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Page 1: Research Article First Evaluation of the Biologically Active ...We investigated the biologically active substances contained in RVA (regrowth velvet antler) by comparing the composition

Research ArticleFirst Evaluation of the Biologically Active Substances andAntioxidant Potential of Regrowth Velvet Antler by means ofMultiple Biochemical Assays

Yujiao Tang123 Byong-Tae Jeon12 Yanmei Wang3 Eun-Ju Choi4 Pyo-Jam Park25

Hye-Jin Seong12 Sang Ho Moon12 and Eun-Kyung Kim12

1Division of Food Bio Science College of Biomedical and Health Sciences Konkuk University Chungju 380-701 Republic of Korea2Korea Nokyong Research Center Konkuk University Chungju 380-701 Republic of Korea3Jilin Sino-ROK Institute of Animal Science Changchun 130-600 China4Division of Sport Science College of Biomedical and Health Sciences Konkuk University Chungju 380-701 Republic of Korea5Department of Biotechnology Konkuk University Chungju 380-701 Republic of Korea

Correspondence should be addressed to Sang Ho Moon moon0204kkuackr and Eun-Kyung Kim eunkyungkimkkuackr

Received 31 May 2015 Accepted 2 July 2015

Academic Editor Patricia Valentao

Copyright copy 2015 Yujiao Tang 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

We investigated the biologically active substances contained in RVA (regrowth velvet antler) by comparing the composition ofbiologically active substances and antioxidant potential of different antler segments RVA was subjected to extraction using DW(distilled water) RVA was divided into 3 segments T-RVA (top RVA) M-RVA (middle RVA) and B-RVA (base RVA) The T-RVAsection possessed the greatest amounts of uronic acid (36251mgg) sulfated GAGs (sulfated glycosaminoglycans) (55576mgg)sialic acid (111276mgg) uridine (0957mgg) uracil (1084mgg) and hypoxanthine (12631mgg) In addition the T-RVA sectionpossessed the strongest antioxidant capacity as determined by DPPH H

2O2(hydrogen peroxide) hydroxyl and ABTS (221015840-

azinobis-3-ethylbenzthiazoline-6-sulphonate) radical scavenging activity as well as FRAP (ferric reducing antioxidant power) andORAC (oxygen radical absorbance capacity) The values of those were 5344 2309 3412 6031 and 3581 TE120583M at 1mgmLand 11357 TE120583M at 20120583gmL These results indicate that the T-RVA section possesses the greatest amount of biologically activesubstances and highest antioxidant potential This is the first report on the biologically active substances and antioxidant potentialof RVA

1 Introduction

Velvet antler consists of the cartilaginous prequalified antlersof moose elk and sika deer which regrow yearlyThe growthof deer antlers is one of the fastest types of tissue growth inmammals Growing antlers contain nerves and blood vesselsand are covered with a hairy skin covering tissue commonlyknown as ldquovelvetrdquo [1] Velvet antler is a widely used traditionalAsian medicine that has been used clinically in East Asia formillennia to treat various diseases and as a tonic [2] Velvetantler is generally harvested twice per year The first velvetantler harvest occurs after 40ndash45 days of growth while asecond harvest occurs after 50ndash55 days of regrowth at whichpoint the harvested velvet antler is known as RVA Although

studies have been conducted on the chemical composition ofRVA [3 4] there have been no comprehensive reports on thecomposition of biologically active substances and antioxidantpotential of RVA

Numerous studies have demonstrated that free radicalsare generated by oxidative damage to biomolecules suchas lipids nucleic acids proteins and carbohydrates [5ndash7] Overproduction of free radicals and reactive oxygenspecies is believed to be associated with cellular and tissuepathogenesis which leads to several chronic diseases suchas cancer diabetes mellitus and neurodegenerative andinflammatory diseases [8] Manymedical reports and clinicalobservations convincingly show that disease-resistance canbe conferred by enhancing antioxidative processes [9ndash14]

Hindawi Publishing CorporationJournal of ChemistryVolume 2015 Article ID 975292 7 pageshttpdxdoiorg1011552015975292

2 Journal of Chemistry

Therefore antioxidant supplementation could prevent orinhibit oxidative stress induced by ROS Antioxidants ter-minate free radical chain reactions by removing free radicalintermediates while inhibiting other oxidation reactionsBecause of the clinical potential of antioxidants significantinterest has been focusing on the development of naturalantioxidants that are safe and effective

In this study RVA was subjected to extraction by DWto allow determination of its constituent biologically activesubstances including uronic acid sulfated GAGs sialic aciduracil hypoxanthine and uridine In addition the antioxi-dant activities of RVA were determined by assessing DPPHH2O2 hydroxyl and ABTS radical scavenging activity as well

as FRAP and ORAC

2 Materials and Methods

21 Materials Seven specimens of sika deer (Cervus nip-pon) RVA were collected at the same farm (Fanrong farmChina) Carbazole sodium tetraborate dimethylmethyleneblue glycine sodium thiosulfate acetoacetanilide uracilhypoxanthine uridine DPPH ABTS potassium persulfateTPTZ (246-tris(2-pyridyl)-s-triazine) FL and AAPH (221015840-azobis(2-amidinopropane) dihydrochloride) were purchasedfrom Sigma-Aldrich (St Louis MO USA)

22 Preparation of Samples The RVA specimens weredivided into 3 sections T-RVA M-RVA and B-RVA lyophil-ized and homogenized with a grinder Next 10 g of eachsegment was added to 100mL of DW and subjected toextraction in boiling DW for 1 h The RVA extracts werefiltered (025 120583m pore size) and lyophilized (yields T-RVA387 M-RVA 361 B-RVA 266) in a freeze dryer for 5days

23 Analysis of Bioactive Compounds

231 Uronic Acid Uronic acid content was determined bythe carbazole reaction [15] Briefly a 50 120583L serial dilutionof the standards or samples was placed in a 96-well plateafter which 200120583L of 25mM sodium tetraborate in sulfuricacid was added to each well The plate was heated for 10minat 100∘C in an oven After cooling at room temperature for15min 50 120583L of 0125 carbazole in absolute ethanol wascarefully added After heating at 100∘C for 10min in an ovenand cooling at room temperature for 15min the plate wasread in a microplate reader at a wavelength of 550 nm

232 Sulfated GAGs GAGs content was determined by theDMB (dimethylmethylene blue) dye binding method [16]Briefly the color reagent was prepared by dissolving 0008 gof DMB in a solution containing 1185 g NaCl 1520 g glycine047mLHCl (12M) and 500mLDWEach samplewasmixedinto 1mL of color reagent and the absorbance was readimmediately at 525 nm

233 Sialic Acid Sialic acid content was determined basedon the procedures described by Matsuno and Suzuki [17] Allsolutions were precooled in an ice bath Sodium periodate

solution (10mM 20120583L) was added to 200120583L of a glycoconju-gate sample in a 15mL polypropylene test tube The solutionwas chilled in an ice bath for 45minThe reaction was termi-nated by the addition of 100 120583L of 50mM sodium thiosulfatesolution Next 500 120583L of 40M ammonium acetate (pH 75)and 400 120583L of ethanolic solution of 100mM acetoacetanilidewere added to the solution which was left standing for10min at room temperature The fluorescence intensity ofthe solution was measured at 471 nm with an excitationwavelength of 388 nm

234 Uracil Hypoxanthine and Uridine Uracil hypoxan-thine and uridine were determined as described previously[18] 1mg of the DW extract was dissolved in 1mL of3 methanol solution after which 1mL of the resultingsolution was filtered for HPLC analysis The analysis wasperformed on an HPLC system equipped with an isocraticpump (Kyoto Japan) and RI (refractive index) detector (LabAlliance model 500) The separation was conducted on aZORBAX Eclipse Plus C18 column (46 times 150mm 5 120583mAgilent Technologies USA) The mobile phase was 007acetic acid methanol water (3 97 vv pH 35) at a flow rateof 10mLmin A series of standards of uracil hypoxanthineand uridine in the range of 0625ndash4000 ppm were preparedin the mobile phase Quantification was carried out by inte-gration of the peak areas using external standard calibrationA linear response with a correlation coefficient of 0999(119899 = 6) was obtained for the standards For all experimentsthe extracts and standards were filtered through a 045 120583mcellulose ester membrane before injection into the HPLCsystem Detection was performed at a wavelength of 254 nm

24 Antioxidant Activity

241 DPPHRadical Scavenging Activity TheDPPH scaveng-ing activity of each antler extract was measured accordingto a slightly modified version of the method of Blois [19]DPPH solutions (15 times 10minus4M 100 120583L) were mixed with andwithout each extract (100 120583L) after which the mixtures wereincubated at room temperature for 30min After standingfor 30min absorbance was recorded at 540 nm using amicroplate readerThe scavenging activity was calculated as apercentage using the following equation

Inhibition () =(119860control minus 119860 sample)

119860controltimes 100 (1)

where 119860control was the absorbance of the reaction mixturewithout an RVA sample and 119860 sample was the absorbance ofthe reaction mixture with an RVA sample

242 Hydrogen Peroxide Radical Scavenging Activity Hydro-gen peroxide scavenging activity was determined accordingto the method of Muller [20] A 100 120583L of 01M phosphatebuffer (pH 50) wasmixedwith each extract in a 96-microwellplate A 20 120583L of hydrogen peroxide was added to themixture and then incubated at 37∘C for 5min After theincubation 30120583L of 125mM ABTS and 30 120583L of peroxidase(1 unitmL) were added to the mixture and then incubated

Journal of Chemistry 3

at 37∘C for 10minThe absorbancewas recorded at 405 nmbymicroplate reader and the percentage of scavenging activitywas calculated using (1)

243 Hydroxyl Radical Scavenging Activity The hydroxylradical scavenging activity of each antler extract was deter-mined according to themethod of Chung et al [21] Hydroxylradicals were generated by the Fenton reaction in the pres-ence of FeSO

4 A reaction mixture containing 01mL of

10mM FeSO4 10mM EDTA and 10mM 2-deoxyribose was

mixed with 01mL of the extract solution after which 01Mphosphate buffer (pH 74) was added to the reaction mixtureto reach a total volume of 09mL Subsequently 01mL of10mM H

2O2was added to the reaction mixture which was

incubated at 37∘C for 4 h After incubation 05mL of 28TCA and 10 TBA were added to each mixture after whicheach mixture was placed in a boiling water bath for 10minAbsorbance was measured at 532 nm Hydroxyl radical scav-enging activity was calculated as a percentage using (1)

244 ABTS Radical Scavenging Activity The ABTS scaveng-ing activity of each antler extract was assessed following themethod of Arnao et al [22] Stock solutions included ABTS∙+solution and potassium persulfate solutions A workingsolutionwas prepared bymixing the 2 stock solutions in equalquantities and allowing them to react for 12 h The workingsolution was diluted with fresh ABTS∙+ solution and mixedwith or without each extract After incubation for 2 h theabsorbance of each solution was recorded at 735 nm Thescavenging activity was calculated as a percentage using (1)

245 FRAP Assay The FRAP assay was performed accord-ing to the method of Benzie and Strain [23] Fresh work-ing solution was prepared by mixing acetate buffer TPTZsolution and FeCl

3sdot6H2O solution and warmed at 37∘C

before use Each extract was allowed to react with the FRAPsolution in a dark room at room temperature for 30minTheabsorbance of the colored product was measured at 595 nmScavenging activity was calculated as a percentage using (1)

246 ORAC Assay For ORAC assay the method of Ou et alwas used with some slightly modification [24] The workingsolution of FL and AAPH radical was prepared daily Sampleblank or standard was placed in 96-microwell plate and theplate was heated to 37∘C for 15min prior to the additionof AAPH The fluorescence was measured immediately afterthe AAPH addition and measurements with fluorescencefilters for an excitationwavelength of 485 nm and an emissionwavelength of 535 nm were taken every 5min until therelative fluorescence intensity was less than 5 of the valueof the initial reading

The ORAC values expressed as 120583M Trolox equivalents(120583MTEmg) were calculated by applying the following for-mula

ORAC (120583MTE)

=(119862Trolox times (AUCsample minus AUCblank) times 119896)

(AUCsample minus AUCblank)

(2)

where 119862Trolox is the concentration of Trolox (20120583M) 119896 isthe sample dilution factor and AUC is the area below thefluorescence decay curve of the sample blank and Troloxrespectively calculated by applying the following formula in aMicrosoft Excel spreadsheet (Microsoft Washington USA)

AUC = (05 +1198915

1198910

+11989110

1198910

+ sdot sdot sdot + 119891119899+5

1198910

) times 5 (3)

where 1198910is the initial fluorescence and 119891

119899is the fluorescence

at time 119899

25 Statistical Analysis The results shown are summaries ofthe data from at least 3 experiments All data are presented asmeanplusmn SEM (standard error of themean) Statistical analyseswere performed using SAS statistical software (SAS InstituteInc Cary NC USA) Treatment effects were analyzed usingone-way ANOVA followed by Dunnettrsquos multiple range testResults of 119875 lt 005 indicated statistical significance

3 Results and Discussion

31 Bioactive Composition Thebiologically active substancescontained in the 3 RVA segments including uronic acidsulfated GAGs sialic acid uridine uracil and hypoxanthineare listed in Tables 1 and 2

The uronic acid content sulfated GAGs content andsialic acid content of the T-RVA and M-RVA sections weresignificantly greater than those of the B-RVA section (119875 lt005) The DW extract of the T-RVA section contained3625mgg uronic acid 55576mgg sulfated GAGs and11128mgg sialic acid (Table 1) The DW extract of theRVA contained 0957mgg uridine 1084mgg uracil and1263mgg hypoxanthine (Table 2)

Uronic acid has been reported to improve circulation anddecrease stroke risk [25] therefore our chemical analysesindicate that the DW extract of T-RVA might possess sim-ilar activities Sulfated GAGs particularly CS (chondroitinsulfate) are of particular interest to physicians and phar-macists Sulfated GAGs are composed of units of aminosugar including D-glucosamine and D-galactosamine andbond with core proteins to form proteoglycans Cartilageproteoglycans regulate water retention and are integral to thedifferentiation and proliferation of chondrocytes The mostprominent sulfated GAG in velvet antler tissue is chondroitinsulfate [26] Sialic acid is a water soluble component thatwas efficiently extracted by DW and showed significantaccumulation in the T-RVA section Our findings are similarto those of a previous report [27] which showed that the T-RVA or ldquowax piecerdquo contains sialic acid levels higher thanthose of other antler regions Uracil is a primary mediatorof MAO (monoamine oxidase) inhibition by velvet antlerextract [28] Our data indicate that the T-RVA section maycontribute the majority of the inhibitory effect on MAOactivity produced by velvet antler In a report by Wang et al[29] aimed at identifying the active compound in velvetantler responsible for inhibiting MAO-B activity the authorsuggested that the main antiaging compound in velvet antleris hypoxanthine Zhou et al [28] showed that uridine was

4 Journal of Chemistry

Table 1 Sulfated GAGs sialic acid and uronic acid contents ofregrowth velvet antler extracts

T-RVA M-RVA B-RVAmgg mgg mgg

Sulfated GAGs 55576 plusmn 1248 36930 plusmn 1981 22898 plusmn 2442Sialic acid 11128 plusmn 427 7950 plusmn 520 7296 plusmn 150Uronic acid 3625 plusmn 296 2590 plusmn 229 2311 plusmn 240

Table 2 Hypoxanthine uridine and uracil contents of regrowthvelvet antler extracts

T-RVA M-RVA B-RVAmgg mgg mgg

Hypoxanthine 108 plusmn 003 100 plusmn 001 092 plusmn 001Uridine 126 plusmn 003 114 plusmn 004 106 plusmn 003Uracil 096 plusmn 008 0092 plusmn 003 0845 plusmn 004

responsible for 3475 of the Fe2+-chelating activity of velvetantler Therefore the DW extract of T-RVA is expected toshow strong antioxidant activity due to its abundance ofuridine Zhou and Li [18] investigated the amounts of uridineuracil and hypoxanthine from ethanol extracted velvet antlerand the values were 37 36 and 39mgg respectively Theirvalues were higher than ours This may be attributed toextraction method

32 Antioxidant Activity The antioxidant activities of RVAmay not be attributed to a single mechanism Therefore6 methods were used to evaluate different aspects of theantioxidant activities of RVA

The antioxidant activities of the DWextracts of RVAwereevaluated by assessing DPPH H

2O2 ABTS and hydroxyl

radical scavenging activity In addition FRAP and ORACwere estimated

The antioxidant activity of the DW extract of T-RVA wassignificantly better than those of the M-RVA and B-RVAsections (119875 lt 005) and appeared to be dose-dependent TheDPPH radical scavenging activity was highest for the T-RVAsection (5344 120583MTEmg IC

500853mgmL) and lowest

for the B-RVA section (Figure 1) H2O2(3220 120583MTEmg

Figure 2) and ABTS (6031 120583MTEmg Figure 3) radicalscavenging activities were also highest for the T-RVA sec-tion The hydroxyl radical scavenging activity was high-est for the T-RVA section (2309120583MTEmg) whereas theactivities of M-RVA and B-RVA were similar (Figure 4)The T-RVA section was the most effective section inthe FRAP assay (3581 120583MTEmg) whereas the activitiesof B-RVA and M-RVA were similar (Figure 5) In theORAC assay 1000mgmL T-RVA showed excellent activity(12158 120583MTEmg) (Figure 6)

DPPH radical scavenging activity is often used as amethod of evaluating antioxidant activity DPPH is a stableradical that accepts an electron andor hydrogen radical fromdonor molecules to form a stable diamagnetic moleculeTherefore the extracts of velvet antler may have provided anelectron andor hydrogen radical to neutralize DPPH [30] Ina report by Lee and Chung [31] the DPPH radical scavenging

0

10

20

30

40

50

60

T-RVA M-RVA B-RVA

DPP

H ra

dica

l sca

veng

ing

activ

ity

a

b

c

e

h

i

d

f

h

c

e

g

(120583M

TE

mg)

Figure 1 The effect of RVA on DPPH radical scavenging activityandashiValues not sharing a common letter are significantly different at119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000 120583gmL

0

5

10

15

20

25

30

ab

d

e

dd

eg

i hg

j

H2O

2ra

dica

l sca

veng

ing

activ

ity

T-RVA M-RVA B-RVA

(120583M

TE

mg)

Figure 2 The effect of RVA on H2O2radical scavenging activity

andashiValues not sharing a common letter are significantly different at119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000 120583gmL

0

5

10

15

20

25

30

35

40

T-RVA M-RVA B-RVA

ab

c

ddc

e

f gg g g

Hyd

roxy

l rad

ical

scav

engi

ng ac

tivity

(120583

M T

Em

g)

Figure 3The effect of RVA on hydroxyl radical scavenging activityandashgValues not sharing a common letter are significantly differentat 119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000 120583gmL

Journal of Chemistry 5

0

10

20

30

40

50

60

70

ab

cddd

e ef

fg

h

ABT

S ra

dica

l sca

veng

ing

activ

ity(120583

M T

Em

g)

T-RVA M-RVA B-RVA

Figure 4 The effect of RVA on ABYS radical scavenging activityandashgValues not sharing a common letter are significantly differentat 119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000120583gmL

0

5

10

15

20

25

30

35

40

T-RVA M-RVA B-RVA

a

b bd

de

e fgg g

hFRA

P (120583

M T

Em

g)

Figure 5 The effect of RVA on FRAP assay andashgValues not sharing acommon letter are significantly different at 119875 lt 005 by Dunnettrsquosmultiple range tests Light gray square 125 120583gmL gray square250 120583gmL dark grey square 500120583gmL black square 1000 120583gmL

0

20

40

60

80

100

120

140

T-RVA M-RVA B-RVA

a

bcc

b

d

fg

fee e

ORA

C (120583

M T

Em

g)

Figure 6 The effect of RVA on ORAC andashgValues not sharing acommon letter are significantly different at 119875 lt 005 by Dunnettrsquosmultiple range tests Light gray square 125 120583gmL gray square250 120583gmL dark grey square 500120583gmL black square 1000 120583gmL

activity of velvet antler extract obtained from the uppersection was reported to be 671 at an extract concentrationof 100mgmL which was lower than the activity measuredin our analysis H

2O2is a reactive nonradical and a clinically

important compound due to its ability to penetrate biologicalmembranes H

2O2can be converted into more reactive

species such as singlet oxygen and hydroxyl radicals therebycausing lipid peroxidation or toxicity to cells Thereforescavenging of hydrogen peroxide can decrease prooxidantsrsquolevels Our analysis of H

2O2scavenging by velvet antler

produced results similar to those reported by Je et al [30]Hydroxyl radicals are extremely reactive and easily reactwith amino acids DNA and membrane components In thisstudy the hydroxyl radical scavenging activity of RVA washigher than that of velvet antler as reported by Je et al[32] In addition our analysis of ABTS radical scavengingactivity by RVA identified activity higher than that reportedby Zhao et al [33] The FRAP assay treats the antioxidantscontained in the samples as reductants in a redox-linkedcolorimetric reaction allowing assessment of the reducingpower of antioxidants [34] Zhao et al [33] reported activityof 858 plusmn 002 by 5mgmL velvet antler extract in the FARPassay which was lower than the activity measured in ouranalysis The ORAC assay has been applied extensively toevaluate the antioxidant activities of fruits vegetables leavesstems herbs and spices As a result the ORAC assay iscommonly mentioned in scientific publications and healthfood publications [35] However the antioxidant activity ofRVAhas not been evaluated using theORACassayThereforethis is the first report of an assessment of the antioxidantactivity of RVA using the ORAC assay ORAC value of gallicacid was shown 161 plusmn 48 by Zulueta et al [36] which washigher than the activity of RVA found in our study

4 Conclusions

In the present study we provided the first comprehensiveevaluation of the biologically active substances of RVA andthe antioxidant potential of different RVA segments Futurestudies are required to further elucidate the other biologicalactivities of the T-RVA M-RVA and B-RVA sections and thebiological mechanisms underlying their effects

Conflict of Interests

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

Acknowledgment

This paper was supported by Konkuk University in 2015

References

[1] C Li ldquoDevelopment of deer antler model for biomedicalresearchrdquo Recent Advancesamp Research Updates vol 4 no 2 pp255ndash274 2003

[2] Z Q Zhang Y Zhang B X Wang H O Zhou Y Wangand H Zhang ldquoPurification and partial characterization of

6 Journal of Chemistry

anti-inflammatory peptide from pilose antler of Cervus nipponTemminckrdquo Acta Pharmaceutica Sinica vol 27 no 5 pp 321ndash324 1992

[3] Z F Ma D S Zhao Q D Zhou and S Sun ldquoStudy onraising yield of reproductive pilose antler of sika deerrdquo Journalof Northeast Forestry University vol 22 no 6 pp 41ndash47 1994

[4] J F He Y Z Liu S B Zhu and L Shang ldquoStudy on increasingquality and quantity of reborn antlers of cervus elaphuslinnaeusand cervus Nippon hortulorun swinchonerdquo Natural ScienceJournal of Harbin Normal University vol 20 no 4 pp 91ndash942004

[5] D A Butterfield A Castegna C M Lauderback and J DrakeldquoEvidence that amyloid beta-peptide-induced lipid peroxida-tion and its sequelae in Alzheimerrsquos disease brain contribute toneuronal deathrdquo Neurobiology of Aging vol 23 no 5 pp 655ndash664 2002

[6] W A Pryor and N Y Ann ldquoFree radical biology xenobioticscancer and agingrdquo Annals of the New York Academy of Sciencesvol 393 no 1 pp 1ndash22 1982

[7] E-K Kim S-J Lee J-W Hwang et al ldquoIn vitro investigationon antioxidative effect of Inonotus obliquus extracts againstoxidative stress on PC12 cellsrdquo Journal of the Korean Society forApplied Biological Chemistry vol 54 no 1 pp 112ndash117 2011

[8] J-WHwang E-K Kim S-J Lee et al ldquoAntioxidant activity andprotective effect of anthocyanin oligomers on H

2O2-triggered

G2M arrest in retinal cellsrdquo Journal of Agricultural and FoodChemistry vol 60 no 17 pp 4282ndash4288 2012

[9] B R Bhavnani ldquoPharmacology of hormonal therapeuticagentsrdquo in The Menopause Comprehensive Management B AEskin Ed pp 229ndash256 Parthenon Press New York NY USA2000

[10] S S Ditchkoff L J Spicer R E Masters and R L LochmillerldquoConcentrations of insulin-like growth factor-I in adult malewhite-tailed deer (Odocoileus virginianus) associations withserum testosterone morphometrics and age during and afterthe breeding seasonrdquo Comparative Biochemistry and PhysiologyA Physiology vol 129 no 4 pp 887ndash895 2001

[11] J A Gomez A J Garcıa T Landete-Castillejos and L GallegoldquoEffect of advancing births on testosterone until 25 years of ageand puberty in Iberian red deer (Cervus elaphus hispanicus)rdquoAnimal Reproduction Science vol 96 no 1-2 pp 79ndash88 2006

[12] C Li Z Jiang G Jiang and J Fang ldquoSeasonal changes ofreproductive behavior and fecal steroid concentrations in PereDavidrsquos deerrdquo Hormones and Behavior vol 40 no 4 pp 518ndash525 2001

[13] Y-J Li T-H Kim H B Kwak Z H Lee S-Y Lee and G-J Jhon ldquoChloroform extract of deer antler inhibits osteoclastdifferentiation and bone resorptionrdquo Journal of Ethnopharma-cology vol 113 no 2 pp 191ndash198 2007

[14] H H Sunwoo T Nakano and J S Sim ldquoEffect of water-solubleextract from antler of wapiti (Cervus elaphus) on the growth offibroblastsrdquo Canadian Journal of Animal Science vol 77 no 2pp 343ndash345 1997

[15] M Cesaretti E Luppi FMaccari andN Volpi ldquoA 96-well assayfor uronic acid carbazole reactionrdquo Carbohydrate Polymers vol54 no 1 pp 59ndash61 2003

[16] RW Farndale D J Buttle and A J Barrett ldquoImproved quanti-tation and discrimination of sulphated glycosaminoglycans byuse of dimethylmethylene bluerdquo Biochimica Biophysica Actavol 883 no 2 pp 173ndash177 1986

[17] K Matsuno and S Suzuki ldquoSimple fluorimetric method forquantification of sialic acids in glycoproteinsrdquo Analytical Bio-chemistry vol 375 no 1 pp 53ndash59 2008

[18] R Zhou and S F Li ldquoIn vitro antioxidant analysis andcharacterisation of antler velvet extractrdquo Food Chemistry vol114 no 4 pp 1321ndash1327 2009

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

[20] H E Muller ldquoDetection of hydrogen peroxide produced bymicroorganism on ABTS-peroxidase mediumrdquo Zentralblatt furBakteriologieMikrobiologie undHygiene vol 259 no 2 pp 151ndash158 1985

[21] S-K Chung T Osawa and S Kawakishi ldquoHydroxyl radical-scavenging effects of spices and scavengers frombrownmustard(Brassica nigra)rdquo Bioscience Biotechnology and Biochemistryvol 61 no 1 pp 118ndash123 1997

[22] M B Arnao A Cano and M Acosta ldquoThe hydrophilicand lipophilic contribution to total antioxidant activityrdquo FoodChemistry vol 73 no 2 pp 239ndash244 2001

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

[24] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal ofAgricultural and Food Chemistry vol 49 no 10 pp 4619ndash46262001

[25] M A Moskowitz E H Lo and C Iadecola ldquoThe science ofstrokemechanisms in search of treatmentsrdquoNeuron vol 67 no2 pp 181ndash198 2010

[26] Y W Ha B T Jeon S H Moon et al ldquoCharacterization ofheparan sulfate from the unossified antler of Cervus elaphusrdquoCarbohydrate Research vol 340 no 3 pp 411ndash416 2005

[27] B Jeon S Kim S Lee et al ldquoEffect of antler growth period onthe chemical composition of velvet antler in sika deer (Cervusnippon)rdquoMammalian Biology vol 74 no 5 pp 374ndash380 2009

[28] R Zhou JWang S Li andY Liu ldquoSupercritical fluid extractionof monoamine oxidase inhibitor from antler velvetrdquo Separationand Purification Technology vol 65 no 3 pp 275ndash281 2009

[29] B X Wang X H Zhao X W Yang et al ldquoInhibition oflipid peroxidation of deer antler (Rokujo) extract in vivo andin vitrordquo Journal of Medical and Pharmaceutical Society forWAKAN-YAKU vol 5 pp 123ndash128 1988

[30] J Y Je P J Park D H Lim B T Jeon K H Kho and C B AhnldquoAntioxidant anti-acetylcholinesterase and composition of bio-chemical components of Russian deer velvet antler extractsrdquoKorean Journal for Food Science of Animal Resources vol 31 no3 pp 349ndash355 2011

[31] K A Lee andH Y Chung ldquoThebiological activity of deer antlerextract in vitrordquoThe Korean Journal of Food and Nutrition vol20 no 2 pp 114ndash119 2007

[32] J-Y Je P-J Park E-K Kim et al ldquoComposition of biologicallyactive substances and antioxidant activity of New Zealand deervelvet antler extractsrdquoKorean Journal for Food Science of AnimalResources vol 30 no 1 pp 20ndash27 2010

[33] L Zhao Y-C Luo C-T Wang and B-P Ji ldquoAntioxidantactivity of protein hydrolysates from aqueous extract of velvetantler (Cervus elaphus) as influenced by molecular weight andenzymesrdquo Natural Product Communications vol 6 no 11 pp1683ndash1688 2011

Journal of Chemistry 7

[34] S-Q Huang S D Ding and L P Fan ldquoAntioxidant activitiesof five polysaccharides from Inonotus obliquusrdquo InternationalJournal of BiologicalMacromolecules vol 50 no 5 pp 1183ndash11872012

[35] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006

[36] A Zulueta M J Esteve and A Frıgola ldquoORAC and TEACassays comparison to measure the antioxidant capacity of foodproductsrdquo Food Chemistry vol 114 no 1 pp 310ndash316 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

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Advances in

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Analytical Methods in Chemistry

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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

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CatalystsJournal of

Page 2: Research Article First Evaluation of the Biologically Active ...We investigated the biologically active substances contained in RVA (regrowth velvet antler) by comparing the composition

2 Journal of Chemistry

Therefore antioxidant supplementation could prevent orinhibit oxidative stress induced by ROS Antioxidants ter-minate free radical chain reactions by removing free radicalintermediates while inhibiting other oxidation reactionsBecause of the clinical potential of antioxidants significantinterest has been focusing on the development of naturalantioxidants that are safe and effective

In this study RVA was subjected to extraction by DWto allow determination of its constituent biologically activesubstances including uronic acid sulfated GAGs sialic aciduracil hypoxanthine and uridine In addition the antioxi-dant activities of RVA were determined by assessing DPPHH2O2 hydroxyl and ABTS radical scavenging activity as well

as FRAP and ORAC

2 Materials and Methods

21 Materials Seven specimens of sika deer (Cervus nip-pon) RVA were collected at the same farm (Fanrong farmChina) Carbazole sodium tetraborate dimethylmethyleneblue glycine sodium thiosulfate acetoacetanilide uracilhypoxanthine uridine DPPH ABTS potassium persulfateTPTZ (246-tris(2-pyridyl)-s-triazine) FL and AAPH (221015840-azobis(2-amidinopropane) dihydrochloride) were purchasedfrom Sigma-Aldrich (St Louis MO USA)

22 Preparation of Samples The RVA specimens weredivided into 3 sections T-RVA M-RVA and B-RVA lyophil-ized and homogenized with a grinder Next 10 g of eachsegment was added to 100mL of DW and subjected toextraction in boiling DW for 1 h The RVA extracts werefiltered (025 120583m pore size) and lyophilized (yields T-RVA387 M-RVA 361 B-RVA 266) in a freeze dryer for 5days

23 Analysis of Bioactive Compounds

231 Uronic Acid Uronic acid content was determined bythe carbazole reaction [15] Briefly a 50 120583L serial dilutionof the standards or samples was placed in a 96-well plateafter which 200120583L of 25mM sodium tetraborate in sulfuricacid was added to each well The plate was heated for 10minat 100∘C in an oven After cooling at room temperature for15min 50 120583L of 0125 carbazole in absolute ethanol wascarefully added After heating at 100∘C for 10min in an ovenand cooling at room temperature for 15min the plate wasread in a microplate reader at a wavelength of 550 nm

232 Sulfated GAGs GAGs content was determined by theDMB (dimethylmethylene blue) dye binding method [16]Briefly the color reagent was prepared by dissolving 0008 gof DMB in a solution containing 1185 g NaCl 1520 g glycine047mLHCl (12M) and 500mLDWEach samplewasmixedinto 1mL of color reagent and the absorbance was readimmediately at 525 nm

233 Sialic Acid Sialic acid content was determined basedon the procedures described by Matsuno and Suzuki [17] Allsolutions were precooled in an ice bath Sodium periodate

solution (10mM 20120583L) was added to 200120583L of a glycoconju-gate sample in a 15mL polypropylene test tube The solutionwas chilled in an ice bath for 45minThe reaction was termi-nated by the addition of 100 120583L of 50mM sodium thiosulfatesolution Next 500 120583L of 40M ammonium acetate (pH 75)and 400 120583L of ethanolic solution of 100mM acetoacetanilidewere added to the solution which was left standing for10min at room temperature The fluorescence intensity ofthe solution was measured at 471 nm with an excitationwavelength of 388 nm

234 Uracil Hypoxanthine and Uridine Uracil hypoxan-thine and uridine were determined as described previously[18] 1mg of the DW extract was dissolved in 1mL of3 methanol solution after which 1mL of the resultingsolution was filtered for HPLC analysis The analysis wasperformed on an HPLC system equipped with an isocraticpump (Kyoto Japan) and RI (refractive index) detector (LabAlliance model 500) The separation was conducted on aZORBAX Eclipse Plus C18 column (46 times 150mm 5 120583mAgilent Technologies USA) The mobile phase was 007acetic acid methanol water (3 97 vv pH 35) at a flow rateof 10mLmin A series of standards of uracil hypoxanthineand uridine in the range of 0625ndash4000 ppm were preparedin the mobile phase Quantification was carried out by inte-gration of the peak areas using external standard calibrationA linear response with a correlation coefficient of 0999(119899 = 6) was obtained for the standards For all experimentsthe extracts and standards were filtered through a 045 120583mcellulose ester membrane before injection into the HPLCsystem Detection was performed at a wavelength of 254 nm

24 Antioxidant Activity

241 DPPHRadical Scavenging Activity TheDPPH scaveng-ing activity of each antler extract was measured accordingto a slightly modified version of the method of Blois [19]DPPH solutions (15 times 10minus4M 100 120583L) were mixed with andwithout each extract (100 120583L) after which the mixtures wereincubated at room temperature for 30min After standingfor 30min absorbance was recorded at 540 nm using amicroplate readerThe scavenging activity was calculated as apercentage using the following equation

Inhibition () =(119860control minus 119860 sample)

119860controltimes 100 (1)

where 119860control was the absorbance of the reaction mixturewithout an RVA sample and 119860 sample was the absorbance ofthe reaction mixture with an RVA sample

242 Hydrogen Peroxide Radical Scavenging Activity Hydro-gen peroxide scavenging activity was determined accordingto the method of Muller [20] A 100 120583L of 01M phosphatebuffer (pH 50) wasmixedwith each extract in a 96-microwellplate A 20 120583L of hydrogen peroxide was added to themixture and then incubated at 37∘C for 5min After theincubation 30120583L of 125mM ABTS and 30 120583L of peroxidase(1 unitmL) were added to the mixture and then incubated

Journal of Chemistry 3

at 37∘C for 10minThe absorbancewas recorded at 405 nmbymicroplate reader and the percentage of scavenging activitywas calculated using (1)

243 Hydroxyl Radical Scavenging Activity The hydroxylradical scavenging activity of each antler extract was deter-mined according to themethod of Chung et al [21] Hydroxylradicals were generated by the Fenton reaction in the pres-ence of FeSO

4 A reaction mixture containing 01mL of

10mM FeSO4 10mM EDTA and 10mM 2-deoxyribose was

mixed with 01mL of the extract solution after which 01Mphosphate buffer (pH 74) was added to the reaction mixtureto reach a total volume of 09mL Subsequently 01mL of10mM H

2O2was added to the reaction mixture which was

incubated at 37∘C for 4 h After incubation 05mL of 28TCA and 10 TBA were added to each mixture after whicheach mixture was placed in a boiling water bath for 10minAbsorbance was measured at 532 nm Hydroxyl radical scav-enging activity was calculated as a percentage using (1)

244 ABTS Radical Scavenging Activity The ABTS scaveng-ing activity of each antler extract was assessed following themethod of Arnao et al [22] Stock solutions included ABTS∙+solution and potassium persulfate solutions A workingsolutionwas prepared bymixing the 2 stock solutions in equalquantities and allowing them to react for 12 h The workingsolution was diluted with fresh ABTS∙+ solution and mixedwith or without each extract After incubation for 2 h theabsorbance of each solution was recorded at 735 nm Thescavenging activity was calculated as a percentage using (1)

245 FRAP Assay The FRAP assay was performed accord-ing to the method of Benzie and Strain [23] Fresh work-ing solution was prepared by mixing acetate buffer TPTZsolution and FeCl

3sdot6H2O solution and warmed at 37∘C

before use Each extract was allowed to react with the FRAPsolution in a dark room at room temperature for 30minTheabsorbance of the colored product was measured at 595 nmScavenging activity was calculated as a percentage using (1)

246 ORAC Assay For ORAC assay the method of Ou et alwas used with some slightly modification [24] The workingsolution of FL and AAPH radical was prepared daily Sampleblank or standard was placed in 96-microwell plate and theplate was heated to 37∘C for 15min prior to the additionof AAPH The fluorescence was measured immediately afterthe AAPH addition and measurements with fluorescencefilters for an excitationwavelength of 485 nm and an emissionwavelength of 535 nm were taken every 5min until therelative fluorescence intensity was less than 5 of the valueof the initial reading

The ORAC values expressed as 120583M Trolox equivalents(120583MTEmg) were calculated by applying the following for-mula

ORAC (120583MTE)

=(119862Trolox times (AUCsample minus AUCblank) times 119896)

(AUCsample minus AUCblank)

(2)

where 119862Trolox is the concentration of Trolox (20120583M) 119896 isthe sample dilution factor and AUC is the area below thefluorescence decay curve of the sample blank and Troloxrespectively calculated by applying the following formula in aMicrosoft Excel spreadsheet (Microsoft Washington USA)

AUC = (05 +1198915

1198910

+11989110

1198910

+ sdot sdot sdot + 119891119899+5

1198910

) times 5 (3)

where 1198910is the initial fluorescence and 119891

119899is the fluorescence

at time 119899

25 Statistical Analysis The results shown are summaries ofthe data from at least 3 experiments All data are presented asmeanplusmn SEM (standard error of themean) Statistical analyseswere performed using SAS statistical software (SAS InstituteInc Cary NC USA) Treatment effects were analyzed usingone-way ANOVA followed by Dunnettrsquos multiple range testResults of 119875 lt 005 indicated statistical significance

3 Results and Discussion

31 Bioactive Composition Thebiologically active substancescontained in the 3 RVA segments including uronic acidsulfated GAGs sialic acid uridine uracil and hypoxanthineare listed in Tables 1 and 2

The uronic acid content sulfated GAGs content andsialic acid content of the T-RVA and M-RVA sections weresignificantly greater than those of the B-RVA section (119875 lt005) The DW extract of the T-RVA section contained3625mgg uronic acid 55576mgg sulfated GAGs and11128mgg sialic acid (Table 1) The DW extract of theRVA contained 0957mgg uridine 1084mgg uracil and1263mgg hypoxanthine (Table 2)

Uronic acid has been reported to improve circulation anddecrease stroke risk [25] therefore our chemical analysesindicate that the DW extract of T-RVA might possess sim-ilar activities Sulfated GAGs particularly CS (chondroitinsulfate) are of particular interest to physicians and phar-macists Sulfated GAGs are composed of units of aminosugar including D-glucosamine and D-galactosamine andbond with core proteins to form proteoglycans Cartilageproteoglycans regulate water retention and are integral to thedifferentiation and proliferation of chondrocytes The mostprominent sulfated GAG in velvet antler tissue is chondroitinsulfate [26] Sialic acid is a water soluble component thatwas efficiently extracted by DW and showed significantaccumulation in the T-RVA section Our findings are similarto those of a previous report [27] which showed that the T-RVA or ldquowax piecerdquo contains sialic acid levels higher thanthose of other antler regions Uracil is a primary mediatorof MAO (monoamine oxidase) inhibition by velvet antlerextract [28] Our data indicate that the T-RVA section maycontribute the majority of the inhibitory effect on MAOactivity produced by velvet antler In a report by Wang et al[29] aimed at identifying the active compound in velvetantler responsible for inhibiting MAO-B activity the authorsuggested that the main antiaging compound in velvet antleris hypoxanthine Zhou et al [28] showed that uridine was

4 Journal of Chemistry

Table 1 Sulfated GAGs sialic acid and uronic acid contents ofregrowth velvet antler extracts

T-RVA M-RVA B-RVAmgg mgg mgg

Sulfated GAGs 55576 plusmn 1248 36930 plusmn 1981 22898 plusmn 2442Sialic acid 11128 plusmn 427 7950 plusmn 520 7296 plusmn 150Uronic acid 3625 plusmn 296 2590 plusmn 229 2311 plusmn 240

Table 2 Hypoxanthine uridine and uracil contents of regrowthvelvet antler extracts

T-RVA M-RVA B-RVAmgg mgg mgg

Hypoxanthine 108 plusmn 003 100 plusmn 001 092 plusmn 001Uridine 126 plusmn 003 114 plusmn 004 106 plusmn 003Uracil 096 plusmn 008 0092 plusmn 003 0845 plusmn 004

responsible for 3475 of the Fe2+-chelating activity of velvetantler Therefore the DW extract of T-RVA is expected toshow strong antioxidant activity due to its abundance ofuridine Zhou and Li [18] investigated the amounts of uridineuracil and hypoxanthine from ethanol extracted velvet antlerand the values were 37 36 and 39mgg respectively Theirvalues were higher than ours This may be attributed toextraction method

32 Antioxidant Activity The antioxidant activities of RVAmay not be attributed to a single mechanism Therefore6 methods were used to evaluate different aspects of theantioxidant activities of RVA

The antioxidant activities of the DWextracts of RVAwereevaluated by assessing DPPH H

2O2 ABTS and hydroxyl

radical scavenging activity In addition FRAP and ORACwere estimated

The antioxidant activity of the DW extract of T-RVA wassignificantly better than those of the M-RVA and B-RVAsections (119875 lt 005) and appeared to be dose-dependent TheDPPH radical scavenging activity was highest for the T-RVAsection (5344 120583MTEmg IC

500853mgmL) and lowest

for the B-RVA section (Figure 1) H2O2(3220 120583MTEmg

Figure 2) and ABTS (6031 120583MTEmg Figure 3) radicalscavenging activities were also highest for the T-RVA sec-tion The hydroxyl radical scavenging activity was high-est for the T-RVA section (2309120583MTEmg) whereas theactivities of M-RVA and B-RVA were similar (Figure 4)The T-RVA section was the most effective section inthe FRAP assay (3581 120583MTEmg) whereas the activitiesof B-RVA and M-RVA were similar (Figure 5) In theORAC assay 1000mgmL T-RVA showed excellent activity(12158 120583MTEmg) (Figure 6)

DPPH radical scavenging activity is often used as amethod of evaluating antioxidant activity DPPH is a stableradical that accepts an electron andor hydrogen radical fromdonor molecules to form a stable diamagnetic moleculeTherefore the extracts of velvet antler may have provided anelectron andor hydrogen radical to neutralize DPPH [30] Ina report by Lee and Chung [31] the DPPH radical scavenging

0

10

20

30

40

50

60

T-RVA M-RVA B-RVA

DPP

H ra

dica

l sca

veng

ing

activ

ity

a

b

c

e

h

i

d

f

h

c

e

g

(120583M

TE

mg)

Figure 1 The effect of RVA on DPPH radical scavenging activityandashiValues not sharing a common letter are significantly different at119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000 120583gmL

0

5

10

15

20

25

30

ab

d

e

dd

eg

i hg

j

H2O

2ra

dica

l sca

veng

ing

activ

ity

T-RVA M-RVA B-RVA

(120583M

TE

mg)

Figure 2 The effect of RVA on H2O2radical scavenging activity

andashiValues not sharing a common letter are significantly different at119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000 120583gmL

0

5

10

15

20

25

30

35

40

T-RVA M-RVA B-RVA

ab

c

ddc

e

f gg g g

Hyd

roxy

l rad

ical

scav

engi

ng ac

tivity

(120583

M T

Em

g)

Figure 3The effect of RVA on hydroxyl radical scavenging activityandashgValues not sharing a common letter are significantly differentat 119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000 120583gmL

Journal of Chemistry 5

0

10

20

30

40

50

60

70

ab

cddd

e ef

fg

h

ABT

S ra

dica

l sca

veng

ing

activ

ity(120583

M T

Em

g)

T-RVA M-RVA B-RVA

Figure 4 The effect of RVA on ABYS radical scavenging activityandashgValues not sharing a common letter are significantly differentat 119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000120583gmL

0

5

10

15

20

25

30

35

40

T-RVA M-RVA B-RVA

a

b bd

de

e fgg g

hFRA

P (120583

M T

Em

g)

Figure 5 The effect of RVA on FRAP assay andashgValues not sharing acommon letter are significantly different at 119875 lt 005 by Dunnettrsquosmultiple range tests Light gray square 125 120583gmL gray square250 120583gmL dark grey square 500120583gmL black square 1000 120583gmL

0

20

40

60

80

100

120

140

T-RVA M-RVA B-RVA

a

bcc

b

d

fg

fee e

ORA

C (120583

M T

Em

g)

Figure 6 The effect of RVA on ORAC andashgValues not sharing acommon letter are significantly different at 119875 lt 005 by Dunnettrsquosmultiple range tests Light gray square 125 120583gmL gray square250 120583gmL dark grey square 500120583gmL black square 1000 120583gmL

activity of velvet antler extract obtained from the uppersection was reported to be 671 at an extract concentrationof 100mgmL which was lower than the activity measuredin our analysis H

2O2is a reactive nonradical and a clinically

important compound due to its ability to penetrate biologicalmembranes H

2O2can be converted into more reactive

species such as singlet oxygen and hydroxyl radicals therebycausing lipid peroxidation or toxicity to cells Thereforescavenging of hydrogen peroxide can decrease prooxidantsrsquolevels Our analysis of H

2O2scavenging by velvet antler

produced results similar to those reported by Je et al [30]Hydroxyl radicals are extremely reactive and easily reactwith amino acids DNA and membrane components In thisstudy the hydroxyl radical scavenging activity of RVA washigher than that of velvet antler as reported by Je et al[32] In addition our analysis of ABTS radical scavengingactivity by RVA identified activity higher than that reportedby Zhao et al [33] The FRAP assay treats the antioxidantscontained in the samples as reductants in a redox-linkedcolorimetric reaction allowing assessment of the reducingpower of antioxidants [34] Zhao et al [33] reported activityof 858 plusmn 002 by 5mgmL velvet antler extract in the FARPassay which was lower than the activity measured in ouranalysis The ORAC assay has been applied extensively toevaluate the antioxidant activities of fruits vegetables leavesstems herbs and spices As a result the ORAC assay iscommonly mentioned in scientific publications and healthfood publications [35] However the antioxidant activity ofRVAhas not been evaluated using theORACassayThereforethis is the first report of an assessment of the antioxidantactivity of RVA using the ORAC assay ORAC value of gallicacid was shown 161 plusmn 48 by Zulueta et al [36] which washigher than the activity of RVA found in our study

4 Conclusions

In the present study we provided the first comprehensiveevaluation of the biologically active substances of RVA andthe antioxidant potential of different RVA segments Futurestudies are required to further elucidate the other biologicalactivities of the T-RVA M-RVA and B-RVA sections and thebiological mechanisms underlying their effects

Conflict of Interests

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

Acknowledgment

This paper was supported by Konkuk University in 2015

References

[1] C Li ldquoDevelopment of deer antler model for biomedicalresearchrdquo Recent Advancesamp Research Updates vol 4 no 2 pp255ndash274 2003

[2] Z Q Zhang Y Zhang B X Wang H O Zhou Y Wangand H Zhang ldquoPurification and partial characterization of

6 Journal of Chemistry

anti-inflammatory peptide from pilose antler of Cervus nipponTemminckrdquo Acta Pharmaceutica Sinica vol 27 no 5 pp 321ndash324 1992

[3] Z F Ma D S Zhao Q D Zhou and S Sun ldquoStudy onraising yield of reproductive pilose antler of sika deerrdquo Journalof Northeast Forestry University vol 22 no 6 pp 41ndash47 1994

[4] J F He Y Z Liu S B Zhu and L Shang ldquoStudy on increasingquality and quantity of reborn antlers of cervus elaphuslinnaeusand cervus Nippon hortulorun swinchonerdquo Natural ScienceJournal of Harbin Normal University vol 20 no 4 pp 91ndash942004

[5] D A Butterfield A Castegna C M Lauderback and J DrakeldquoEvidence that amyloid beta-peptide-induced lipid peroxida-tion and its sequelae in Alzheimerrsquos disease brain contribute toneuronal deathrdquo Neurobiology of Aging vol 23 no 5 pp 655ndash664 2002

[6] W A Pryor and N Y Ann ldquoFree radical biology xenobioticscancer and agingrdquo Annals of the New York Academy of Sciencesvol 393 no 1 pp 1ndash22 1982

[7] E-K Kim S-J Lee J-W Hwang et al ldquoIn vitro investigationon antioxidative effect of Inonotus obliquus extracts againstoxidative stress on PC12 cellsrdquo Journal of the Korean Society forApplied Biological Chemistry vol 54 no 1 pp 112ndash117 2011

[8] J-WHwang E-K Kim S-J Lee et al ldquoAntioxidant activity andprotective effect of anthocyanin oligomers on H

2O2-triggered

G2M arrest in retinal cellsrdquo Journal of Agricultural and FoodChemistry vol 60 no 17 pp 4282ndash4288 2012

[9] B R Bhavnani ldquoPharmacology of hormonal therapeuticagentsrdquo in The Menopause Comprehensive Management B AEskin Ed pp 229ndash256 Parthenon Press New York NY USA2000

[10] S S Ditchkoff L J Spicer R E Masters and R L LochmillerldquoConcentrations of insulin-like growth factor-I in adult malewhite-tailed deer (Odocoileus virginianus) associations withserum testosterone morphometrics and age during and afterthe breeding seasonrdquo Comparative Biochemistry and PhysiologyA Physiology vol 129 no 4 pp 887ndash895 2001

[11] J A Gomez A J Garcıa T Landete-Castillejos and L GallegoldquoEffect of advancing births on testosterone until 25 years of ageand puberty in Iberian red deer (Cervus elaphus hispanicus)rdquoAnimal Reproduction Science vol 96 no 1-2 pp 79ndash88 2006

[12] C Li Z Jiang G Jiang and J Fang ldquoSeasonal changes ofreproductive behavior and fecal steroid concentrations in PereDavidrsquos deerrdquo Hormones and Behavior vol 40 no 4 pp 518ndash525 2001

[13] Y-J Li T-H Kim H B Kwak Z H Lee S-Y Lee and G-J Jhon ldquoChloroform extract of deer antler inhibits osteoclastdifferentiation and bone resorptionrdquo Journal of Ethnopharma-cology vol 113 no 2 pp 191ndash198 2007

[14] H H Sunwoo T Nakano and J S Sim ldquoEffect of water-solubleextract from antler of wapiti (Cervus elaphus) on the growth offibroblastsrdquo Canadian Journal of Animal Science vol 77 no 2pp 343ndash345 1997

[15] M Cesaretti E Luppi FMaccari andN Volpi ldquoA 96-well assayfor uronic acid carbazole reactionrdquo Carbohydrate Polymers vol54 no 1 pp 59ndash61 2003

[16] RW Farndale D J Buttle and A J Barrett ldquoImproved quanti-tation and discrimination of sulphated glycosaminoglycans byuse of dimethylmethylene bluerdquo Biochimica Biophysica Actavol 883 no 2 pp 173ndash177 1986

[17] K Matsuno and S Suzuki ldquoSimple fluorimetric method forquantification of sialic acids in glycoproteinsrdquo Analytical Bio-chemistry vol 375 no 1 pp 53ndash59 2008

[18] R Zhou and S F Li ldquoIn vitro antioxidant analysis andcharacterisation of antler velvet extractrdquo Food Chemistry vol114 no 4 pp 1321ndash1327 2009

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

[20] H E Muller ldquoDetection of hydrogen peroxide produced bymicroorganism on ABTS-peroxidase mediumrdquo Zentralblatt furBakteriologieMikrobiologie undHygiene vol 259 no 2 pp 151ndash158 1985

[21] S-K Chung T Osawa and S Kawakishi ldquoHydroxyl radical-scavenging effects of spices and scavengers frombrownmustard(Brassica nigra)rdquo Bioscience Biotechnology and Biochemistryvol 61 no 1 pp 118ndash123 1997

[22] M B Arnao A Cano and M Acosta ldquoThe hydrophilicand lipophilic contribution to total antioxidant activityrdquo FoodChemistry vol 73 no 2 pp 239ndash244 2001

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

[24] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal ofAgricultural and Food Chemistry vol 49 no 10 pp 4619ndash46262001

[25] M A Moskowitz E H Lo and C Iadecola ldquoThe science ofstrokemechanisms in search of treatmentsrdquoNeuron vol 67 no2 pp 181ndash198 2010

[26] Y W Ha B T Jeon S H Moon et al ldquoCharacterization ofheparan sulfate from the unossified antler of Cervus elaphusrdquoCarbohydrate Research vol 340 no 3 pp 411ndash416 2005

[27] B Jeon S Kim S Lee et al ldquoEffect of antler growth period onthe chemical composition of velvet antler in sika deer (Cervusnippon)rdquoMammalian Biology vol 74 no 5 pp 374ndash380 2009

[28] R Zhou JWang S Li andY Liu ldquoSupercritical fluid extractionof monoamine oxidase inhibitor from antler velvetrdquo Separationand Purification Technology vol 65 no 3 pp 275ndash281 2009

[29] B X Wang X H Zhao X W Yang et al ldquoInhibition oflipid peroxidation of deer antler (Rokujo) extract in vivo andin vitrordquo Journal of Medical and Pharmaceutical Society forWAKAN-YAKU vol 5 pp 123ndash128 1988

[30] J Y Je P J Park D H Lim B T Jeon K H Kho and C B AhnldquoAntioxidant anti-acetylcholinesterase and composition of bio-chemical components of Russian deer velvet antler extractsrdquoKorean Journal for Food Science of Animal Resources vol 31 no3 pp 349ndash355 2011

[31] K A Lee andH Y Chung ldquoThebiological activity of deer antlerextract in vitrordquoThe Korean Journal of Food and Nutrition vol20 no 2 pp 114ndash119 2007

[32] J-Y Je P-J Park E-K Kim et al ldquoComposition of biologicallyactive substances and antioxidant activity of New Zealand deervelvet antler extractsrdquoKorean Journal for Food Science of AnimalResources vol 30 no 1 pp 20ndash27 2010

[33] L Zhao Y-C Luo C-T Wang and B-P Ji ldquoAntioxidantactivity of protein hydrolysates from aqueous extract of velvetantler (Cervus elaphus) as influenced by molecular weight andenzymesrdquo Natural Product Communications vol 6 no 11 pp1683ndash1688 2011

Journal of Chemistry 7

[34] S-Q Huang S D Ding and L P Fan ldquoAntioxidant activitiesof five polysaccharides from Inonotus obliquusrdquo InternationalJournal of BiologicalMacromolecules vol 50 no 5 pp 1183ndash11872012

[35] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006

[36] A Zulueta M J Esteve and A Frıgola ldquoORAC and TEACassays comparison to measure the antioxidant capacity of foodproductsrdquo Food Chemistry vol 114 no 1 pp 310ndash316 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Page 3: Research Article First Evaluation of the Biologically Active ...We investigated the biologically active substances contained in RVA (regrowth velvet antler) by comparing the composition

Journal of Chemistry 3

at 37∘C for 10minThe absorbancewas recorded at 405 nmbymicroplate reader and the percentage of scavenging activitywas calculated using (1)

243 Hydroxyl Radical Scavenging Activity The hydroxylradical scavenging activity of each antler extract was deter-mined according to themethod of Chung et al [21] Hydroxylradicals were generated by the Fenton reaction in the pres-ence of FeSO

4 A reaction mixture containing 01mL of

10mM FeSO4 10mM EDTA and 10mM 2-deoxyribose was

mixed with 01mL of the extract solution after which 01Mphosphate buffer (pH 74) was added to the reaction mixtureto reach a total volume of 09mL Subsequently 01mL of10mM H

2O2was added to the reaction mixture which was

incubated at 37∘C for 4 h After incubation 05mL of 28TCA and 10 TBA were added to each mixture after whicheach mixture was placed in a boiling water bath for 10minAbsorbance was measured at 532 nm Hydroxyl radical scav-enging activity was calculated as a percentage using (1)

244 ABTS Radical Scavenging Activity The ABTS scaveng-ing activity of each antler extract was assessed following themethod of Arnao et al [22] Stock solutions included ABTS∙+solution and potassium persulfate solutions A workingsolutionwas prepared bymixing the 2 stock solutions in equalquantities and allowing them to react for 12 h The workingsolution was diluted with fresh ABTS∙+ solution and mixedwith or without each extract After incubation for 2 h theabsorbance of each solution was recorded at 735 nm Thescavenging activity was calculated as a percentage using (1)

245 FRAP Assay The FRAP assay was performed accord-ing to the method of Benzie and Strain [23] Fresh work-ing solution was prepared by mixing acetate buffer TPTZsolution and FeCl

3sdot6H2O solution and warmed at 37∘C

before use Each extract was allowed to react with the FRAPsolution in a dark room at room temperature for 30minTheabsorbance of the colored product was measured at 595 nmScavenging activity was calculated as a percentage using (1)

246 ORAC Assay For ORAC assay the method of Ou et alwas used with some slightly modification [24] The workingsolution of FL and AAPH radical was prepared daily Sampleblank or standard was placed in 96-microwell plate and theplate was heated to 37∘C for 15min prior to the additionof AAPH The fluorescence was measured immediately afterthe AAPH addition and measurements with fluorescencefilters for an excitationwavelength of 485 nm and an emissionwavelength of 535 nm were taken every 5min until therelative fluorescence intensity was less than 5 of the valueof the initial reading

The ORAC values expressed as 120583M Trolox equivalents(120583MTEmg) were calculated by applying the following for-mula

ORAC (120583MTE)

=(119862Trolox times (AUCsample minus AUCblank) times 119896)

(AUCsample minus AUCblank)

(2)

where 119862Trolox is the concentration of Trolox (20120583M) 119896 isthe sample dilution factor and AUC is the area below thefluorescence decay curve of the sample blank and Troloxrespectively calculated by applying the following formula in aMicrosoft Excel spreadsheet (Microsoft Washington USA)

AUC = (05 +1198915

1198910

+11989110

1198910

+ sdot sdot sdot + 119891119899+5

1198910

) times 5 (3)

where 1198910is the initial fluorescence and 119891

119899is the fluorescence

at time 119899

25 Statistical Analysis The results shown are summaries ofthe data from at least 3 experiments All data are presented asmeanplusmn SEM (standard error of themean) Statistical analyseswere performed using SAS statistical software (SAS InstituteInc Cary NC USA) Treatment effects were analyzed usingone-way ANOVA followed by Dunnettrsquos multiple range testResults of 119875 lt 005 indicated statistical significance

3 Results and Discussion

31 Bioactive Composition Thebiologically active substancescontained in the 3 RVA segments including uronic acidsulfated GAGs sialic acid uridine uracil and hypoxanthineare listed in Tables 1 and 2

The uronic acid content sulfated GAGs content andsialic acid content of the T-RVA and M-RVA sections weresignificantly greater than those of the B-RVA section (119875 lt005) The DW extract of the T-RVA section contained3625mgg uronic acid 55576mgg sulfated GAGs and11128mgg sialic acid (Table 1) The DW extract of theRVA contained 0957mgg uridine 1084mgg uracil and1263mgg hypoxanthine (Table 2)

Uronic acid has been reported to improve circulation anddecrease stroke risk [25] therefore our chemical analysesindicate that the DW extract of T-RVA might possess sim-ilar activities Sulfated GAGs particularly CS (chondroitinsulfate) are of particular interest to physicians and phar-macists Sulfated GAGs are composed of units of aminosugar including D-glucosamine and D-galactosamine andbond with core proteins to form proteoglycans Cartilageproteoglycans regulate water retention and are integral to thedifferentiation and proliferation of chondrocytes The mostprominent sulfated GAG in velvet antler tissue is chondroitinsulfate [26] Sialic acid is a water soluble component thatwas efficiently extracted by DW and showed significantaccumulation in the T-RVA section Our findings are similarto those of a previous report [27] which showed that the T-RVA or ldquowax piecerdquo contains sialic acid levels higher thanthose of other antler regions Uracil is a primary mediatorof MAO (monoamine oxidase) inhibition by velvet antlerextract [28] Our data indicate that the T-RVA section maycontribute the majority of the inhibitory effect on MAOactivity produced by velvet antler In a report by Wang et al[29] aimed at identifying the active compound in velvetantler responsible for inhibiting MAO-B activity the authorsuggested that the main antiaging compound in velvet antleris hypoxanthine Zhou et al [28] showed that uridine was

4 Journal of Chemistry

Table 1 Sulfated GAGs sialic acid and uronic acid contents ofregrowth velvet antler extracts

T-RVA M-RVA B-RVAmgg mgg mgg

Sulfated GAGs 55576 plusmn 1248 36930 plusmn 1981 22898 plusmn 2442Sialic acid 11128 plusmn 427 7950 plusmn 520 7296 plusmn 150Uronic acid 3625 plusmn 296 2590 plusmn 229 2311 plusmn 240

Table 2 Hypoxanthine uridine and uracil contents of regrowthvelvet antler extracts

T-RVA M-RVA B-RVAmgg mgg mgg

Hypoxanthine 108 plusmn 003 100 plusmn 001 092 plusmn 001Uridine 126 plusmn 003 114 plusmn 004 106 plusmn 003Uracil 096 plusmn 008 0092 plusmn 003 0845 plusmn 004

responsible for 3475 of the Fe2+-chelating activity of velvetantler Therefore the DW extract of T-RVA is expected toshow strong antioxidant activity due to its abundance ofuridine Zhou and Li [18] investigated the amounts of uridineuracil and hypoxanthine from ethanol extracted velvet antlerand the values were 37 36 and 39mgg respectively Theirvalues were higher than ours This may be attributed toextraction method

32 Antioxidant Activity The antioxidant activities of RVAmay not be attributed to a single mechanism Therefore6 methods were used to evaluate different aspects of theantioxidant activities of RVA

The antioxidant activities of the DWextracts of RVAwereevaluated by assessing DPPH H

2O2 ABTS and hydroxyl

radical scavenging activity In addition FRAP and ORACwere estimated

The antioxidant activity of the DW extract of T-RVA wassignificantly better than those of the M-RVA and B-RVAsections (119875 lt 005) and appeared to be dose-dependent TheDPPH radical scavenging activity was highest for the T-RVAsection (5344 120583MTEmg IC

500853mgmL) and lowest

for the B-RVA section (Figure 1) H2O2(3220 120583MTEmg

Figure 2) and ABTS (6031 120583MTEmg Figure 3) radicalscavenging activities were also highest for the T-RVA sec-tion The hydroxyl radical scavenging activity was high-est for the T-RVA section (2309120583MTEmg) whereas theactivities of M-RVA and B-RVA were similar (Figure 4)The T-RVA section was the most effective section inthe FRAP assay (3581 120583MTEmg) whereas the activitiesof B-RVA and M-RVA were similar (Figure 5) In theORAC assay 1000mgmL T-RVA showed excellent activity(12158 120583MTEmg) (Figure 6)

DPPH radical scavenging activity is often used as amethod of evaluating antioxidant activity DPPH is a stableradical that accepts an electron andor hydrogen radical fromdonor molecules to form a stable diamagnetic moleculeTherefore the extracts of velvet antler may have provided anelectron andor hydrogen radical to neutralize DPPH [30] Ina report by Lee and Chung [31] the DPPH radical scavenging

0

10

20

30

40

50

60

T-RVA M-RVA B-RVA

DPP

H ra

dica

l sca

veng

ing

activ

ity

a

b

c

e

h

i

d

f

h

c

e

g

(120583M

TE

mg)

Figure 1 The effect of RVA on DPPH radical scavenging activityandashiValues not sharing a common letter are significantly different at119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000 120583gmL

0

5

10

15

20

25

30

ab

d

e

dd

eg

i hg

j

H2O

2ra

dica

l sca

veng

ing

activ

ity

T-RVA M-RVA B-RVA

(120583M

TE

mg)

Figure 2 The effect of RVA on H2O2radical scavenging activity

andashiValues not sharing a common letter are significantly different at119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000 120583gmL

0

5

10

15

20

25

30

35

40

T-RVA M-RVA B-RVA

ab

c

ddc

e

f gg g g

Hyd

roxy

l rad

ical

scav

engi

ng ac

tivity

(120583

M T

Em

g)

Figure 3The effect of RVA on hydroxyl radical scavenging activityandashgValues not sharing a common letter are significantly differentat 119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000 120583gmL

Journal of Chemistry 5

0

10

20

30

40

50

60

70

ab

cddd

e ef

fg

h

ABT

S ra

dica

l sca

veng

ing

activ

ity(120583

M T

Em

g)

T-RVA M-RVA B-RVA

Figure 4 The effect of RVA on ABYS radical scavenging activityandashgValues not sharing a common letter are significantly differentat 119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000120583gmL

0

5

10

15

20

25

30

35

40

T-RVA M-RVA B-RVA

a

b bd

de

e fgg g

hFRA

P (120583

M T

Em

g)

Figure 5 The effect of RVA on FRAP assay andashgValues not sharing acommon letter are significantly different at 119875 lt 005 by Dunnettrsquosmultiple range tests Light gray square 125 120583gmL gray square250 120583gmL dark grey square 500120583gmL black square 1000 120583gmL

0

20

40

60

80

100

120

140

T-RVA M-RVA B-RVA

a

bcc

b

d

fg

fee e

ORA

C (120583

M T

Em

g)

Figure 6 The effect of RVA on ORAC andashgValues not sharing acommon letter are significantly different at 119875 lt 005 by Dunnettrsquosmultiple range tests Light gray square 125 120583gmL gray square250 120583gmL dark grey square 500120583gmL black square 1000 120583gmL

activity of velvet antler extract obtained from the uppersection was reported to be 671 at an extract concentrationof 100mgmL which was lower than the activity measuredin our analysis H

2O2is a reactive nonradical and a clinically

important compound due to its ability to penetrate biologicalmembranes H

2O2can be converted into more reactive

species such as singlet oxygen and hydroxyl radicals therebycausing lipid peroxidation or toxicity to cells Thereforescavenging of hydrogen peroxide can decrease prooxidantsrsquolevels Our analysis of H

2O2scavenging by velvet antler

produced results similar to those reported by Je et al [30]Hydroxyl radicals are extremely reactive and easily reactwith amino acids DNA and membrane components In thisstudy the hydroxyl radical scavenging activity of RVA washigher than that of velvet antler as reported by Je et al[32] In addition our analysis of ABTS radical scavengingactivity by RVA identified activity higher than that reportedby Zhao et al [33] The FRAP assay treats the antioxidantscontained in the samples as reductants in a redox-linkedcolorimetric reaction allowing assessment of the reducingpower of antioxidants [34] Zhao et al [33] reported activityof 858 plusmn 002 by 5mgmL velvet antler extract in the FARPassay which was lower than the activity measured in ouranalysis The ORAC assay has been applied extensively toevaluate the antioxidant activities of fruits vegetables leavesstems herbs and spices As a result the ORAC assay iscommonly mentioned in scientific publications and healthfood publications [35] However the antioxidant activity ofRVAhas not been evaluated using theORACassayThereforethis is the first report of an assessment of the antioxidantactivity of RVA using the ORAC assay ORAC value of gallicacid was shown 161 plusmn 48 by Zulueta et al [36] which washigher than the activity of RVA found in our study

4 Conclusions

In the present study we provided the first comprehensiveevaluation of the biologically active substances of RVA andthe antioxidant potential of different RVA segments Futurestudies are required to further elucidate the other biologicalactivities of the T-RVA M-RVA and B-RVA sections and thebiological mechanisms underlying their effects

Conflict of Interests

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

Acknowledgment

This paper was supported by Konkuk University in 2015

References

[1] C Li ldquoDevelopment of deer antler model for biomedicalresearchrdquo Recent Advancesamp Research Updates vol 4 no 2 pp255ndash274 2003

[2] Z Q Zhang Y Zhang B X Wang H O Zhou Y Wangand H Zhang ldquoPurification and partial characterization of

6 Journal of Chemistry

anti-inflammatory peptide from pilose antler of Cervus nipponTemminckrdquo Acta Pharmaceutica Sinica vol 27 no 5 pp 321ndash324 1992

[3] Z F Ma D S Zhao Q D Zhou and S Sun ldquoStudy onraising yield of reproductive pilose antler of sika deerrdquo Journalof Northeast Forestry University vol 22 no 6 pp 41ndash47 1994

[4] J F He Y Z Liu S B Zhu and L Shang ldquoStudy on increasingquality and quantity of reborn antlers of cervus elaphuslinnaeusand cervus Nippon hortulorun swinchonerdquo Natural ScienceJournal of Harbin Normal University vol 20 no 4 pp 91ndash942004

[5] D A Butterfield A Castegna C M Lauderback and J DrakeldquoEvidence that amyloid beta-peptide-induced lipid peroxida-tion and its sequelae in Alzheimerrsquos disease brain contribute toneuronal deathrdquo Neurobiology of Aging vol 23 no 5 pp 655ndash664 2002

[6] W A Pryor and N Y Ann ldquoFree radical biology xenobioticscancer and agingrdquo Annals of the New York Academy of Sciencesvol 393 no 1 pp 1ndash22 1982

[7] E-K Kim S-J Lee J-W Hwang et al ldquoIn vitro investigationon antioxidative effect of Inonotus obliquus extracts againstoxidative stress on PC12 cellsrdquo Journal of the Korean Society forApplied Biological Chemistry vol 54 no 1 pp 112ndash117 2011

[8] J-WHwang E-K Kim S-J Lee et al ldquoAntioxidant activity andprotective effect of anthocyanin oligomers on H

2O2-triggered

G2M arrest in retinal cellsrdquo Journal of Agricultural and FoodChemistry vol 60 no 17 pp 4282ndash4288 2012

[9] B R Bhavnani ldquoPharmacology of hormonal therapeuticagentsrdquo in The Menopause Comprehensive Management B AEskin Ed pp 229ndash256 Parthenon Press New York NY USA2000

[10] S S Ditchkoff L J Spicer R E Masters and R L LochmillerldquoConcentrations of insulin-like growth factor-I in adult malewhite-tailed deer (Odocoileus virginianus) associations withserum testosterone morphometrics and age during and afterthe breeding seasonrdquo Comparative Biochemistry and PhysiologyA Physiology vol 129 no 4 pp 887ndash895 2001

[11] J A Gomez A J Garcıa T Landete-Castillejos and L GallegoldquoEffect of advancing births on testosterone until 25 years of ageand puberty in Iberian red deer (Cervus elaphus hispanicus)rdquoAnimal Reproduction Science vol 96 no 1-2 pp 79ndash88 2006

[12] C Li Z Jiang G Jiang and J Fang ldquoSeasonal changes ofreproductive behavior and fecal steroid concentrations in PereDavidrsquos deerrdquo Hormones and Behavior vol 40 no 4 pp 518ndash525 2001

[13] Y-J Li T-H Kim H B Kwak Z H Lee S-Y Lee and G-J Jhon ldquoChloroform extract of deer antler inhibits osteoclastdifferentiation and bone resorptionrdquo Journal of Ethnopharma-cology vol 113 no 2 pp 191ndash198 2007

[14] H H Sunwoo T Nakano and J S Sim ldquoEffect of water-solubleextract from antler of wapiti (Cervus elaphus) on the growth offibroblastsrdquo Canadian Journal of Animal Science vol 77 no 2pp 343ndash345 1997

[15] M Cesaretti E Luppi FMaccari andN Volpi ldquoA 96-well assayfor uronic acid carbazole reactionrdquo Carbohydrate Polymers vol54 no 1 pp 59ndash61 2003

[16] RW Farndale D J Buttle and A J Barrett ldquoImproved quanti-tation and discrimination of sulphated glycosaminoglycans byuse of dimethylmethylene bluerdquo Biochimica Biophysica Actavol 883 no 2 pp 173ndash177 1986

[17] K Matsuno and S Suzuki ldquoSimple fluorimetric method forquantification of sialic acids in glycoproteinsrdquo Analytical Bio-chemistry vol 375 no 1 pp 53ndash59 2008

[18] R Zhou and S F Li ldquoIn vitro antioxidant analysis andcharacterisation of antler velvet extractrdquo Food Chemistry vol114 no 4 pp 1321ndash1327 2009

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

[20] H E Muller ldquoDetection of hydrogen peroxide produced bymicroorganism on ABTS-peroxidase mediumrdquo Zentralblatt furBakteriologieMikrobiologie undHygiene vol 259 no 2 pp 151ndash158 1985

[21] S-K Chung T Osawa and S Kawakishi ldquoHydroxyl radical-scavenging effects of spices and scavengers frombrownmustard(Brassica nigra)rdquo Bioscience Biotechnology and Biochemistryvol 61 no 1 pp 118ndash123 1997

[22] M B Arnao A Cano and M Acosta ldquoThe hydrophilicand lipophilic contribution to total antioxidant activityrdquo FoodChemistry vol 73 no 2 pp 239ndash244 2001

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

[24] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal ofAgricultural and Food Chemistry vol 49 no 10 pp 4619ndash46262001

[25] M A Moskowitz E H Lo and C Iadecola ldquoThe science ofstrokemechanisms in search of treatmentsrdquoNeuron vol 67 no2 pp 181ndash198 2010

[26] Y W Ha B T Jeon S H Moon et al ldquoCharacterization ofheparan sulfate from the unossified antler of Cervus elaphusrdquoCarbohydrate Research vol 340 no 3 pp 411ndash416 2005

[27] B Jeon S Kim S Lee et al ldquoEffect of antler growth period onthe chemical composition of velvet antler in sika deer (Cervusnippon)rdquoMammalian Biology vol 74 no 5 pp 374ndash380 2009

[28] R Zhou JWang S Li andY Liu ldquoSupercritical fluid extractionof monoamine oxidase inhibitor from antler velvetrdquo Separationand Purification Technology vol 65 no 3 pp 275ndash281 2009

[29] B X Wang X H Zhao X W Yang et al ldquoInhibition oflipid peroxidation of deer antler (Rokujo) extract in vivo andin vitrordquo Journal of Medical and Pharmaceutical Society forWAKAN-YAKU vol 5 pp 123ndash128 1988

[30] J Y Je P J Park D H Lim B T Jeon K H Kho and C B AhnldquoAntioxidant anti-acetylcholinesterase and composition of bio-chemical components of Russian deer velvet antler extractsrdquoKorean Journal for Food Science of Animal Resources vol 31 no3 pp 349ndash355 2011

[31] K A Lee andH Y Chung ldquoThebiological activity of deer antlerextract in vitrordquoThe Korean Journal of Food and Nutrition vol20 no 2 pp 114ndash119 2007

[32] J-Y Je P-J Park E-K Kim et al ldquoComposition of biologicallyactive substances and antioxidant activity of New Zealand deervelvet antler extractsrdquoKorean Journal for Food Science of AnimalResources vol 30 no 1 pp 20ndash27 2010

[33] L Zhao Y-C Luo C-T Wang and B-P Ji ldquoAntioxidantactivity of protein hydrolysates from aqueous extract of velvetantler (Cervus elaphus) as influenced by molecular weight andenzymesrdquo Natural Product Communications vol 6 no 11 pp1683ndash1688 2011

Journal of Chemistry 7

[34] S-Q Huang S D Ding and L P Fan ldquoAntioxidant activitiesof five polysaccharides from Inonotus obliquusrdquo InternationalJournal of BiologicalMacromolecules vol 50 no 5 pp 1183ndash11872012

[35] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006

[36] A Zulueta M J Esteve and A Frıgola ldquoORAC and TEACassays comparison to measure the antioxidant capacity of foodproductsrdquo Food Chemistry vol 114 no 1 pp 310ndash316 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Page 4: Research Article First Evaluation of the Biologically Active ...We investigated the biologically active substances contained in RVA (regrowth velvet antler) by comparing the composition

4 Journal of Chemistry

Table 1 Sulfated GAGs sialic acid and uronic acid contents ofregrowth velvet antler extracts

T-RVA M-RVA B-RVAmgg mgg mgg

Sulfated GAGs 55576 plusmn 1248 36930 plusmn 1981 22898 plusmn 2442Sialic acid 11128 plusmn 427 7950 plusmn 520 7296 plusmn 150Uronic acid 3625 plusmn 296 2590 plusmn 229 2311 plusmn 240

Table 2 Hypoxanthine uridine and uracil contents of regrowthvelvet antler extracts

T-RVA M-RVA B-RVAmgg mgg mgg

Hypoxanthine 108 plusmn 003 100 plusmn 001 092 plusmn 001Uridine 126 plusmn 003 114 plusmn 004 106 plusmn 003Uracil 096 plusmn 008 0092 plusmn 003 0845 plusmn 004

responsible for 3475 of the Fe2+-chelating activity of velvetantler Therefore the DW extract of T-RVA is expected toshow strong antioxidant activity due to its abundance ofuridine Zhou and Li [18] investigated the amounts of uridineuracil and hypoxanthine from ethanol extracted velvet antlerand the values were 37 36 and 39mgg respectively Theirvalues were higher than ours This may be attributed toextraction method

32 Antioxidant Activity The antioxidant activities of RVAmay not be attributed to a single mechanism Therefore6 methods were used to evaluate different aspects of theantioxidant activities of RVA

The antioxidant activities of the DWextracts of RVAwereevaluated by assessing DPPH H

2O2 ABTS and hydroxyl

radical scavenging activity In addition FRAP and ORACwere estimated

The antioxidant activity of the DW extract of T-RVA wassignificantly better than those of the M-RVA and B-RVAsections (119875 lt 005) and appeared to be dose-dependent TheDPPH radical scavenging activity was highest for the T-RVAsection (5344 120583MTEmg IC

500853mgmL) and lowest

for the B-RVA section (Figure 1) H2O2(3220 120583MTEmg

Figure 2) and ABTS (6031 120583MTEmg Figure 3) radicalscavenging activities were also highest for the T-RVA sec-tion The hydroxyl radical scavenging activity was high-est for the T-RVA section (2309120583MTEmg) whereas theactivities of M-RVA and B-RVA were similar (Figure 4)The T-RVA section was the most effective section inthe FRAP assay (3581 120583MTEmg) whereas the activitiesof B-RVA and M-RVA were similar (Figure 5) In theORAC assay 1000mgmL T-RVA showed excellent activity(12158 120583MTEmg) (Figure 6)

DPPH radical scavenging activity is often used as amethod of evaluating antioxidant activity DPPH is a stableradical that accepts an electron andor hydrogen radical fromdonor molecules to form a stable diamagnetic moleculeTherefore the extracts of velvet antler may have provided anelectron andor hydrogen radical to neutralize DPPH [30] Ina report by Lee and Chung [31] the DPPH radical scavenging

0

10

20

30

40

50

60

T-RVA M-RVA B-RVA

DPP

H ra

dica

l sca

veng

ing

activ

ity

a

b

c

e

h

i

d

f

h

c

e

g

(120583M

TE

mg)

Figure 1 The effect of RVA on DPPH radical scavenging activityandashiValues not sharing a common letter are significantly different at119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000 120583gmL

0

5

10

15

20

25

30

ab

d

e

dd

eg

i hg

j

H2O

2ra

dica

l sca

veng

ing

activ

ity

T-RVA M-RVA B-RVA

(120583M

TE

mg)

Figure 2 The effect of RVA on H2O2radical scavenging activity

andashiValues not sharing a common letter are significantly different at119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000 120583gmL

0

5

10

15

20

25

30

35

40

T-RVA M-RVA B-RVA

ab

c

ddc

e

f gg g g

Hyd

roxy

l rad

ical

scav

engi

ng ac

tivity

(120583

M T

Em

g)

Figure 3The effect of RVA on hydroxyl radical scavenging activityandashgValues not sharing a common letter are significantly differentat 119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000 120583gmL

Journal of Chemistry 5

0

10

20

30

40

50

60

70

ab

cddd

e ef

fg

h

ABT

S ra

dica

l sca

veng

ing

activ

ity(120583

M T

Em

g)

T-RVA M-RVA B-RVA

Figure 4 The effect of RVA on ABYS radical scavenging activityandashgValues not sharing a common letter are significantly differentat 119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000120583gmL

0

5

10

15

20

25

30

35

40

T-RVA M-RVA B-RVA

a

b bd

de

e fgg g

hFRA

P (120583

M T

Em

g)

Figure 5 The effect of RVA on FRAP assay andashgValues not sharing acommon letter are significantly different at 119875 lt 005 by Dunnettrsquosmultiple range tests Light gray square 125 120583gmL gray square250 120583gmL dark grey square 500120583gmL black square 1000 120583gmL

0

20

40

60

80

100

120

140

T-RVA M-RVA B-RVA

a

bcc

b

d

fg

fee e

ORA

C (120583

M T

Em

g)

Figure 6 The effect of RVA on ORAC andashgValues not sharing acommon letter are significantly different at 119875 lt 005 by Dunnettrsquosmultiple range tests Light gray square 125 120583gmL gray square250 120583gmL dark grey square 500120583gmL black square 1000 120583gmL

activity of velvet antler extract obtained from the uppersection was reported to be 671 at an extract concentrationof 100mgmL which was lower than the activity measuredin our analysis H

2O2is a reactive nonradical and a clinically

important compound due to its ability to penetrate biologicalmembranes H

2O2can be converted into more reactive

species such as singlet oxygen and hydroxyl radicals therebycausing lipid peroxidation or toxicity to cells Thereforescavenging of hydrogen peroxide can decrease prooxidantsrsquolevels Our analysis of H

2O2scavenging by velvet antler

produced results similar to those reported by Je et al [30]Hydroxyl radicals are extremely reactive and easily reactwith amino acids DNA and membrane components In thisstudy the hydroxyl radical scavenging activity of RVA washigher than that of velvet antler as reported by Je et al[32] In addition our analysis of ABTS radical scavengingactivity by RVA identified activity higher than that reportedby Zhao et al [33] The FRAP assay treats the antioxidantscontained in the samples as reductants in a redox-linkedcolorimetric reaction allowing assessment of the reducingpower of antioxidants [34] Zhao et al [33] reported activityof 858 plusmn 002 by 5mgmL velvet antler extract in the FARPassay which was lower than the activity measured in ouranalysis The ORAC assay has been applied extensively toevaluate the antioxidant activities of fruits vegetables leavesstems herbs and spices As a result the ORAC assay iscommonly mentioned in scientific publications and healthfood publications [35] However the antioxidant activity ofRVAhas not been evaluated using theORACassayThereforethis is the first report of an assessment of the antioxidantactivity of RVA using the ORAC assay ORAC value of gallicacid was shown 161 plusmn 48 by Zulueta et al [36] which washigher than the activity of RVA found in our study

4 Conclusions

In the present study we provided the first comprehensiveevaluation of the biologically active substances of RVA andthe antioxidant potential of different RVA segments Futurestudies are required to further elucidate the other biologicalactivities of the T-RVA M-RVA and B-RVA sections and thebiological mechanisms underlying their effects

Conflict of Interests

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

Acknowledgment

This paper was supported by Konkuk University in 2015

References

[1] C Li ldquoDevelopment of deer antler model for biomedicalresearchrdquo Recent Advancesamp Research Updates vol 4 no 2 pp255ndash274 2003

[2] Z Q Zhang Y Zhang B X Wang H O Zhou Y Wangand H Zhang ldquoPurification and partial characterization of

6 Journal of Chemistry

anti-inflammatory peptide from pilose antler of Cervus nipponTemminckrdquo Acta Pharmaceutica Sinica vol 27 no 5 pp 321ndash324 1992

[3] Z F Ma D S Zhao Q D Zhou and S Sun ldquoStudy onraising yield of reproductive pilose antler of sika deerrdquo Journalof Northeast Forestry University vol 22 no 6 pp 41ndash47 1994

[4] J F He Y Z Liu S B Zhu and L Shang ldquoStudy on increasingquality and quantity of reborn antlers of cervus elaphuslinnaeusand cervus Nippon hortulorun swinchonerdquo Natural ScienceJournal of Harbin Normal University vol 20 no 4 pp 91ndash942004

[5] D A Butterfield A Castegna C M Lauderback and J DrakeldquoEvidence that amyloid beta-peptide-induced lipid peroxida-tion and its sequelae in Alzheimerrsquos disease brain contribute toneuronal deathrdquo Neurobiology of Aging vol 23 no 5 pp 655ndash664 2002

[6] W A Pryor and N Y Ann ldquoFree radical biology xenobioticscancer and agingrdquo Annals of the New York Academy of Sciencesvol 393 no 1 pp 1ndash22 1982

[7] E-K Kim S-J Lee J-W Hwang et al ldquoIn vitro investigationon antioxidative effect of Inonotus obliquus extracts againstoxidative stress on PC12 cellsrdquo Journal of the Korean Society forApplied Biological Chemistry vol 54 no 1 pp 112ndash117 2011

[8] J-WHwang E-K Kim S-J Lee et al ldquoAntioxidant activity andprotective effect of anthocyanin oligomers on H

2O2-triggered

G2M arrest in retinal cellsrdquo Journal of Agricultural and FoodChemistry vol 60 no 17 pp 4282ndash4288 2012

[9] B R Bhavnani ldquoPharmacology of hormonal therapeuticagentsrdquo in The Menopause Comprehensive Management B AEskin Ed pp 229ndash256 Parthenon Press New York NY USA2000

[10] S S Ditchkoff L J Spicer R E Masters and R L LochmillerldquoConcentrations of insulin-like growth factor-I in adult malewhite-tailed deer (Odocoileus virginianus) associations withserum testosterone morphometrics and age during and afterthe breeding seasonrdquo Comparative Biochemistry and PhysiologyA Physiology vol 129 no 4 pp 887ndash895 2001

[11] J A Gomez A J Garcıa T Landete-Castillejos and L GallegoldquoEffect of advancing births on testosterone until 25 years of ageand puberty in Iberian red deer (Cervus elaphus hispanicus)rdquoAnimal Reproduction Science vol 96 no 1-2 pp 79ndash88 2006

[12] C Li Z Jiang G Jiang and J Fang ldquoSeasonal changes ofreproductive behavior and fecal steroid concentrations in PereDavidrsquos deerrdquo Hormones and Behavior vol 40 no 4 pp 518ndash525 2001

[13] Y-J Li T-H Kim H B Kwak Z H Lee S-Y Lee and G-J Jhon ldquoChloroform extract of deer antler inhibits osteoclastdifferentiation and bone resorptionrdquo Journal of Ethnopharma-cology vol 113 no 2 pp 191ndash198 2007

[14] H H Sunwoo T Nakano and J S Sim ldquoEffect of water-solubleextract from antler of wapiti (Cervus elaphus) on the growth offibroblastsrdquo Canadian Journal of Animal Science vol 77 no 2pp 343ndash345 1997

[15] M Cesaretti E Luppi FMaccari andN Volpi ldquoA 96-well assayfor uronic acid carbazole reactionrdquo Carbohydrate Polymers vol54 no 1 pp 59ndash61 2003

[16] RW Farndale D J Buttle and A J Barrett ldquoImproved quanti-tation and discrimination of sulphated glycosaminoglycans byuse of dimethylmethylene bluerdquo Biochimica Biophysica Actavol 883 no 2 pp 173ndash177 1986

[17] K Matsuno and S Suzuki ldquoSimple fluorimetric method forquantification of sialic acids in glycoproteinsrdquo Analytical Bio-chemistry vol 375 no 1 pp 53ndash59 2008

[18] R Zhou and S F Li ldquoIn vitro antioxidant analysis andcharacterisation of antler velvet extractrdquo Food Chemistry vol114 no 4 pp 1321ndash1327 2009

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

[20] H E Muller ldquoDetection of hydrogen peroxide produced bymicroorganism on ABTS-peroxidase mediumrdquo Zentralblatt furBakteriologieMikrobiologie undHygiene vol 259 no 2 pp 151ndash158 1985

[21] S-K Chung T Osawa and S Kawakishi ldquoHydroxyl radical-scavenging effects of spices and scavengers frombrownmustard(Brassica nigra)rdquo Bioscience Biotechnology and Biochemistryvol 61 no 1 pp 118ndash123 1997

[22] M B Arnao A Cano and M Acosta ldquoThe hydrophilicand lipophilic contribution to total antioxidant activityrdquo FoodChemistry vol 73 no 2 pp 239ndash244 2001

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

[24] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal ofAgricultural and Food Chemistry vol 49 no 10 pp 4619ndash46262001

[25] M A Moskowitz E H Lo and C Iadecola ldquoThe science ofstrokemechanisms in search of treatmentsrdquoNeuron vol 67 no2 pp 181ndash198 2010

[26] Y W Ha B T Jeon S H Moon et al ldquoCharacterization ofheparan sulfate from the unossified antler of Cervus elaphusrdquoCarbohydrate Research vol 340 no 3 pp 411ndash416 2005

[27] B Jeon S Kim S Lee et al ldquoEffect of antler growth period onthe chemical composition of velvet antler in sika deer (Cervusnippon)rdquoMammalian Biology vol 74 no 5 pp 374ndash380 2009

[28] R Zhou JWang S Li andY Liu ldquoSupercritical fluid extractionof monoamine oxidase inhibitor from antler velvetrdquo Separationand Purification Technology vol 65 no 3 pp 275ndash281 2009

[29] B X Wang X H Zhao X W Yang et al ldquoInhibition oflipid peroxidation of deer antler (Rokujo) extract in vivo andin vitrordquo Journal of Medical and Pharmaceutical Society forWAKAN-YAKU vol 5 pp 123ndash128 1988

[30] J Y Je P J Park D H Lim B T Jeon K H Kho and C B AhnldquoAntioxidant anti-acetylcholinesterase and composition of bio-chemical components of Russian deer velvet antler extractsrdquoKorean Journal for Food Science of Animal Resources vol 31 no3 pp 349ndash355 2011

[31] K A Lee andH Y Chung ldquoThebiological activity of deer antlerextract in vitrordquoThe Korean Journal of Food and Nutrition vol20 no 2 pp 114ndash119 2007

[32] J-Y Je P-J Park E-K Kim et al ldquoComposition of biologicallyactive substances and antioxidant activity of New Zealand deervelvet antler extractsrdquoKorean Journal for Food Science of AnimalResources vol 30 no 1 pp 20ndash27 2010

[33] L Zhao Y-C Luo C-T Wang and B-P Ji ldquoAntioxidantactivity of protein hydrolysates from aqueous extract of velvetantler (Cervus elaphus) as influenced by molecular weight andenzymesrdquo Natural Product Communications vol 6 no 11 pp1683ndash1688 2011

Journal of Chemistry 7

[34] S-Q Huang S D Ding and L P Fan ldquoAntioxidant activitiesof five polysaccharides from Inonotus obliquusrdquo InternationalJournal of BiologicalMacromolecules vol 50 no 5 pp 1183ndash11872012

[35] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006

[36] A Zulueta M J Esteve and A Frıgola ldquoORAC and TEACassays comparison to measure the antioxidant capacity of foodproductsrdquo Food Chemistry vol 114 no 1 pp 310ndash316 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Page 5: Research Article First Evaluation of the Biologically Active ...We investigated the biologically active substances contained in RVA (regrowth velvet antler) by comparing the composition

Journal of Chemistry 5

0

10

20

30

40

50

60

70

ab

cddd

e ef

fg

h

ABT

S ra

dica

l sca

veng

ing

activ

ity(120583

M T

Em

g)

T-RVA M-RVA B-RVA

Figure 4 The effect of RVA on ABYS radical scavenging activityandashgValues not sharing a common letter are significantly differentat 119875 lt 005 by Dunnettrsquos multiple range tests Light gray square125 120583gmL gray square 250 120583gmL dark grey square 500120583gmLblack square 1000120583gmL

0

5

10

15

20

25

30

35

40

T-RVA M-RVA B-RVA

a

b bd

de

e fgg g

hFRA

P (120583

M T

Em

g)

Figure 5 The effect of RVA on FRAP assay andashgValues not sharing acommon letter are significantly different at 119875 lt 005 by Dunnettrsquosmultiple range tests Light gray square 125 120583gmL gray square250 120583gmL dark grey square 500120583gmL black square 1000 120583gmL

0

20

40

60

80

100

120

140

T-RVA M-RVA B-RVA

a

bcc

b

d

fg

fee e

ORA

C (120583

M T

Em

g)

Figure 6 The effect of RVA on ORAC andashgValues not sharing acommon letter are significantly different at 119875 lt 005 by Dunnettrsquosmultiple range tests Light gray square 125 120583gmL gray square250 120583gmL dark grey square 500120583gmL black square 1000 120583gmL

activity of velvet antler extract obtained from the uppersection was reported to be 671 at an extract concentrationof 100mgmL which was lower than the activity measuredin our analysis H

2O2is a reactive nonradical and a clinically

important compound due to its ability to penetrate biologicalmembranes H

2O2can be converted into more reactive

species such as singlet oxygen and hydroxyl radicals therebycausing lipid peroxidation or toxicity to cells Thereforescavenging of hydrogen peroxide can decrease prooxidantsrsquolevels Our analysis of H

2O2scavenging by velvet antler

produced results similar to those reported by Je et al [30]Hydroxyl radicals are extremely reactive and easily reactwith amino acids DNA and membrane components In thisstudy the hydroxyl radical scavenging activity of RVA washigher than that of velvet antler as reported by Je et al[32] In addition our analysis of ABTS radical scavengingactivity by RVA identified activity higher than that reportedby Zhao et al [33] The FRAP assay treats the antioxidantscontained in the samples as reductants in a redox-linkedcolorimetric reaction allowing assessment of the reducingpower of antioxidants [34] Zhao et al [33] reported activityof 858 plusmn 002 by 5mgmL velvet antler extract in the FARPassay which was lower than the activity measured in ouranalysis The ORAC assay has been applied extensively toevaluate the antioxidant activities of fruits vegetables leavesstems herbs and spices As a result the ORAC assay iscommonly mentioned in scientific publications and healthfood publications [35] However the antioxidant activity ofRVAhas not been evaluated using theORACassayThereforethis is the first report of an assessment of the antioxidantactivity of RVA using the ORAC assay ORAC value of gallicacid was shown 161 plusmn 48 by Zulueta et al [36] which washigher than the activity of RVA found in our study

4 Conclusions

In the present study we provided the first comprehensiveevaluation of the biologically active substances of RVA andthe antioxidant potential of different RVA segments Futurestudies are required to further elucidate the other biologicalactivities of the T-RVA M-RVA and B-RVA sections and thebiological mechanisms underlying their effects

Conflict of Interests

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

Acknowledgment

This paper was supported by Konkuk University in 2015

References

[1] C Li ldquoDevelopment of deer antler model for biomedicalresearchrdquo Recent Advancesamp Research Updates vol 4 no 2 pp255ndash274 2003

[2] Z Q Zhang Y Zhang B X Wang H O Zhou Y Wangand H Zhang ldquoPurification and partial characterization of

6 Journal of Chemistry

anti-inflammatory peptide from pilose antler of Cervus nipponTemminckrdquo Acta Pharmaceutica Sinica vol 27 no 5 pp 321ndash324 1992

[3] Z F Ma D S Zhao Q D Zhou and S Sun ldquoStudy onraising yield of reproductive pilose antler of sika deerrdquo Journalof Northeast Forestry University vol 22 no 6 pp 41ndash47 1994

[4] J F He Y Z Liu S B Zhu and L Shang ldquoStudy on increasingquality and quantity of reborn antlers of cervus elaphuslinnaeusand cervus Nippon hortulorun swinchonerdquo Natural ScienceJournal of Harbin Normal University vol 20 no 4 pp 91ndash942004

[5] D A Butterfield A Castegna C M Lauderback and J DrakeldquoEvidence that amyloid beta-peptide-induced lipid peroxida-tion and its sequelae in Alzheimerrsquos disease brain contribute toneuronal deathrdquo Neurobiology of Aging vol 23 no 5 pp 655ndash664 2002

[6] W A Pryor and N Y Ann ldquoFree radical biology xenobioticscancer and agingrdquo Annals of the New York Academy of Sciencesvol 393 no 1 pp 1ndash22 1982

[7] E-K Kim S-J Lee J-W Hwang et al ldquoIn vitro investigationon antioxidative effect of Inonotus obliquus extracts againstoxidative stress on PC12 cellsrdquo Journal of the Korean Society forApplied Biological Chemistry vol 54 no 1 pp 112ndash117 2011

[8] J-WHwang E-K Kim S-J Lee et al ldquoAntioxidant activity andprotective effect of anthocyanin oligomers on H

2O2-triggered

G2M arrest in retinal cellsrdquo Journal of Agricultural and FoodChemistry vol 60 no 17 pp 4282ndash4288 2012

[9] B R Bhavnani ldquoPharmacology of hormonal therapeuticagentsrdquo in The Menopause Comprehensive Management B AEskin Ed pp 229ndash256 Parthenon Press New York NY USA2000

[10] S S Ditchkoff L J Spicer R E Masters and R L LochmillerldquoConcentrations of insulin-like growth factor-I in adult malewhite-tailed deer (Odocoileus virginianus) associations withserum testosterone morphometrics and age during and afterthe breeding seasonrdquo Comparative Biochemistry and PhysiologyA Physiology vol 129 no 4 pp 887ndash895 2001

[11] J A Gomez A J Garcıa T Landete-Castillejos and L GallegoldquoEffect of advancing births on testosterone until 25 years of ageand puberty in Iberian red deer (Cervus elaphus hispanicus)rdquoAnimal Reproduction Science vol 96 no 1-2 pp 79ndash88 2006

[12] C Li Z Jiang G Jiang and J Fang ldquoSeasonal changes ofreproductive behavior and fecal steroid concentrations in PereDavidrsquos deerrdquo Hormones and Behavior vol 40 no 4 pp 518ndash525 2001

[13] Y-J Li T-H Kim H B Kwak Z H Lee S-Y Lee and G-J Jhon ldquoChloroform extract of deer antler inhibits osteoclastdifferentiation and bone resorptionrdquo Journal of Ethnopharma-cology vol 113 no 2 pp 191ndash198 2007

[14] H H Sunwoo T Nakano and J S Sim ldquoEffect of water-solubleextract from antler of wapiti (Cervus elaphus) on the growth offibroblastsrdquo Canadian Journal of Animal Science vol 77 no 2pp 343ndash345 1997

[15] M Cesaretti E Luppi FMaccari andN Volpi ldquoA 96-well assayfor uronic acid carbazole reactionrdquo Carbohydrate Polymers vol54 no 1 pp 59ndash61 2003

[16] RW Farndale D J Buttle and A J Barrett ldquoImproved quanti-tation and discrimination of sulphated glycosaminoglycans byuse of dimethylmethylene bluerdquo Biochimica Biophysica Actavol 883 no 2 pp 173ndash177 1986

[17] K Matsuno and S Suzuki ldquoSimple fluorimetric method forquantification of sialic acids in glycoproteinsrdquo Analytical Bio-chemistry vol 375 no 1 pp 53ndash59 2008

[18] R Zhou and S F Li ldquoIn vitro antioxidant analysis andcharacterisation of antler velvet extractrdquo Food Chemistry vol114 no 4 pp 1321ndash1327 2009

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

[20] H E Muller ldquoDetection of hydrogen peroxide produced bymicroorganism on ABTS-peroxidase mediumrdquo Zentralblatt furBakteriologieMikrobiologie undHygiene vol 259 no 2 pp 151ndash158 1985

[21] S-K Chung T Osawa and S Kawakishi ldquoHydroxyl radical-scavenging effects of spices and scavengers frombrownmustard(Brassica nigra)rdquo Bioscience Biotechnology and Biochemistryvol 61 no 1 pp 118ndash123 1997

[22] M B Arnao A Cano and M Acosta ldquoThe hydrophilicand lipophilic contribution to total antioxidant activityrdquo FoodChemistry vol 73 no 2 pp 239ndash244 2001

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

[24] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal ofAgricultural and Food Chemistry vol 49 no 10 pp 4619ndash46262001

[25] M A Moskowitz E H Lo and C Iadecola ldquoThe science ofstrokemechanisms in search of treatmentsrdquoNeuron vol 67 no2 pp 181ndash198 2010

[26] Y W Ha B T Jeon S H Moon et al ldquoCharacterization ofheparan sulfate from the unossified antler of Cervus elaphusrdquoCarbohydrate Research vol 340 no 3 pp 411ndash416 2005

[27] B Jeon S Kim S Lee et al ldquoEffect of antler growth period onthe chemical composition of velvet antler in sika deer (Cervusnippon)rdquoMammalian Biology vol 74 no 5 pp 374ndash380 2009

[28] R Zhou JWang S Li andY Liu ldquoSupercritical fluid extractionof monoamine oxidase inhibitor from antler velvetrdquo Separationand Purification Technology vol 65 no 3 pp 275ndash281 2009

[29] B X Wang X H Zhao X W Yang et al ldquoInhibition oflipid peroxidation of deer antler (Rokujo) extract in vivo andin vitrordquo Journal of Medical and Pharmaceutical Society forWAKAN-YAKU vol 5 pp 123ndash128 1988

[30] J Y Je P J Park D H Lim B T Jeon K H Kho and C B AhnldquoAntioxidant anti-acetylcholinesterase and composition of bio-chemical components of Russian deer velvet antler extractsrdquoKorean Journal for Food Science of Animal Resources vol 31 no3 pp 349ndash355 2011

[31] K A Lee andH Y Chung ldquoThebiological activity of deer antlerextract in vitrordquoThe Korean Journal of Food and Nutrition vol20 no 2 pp 114ndash119 2007

[32] J-Y Je P-J Park E-K Kim et al ldquoComposition of biologicallyactive substances and antioxidant activity of New Zealand deervelvet antler extractsrdquoKorean Journal for Food Science of AnimalResources vol 30 no 1 pp 20ndash27 2010

[33] L Zhao Y-C Luo C-T Wang and B-P Ji ldquoAntioxidantactivity of protein hydrolysates from aqueous extract of velvetantler (Cervus elaphus) as influenced by molecular weight andenzymesrdquo Natural Product Communications vol 6 no 11 pp1683ndash1688 2011

Journal of Chemistry 7

[34] S-Q Huang S D Ding and L P Fan ldquoAntioxidant activitiesof five polysaccharides from Inonotus obliquusrdquo InternationalJournal of BiologicalMacromolecules vol 50 no 5 pp 1183ndash11872012

[35] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006

[36] A Zulueta M J Esteve and A Frıgola ldquoORAC and TEACassays comparison to measure the antioxidant capacity of foodproductsrdquo Food Chemistry vol 114 no 1 pp 310ndash316 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Page 6: Research Article First Evaluation of the Biologically Active ...We investigated the biologically active substances contained in RVA (regrowth velvet antler) by comparing the composition

6 Journal of Chemistry

anti-inflammatory peptide from pilose antler of Cervus nipponTemminckrdquo Acta Pharmaceutica Sinica vol 27 no 5 pp 321ndash324 1992

[3] Z F Ma D S Zhao Q D Zhou and S Sun ldquoStudy onraising yield of reproductive pilose antler of sika deerrdquo Journalof Northeast Forestry University vol 22 no 6 pp 41ndash47 1994

[4] J F He Y Z Liu S B Zhu and L Shang ldquoStudy on increasingquality and quantity of reborn antlers of cervus elaphuslinnaeusand cervus Nippon hortulorun swinchonerdquo Natural ScienceJournal of Harbin Normal University vol 20 no 4 pp 91ndash942004

[5] D A Butterfield A Castegna C M Lauderback and J DrakeldquoEvidence that amyloid beta-peptide-induced lipid peroxida-tion and its sequelae in Alzheimerrsquos disease brain contribute toneuronal deathrdquo Neurobiology of Aging vol 23 no 5 pp 655ndash664 2002

[6] W A Pryor and N Y Ann ldquoFree radical biology xenobioticscancer and agingrdquo Annals of the New York Academy of Sciencesvol 393 no 1 pp 1ndash22 1982

[7] E-K Kim S-J Lee J-W Hwang et al ldquoIn vitro investigationon antioxidative effect of Inonotus obliquus extracts againstoxidative stress on PC12 cellsrdquo Journal of the Korean Society forApplied Biological Chemistry vol 54 no 1 pp 112ndash117 2011

[8] J-WHwang E-K Kim S-J Lee et al ldquoAntioxidant activity andprotective effect of anthocyanin oligomers on H

2O2-triggered

G2M arrest in retinal cellsrdquo Journal of Agricultural and FoodChemistry vol 60 no 17 pp 4282ndash4288 2012

[9] B R Bhavnani ldquoPharmacology of hormonal therapeuticagentsrdquo in The Menopause Comprehensive Management B AEskin Ed pp 229ndash256 Parthenon Press New York NY USA2000

[10] S S Ditchkoff L J Spicer R E Masters and R L LochmillerldquoConcentrations of insulin-like growth factor-I in adult malewhite-tailed deer (Odocoileus virginianus) associations withserum testosterone morphometrics and age during and afterthe breeding seasonrdquo Comparative Biochemistry and PhysiologyA Physiology vol 129 no 4 pp 887ndash895 2001

[11] J A Gomez A J Garcıa T Landete-Castillejos and L GallegoldquoEffect of advancing births on testosterone until 25 years of ageand puberty in Iberian red deer (Cervus elaphus hispanicus)rdquoAnimal Reproduction Science vol 96 no 1-2 pp 79ndash88 2006

[12] C Li Z Jiang G Jiang and J Fang ldquoSeasonal changes ofreproductive behavior and fecal steroid concentrations in PereDavidrsquos deerrdquo Hormones and Behavior vol 40 no 4 pp 518ndash525 2001

[13] Y-J Li T-H Kim H B Kwak Z H Lee S-Y Lee and G-J Jhon ldquoChloroform extract of deer antler inhibits osteoclastdifferentiation and bone resorptionrdquo Journal of Ethnopharma-cology vol 113 no 2 pp 191ndash198 2007

[14] H H Sunwoo T Nakano and J S Sim ldquoEffect of water-solubleextract from antler of wapiti (Cervus elaphus) on the growth offibroblastsrdquo Canadian Journal of Animal Science vol 77 no 2pp 343ndash345 1997

[15] M Cesaretti E Luppi FMaccari andN Volpi ldquoA 96-well assayfor uronic acid carbazole reactionrdquo Carbohydrate Polymers vol54 no 1 pp 59ndash61 2003

[16] RW Farndale D J Buttle and A J Barrett ldquoImproved quanti-tation and discrimination of sulphated glycosaminoglycans byuse of dimethylmethylene bluerdquo Biochimica Biophysica Actavol 883 no 2 pp 173ndash177 1986

[17] K Matsuno and S Suzuki ldquoSimple fluorimetric method forquantification of sialic acids in glycoproteinsrdquo Analytical Bio-chemistry vol 375 no 1 pp 53ndash59 2008

[18] R Zhou and S F Li ldquoIn vitro antioxidant analysis andcharacterisation of antler velvet extractrdquo Food Chemistry vol114 no 4 pp 1321ndash1327 2009

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

[20] H E Muller ldquoDetection of hydrogen peroxide produced bymicroorganism on ABTS-peroxidase mediumrdquo Zentralblatt furBakteriologieMikrobiologie undHygiene vol 259 no 2 pp 151ndash158 1985

[21] S-K Chung T Osawa and S Kawakishi ldquoHydroxyl radical-scavenging effects of spices and scavengers frombrownmustard(Brassica nigra)rdquo Bioscience Biotechnology and Biochemistryvol 61 no 1 pp 118ndash123 1997

[22] M B Arnao A Cano and M Acosta ldquoThe hydrophilicand lipophilic contribution to total antioxidant activityrdquo FoodChemistry vol 73 no 2 pp 239ndash244 2001

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

[24] BOuMHampsch-Woodill andR L Prior ldquoDevelopment andvalidation of an improved oxygen radical absorbance capacityassay using fluorescein as the fluorescent proberdquo Journal ofAgricultural and Food Chemistry vol 49 no 10 pp 4619ndash46262001

[25] M A Moskowitz E H Lo and C Iadecola ldquoThe science ofstrokemechanisms in search of treatmentsrdquoNeuron vol 67 no2 pp 181ndash198 2010

[26] Y W Ha B T Jeon S H Moon et al ldquoCharacterization ofheparan sulfate from the unossified antler of Cervus elaphusrdquoCarbohydrate Research vol 340 no 3 pp 411ndash416 2005

[27] B Jeon S Kim S Lee et al ldquoEffect of antler growth period onthe chemical composition of velvet antler in sika deer (Cervusnippon)rdquoMammalian Biology vol 74 no 5 pp 374ndash380 2009

[28] R Zhou JWang S Li andY Liu ldquoSupercritical fluid extractionof monoamine oxidase inhibitor from antler velvetrdquo Separationand Purification Technology vol 65 no 3 pp 275ndash281 2009

[29] B X Wang X H Zhao X W Yang et al ldquoInhibition oflipid peroxidation of deer antler (Rokujo) extract in vivo andin vitrordquo Journal of Medical and Pharmaceutical Society forWAKAN-YAKU vol 5 pp 123ndash128 1988

[30] J Y Je P J Park D H Lim B T Jeon K H Kho and C B AhnldquoAntioxidant anti-acetylcholinesterase and composition of bio-chemical components of Russian deer velvet antler extractsrdquoKorean Journal for Food Science of Animal Resources vol 31 no3 pp 349ndash355 2011

[31] K A Lee andH Y Chung ldquoThebiological activity of deer antlerextract in vitrordquoThe Korean Journal of Food and Nutrition vol20 no 2 pp 114ndash119 2007

[32] J-Y Je P-J Park E-K Kim et al ldquoComposition of biologicallyactive substances and antioxidant activity of New Zealand deervelvet antler extractsrdquoKorean Journal for Food Science of AnimalResources vol 30 no 1 pp 20ndash27 2010

[33] L Zhao Y-C Luo C-T Wang and B-P Ji ldquoAntioxidantactivity of protein hydrolysates from aqueous extract of velvetantler (Cervus elaphus) as influenced by molecular weight andenzymesrdquo Natural Product Communications vol 6 no 11 pp1683ndash1688 2011

Journal of Chemistry 7

[34] S-Q Huang S D Ding and L P Fan ldquoAntioxidant activitiesof five polysaccharides from Inonotus obliquusrdquo InternationalJournal of BiologicalMacromolecules vol 50 no 5 pp 1183ndash11872012

[35] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006

[36] A Zulueta M J Esteve and A Frıgola ldquoORAC and TEACassays comparison to measure the antioxidant capacity of foodproductsrdquo Food Chemistry vol 114 no 1 pp 310ndash316 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Page 7: Research Article First Evaluation of the Biologically Active ...We investigated the biologically active substances contained in RVA (regrowth velvet antler) by comparing the composition

Journal of Chemistry 7

[34] S-Q Huang S D Ding and L P Fan ldquoAntioxidant activitiesof five polysaccharides from Inonotus obliquusrdquo InternationalJournal of BiologicalMacromolecules vol 50 no 5 pp 1183ndash11872012

[35] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D H Byrne ldquoComparison of ABTS DPPH FRAP andORAC assays for estimating antioxidant activity from guavafruit extractsrdquo Journal of Food Composition and Analysis vol19 no 6-7 pp 669ndash675 2006

[36] A Zulueta M J Esteve and A Frıgola ldquoORAC and TEACassays comparison to measure the antioxidant capacity of foodproductsrdquo Food Chemistry vol 114 no 1 pp 310ndash316 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Page 8: Research Article First Evaluation of the Biologically Active ...We investigated the biologically active substances contained in RVA (regrowth velvet antler) by comparing the composition

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of


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