPC) and total

flavonoid content (TFC) for kenaf seeds extracted with 100ethanol decreased as the extraction time increasedThis trendcan be explained by the potential extraction time increase bythe loss of antioxidants following heat or oxygen exposure[34] The decrease of antioxidant activity and phenolic con-tent only appeared in 100 ethanol concentration extractionbut did not appear in 60 and 80 ethanol concentrationextraction when the extraction time increased from 5 to15minThemaximum temperature reached for 60 and 80ethanol concentration extraction after 15min was lower than70∘Cwhile the temperature of 100 ethanol increases rapidlyduring the extraction and reached a temperature above 70∘CIt is generally believed that most of the antioxidant com-pounds have shown rapid degradation when the temperaturewas above 70∘C [24] In a complex polyphenol systema temperature increase can promote molecular collisionsfavouring polymerisation and reducing antioxidant capacity[35] The difference in optimum extraction times for TPCand TFC may be explained by different degrees of phenolicpolymerisation the phenolic compound solubility and theirinteractions with other food constituents which then ledto different time requirements for reaching the equilibriumbetween the solution in the solid matrix (M citrifolia) and inthe bulk solution (ethanol) [36]

This study showed that the optimum extraction time forantioxidant compounds varies with the antioxidant capac-ity This phenomenon postulated that the estimation ofABTS and DPPH radical-scavenging capacities is not solelydependent on a single group of antioxidant compoundsABTS and DPPH radical-scavenging capacities are based onthe capacity of existing compounds to scavenge ABTS orDPPH radicals [37] These data highlighted the impact of thesolvent choice on the extraction efficiency Other studies haveshown that the ethanol concentration extraction time andtemperature affect the recovery of phenolic compounds andthe antioxidant capacity of plant extracts [38]


Table 2 Phenolic compounds in kenaf seed extract extracted by80 ethanol for 15min

Phenolic compounds Concentration (mg100 g)Gallic acid 12336 plusmn 031

Tannic acid 230220 plusmn 1648

Catechin hydrate 50273 plusmn 198

4-Hydroxybenzaldehyde 11614 plusmn 787

4-Hydroxybenzoic acid 25584 plusmn 774

Syringic acid 6156 plusmn 597

Sinapic acid 119822 plusmn 2182

Ferulic acid 28838 plusmn 1405

Naringin 4379 plusmn 319

Protocatechuic acid 17083 plusmn 822

Total 588056Value are presented in means plusmn standard deviation (119899 = 4)

33 Phenolic Compounds The HPLC analysis of phenoliccompounds has become one of the most dominant analyticalprocedures because of its advantages The advantages ofHPLC are simple sample treatment possible preseparationand impurity removal capacity to change the polarity ofthe mobile phase during analysis and high reproducibilityIn this study the wavelength of the detector was set as210 nm since the maximum absorbance for most of phenoliccompounds is at 210 nm Moreover the research done by Duand Chen [39] showed that at wavelength below 210 nm thebaseline becomes unstable because of the strong absorptionof methanol which caused the interferences to the detectionwhile at wavelength above 215 nm the adsorption of analytesdiminished significantly The phenolic compounds in kenafseed extract were extracted with 80 ethanol for 15min anddetermined by HPLC-DAD (Table 2) revealing the presenceof gallic acid tannic acid catechin benzaldehyde benzoicacid syringic acid sinapic acid ferulic acid naringin acidand protocatechuic acid as some of the phenolic compoundsthat were found in kenaf seed oil [40]

Tannic acid (230220mg100 g extract) and sinapic acid(119822mg100 g extract) were the major compounds inkenaf seed extract Tannic acid has been shown to possessantioxidant antimutagenic and anticarcinogenic properties[41] Sinapic acid is a cinnamic acid derivative and itpossesses 35-dimethoxy and 4-hydroxyl substitutions inthe phenyl group of cinnamic acid Sinapic acid is widelydistributed in the plant kingdom and can be obtained fromvarious sources such as rye broccoli cabbage and kale [42]Sinapic acid has already been pharmacologically evaluatedfor its potent antioxidant anxiolytic anti-inflammatory andperoxynitrite scavenging effects and neuroprotective effects[43] Apart from that Nyam et al [44] proved that kenafseed extract showed greater effect than synthetic antioxidant(BHA) in prevention of sunflower oil oxidation Thereforewe suggested that kenaf seed extract can be used as an alter-native to synthetic antioxidants for the efficient large-scaleproduction of tannic acid and other high-value secondarymetabolites

4 Conclusion

This study showed that kenaf seeds extracted with 80ethanol for 15min had the highest DPPH ABTS and FRAPassay values Therefore the best compromise between highantioxidant compound extraction yield and cost as well aspracticality was found for a 15min extraction in 80 forthe kenaf seed extract It was also found that the kenaf seedextract primarily contained tannic acid and sinapic acidwhich can be used as alternative antioxidants in the industrialsector

Conflict of Interests

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

Acknowledgment

Financial support of this work by internal funding fromCREVIE UCSI University (Proj-In-FAS-003) is gratefullyacknowledged

References

[1] N Tomas K Vladimir B Vladimir H Martin D Petr andM Ladislav ldquoDecrease in the antioxidant capacity in beveragescontaining tea extracts during storagerdquo The Scientific WorldJournal vol 2012 Article ID 361698 5 pages 2012

[2] G Sanjay A K Tiku K Apurva G Sahil S Gurjinder andV K Razdan ldquoAntioxidant and antimicrobial properties ofthe essential oil and extracts of Zanthoxylum alatum grownin North-Western Himalayardquo The Scientific World Journal vol2013 Article ID 790580 9 pages 2013

[3] J C M Barreira A L Morais I C F R Ferreira andM B P POliveira ldquoInsights on the formulation of herbal beverages withmedicinal claims according with their antioxidant propertiesrdquoMolecules vol 18 no 3 pp 2851ndash2863 2013

[4] AM L Seca AM S Silva A J D Silvestre J A S Cavaleiro FM J Domingues and C Pascoal-Neto ldquoPhenolic constituentsfrom the core of kenaf (Hibiscus cannabinus)rdquo Phytochemistryvol 56 no 7 pp 759ndash767 2001

[5] Z Cheng B-R Lu B S Baldwin K Sameshima and J-KChen ldquoComparative studies of genetic diversity in kenaf (Hibis-cus cannabinus L) varieties based on analysis of agronomic andRAPD datardquo Hereditas vol 136 no 3 pp 231ndash239 2002

[6] S Keshk W Suwinarti and K Sameshima ldquoPhysicochemicalcharacterization of different treatment sequences on kenaf bastfiberrdquo Carbohydrate Polymers vol 65 no 2 pp 202ndash206 2006

[7] H S Lee ldquoHPLC analysis of phenolic compoundsrdquo in FoodAnalysis by HPLC L M L Nollet Ed Marcel Dekker NewYork NY USA 2000

[8] K L Nyam C P Tan O M Lai K Long and Y B CheMan ldquoPhysicochemical properties and bioactive compounds ofselected seed oilsrdquo LWTmdashFood Science and Technology vol 42no 8 pp 1396ndash1403 2009

[9] M A Rostagno M Palma and C G Barroso ldquoUltrasound-assisted extraction of soy isoflavonesrdquo Journal of Chromatogra-phy A vol 1012 no 2 pp 119ndash128 2003


[10] W Jin Z Yong-Ming T Ya-Ting Y Chun-Lin and G Chun-Yan ldquoUltrasound-assisted extraction of total phenolic com-pounds from Inula heleniumrdquoThe Scientific World Journal vol2013 Article ID 157527 5 pages 2013

[11] S K Ng L L Y Jessie C P Tan K Long and K L NyamldquoEffect of accelerated storage on microencapsulated kenaf seedoilrdquo Journal of the American Oil Chemistsrsquo Society vol 90 no 7pp 1023ndash1029 2013

[12] K L Nyam C P Tan O M Lai K Long and Y B Che ManldquoOptimization of supercritical fluid extraction of phytosterolfrom roselle seeds with a central composite designmodelrdquo Foodand Bioproducts Processing vol 88 no 2-3 pp 239ndash246 2010

[13] N M Yusri K W Chan S Igbal and M Ismail ldquoPhenoliccontent and antioxidant activity of Hibiscus cannabinus L seedextracts after sequential solvent extractionrdquo Molecules vol 17no 11 pp 12612ndash12621 2012

[14] X Liu M Dong X Chen M Jiang X Lv and G YanldquoAntioxidant activity and phenolics of an endophyticXylaria spfrom Ginkgo bilobardquo Food Chemistry vol 105 no 2 pp 548ndash554 2007

[15] A Floegel D-O Kim S-J Chung S I Koo and O K ChunldquoComparison of ABTSDPPH assays to measure antioxidantcapacity in popular antioxidant-rich US foodsrdquo Journal of FoodComposition and Analysis vol 24 no 7 pp 1043ndash1048 2011

[16] L Barros S Falcao P Baptista C Freire M Vilas-Boasand I C F R Ferreira ldquoAntioxidant activity of Agaricussp mushrooms by chemical biochemical and electrochemicalassaysrdquo Food Chemistry vol 111 no 1 pp 61ndash66 2008

[17] P C Wootton-Beard A Moran and L Ryan ldquoStability ofthe total antioxidant capacity and total polyphenol content of23 commercially available vegetable juices before and after invitro digestion measured by FRAP DPPH ABTS and Folin-Ciocalteu methodsrdquo Food Research International vol 44 no 1pp 217ndash224 2011

[18] Y Y Lim T T Lim and J J Tee ldquoAntioxidant properties ofseveral tropical fruits a comparative studyrdquoFoodChemistry vol103 no 3 pp 1003ndash1008 2007

[19] E Verzelloni D Tagliazucchi and A Conte ldquoRelationshipbetween the antioxidant properties and the phenolic andflavonoid content in traditional balsamic vinegarrdquo Food Chem-istry vol 105 no 2 pp 564ndash571 2007

[20] NG BaydarO Sagdic GOzkan and S Cetin ldquoDeterminationof antibacterial effects and total phenolic contents of grape (Vitisvinifera L) seed extractsrdquo International Journal of Food Scienceamp Technology vol 41 no 7 pp 799ndash804 2006

[21] B Sultana F Anwar and R Przybylski ldquoAntioxidant activityof phenolic components present in barks of Azadirachta indicaTerminalia arjunaAcacia nilotica andEugenia jambolana Lamtreesrdquo Food Chemistry vol 104 no 3 pp 1106ndash1114 2007

[22] 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

[23] Z-S Zhang D Li L-J Wang et al ldquoOptimization of ethanol-water extraction of lignans from flaxseedrdquo Separation andPurification Technology vol 57 no 1 pp 17ndash24 2007

[24] J Dai and R J Mumper ldquoPlant phenolics extraction analysisand their antioxidant and anticancer propertiesrdquoMolecules vol15 no 10 pp 7313ndash7352 2010

[25] M K Khan M Abert-Vian A-S Fabiano-Tixier O Danglesand F Chemat ldquoUltrasound-assisted extraction of polyphenols(flavanone glycosides) from orange (Citrus sinensis L) peelrdquoFood Chemistry vol 119 no 2 pp 851ndash858 2010

[26] C Lu H Wang W Lv et al ldquoIonic liquid-basedultrasonicmicrowave-assisted extraction combined withUPLC-MS-MS for the determination of tannins in Gallachinensisrdquo Natural Product Research vol 26 no 19 pp1842ndash1847 2012

[27] Y Wu X Wang and E Fan ldquoOptimisation of ultrasound-assisted extraction of puerarin and total isoflavones fromPuerariae lobatae Radix (Pueraria lobata (Wild) Ohwi) withresponse surface methodologyrdquo Phytochemical Analysis vol 23no 5 pp 513ndash519 2012

[28] N Trabelsi W Megdiche R Ksouri et al ldquoSolvent effects onphenolic contents and biological activities of the halophyteLimoniastrum monopetalum leavesrdquo LWTmdashFood Science andTechnology vol 43 no 4 pp 632ndash639 2010

[29] H J D Dorman and R Hiltunen ldquoFe(III) reductive andfree radical-scavenging properties of summer savory (Saturejahortensis L) extract and subfractionsrdquo Food Chemistry vol 88no 2 pp 193ndash199 2004

[30] G Oboh and J B T Rocha ldquoWater extractable phytochemicalsfrom Capsicum pubescens (tree pepper) inhibit lipid peroxida-tion induced by different pro-oxidant agents in brain in vitrordquoEuropean Food Research andTechnology vol 226 no 4 pp 707ndash713 2008

[31] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[32] T Sun and C-T Ho ldquoAntioxidant activities of buckwheatextractsrdquo Food Chemistry vol 90 no 4 pp 743ndash749 2005

[33] N E Durling O J Catchpole J B Grey et al ldquoExtraction ofphenolics and essential oil from dried sage (Salvia officinalis)using ethanolndashwater mixturesrdquo Food Chemistry vol 101 no 4pp 1417ndash1424 2007

[34] Y Y Thoo S K Ho J Y Liang C W Ho and C PTan ldquoEffects of binary solvent extraction system extractiontime and extraction temperature on phenolic antioxidants andantioxidant capacity frommengkudu (Morinda citrifolia)rdquo FoodChemistry vol 120 no 1 pp 290ndash295 2010

[35] M Pinelo L Manzocco M J Nunez and M C NicolildquoInteraction among phenols in food fortification negativesynergism on antioxidant capacityrdquo Journal of Agricultural andFood Chemistry vol 52 no 5 pp 1177ndash1180 2004

[36] E M Silva H Rogez and Y Larondelle ldquoOptimization ofextraction of phenolics from Inga edulis leaves using responsesurface methodologyrdquo Separation and Purification Technologyvol 55 no 3 pp 381ndash387 2007

[37] R L Prior X Wu and K Schaich ldquoStandardized methodsfor the determination of antioxidant capacity and phenolicsin foods and dietary supplementsrdquo Journal of Agricultural andFood Chemistry vol 53 no 10 pp 4290ndash4302 2005

[38] K K ChewM Z Khoo S Y Ng Y YThooWMWAida andC W Ho ldquoEffect of ethanol concentration extraction time andextraction temperature on the recovery of phenolic compoundsand antioxidant capacity of Orthosiphon stamineus extractsrdquoInternational Food Research Journal vol 18 no 4 pp 1427ndash14352011

[39] H Du and X Q Chen ldquoA comparative study of the separationof oleanolic acid and ursolic acid in Prunella vulgaris byhigh-performance liquid chromatography and cyclodextrin-modified micellar electrokinetic chromatographyrdquo Journal ofthe Iranian Chemical Society vol 6 no 2 pp 334ndash340 2009



[41] I Gulcin Z Huyut M Elmastas and H Y Aboul-EneinldquoRadical scavenging and antioxidant activity of tannic acidrdquoArabian Journal of Chemistry vol 3 no 1 pp 43ndash53 2010

[42] C Lu S Yao and N Lin ldquoStudies on reactions of oxidizingsulfur-sulfur three-electron-bond complexes and reducing 120572-amino radicals derived from OH reaction with methionine inaqueous solutionrdquo Biochimica et Biophysica Acta vol 1525 no1-2 pp 89ndash96 2001

[43] L Pari and A M Jalaludeen ldquoProtective role of sinapic acidagainst arsenicmdashinduced toxicity in ratsrdquo Chemico-BiologicalInteractions vol 194 no 1 pp 40ndash47 2011

[44] K L Nyam M M Wong K Long and C P Tan ldquoOxidativestability of sunflower oils supplemented with kenaf seedsextract roselle seeds extract and roselle extract respectivelyunder accelerated storagerdquo International Food Research Journalvol 20 no 2 pp 695ndash701 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Zoology


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from the solid phase to the solvent [9 10] Kenaf seed oilhas normally been extracted by Soxhlet extraction [11] andsupercritical carbon dioxide fluid extraction [12] Howeverthere have been limited studies on the extraction of kenaf seedextract Yusri et al [13] have extracted phenolic compoundsfrom kenaf seed by normal solvent extraction There is noinformation regarding the ultrasonic-assisted extraction ofkenaf seeds Therefore pulse ultrasound-assisted extraction(PUAE) was chosen to extract kenaf seeds in this studyPUAE required lower electrical energy consumption highextraction time reduction higher antioxidant yield andincreased antioxidant activity

The objectives of this study were to determine thebest parameters (ethanol concentration and extractiontime) for extracting phenolic-enriched kenaf seeds bypulsed ultrasonic-assisted extraction (PUAE) to compare theantioxidant properties among different extraction parame-ters and to determine the phenolic compounds in kenaf seedextract which were extracted by using the best parameters

2 Materials and Methods

21 Sample Dried kenaf (Hibiscus cannabinus L) seed wasobtained from the Malaysian Agricultural Research andDevelopment Institute (MARDI Serdang Malaysia) and wasground into powder with a grinder (SHARP Japan) Thekenaf seed powder particle size was 1mm

22 Sample Extraction

221 Pulsed Ultrasound-Assisted Extraction (PUAE) Nine500mL Scott bottles were prepared Each set of three bottleswas filled with 60 80 and 100 ethanol Fifty gramsof ground kenaf seed was added to the solvent to undergoultrasonic extraction (Ultrasonic Homogeniser Labsonic P400W Sartorius AG) with a 5min pulse duration period and5min pulse interval period A 5min pulse duration periodand 5min pulse interval period were considered as 1 extrac-tion cycle Extractions were carried out with 1 extractioncycle 2 extraction cycles and 3 extraction cycles for eachethanol concentration The resulting kenaf seed extract wascentrifuged at 3500 rpm for 10min The supernatant of thekenaf seed extract was collected and underwent filtration andthe pellet was discarded The filtered extract was evaporatedby a rotary evaporator (Rotavapor R-200 Buchi Switzerland)and left in the centrifuge tube which was wrapped withaluminium foil

23 Antioxidant Activities

231 22-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scav-enging Capacity Assay The 22-diphenyl-1-picrylhydrazyl(DPPH) radical scavenging capacity assay for all nine kenafseed extracts was determined according to Liu et al [14] withslight modifications The kenaf seed extract (200120583L) withconcentration of 1mgmLwas added to 28mL of ethanol and1mL of 0004 of DPPH solution After 30min of incuba-tion the absorbance was measured against a blank reagent

(ethanol) at 517 nm by using a UV-Vis spectrophotometer(Model XTD 5 Secomam Domont France) All DPPHradical scavenging activities of the KSE were expressed aspercentage inhibition Inhibition percentage was calculatedby using the formula below

Inhibition percentage (IP) = (blank minus KSE)blank

times 100 (1)

where blank and KSEwere the absorbance values of the blankand the KSE

232 221015840-Azino-bis(3-ethylbenzothiazoline-6-sulphonicAcid) (ABTS) Radical Scavenging Assay ABTS was carriedout according to Floegel et al [15] with slight modificationsABTS (7mM) and 245mM of potassium persulphate(K2O8S2) were mixed and kept in the dark at room

temperature for 12ndash16 h for activation The activated mixturewas then adjusted with ethanol at 734 nm to 07 AU (plusmn002)Fifty milliliters of the sample was added to 1950120583L of ABTSand allowed to react for 6min The absorbance at 734 nmwas measured by using a UV-Vis spectrophotometer (ModelXTD 5 Secomam Domont France) against a blank reagent

All ABTS radical scavenging activities of the KSE wereexpressed as percentage inhibition Inhibition percentagewascalculated by using the formula below

Inhibition Percentage (IP) = (blank minus KSE)blank

times 100 (2)

where blank and KSE are the absorbance values of the blankand the KSE

233 120573-Carotene Bleaching (BCB) The 120573-carotene bleachingactivity of the extracts was evaluated by the 120573-carotenelinoleate model system [16] A solution of 120573-carotene wasprepared by dissolving 120573-carotene (5mg) in chloroform(50mL) Three milliliters of this solution was pipetted intoa round-bottom flask Then the chloroform was removedat 40∘C under vacuum The linoleic acid (40mg) Tween 20emulsifier (400mg) and distilled water (100mL) were addedto the flask with vigorous shaking An aliquot (3mL) of thisemulsion was transferred into different test tubes containingdifferent concentrations of the extracts (01mL) The tubeswere shaken and incubated at 50∘C for 30min in a waterbath As soon as the emulsion was added to each tube thezero time absorbance was measured at 470 nm using a UV-Vis spectrophotometer (Model XTD 5 Secomam DomontFrance) The BCB antioxidant activity was expressed as inhi-bition percentage with reference to the control after 30minstandardized incubation time with the following formula

AA = 100 times(DRcontrol minus DRsample)

DRcontrol (3)

where AA is the antioxidant activity DRcontrol is the degra-dation rate of the control = [(119886119887)30] DRsample is thedegradation rate in the presence of the sample = [(119886119887)30]where 119886 is the absorbance at zero time (first result recorded)while 119887 is the absorbance at 30min (second result recorded)







3)











(mgGAE100 g)TFC

(mgCE100 g)FRAP


Time(min)































4 Conclusion




Acknowledgment


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







3)











(mgGAE100 g)TFC

(mgCE100 g)FRAP


Time(min)































4 Conclusion




Acknowledgment


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




(mgGAE100 g)TFC

(mgCE100 g)FRAP


Time(min)































4 Conclusion




Acknowledgment


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















4 Conclusion




Acknowledgment


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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