Protection of Clitoria ternatea flower petal extract against free radical-induced hemolysis and oxidative damage in canine erythrocytesWathuwan Phrueksanan ab Sirinthorn Yibchok-anun ab Sirichai Adisakwattana bca Department of Pharmacology Faculty of Veterinary Sciences Chulalongkorn University 10330 Thailandb Research Group of Herbal Medicine for Prevention and Therapeutic of Metabolic Diseases Chulalongkorn University 10330 Thailandc Department of Nutrition and Dietetics Faculty of Allied Health Sciences Chulalongkorn University 10330 Thailand

A R T I C L E I N F O

Article historyReceived 3 March 2014Accepted 29 August 2014

KeywordsAnthocyaninAntioxidantErythrocytesClitoria ternatea flower petalFree radicalHemolysisOxidative damage

A B S T R A C T

The present study assessed the antioxidant activity and protective ability of Clitoria ternatea flower petalextract (CTE) against in vitro 22prime-azobis-2-methyl-propanimidamide dihydrochloride (AAPH)-inducedhemolysis and oxidative damage of canine erythrocytes From the phytochemical analysis CTE con-tained phenolic compounds flavonoids and anthocyanins In addition CTE showed antioxidant activityas measured by oxygen radical absorbance capacity (ORAC) method and 22-diphenyl-1-picrylhydrazyl(DPPH) radical scavenging assay CTE (400 μgml) remarkably protected erythrocytes against AAPH-induced hemolysis at 4 h of incubation Moreover CTE (400 μgml) reduced membrane lipid peroxidationand protein carbonyl group formation and prevented the reduction of glutathione concentration in AAPH-induced oxidation of erythrocytes The AAPH-induced morphological alteration of erythrocytes from asmooth discoid to an echinocytic form was effectively protected by CTE The present results contributeimportant insights that CTE may have the potential to act as a natural antioxidant to prevent free radical-induced hemolysis protein oxidation and lipid peroxidation in erythrocytes

copy 2014 Elsevier Ltd All rights reserved

1 Introduction

Oxidative stress is an imbalance between the production of re-active oxygen species and antioxidant defense mechanisms Reactiveoxygen species (ROS) such as hydrogen peroxide superoxide anionand hydroxyl radical are normally generated under aerobic meta-bolic pathways in the human body Excessive generation of ROScauses oxidative damage to the cellular biomolecules including DNAprotein nucleic acid and membrane lipids The increased oxida-tive stress of cellular physiology has been implicated in thepathogenesis of many diseases such as cancer chronic kidney diseaseand neurodegenerative diseases (Dimakopoulos and Mayer 2002Macotpet et al 2013 Silva et al 2013) Recent studies reveal thatoxidative stress may play a significant role in the initiation and reg-ulation of cardiomyocyte apoptosis in a variety of cardiac diseases(Corcoran et al 2004)

Erythrocytes also known as red blood cells (RBCs) have a uniqueshape and inner components that allow them to efficientlytransport oxygen and direct the elimination of carbon dioxideBesides their specific roles as oxygen carriers they are also highly

susceptible to endogenous oxidative damage Especially the poly-unsaturated fatty acids (PUFAs) of the erythrocyte membrane andthe redox active proteins of hemoglobin are key targets for freeradical-induced hemolysis and oxidative damage (Loacutepez-Revueltaet al 2006) The alteration can cause changes in their shape andthe loss of functional membrane integrity leading to the onset ofacute and chronic diseases (Iyer et al 2013)

Numerous studies suggest that antioxidants have gained world-wide popularity for the prevention of oxidative stress-related diseases(Esfahani et al 2011 Landete 2013) It has recently been re-ported that antioxidant supplementations exert a marked protectiveeffect on damage to DNA in dogs (Heaton et al 2002) Recentlyedible plants containing antioxidants have become a major area ofscientific research because they have greater health benefits withvarious pharmacological activities Clitoria ternatea L (familyFabaceae) commonly known as ldquoButterfly peardquo is widely culti-vated in the Caribbean area Central America Africa and SoutheastAsia The flower petal of this plant is recognized as a good sourceof dietary anthocyanins and used as a natural blue colorant in avariety of foods (Mukherjee et al 2008) The flower petal of Clitoriaternatea contains ternatins a group of delphinidin glycosides Thesix major ternatins were isolated from the flower and character-ized as A1 A2 B1 B2 D1 and D2 (Terahara et al 1996 1998) Inaddition other phytochemical compounds including triterpenoidsflavonol glycosides and steroids have been isolated from Clitoriaternatea Linn The Clitoria ternatea extract possesses a wide rangeof pharmacological activities including anti-inflammatory

Abbreviations AAPH 22prime-azobis-2-methyl-propanimidamide dihydrochlorideCTE Clitoria ternatea flower petal extract DPPH 22-diphenyl-1-picrylhydrazyl MDAmalondialdehyde GSH glutathione

Corresponding author Tel +66 2 2181067 fax +66 2 2181076E-mail address Sirichaiachulaacth (S Adisakwattana)

httpdxdoiorg101016jrvsc2014080100034-5288copy 2014 Elsevier Ltd All rights reserved

Research in Veterinary Science (2014) ndash

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anti-diabetic anti-microbial and antioxidant activities (Kamkaenand Wilkinson 2009 Mukherjee et al 2008) Although pharma-cological activities of Clitoria ternatea extract were well investigatedstudies regarding its protective effects against free radical-inducedhemolysis and oxidative damage have not been undertaken The aimof this study was to evaluate the effect of Clitoria ternatea flowerpetal extract (CTE) against AAPH-induced hemolysis and oxida-tive damage in dog erythrocytes In addition the phytochemicalanalyses of Clitoria ternatea flower petal and its antioxidant capaci-ties were also determined

2 Materials and method

21 Chemicals

Thiobarbituric acid (TBA) 22-diphenyl-1-picrylhydrazyl troloxtrichloroacetic acid (TCA) 22prime-azobis-2-methyl-propanimidamidedihydrochloride (AAPH) 24-dihydrophenylhydrazine potassiumcyanide guanidine hydrochloride 55prime-dithiobis(2-nitrobenzoic acid)(DTNB) malondialdehyde (MDA) potassium ferricyanide and tri-chloroacetic acid (TCA) were from Sigma-Aldrich Co (St Louis MOUSA) All chemicals and solvents used were analytical grade

22 Animals

Five healthy male dogs (5ndash10 years) were obtained fromChulalongkorn University Small Animal Hospital This study has beenreviewed and approved by Certification of Institutional Animal Careand Use Committee (IACUC) in accordance with university regula-tions and policies governing the care and use of laboratory animalsThe review has followed guidelines documented in Ethical Prin-ciples and Guidelines for the Use of Animals for Scientific Proposesedited by the National Research Council of Thailand (Approval no12310041 3042011)

23 Plant materials

The dried flower petals of Clitoria ternatea were obtained froma local market in Thailand The flower petals (05 kg) were boiledin 3 l of distilled water for 2 h Then the solution was filtrated throughWhatman 70 mm filter paper Finally the aqueous solution was driedusing spray dryer SD-100 (Eyela World Tokyo Rikakikai Co LTDJapan) Spray drying conditions were inletoutlet temperature (90 degC178 degC) blow rate (08 m3min) and atomizing (80ndash90 kPa) The driedextract of Clitoria ternatea was stored under refrigeration (4 degC) untilused for further analysis

24 The phytochemical analysis

The measurement of total phenolic content in CTE was modi-fied according to a previously published method (Maumlkynen et al2013) The extract was dissolved with distilled water A sample(20 μl) was mixed with 100 μl of the FolicinndashCiocalteu reagent (pre-viously diluted 10-fold with distilled water) followed by 80 μl ofaqueous Na2CO3 (60 gl) The absorbance was then measured at725 nm after incubation for 90 min Total phenolic content was ex-pressed as milligram gallic acid equivalentsgram dry weight ofextract Total flavonoid content in CTE was measured using the alu-minum chloride colorimetric assay (Adisakwattana et al 2012) Thesample solution (100 μl) was added to 30 μl of AlCl3 solution (10 wv) 30 μl of NaNO2 (15 wv) 400 μl of 4 NaOH and 440 μl ofdistilled water After incubation at room temperature for 10 minthe absorbance was measured immediately at 510 nm Total flavo-noid content was calculated from a calibration curve of catechin andexpressed as milligram catechin equivalentsgram dry weight ofextract The total anthocyanin content in CTE was analyzed by a pH

differential method (Moldovan et al 2012) The absorption of thesamples developed through pH 1 and pH 45 buffers was mea-sured in terms of cyanidin-3-glucoside at 510 and 700 nm for 15 minat room temperature The monomeric anthocyanin pigment con-centration was calculated according to the following equationMonomeric anthocyanin pigment (mgl) = (A times MW times DF times 1000)(ε times 1) where A = (A510 minus A700) pH 10 minus (A516 minus A700) pH 45 MW is themolecular weight of cyanidin-3-glucoside (4492) ε is the molar ab-sorptivity (26900) and DF is the dilution factor The totalanthocyanin content was expressed as milligram cyanidin-3-glucoside equivalentsgram dry weight of extract

25 Antioxidant activity

22-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activ-ity was measured according to a previously described method(Maumlkynen et al 2013) Briefly the sample (500 μl) was added to500 μl DPPH solution (02 mM in ethanol) and incubated for 30 minat room temperature The decrease in the solution absorbance wasmeasured at 515 nm and the percent inhibition was calculated usingthe following formula The percent inhibition = (A0 minus Asample)A0 times 100 where A0 is the absorbance of the control (blank) and Asample

is the absorbance of tested extract The DPPH radical scavenging ac-tivity was expressed as IC50 and calculated from the plot of thepercent inhibition against sample concentration L-ascorbic acid wasused as a positive control

The oxygen radical absorbance capacity (ORAC) assay was mea-sured according to a previously published method (Maumlkynen et al2013) Briefly 25 μl of the extract was mixed with 150 μl of 48 nMfluorescein solution and placed in the wells of a microplate Themixture was preincubated for 10 min at room temperature A freeradical generator solution (22prime-azobis-2-methyl-propanimidamidedihydrochloride AAPH 25 μl 64 mM) was added into the solu-tion The fluorescent intensity was recorded every 2 min for 60 minwith emission and excitation at 535 and 485 nm respectively A stan-dard curve was generated with a trolox concentration range from0024 to 3125 μM The ORAC value was calculated as the area underthe curve (AUC) and expressed as micromoles of trolox equivalent(TE) per gram of dry extract

26 Preparation of erythrocytes suspension

Fresh whole blood (5 ml) was obtained from healthy dogs viacephalic venipuncture The whole blood was centrifuged at 1500 gfor 10 min at 4 degC using a refrigerated centrifuge The separatederythrocytes were washed three times in 10 mM phosphate buffersaline (PBS) pH 74 After centrifugation the supernatant and thebuffy coat were carefully removed with each wash Washed eryth-rocytes were finally re-suspended to the desired hematocrit levelin 10 mM PBS The erythrocytes were stored at 4 degC and used within2 h of sample preparation

27 Erythrocyte hemolysis assay

The inhibition of free radical-induced erythrocyte hemolysis wasperformed according to a previously published method (Wang et al2009) The erythrocyte hemolysis was induced by thermal decom-position of AAPH Briefly 10 of erythrocyte suspension in PBS waspreincubated for 5 min at 37 degC with CTE (50ndash400 μgml) and trolox(100 μgml) followed by incubation with or without 50 mM AAPHsolution for 6 h with gentle shaking At 60 min intervals aliquotsof the reaction mixture (50 μl) were taken out and diluted with1000 μl of PBS After centrifugation at 2000 g for 10 min the ab-sorbance (A) of the supernatant was measured at 540 nm using aspectrophotometer The reference values were determined by usingthe same amount of erythrocyte in distilled water to obtain a

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2 W Phrueksanan et alResearch in Veterinary Science (2014) ndash

complete hemolysis (B) The percentage of hemolysis was then cal-culated as AB times 100

28 Quantitative estimation of lipid peroxidation

Lipid peroxidation was assessed through determination of themalondialdehyde (MDA) concentration in the erythrocyte mem-brane according to a previously published report (Wang et al 2009)The erythrocyte suspension (75 μl) was diluted to 250 μl with 10 mMPBS and then 130 μl of 28 (wv) trichloroacetic acid (TCA) solu-tion added to the solution with vigorous mixing After centrifugationat 12000 g for 5 min the obtained supernatant (200 μl) was sepa-rated and reacted with 250 μl of 1 TBA for 15 min at 100 degC in aheating block After cooling absorbance was measured using a spec-trophotometer at 532 nm MDA values in erythrocytes weredetermined from the calibration curve of MDA (0ndash6 nmolml) andpresented as nmolmg hemoglobin (Hb) The concentration of Hbwas determined using the Darbkin method (Darbkin 1946)

29 Determination of glutathione content

Intracellular glutathione (GSH) was measured according to a pre-viously published report (Van den Berg et al 1992) Briefly thedistilled water (60 μl) was added into the erythrocyte suspension(100 μl) for cell lysate The lysate cell was precipitated by 10 (wv) TCA (130 μl) for 5 min After centrifugation at 18000 g for 5 min30 μl of DTNB solution (20 mg of DTNB diluted in 1 citrate solu-tion 100 ml) was added to the supernatant (135 μl) in a microplateThe absorbance of the samples was measured at 412 nm usinga spectrophotometer The content of GSH was expressed asμmolg Hb

210 Determination of protein carbonyl content

The protein carbonyl content in erythrocytes was done accord-ing to a previously published method with minor modifications(Levine et al 1990) Briefly the erythrocyte suspension (50 μl) wasprecipitated by 10 (wv) TCA and centrifuged at 12000 g for 10 minThe reaction mixture (50 μl) was taken in two tubes as test sampleand control The test sample was reacted with 400 μl of 2 (wv)24-dinitrophenylhydrazine in 2 M HCl and 400 μl of 2 M HCl wasadded to the control sample Thereafter 10 (wv) TCA was addedto both tubes and the mixture left in ice for 1 h The tubes were thencentrifuged at 10000 g for 10 min to obtain the protein pellets Thesupernatant was carefully aspirated and discarded The protein pelletswere washed three times with ethanolethyl acetate (11 vv) so-lution to remove unreacted DNPH and lipid remnants Finally proteinpellets were dissolved in 6 M guanidine hydrochloride (250 μl) andincubated for 10 min at 37 degC The carbonyl content was calcu-lated by using a molar extinction coefficient of 22000 Mminus1 cmminus1 andexpressed as nmol carbonylmg protein

211 Determination of erythrocyte shape

After 4 h of incubation erythrocytes were centrifuged at 1500 gfor 10 min For scanning electron microscope (SEM) obser-vations the cells were washed twice with PBS and fixed in 25

glutaraldehyde in 10 mM PBS pH 74 for 24 h at 4 degC The stored cellswere pelleted and post-fixed with 1 osmium tetroxide at 4 degC toachieve final hematocrit of about 50 Fixed cells were allowed tosettle on standard microscopic cover glasses After 1 h of incuba-tion cover glasses were washed three times with the samephosphate buffer Subsequently samples were dehydrated with suc-cessive washes in ascending ethanol series (30 50 70 953 mineach and 100 vv 3 mineach for three times) and finallydried with CO2 in the triple-point (Critical point dryer Balzersregmodel CPD 020) and coated with goldndashpalladium (Sputter coaterBlazersreg model SCD 040) Finally the samples were observed usinga SEM (JEOLreg model JSM-5410LV)

212 Statistical analysis

Data were expressed as mean plusmn SEM (standard error of mean)of five different experiments The statistical analysis was per-formed using a one-way ANOVA followed by a least significantdifference (LSD) test A value of p lt 005 was considered to besignificant

3 Results

31 The phytochemical analysis of CTE

As shown in Table 1 the total phenolic compound and flavo-noid contents in CTE were 5300 plusmn 034 mg gallic acid equivalentsgdried extract and 1120 plusmn 033 mg catechin equivalentsg dried extractIn addition CTE had 146 plusmn 004 mg cyanidin-3-glucosideequivalentsg dried extract

32 Determination of in vitro antioxidant activity

In the DPPH assay the IC50 value of CTE and ascorbic acid werefound to be 047 plusmn 001 mgml and 0002 plusmn 0001 mgml respec-tively The results indicated that CTE had less potency than ascorbicacid The ORAC value was 1754 plusmn 042 μg trolox equivalentsmg driedextract

33 Protective effect of CTE on AAPH-induced hemolysis incanine erythrocytes

Fig 1 illustrates AAPH-induced hemolysis of erythrocytes at dif-ferent concentrations (0ndash50 mM) In the absence of AAPHerythrocytes incubated with PBS remained stable and demon-strated a slight hemolysis at 6 h (279 plusmn 088) When AAPH (125ndash50 mM) was added to the erythrocyte suspension hemolysisoccurred after 3 h of incubation The results showed that AAPHinduced hemolysis in a typical time- and concentration-dependentmanner At the concentration of 50 mM AAPH it markedly inducedthe hemolysis at 4 5 and 6 h of incubation ( hemolysis at4 h = 5288 plusmn 243 at 5 h = 6516 plusmn 381 and at 6 h = 7070 plusmn 370)Therefore we selected AAPH at a concentration of 50 mM and theincubation period was 4ndash6 h for hemolysis conditions in this study

The effect of CTE on AAPH-induced hemolysis in erythrocytesis shown in Fig 2 A preliminary study of CTE (400 μgml) did notshow the effects on the induction of erythrocyte hemolysis (Fig 2)

Table 1The phytochemical analysis and antioxidant activity of CTE

Total phenolic compounds Total flavonoids Total anthocyanins DPPH radical scavenging activity ORAC

CTE 5300 plusmn 003 1120 plusmn 033 146 plusmn 004 047 plusmn 001 1754 plusmn 042

Data are expressed as mean plusmn SEM n = 3 Total phenolic compounds (TPC) were expressed as microgram gallic acid equivalentsgram dry weight of extract Total flavonoids(TF) were expressed as milligram catechin equivalentsgram dry weight of extract Total anthocyanins (TA) were expressed as milligram cyanidin-3-glucoside equivalentsgram dry weight of extract DPPH radical scavenging activity was expressed as the IC50 (mgml) ORAC was expressed as microgram troloxmilligram dried weight of extract

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The results demonstrated that CTE (50ndash400 μgml) inhibited he-molysis of erythrocytes in a concentration-dependent manner at3ndash4 h of incubation At 4 h of incubation CTE (50ndash400 μgml) wasshown to decrease the AAPH-challenged erythrocyte membrane from

hemolysis The percentage reduction of hemolysis ranged between2645 and 9625 At 5 h and 6 h of incubation CTE (400 μgml)demonstrated the protection of hemolysis at 7325 and 6025 re-spectively However there was no difference in the hemolysis whenCTE was added at concentrations of 50ndash200 μgml at 5 h and 6 hof incubation periods Under similar conditions trolox (400 μgml) prevented the hemolysis by 9710 (4 h) 5554 (5 h) and 3057(6 h) respectively The results indicated that CTE was less potentthan trolox compared with the same concentration (100 μgml) and4 h of incubation

34 Protective effect of CTE on oxidative damage oncanine erythrocytes

According to the results from the protective effect of CTE the in-cubation time at 4 h was chosen for assessing the concentration ofMDA GSH and protein carbonyl content At 4 h of incubation theMDA concentration of AAPH-untreated and -treated erythrocyteswas 228 plusmn 134 nmolg Hb and 2096 plusmn 391 nmolg Hb respective-ly The results indicated that AAPH led to a 919-fold increase of MDAconcentration in erythrocytes (Table 2) When erythrocytes wereincubated with CTE (400 μgml) in the absence of AAPH the MDAlevel was similar to that of the AAPH-untreated erythrocytes In themeantime the treatment of CTE (200 and 400 μgml) significantlyreduced the formation of intracellular MDA in the AAPH-treatederythrocytes by approximately 509 and 727 respectivelyHowever CTE (50 and 100 μgml) had no significant reduction ofMDA in the AAPH-treated erythrocytes In the case of the AAPH-treated erythrocytes incubated with trolox (100 μgml) theconcentration of MDA was decreased by 6407

As shown in Table 2 the concentrations of GSH in the AAPH-untreated and -treated erythrocytes were 3182 plusmn 300 μmolg Hband 1830 plusmn 386 μmolg Hb respectively The results indicated thatAAPH treatment reduced the concentration of GSH 174-fold whencompared to AAPH-untreated erythrocytes Furthermore CTE(400 μgml) had no effect on the concentration of GSH in erythro-cytes When erythrocytes were incubated with AAPH + CTE (400 μgml) the amount of GSH was significantly increased by 652 ascompared with the AAPH-treated erythrocytes However there wereno significant changes in the concentration of GSH in the AAPH-treated erythrocytes with CTE (50ndash200 μgml) In addition troloxhad no protective effect in the depletion of GSH in the AAPH-treated erythrocytes

The protein carbonyl content is generally considered an indexof protein oxidation in erythrocytes The amount of protein car-bonyl in the AAPH-untreated erythrocytes was 032 plusmn 004 nmolcarbonylmg protein at 4 h of incubation (Table 2) When erythro-cytes were incubated with AAPH the content of protein carbonylwas significantly increased (060 plusmn 012 nmol carbonylmg protein)

Fig 1 The percentage of hemolysis (10 erythrocytes in 10 mM PBS pH 74) in-cubated with various concentrations of AAPH (0ndash50 mM) at 37 degC for 0ndash6 h The resultsare expressed as mean plusmn SEM (n = 5) p lt 005 compared to control (PBS)

Fig 2 The percentage of hemolysis (10 erythrocytes in 10 mM PBS pH 74) in-cubated with AAPH and CTE (50ndash400 μgml) and trolox (100 μgml) at 37 degC for 4 hof incubation The results are expressed as mean plusmn SEM (n = 5) p lt 005 comparedto AAPH

Table 2Effects of CTE on AAPH-induced lipid peroxidation (MDA) protein oxidation (PC) and GSH depletion in erythrocytesErythrocyte suspension at 10 hematocrit was incubated with PBS (control) or preincubated with CTE for 5 min Thenit was incubated with a final concentration of 50 mM AAPH for 4 h at 37 degC Values are expressed as mean plusmn SEM (n = 5)

Treatment MDA (nmolg Hb) GSH (μmolg Hb) PC (nmolmg protein)

PBS (control) 228 plusmn 134 3186 plusmn 295 032 plusmn 004PBS + CTE 400 μgml 314 plusmn 024 2856 plusmn 145 030 plusmn 002AAPH 2096 plusmn 391 1830 plusmn 386 060 plusmn 012AAPH + CTE 50 μgml 1414 plusmn 323 1672 plusmn 472 068 plusmn 010AAPH + CTE 100 μgml 1407 plusmn 395 1792 plusmn 220 043 plusmn 007AAPH + CTE 200 μgml 1030 plusmn 213 2067 plusmn 250 053 plusmn 005AAPH + CTE 400 μgml 572 plusmn 145 3022 plusmn 562 035 plusmn 004

AAPH + trolox 100 μgml 753 plusmn 337 2087 plusmn 543 050 plusmn 005

p lt 005 compared to control (PBS) p lt 005 compared to AAPH

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4 W Phrueksanan et alResearch in Veterinary Science (2014) ndash

These findings indicate a 188-fold increase in the protein car-bonyl content in the AAPH-treated erythrocytes as compared withthe AAPH-untreated erythrocytes In addition CTE alone (400 μgml) did not produce any significant change in the protein carbonylcontent The results showed that the AAPH-treated erythrocytes withCTE (400 μgml) reduced the protein carbonyl level by 417 overthe AAPH-treated erythrocytes However CTE (50ndash200 μgml) andtrolox had no effect on the reduction of protein carbonyl contentin the AAPH-treated erythrocytes

35 Protective effect of CTE on AAPH-induced erythrocytemembrane alterations

The observation revealed that the AAPH-untreated erythro-cytes are normal biconcave shape (Fig 3A) while exposure to AAPHresulted in a significant change in the size and shape of cells nu-merous extrusion protuberances on their surfaces andor cell rufflededges (echinocyte or crenated cells) (Fig 3B) The morphologicalchanges mediated by AAPH were largely prevented when erythro-cytes were treated with CTE (Fig 3CndashE) Importantly the AAPH-treated erythrocytes in the presence of CTE (400 μgml) and trolox(100 μgml) still maintained the normal biconcave shape except avery few cells underwent a slight change in conformation(Fig 3F and G)

4 Discussion

Free radicals attack erythrocyte membrane components such asproteins and lipids and cause the alteration of membrane struc-ture and function which may result in hemolysis AAPH has beenwidely used as a water-soluble source of free radical initiators capableof inducing lipid peroxidation and protein damage The peroxyl radi-cals of AAPH are generated by thermal decomposition of azocompound in oxygen at physiological temperature (Shiva ShankarReddy et al 2007) The results demonstrated that the peroxyl radi-cals were initiated by AAPH-induced hemolysis in time- and

concentration-dependent manners In the present study the incu-bation of erythrocyte together with AAPH led to remarkablehemolysis that was consistent with previous findings (Loacutepez-Revueltaet al 2006 Wang et al 2009 Zhang et al 2014) Moreover theresults from the AAPH-induced hemolysis of canine erythrocyte aresimilar to hemolysis of rat erythrocytes and human erythrocytes(Shiva Shankar Reddy et al 2007 Wang et al 2009) According tothe results observed within 3 h there was a notable lag phase inthe progress curve of hemolysis during incubation with AAPH sug-gesting that hemolysis may be preceded by other key events in theprocess of oxidative damage It suggests that endogenous antioxi-dants in erythrocytes mainly glutathione vitamins (ascorbic acidand α-tocopherol) and enzymes (catalase and superoxidedismutase) can quench peroxyl radicals to protect them from he-molysis and oxidative damage During hemolysis peroxyl radicalsgenerated by AAPH more easily penetrated the cells and hemoglo-bin was oxidized to methemoglobin via the oxidation of ferrous ionto ferric ion leading to conversion into hemichromes (Minetti et al1993) The AAPH-induced hemolysis also caused the morphologi-cal alteration to erythrocytes according to the evidence from theSEM images AAPH obviously converted erythrocytes from a discoidinto an echinocytic form These changes are due to the insertion offoreign molecules in either the inner or outer monolayer of the eryth-rocyte membrane (Lim et al 2002) When erythrocytes are exposedto peroxyl radicals generated by AAPH erythrocyte membrane lipidscould lose a hydrogen atom from an unsaturated fatty acyl chainand initiate the lipid peroxidation that propagates as a chain reac-tion resulting the induction of oxidative stress (Loacutepez-Revuelta et al2006) Lipid peroxidation would alter the membrane permeabili-ty disrupt ionic channels and eventually lead to dysfunction of thewhole erythrocyte (Loacutepez-Revuelta et al 2006) The evidence revealsthat AAPH-induced protein oxidation causes the conversion of aminoacid residues (lysine arginine proline and threonine) intocarbonyl groups in erythrocytes (Berlett and Stadtman 1997) There-fore the protein carbonyl content has been commonly used as amarker for protein oxidative damage The glutathione system plays

A B C

D E F

G

Fig 3 Effect of CTE on morphological changes of erythrocytes for 4 h of incubation (A) Control (PBS) (B) AAPH (C) AAPH + CTE (50 μgml) (D) AAPH + CTE (100 μgml)(E) AAPH + CTE (200 μgml) (F) AAPH + CTE (400 μgml) (G) AAPH + trolox (100 μgml)

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5W Phrueksanan et alResearch in Veterinary Science (2014) ndash

a key role in intracellular anti-oxidation of metabolic and regula-tory enzymes The reduced glutathione (GSH) is considered a primarydefense mechanism against free radicals (Vissers and Winterbourn1995) Several studies demonstrate that the incubation of eryth-rocytes with AAPH also provokes the rapid depletion of intracellularGSH caused by oxidation processes taking place in erythrocyte mem-branes (Wang et al 2009 Ximenes et al 2010)

Antioxidants from natural products are important to protectagainst these radicals The literature has documented the antioxi-dant activity of Clitoria ternatea flower petal extracts in inhibitingthe formation of free radicals generated by the DPPH (Kamkaen andWilkinson 2009) The methanol extract of Clitoria ternatea flowerpetal was found to demonstrate the most active free radical scav-enging followed by chloroform and petroleum ether extracts(Mukhopadhyay et al 2012) The present study exhibits remark-able in vitro DPPH radical scavenging activity of the aqueous formof the Clitoria ternatea flower petal We first report the results re-garding the peroxyl radical scavenger activity of CTE studied byoxygen radical absorbance capacity (ORAC) According to the resultsfrom hemolysis the incubation of erythrocytes containing AAPH sig-nificantly exhibited formation of membrane lipid peroxidation andprotein carbonyl levels associated with rapid depletion of GSH Inthe meantime the treatment of erythrocyte with CTE reduced theformation of membrane lipid peroxidation and protein carbonyl leveland prevented an AAPH-induced decrease in GSH concentration inerythrocytes It also inhibited the formation of the echinocytic formthat helped to restore the normal discoid shape and maintained asmooth cell surface without any protrusions Other studies haveshown oral administration of CTE in streptozotocin-induced dia-betic rats to be effective in reducing lipid peroxidation and preventingcellular glutathione depletion in the brain tissue (Talpate et al 2013)

The exact mechanisms of CTE for the prevention of erythro-cyte hemolysis and oxidation have yet to be determined As ourexperiments showed CTE possesses antioxidant properties and couldserve as free radical scavengers It suggests that CTE under inves-tigation can protect hemolysis membrane lipid and protein oxidationof erythrocytes due to its scavenging activity against free radicalsMany epidemiological studies demonstrate that phytochemicals havebeen shown to possess significant antioxidant activity in various invitro models (Malireddy et al 2012) Polyphenols including flavo-noids and anthocyanins exhibit considerable free radical scavengingactivities and prevent AAPH-induced hemolysis in erythrocytes(Asgary et al 2005 Pandey and Rizvi 2009 Tsuda 2012 Zhanget al 2014) All naturally-occurring anthocyanins have the basicflavylium cation structure that may play an important role in an-tioxidants (Castantildeeda-Ovando et al 2009 Lapidot et al 1999) Acompletely conjugated structure of anthocyanins could stabilize freeradicals through electron delocalization (Rice-Evans et al 1996) Inaddition there are data demonstrating that polyphenols are boundby proteins on the cell surface of erythrocytes rather than intra-cellular structures leading to form the complexes which may serveas a free radical scavenger Interestingly the interaction of antho-cyanins with the lipid membrane of erythrocytes has been describedWhen anthocyanins interact with the lipid membrane of erythro-cytes they can penetrate only the outer part of the erythrocytemembrane Their action causes the protective membrane againstoxidative damage induced by AAPH (Bonarska-Kujawa et al 2012)The actions of anthocyanins are supported by other studies indi-cating that they are able to protect AAPH-induced hemolysis andoxidative damage to erythrocytes (Zhang et al 2014) With regardto antioxidant experiments anthocyanins in CTE most likely quenchthe peroxyl radicals before these radicals attack the biomoleculesof the erythrocyte membrane to cause hemolysis and oxidativedamage preventing lipid peroxidation and GSH depletion Hencethe powerful protection of CTE containing high amounts of antho-cyanins against AAPH-induced hemolysis in the present study might

be explained by the free radical scavenging capability of anthocy-anins and their binding abilities to the lipid membrane oferythrocytes

In conclusion the experimental evidence obtained in the presentstudy indicates that CTE are a rich source of anthocyanins that man-ifest DPPH and peroxyl radical scavenging activity Moreover CTEcan effectively protect AAPH-induced hemolysis and oxidativedamage oxidation in erythrocytes Thus CTE may be a valuablesource of natural antioxidants that may potentially be recom-mended for applications in animal food and nutrition

Acknowledgements

The authors would like to thank the Special Task Force for Ac-tivating Research (STAR) under 100 years Chulalongkorn UniversityFund and the Research Group of Herbal Medicine for Prevention andTherapeutic of Metabolic Diseases for financial support

References

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Asgary S Naderi G Askari N 2005 Protective effect of flavonoids against red bloodcell hemolysis by free radicals Experimental and Clinical Cardiology 10 88ndash90

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Bonarska-Kujawa D Pruchnik H Kleszczynska H 2012 Interaction of selectedanthocyanins with erythrocytes and liposome membranes Cellular and MolecularBiology Letters 17 289ndash308

Castantildeeda-Ovando A Pacheco-Hernaacutendez ML Paacuteez-Hernaacutendez ME RodriacuteguezJA Galaacuten-Vidal CA 2009 Chemical studies of anthocyanins a review FoodChemistry 113 859ndash871

Corcoran BM Black A Anderson H McEwan JD French A Smith P et al 2004Identification of surface morphologic changes in the mitral valve leaflets andchordae tendineae of dogs with myxomatous degeneration American Journalof Veterinary Research 65 198ndash206

Darbkin DL 1946 Spectrophotometric studies XIV the crystallographic and opticalproperties of the hemoglobin of man in comparison with these of other speciesJournal of Biological Chemistry 164 703ndash772

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Esfahani A Wong JM Truan J Villa CR Mirrahimi A Srichaikul K et al 2011Health effects of mixed fruit and vegetable concentrates a systematic reviewof the clinical interventions The Journal of the American College of Nutrition30 285ndash294

Heaton PR Reed CF Mann SJ Ransley R Stevenson J Charlton CJ et al 2002Role of dietary antioxidants to protect against DNA damage in adult dogs Journalof Nutrition 132 (6 Suppl 2) 1720Sndash1724S

Iyer MK Nayak R Colah R Chattopadhyay S 2013 Attenuation of oxidativehemolysis of human red blood cells by the natural phenolic compoundallylpyrocatechol Free Radical Research 47 710ndash717

Kamkaen N Wilkinson J 2009 The antioxidant activity of Clitoria ternatea flowerpetal extracts and eye gel Phytotherapy Research 23 1624ndash1625

Landete JM 2013 Dietary intake of natural antioxidants vitamins and polyphenolsCritical Reviews in Food Science and Nutrition 53 706ndash721

Lapidot T Harel S Akiri B Granit R Kanner J 1999 pH-dependent forms of redwine anthocyanins as antioxidants Journal of Agricultural and Food Chemistry47 67ndash70

Levine R Garland D Oliver C Amici A Climent I Lenz AG et al 1990Determination of carbonyl content in oxidatively modified proteins Methodsin Enzymology 186 464ndash478

Lim HWG Wortis M Mukhopadhyay R 2002 Stomatocyte-discocyte-echinocytesequence of the human red blood cell evidence for the bilayer- couple hypothesisfrom membrane mechanics Proceedings of the National Academy of Sciencesof the United States of America 99 16766ndash16769

Loacutepez-Revuelta A Saacutenchez-Gallego JI Hernaacutendez-Hernaacutendez A Saacutenchez-YaguumleJ Llanillo M 2006 Membrane cholesterol contents influence the protectiveeffects of quercetin and rutin in erythrocytes damaged by oxidative stressChemico-Biological Interactions 161 79ndash91

Macotpet A Suksawat F Sukon P Pimpakdee K PattarapanwichienE Tangrassameeprasert R et al 2013 Oxidative stress in cancer-bearing dogsassessed by measuring serum malondialdehyde BMC Veterinary Research 9 101

Malireddy S Kotha SR Secor JD Gurney TO Abbott JL Maulik G et al 2012Phytochemical antioxidants modulate mammalian cellular epigenomeimplications in health and disease Antioxidants and Redox Signaling 17 327ndash339

Maumlkynen K Jitsaardkul S Tachasamran P Sakai N Puranachoti S NirojsinlapachaiN et al 2013 Cultivar variations in antioxidant and antihyperlipidemic

ARTICLE IN PRESS

Please cite this article in press as Wathuwan Phrueksanan Sirinthorn Yibchok-anun Sirichai Adisakwattana Protection of Clitoria ternatea flower petal extract against free radical-induced hemolysis and oxidative damage in canine erythrocytes Research in Veterinary Science (2014) doi 101016jrvsc201408010

6 W Phrueksanan et alResearch in Veterinary Science (2014) ndash

properties of pomelo pulp (Citrus grandis [L] Osbeck) in Thailand Food Chemistry139 735ndash743

Minetti M Mallozzi C Scorza G Scott MD Kuypers FA Lubin BH 1993 Roleof oxygen and carbon radicals in hemoglobin oxidation Archives of Biochemistryand Biophysics 302 233ndash244

Moldovan B David L Chişbora C Cimpoiu C 2012 Degradation kinetics ofanthocyanins from European cranberrybush (Viburnum opulus L) fruit extractsEffects of temperature pH and storage solvent Molecules 17 11655ndash11666

Mukherjee PK Kumar V Kumar NS Heinrich M 2008 The Ayurvedic medicineClitoria ternateandashFrom traditional use to scientific assessment Journal ofEthnopharmacology 120 291ndash301

Mukhopadhyay R Bhattacharya S Biswas M 2012 In vitro free radical scavengingactivity of Clitorea ternatea leaf extracts Journal of Advanced PharmaceuticalResearch 2 206ndash209

Pandey KB Rizvi SI 2009 Plant polyphenols as dietary antioxidants in humanhealth and disease Oxidative Medicine and Cellular Longevity 2 270ndash278

Rice-Evans CA Miller NJ Paganga G 1996 Structure-antioxidant activityrelationships of flavonoids and phenolic acids Free Radical Biology and Medicine20 933ndash956

Shiva Shankar Reddy C Subramanyam M Vani R Asha Devi S 2007 In vitromodels of oxidative stress in rat erythrocytes effect of antioxidant supplementsToxicology in vitro 21 1355ndash1364

Silva AC de Almeida BF Soeiro CS Ferreira WL de Lima VM Ciarlini PC2013 Oxidative stress superoxide production and apoptosis of neutrophils indogs with chronic kidney disease Canadian Journal of Veterinary Research 77136ndash141

Talpate KA Bhosale UA Zambare MR Somani R 2013 Antihyperglycemic andantioxidant activity of Clitorea ternatea Linn on streptozotocin-induced diabeticrats Ayu 34 433ndash439

Terahara N Oda M Matsui T Osajima Y Saito N Toki K et al 1996 Five newanthocyanins ternatins A3 B4 B3 B2 and D2 from Clitoria ternatea flowersJournal of Natural Products 59 139ndash144

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Tsuda T 2012 Dietary anthocyanin-rich plants biochemical basis and recentprogress in health benefits studies Molecular Nutrition amp Food Research 56159ndash170

Van den Berg J Lubin B Roelofsen B Roelofsen B Kuypers FA 1992 Kineticsand site specificity of hydroperoxide-induced oxidative damage in red blood cellsFree Radical Biology and Medicine 12 487ndash498

Vissers M Winterbourn CC 1995 Oxidation of intracellular glutathione afterexposure of human red blood cells to hypochlorous acid Biochemical Journal307 57ndash62

Wang J Sun B Cao Y Tian Y 2009 Protection of wheat bran feruloyloligosaccharides against free radical-induced oxidative damage in normal humanerythrocytes Food and Chemical Toxicology 47 1591ndash1599

Ximenes VF Lopes MG Petrocircnio MS Regasini LO Silva DH da Fonseca LM2010 Inhibitory effect of gallic acid and its esters on 22prime-azobis(2-amidinopropane)hydrochloride (AAPH)-induced hemolysis and depletion ofintracellular glutathione in erythrocytes Journal of Agricultural and FoodChemistry 58 5355ndash5362

Zhang J Hou X Ahmad H Zhang H Zhang L Wang T 2014 Assessment of freeradicals scavenging activity of seven natural pigments and protective effects inAAPH-challenged chicken erythrocytes Food Chemistry 145 57ndash65

ARTICLE IN PRESS

Please cite this article in press as Wathuwan Phrueksanan Sirinthorn Yibchok-anun Sirichai Adisakwattana Protection of Clitoria ternatea flower petal extract against free radical-induced hemolysis and oxidative damage in canine erythrocytes Research in Veterinary Science (2014) doi 101016jrvsc201408010

7W Phrueksanan et alResearch in Veterinary Science (2014) ndash