Research ArticleLipid Peroxidation and Antioxidant Activities of the AqueousRhizome Extract of Rheum officinale Baillon
Eugene Chang1 and Choon Young Kim 2
1Department of Nutritional Science and Food Management Ewha Womans University Seoul 03760 Republic of Korea2Department of Food and Nutrition Yeungnam University Gyeongsan Gyeongbuk 38541 Republic of Korea
Correspondence should be addressed to Choon Young Kim cykimyuackr
Received 22 January 2018 Accepted 14 May 2018 Published 9 July 2018
Academic Editor Jorge Barros-Velazquez
Copyright copy 2018 Eugene Chang and Choon Young Kimis is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in anymedium provided the original work isproperly cited
Generation of reactive oxygen species (ROS) is associated with dysregulation of antioxidant defense mechanisms and incidence ofhuman diseases e specic aim of this study was to investigate the lipid oxidation and antioxidant activity of aqueous extract ofRheum ocinale Baillon rhizome in order to evaluate its potential as a future novel natural antioxidant resource and a functionalingredient in food and pharmaceutical formations Total phenolic and avonoid contents of Rheum rhizome extract were dosedependently increased Consistent with this radical scavenging activities of Rheum rhizome extract as determined by 22-diphenyl-1-picrylhydrazyl assay and 22prime-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity weresignicantly elevated as the concentration increased In addition the treatment of aqueous Rheum rhizome extract signicantlyincreased ferric reducing and copper chelating activities According to results of thiobarbituric acid reactive substance analysisRheum rhizome extract signicantly delayed lipid oxidation Preincubation with Rheum rhizome extract signicantly inhibitedtert-butyl hydroperoxide- (t-BHP-) induced ROS generation Moreover superoxide anion production was signicantly lower inRheum rhizome extract-treated RAW2647 macrophage cells than t-BHP-incubated cells (plt 005) ese ndings suggest thatRheum ocinale Baillon rhizome extract has a potential as an excellent natural antioxidant agent
1 Introduction
Oxidative stress which is an imbalance between the pro-duction of deleterious reactive oxygen species (ROS) andexisting antioxidant defense system plays a pivotal patho-physiological role in the development of liver disease canceraging autoimmune disorders and cardiovascular and neu-rodegenerative diseases [1ndash5] Overproduction of ROS suchas hydroxyl radical (OHmiddot) superoxide radical (O2
ndashmiddot) hydro-gen peroxide (H2O2) and nitric monoxide (NOmiddot) readilyattacks the polyunsaturated fatty acids in the plasma mem-brane resulting in the oxidative degradation of lipids [6 7]Consequently lipid oxidation induces cellular and tissuedamages through covalent binds resulting in lipid perox-idation DNA injury inammation and subsequent cell death[8] In addition lipid oxidation in foods and food productslowers food quality creating osect-avors and unhealthfulcompounds [9 10] erefore it is of great interest to prevent
ROS production and lipid oxidation for the improvement ofquality and nutrition of foods in the agriculture and foodindustry
Rhubarb (Dahuang) is an herbaceous perennial in thePolygonaceae family and its root stems and leaves havebeen used as Asian traditional herbal medicine for treatmentof constipation jaundice gastrointestinal hemorrhages andulcers Recent accumulating evidence shows that severalspecies of the Rheum genus such as Rheum emodi Rheumundulatum L Rhizoma Rhei Rheum ribes Rheum palma-tum L and Rheum rhaponticum have antiallergic anti-bacterial antioxidant anticancer antiangiogenesis andanti-inammation properties [11ndash20] In relation to anti-oxidant capacity anthraquinone derivates are considered tobe one of the major biologically active constituents of aloe-emodin rhein emodin chrysophanol physcion and dan-thron from the rhizomes of Rheum undulatum L andRheum palmatum L [11 12 19 21] However antioxidant
HindawiJournal of Food QualityVolume 2018 Article ID 5258276 7 pageshttpsdoiorg10115520185258276
capacity of Korean rhubarb Rheum officinale Baillon hasnever been investigated in spite of the rapid growing interestin using natural antioxidants and functional ingredients infoods and dietary supplements Given the close associationbetween food quality oxidative stress and health outcomesthe demand for applying natural antioxidants either in theform of raw extracts or their chemical constituents in foodshas been fueled by a growing consumer preference forhealthy foods -erefore the specific purpose of the studywas to investigate the inhibitory effect of aqueous extractfrom Rheum officinale Baillon on lipid oxidation and oxi-dative stress
2 Materials and Methods
21 Chemicals Potassium persulfate was purchased fromJunsei Chemical (Tokyo Japan) Pyrocatechol violet (PV) and22prime-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)were purchased from TCI (Tokyo Japan) Dimethyl sulfoxide(DMSO) was purchased from Amresco Inc (Solon OHUSA) Fetal bovine serum (FBS) was obtained from GibcoInvitrogen (Grand Island NY USA) Dulbeccorsquos modifiedEaglersquos medium (DMEM) and penicillin-streptomycin (PS)were obtained fromWelgene (Gyeongsan Gyeongsangbuk-doRepublic of Korea) Gum Arabic 1133-tetramethoxypropane(TEP) sodium dodecyl sulfate (SDS) phosphotungstic acidand 2-thiobarbituric acid (TBA) were purchased from Sigma-Aldrich (St Louis MO USA) Perilla oil was food grade andprocured from a local supplier All other chemicals unlessotherwise noted were purchased from Sigma-Aldrich(St Louis MO USA) All chemical reagents used in thisstudy were of the highest purity commercially available
22 Sample Preparation Dried Rheum officinale Baillonrhizome was obtained from a local market (GyeongsanGyeongsangbuk-do Republic of Korea) Rhizome waspulverized using a grinder Hot water extraction was carriedout in a 5 L round-bottomed flask fitted with a coolingcondenser at 121plusmn 1degC for 20min -e cooled extract wasfiltered through double layers of filter paper (No 1Whatman International Ltd UK) -e filtered extract wascentrifuged at 11000timesg for 20min and this process wasrepeated three times Supernatant was collected and stored at4degC for further analysis
23 Measurement of Total Phenolic Contents Total phenolconcentration of Rheum rhizome extract was determinedusing the FolinndashCiocalteu method [22] In brief 10 μL ofdifferent concentrations of Rheum extract sample (025 0510 25 and 50mgmL) was added to a 96-well platecontaining 10 FolinndashCiocalteu reagent (20 μL) After5min 80 μL of 700mM sodium carbonate was added to themixture and incubated for 1 h at room temperature -eabsorbance was measured by a microplate spectropho-tometer (Epoch BioTek Winooski VT USA) at a wave-length of 765 nm-e concentration of phenolic compoundswas calculated according to the following equation obtainedfrom a standard curve of gallic acid Total phenolic content
was expressed as mg of gallic acid equivalent (GAE) per mLof aqueous Rheum rhizome extract
24 Measurement of Total Flavonoid Contents Total flavo-noid content was measured by the aluminum chloridecolorimetric method 25 μL of Rheum rhizome extract in thefinal concentration range of 025ndash50mgmL was added toa 96-well plate containing 125 μL of distilled water followedby the addition of 10 μL of 5 sodium nitrite solution After6min the reaction was carried out by addition of 15 μL of10 (wv) aluminum chloride After 5min 50 μL of 1mMsodium hydroxide and 275 μL of distilled water were addedand mixed thoroughly -e absorbance was measured ata wavelength of 510 nm-e concentration of total flavonoidcompounds was determined as compared to a standardcurve of catechin as earlier described and expressed as mg ofcatechin per mL of aqueous Rheum rhizome extract
25 DPPH Radical Scavenging Activity Assay Free radicalscavenging activity of Rheum extract was measured by DPPHassay DPPH stock solution was freshly prepared in methanolat a concentration of 002 An aliquot of 50 μL samplesolution at a wide dose range from 025 to 50mgmL wasmixed with 100 μL of DPPH reagent solution After 30min ofincubation at room temperature in the dark the absorbanceof the mixture was measured at 510 nm against a blankScavenging activity was expressed as mg of ascorbic acid (AA)per mL of aqueous Rheum rhizome extract
DPPH radical scavenging activity()
1minusΔABS(sampleminus blank)
ABS(control)1113896 1113897 times 1001113890 1113891
(1)
26 ABTS Radical Scavenging Activity Assay As describedpreviously [23] ABTS stock solution (74mM) was added to26mM potassium persulfate solution in equal quantities andkept for 16 h at room temperature in the dark to yield a darkcolored solution containing ABTS radical cation Before useABTS radical cation was diluted to an initial absorbance ofabout 07plusmn 002 at 734 nm Free radical scavenging activitywas determined by the addition of 10 μL of Rheum rhizomeextract from 5 different final concentrations (025 05 10 25and 50mgmL) to 290 μL of ABTS working solution After6min the absorbance was determined at 734 nm ABSTradical scavenging activity was expressed as mg of troloxequivalent (TE) per mL of aqueous Rheum rhizome extract
ABTS radical scavenging activity()
1minusΔABS(sampleminus blank)
ABS(control)1113896 1113897 times 1001113890 1113891
(2)
27 Ferric Reducing Antioxidant Power (FRAP) AssayReducing power was measured based on conversion ofFe(III) to Fe(II) using the method described by Benzie andStrain [24] Briefly FRAP working reagent was prepared by
2 Journal of Food Quality
addition of 300mM acetate buffer (pH 36) 10mM 246-tripyridyl-s-triazine (TPTZ) solution in 40mM HCl and20mM ferric chloride (FeSO4middot6H2O) solution at a 10 1 1 ratioA wide dose range of 10μL Rheum rhizome aqueous extract(025 05 10 25 and 50mgmL) was mixed with 10μL ofdistilled water followed by the addition of prewarmed FRAPworking reagent (100μL) Optical density of the mixture wasmeasured at a wavelength of 593nm Reducing power wasdescribed as the use of ferrous sulfate (FeSO4middot7H2O)
28 Determination of Copper Chelating Activity Cupricreducing antioxidant capacity of Rheum extract was de-termined according to the method of Megıas [25] In eachwell of a 96-well plate 30 μL hot water extract sample whichfinal concentration was different from 025 to 50mgmLwasmixed with 6 μL of 4mM PV 290 μL of 50mM sodiumacetate buffer (pH 60) and 10 μL of 2mM copper sulfate Inorder to measure copper chelating activity absorbance wasdetermined at 632 nm according to the following equation
Copper chelating activity ()
1minusΔABS(sampleminus blank)
ABS(control)1113896 1113897 times 1001113890 1113891
(3)
29 Measurement of Lipid Peroxidation Oil emulsion wasprepared by mixing raw perilla oil and rhubarb aqueousextract at the ratio of 3 to 1 -en raw perilla oil emulsionswere mixed with 10 gum arabic as an emulsifier andhomogenized for 16min by homogenizer (AM-8 NisseiJapan) Lipid oxidation of the oil emulsion was induced at30degC or 70degC using incubator (JISICO Korea) and waterbath (Shaking Water Bath JEIO Tech Korea) respectivelyHeated raw perilla oil emulsions were collected at 0 7 and10 days of incubation -e lipid oxidation of raw perilla oilwas analyzed by thiobarbituric acid reactive substance(TBARS) assay [26] In brief 10 μL of samples (rhubarb) atthree different concentrations of 0 025 and 5mgmL orstandard solution (1133-tetramethoxypropane TEP) and40 μL of 20mM phosphate buffer (pH 70) were added to anEppendorf tube on ice In each tube 50 μL of 3 sodiumdodecyl sulfate (SDS) 200 μL of 01N HCl 30 μL of 10phosphotungstic acid and 100 μL of 07 of 2-thiobarbituricacid (TBA) were added -e tubes were firmly closed andboiled at 100degC for 30min in water bath -e reactionmixture was mixed with 400 μL of n-butanol and thencentrifuged at 3000 rpm for 10min Supernatants werecollected and loaded in a 96-well plate Fluorescence in-tensity was read at the excitationemission wavelengths of515 nm555 nm using microplate reader (VICTOR Multi-label Plate Reader PerkinElmer Korea)
210 In Vitro RAW2647 Macrophage Study
2101 Measurement of Cell Viability RAW2647 macro-phage cells were cultured at 37degC and 5 CO2 in DMEM with10FBS and 1PS Cell viability was determined by amodified
3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) assay [27] RAW2647 cells were seeded on a 24-wellplate at a density of 1times 105 cellswell and cultured overnightAfter 24h treatment of Rheum extract with different concen-trations (0 01 05 and 1mgmL) RAW2647 cells were in-cubated 05mM tert-butyl hydroperoxide (t-BHP) for 1hFurther incubation with MTT dye solution (05mgmL MTTsolution and medium at a ratio of 1 5) was executed for 1hAfter dissolving purple formazan intoDMSO the absorbance ofthe product was measured at 540nm -e percentage of cellviability was calculated by the following equation
Cell viability(of control) ODsampleminusblank
ODControlminusblanktimes 100 (4)
2102 Determination of Cell Toxicity Effect of aqueousRheum rhizome extract on cell toxicity was measured byneutral red assay [28] RAW2647 cells (1times 105 cellswell ina 24-well plate) were treated with different concentrations ofaqueous Rheum extract (0 01 05 and 1mgmL) for 24 hfollowed by treatment of 05mM t-BHP for an additional 1 hCulture medium was changed to 0004 neutral red solutiondissolved in medium and incubated for 3 h Lysis buffercontaining distilled water ethanol and acetic acid at a ratio of50 49 1 was added to each well and the plate was rocking for15min -e optical density at 540 nm was measured
2103 Measurement of Intracellular Reactive Oxygen Species(ROS) Levels -e level of intracellular ROS was determinedusing 2prime7prime-dichlorofluorescin diacetate (DCFH-DA) [29]RAW2647 cells were planted on a 96-well plate at a density of5times104 cellswell and incubated at 37degC and 5 CO2 for 24 hAfter 24 h of treatment of Rheum extract (0 01 05 and1mgmL) themediumwas changed to PBS containing 120μMDCFH-DA with or without 05mM t-BHP and incubated for60min at 37degC -e absorbance was determined at 488nmexcitation and 525nm emission by using a fluorescence platereader (VICTOR X3 PerkinElmer Turku Singapore)
2104 Measurements of Superoxide Anion ProductionPreincubated RAW2647 cells with Rheum extract (0 0105 and 1mgmL) for 24 h were incubated with or without05mM t-BHP for 1 h NBT solution (05mgmL NBT so-lution and medium at a ratio of 1 5) was added and furtherincubated at 37degC and 5 CO2 for 7 h Medium was re-moved and lysis buffer including DMSO and 2M potassiumhydroxide at a ratio of 1 1 was added -e absorbance wasmeasured at 570 nm
211 Statistical Analysis -e data are presented as themeanplusmn standard deviation (SD) of at least three independenttriplicate experiments Data were analyzed by the ANOVAprocedure using the Statistical Analysis System (SAS 94)software Differences among groups were determined usingBonferroni procedure at the 5 level
Journal of Food Quality 3
3 Results and Discussion
31 Total Phenolic and Flavonoid Contents Polyphenoliccompounds including flavonoids are known as powerfulantioxidants due to their hydroxyl groups and radical scav-enging activities -ese compounds may contribute directly toantioxidant capacity thus having protective functions againstoxidative damage and health benefits [30ndash33] In order toinvestigate antioxidant activity of Rheum aqueous extract wefirst examined concentrations of total phenols and flavonoidsAs shown in Figure 1(a) total phenolic contents of rhizomeextract significantly increased in a dose-dependent mannerwith a significant increase at 05mgmL Total flavonoidconcentration reached a maximum at 5mgmL about 30 foldshigher than 025mgmL (Figure 1(b)) -ese findings suggestthat flavonoids and phenols may be important components ofRheum officinale Baillon and its radical scavenging activitycould be attributed to the presence of these constituents
32 Radical Scavenging and Metal Chelating ActivitiesExcess generation of free radicals or ROS causes oxidativestress and disease [1ndash5] Increased use of naturally occurringantioxidants is considerably regarded as an effective and safe
way to prevent ROS-induced diseases -erefore we deter-mined the antioxidant effect of Rheum extract based on radicalscavenging -e electron donation ability of rhizome extractfrom Rheum was determined by DPPH purple-colored so-lution bleaching assay Aqueous Rheum extract significantlyincreased the degree of color change in a dose-dependentmanner indicating significant free radical scavenging activity(Table 1) In addition the extent of decolorizationmeasured asthe percentage inhibition of ABTS radical cation was assessedafter addition of Rheum rhizome extract (Table 1) Aqueousextract of Rheum rhizome significantly increased both DPPHand ABTS radical scavenging activities in a dose-dependentmanner from a concentration of 025 to 5mgmL indicatingstrong free radical scavenging activity
Copper(I) and iron(II) are regarded as catalysts for thegeneration of highly reactive hydroxyl radicals which causecell or tissue damages and consequently diseases [6 7]-erefore copper chelating capacity and ferric reducingactivity are important markers for antioxidant activity ofnatural resources and functional ingredients [34ndash36] Asshown in Table 1 both ferric reducing capacity and copperchelating activity were significantly increased by Rheumextract in a dose-dependent manner in ranges of 002ndash032mM ferric levels and 3ndash35 copper chelating compared
00
01
02
03
04
05
025 05 1 25 5
Tota
l phe
nol c
once
ntra
tion
(mg G
AE
mL)
Concentration (mgmL)
c
b
a a
d
(a)
00
01
02
03
04
025 05 1 25 5
Flav
onoi
d co
ncen
trat
ion
(mg
mL)
Concentration (mgmL)
d
e
c
b
a
(b)
Figure 1 Effect of aqueous Rheum rhizome extracts on total phenol (a) and flavonoid (b) contents -e concentrations of total phenoliccompounds and flavonoids are calculated according to standard curves of gallic acid and catechin and expressed as milligrams of gallic acidequivalent (GAE) and catechin per milliliter of aqueous Rheum rhizome extract Values are expressed as the meanplusmn standard deviation (SD)(n 9) Bars with different letters indicate significant differences (plt 005)
Table 1 Radical scavenging and metal chelating activities of aqueous Rheum extracts
Values are expressed as the meanplusmn standard deviation (SD) from at least three experiments (n 9) Different letters indicate a significant difference amonggroups according to Bonferroni procedure (plt 005) AA ascorbic acid ABTS 22prime-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid DPPH 22-diphenyl-1-picrylhydrazyl) FRAP ferric reducing antioxidant power TE Trolox equivalent
4 Journal of Food Quality
Table 2 Inhibitory esectect of rhubarb aqueous extract on lipid oxidation over storage time at 30degC and 70degC
Storage time (days) Storage temperature (degC)Concentration of rhubarb (mgmL)
Data are expressed as the meanplusmn standard deviation (SD) from at least three experiments (n 9) Disecterent letters (A B C) within same row indicatea signicant disecterence groups with the same storage time and temperature (plt 005) Small letters (andashe) demonstrate signicant disecterence at a givenconcentration of rhubarb aqueous extract (plt 005)
00 01 05 10 Concentration of Rheum (mgmL)
Controlt-BHP
0
20
40
60
80
100
120
140
Cel
l via
bilit
y (
of c
ontr
ol)
(a)
00 01 05 10 Concentration of Rheum (mgmL)
Controlt-BHP
0
20
40
60
80
100
120
Cell
toxi
city
( o
f con
trol
)
(b)
Figure 2 Esectects of Rheum rhizome extract on cell viability (a) and cell toxicity (b) in 05mM tert-butyl hydroperoxide- (t-BHP-) inducedRAW2647 macrophages Each value represents the meanplusmn standard deviation (SD) from at least three experiments (n 9) Bars withdisecterent letters disecter among aqueous Rheum rhizome extract treatment groups (plt 005)
0
50
100
150
200
250
300
350
00 01 05 10
ROS
gene
ratio
n(
of c
ontr
ol)
Concentration of Rheum (mgmL)
Controlt-BHP
cd
a
e
b
e e
bc
de
(a)
Controlt-BHP
0
20
40
60
80
100
120
140
160
00 01 05 10
Supe
roxi
de an
ion
prod
uctio
n (
of c
ontr
ol)
Concentration of Rheum (mgmL)
abc
a
bc
ab
bc bcbc
c
(b)
Figure 3 Esectects of Rheum rhizome extract on production of intracellular reactive oxygen species (a) and superoxide anion (b) in 05mMtert-butyl hydroperoxide- (t-BHP-) induced RAW2647 macrophages e value of each bar represents the meanplusmn standard deviation (SD)(n 9) Means sharing the same letter are not signicantly disecterent at the 5 level
Journal of Food Quality 5
to control respectively -ese results demonstrated thatRheum rhizome extract has antioxidant capacity mediatedby ferric reducing and copper chelating activities
33 LipidOxidation Increased oxidation process in the foodscontributes to food quality deterioration by increasing oxi-dative rancidity and deleterious food product as well as losingcolor and nutrient value [9 10] -ereby the ways in whichantioxidants inhibit oxidation of food and increase the anti-oxidant efficacy have attracted much attention In the presentstudy inhibitory effect of aqueous Rheum extract on lipidoxidation was measured using thiobarbituric acid reactivesubstances assay Malondialdehyde the end product of lipidoxidation was significantly generated as storage time andtemperature were increased (Table 2) Even though lipidperoxidation in oil emulsion mixture was significantly in-creased according to storage temperature and times treatmentof aqueous extract of Rheum rhizome significantly suppressedmalondialdehyde levels in the raw perilla oil emulsions ina dose-dependent manner -is result supported that aqueousextract of Rheum officinale Baillon inhibits lipid oxidation
34 Antioxidant Properties of Rheum Aqueous Extract int-BHP-Treated RAW2647 Murine Macrophages Antioxidanteffect of Rheum officinale Baillon was determined in in vitrocell culture model t-BHP treated RAW2647 macrophagecells First we measured the cytotoxic effect of Rheumofficinale Baillon rhizome extract After 24 h exposure to 0105 and 1mgmL of Rheum extract viabilities of RAW2647cells were not statistically different from control regardlessof t-BHP treatment (Figure 2(a)) In addition cell toxicitywas not altered by either Rheum extract or t-BHP treatment(Figure 2(b)) -ese results indicate that Rheum extract atany concentration ranging from 01 to 1mgmL had nosignificant cytotoxicity on RAW2647 cell viability
As an indicator of oxidative stress increased productionof ROS promotes the pathogenesis of multiple diseases [1ndash5]In order to evaluate the effect of Rheum extract on oxidativeprocess measurement of ROS and superoxide anion pro-duction were carried out in t-BHP-treated RAW2647 cells Inthe presence of Rheum extract t-BHP-induced ROS (Figure3(a)) and superoxide anion production (Figure 3(b)) weresignificantly diminished in a dose-dependent manner com-pared to t-BHP only-treated cells
4 Conclusions
In the present study analysis of radical scavenging abilitiesmetal chelating activities and total phenolic and flavonoidcontents showed that aqueous extract from Rheum officinaleBaillon rhizome could be a potent source of natural anti-oxidants In addition Rheum aqueous extract significantlyinhibited lipid oxidation in a dose-dependent manner Tothe best of our knowledge this is the first study to suggestthat aqueous extract of Korean Rhubarb (Rheum officinaleBaillon) rhizome may be useful as a natural antioxidant dueto its antioxidant capacities to prevent ROS generation anddelay oxidant degradation of lipids
Data Availability
-e data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
-e authors declare no conflicts of interest
Acknowledgments
-e authors greatly appreciate the technical assistance fromDarae Lee and Naeun Jeon Yeungnam University -iswork was supported by the 2014 Yeungnam UniversityResearch Grant
References
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[2] A H Bhat K B Dar S Anees et al ldquoOxidative stressmitochondrial dysfunction and neurodegenerative diseasesa mechanistic insightrdquo Biomedicine and Pharmacotherapyvol 74 pp 101ndash110 2015
[3] J S Bhatti G K Bhatti and P H Reddy ldquoMitochondrialdysfunction and oxidative stress in metabolic disordersmdashastep towards mitochondria based therapeutic strategiesrdquoBiochimica et Biophysica Acta (BBA)-Molecular Basis ofDisease vol 1863 no 5 pp 1066ndash1077 2017
[4] L A Pham-Huy H He and C Pham-Huy ldquoFree radicalsantioxidants in disease and healthrdquo International Journal ofBiomedical Science vol 4 no 2 pp 89ndash96 2008
[5] B P Yu ldquoCellular defenses against damage from reactiveoxygen speciesrdquo Physiological Reviews vol 74 no 1pp 139ndash162 1994
[6] M Ott V Gogvadze S Orrenius and B ZhivotovskyldquoMitochondria oxidative stress and cell deathrdquo Apoptosisvol 12 no 5 pp 913ndash922 2007
[7] G Stark ldquoFunctional consequences of oxidative membranedamagerdquo Journal of Membrane Biology vol 205 no 1pp 1ndash16 2005
[8] E Birben U M Sahiner C Sackesen S Erzurum andO Kalayci ldquoOxidative stress and antioxidant defenserdquoWorldAllergy Organization Journal vol 5 no 1 pp 9ndash19 2012
[9] A J St Angelo J Vercellotti T Jacks and M LegendreldquoLipid oxidation on foodsrdquo Critical Reviews in Food Scienceand Nutrition vol 36 no 3 pp 175ndash224 1996
[10] I Gulccedilin ldquoAntioxidant activity of food constituents anoverviewrdquo Archives of Toxicology vol 86 no 3 pp 345ndash3912012
[11] Z H He R Zhou M F He et al ldquoAnti-angiogenic effect andmechanism of rhein from Rhizoma Rheirdquo Phytomedicinevol 18 no 6 pp 470ndash478 2011
[12] B Hu H Zhang X Meng F Wang and P Wang ldquoAloe-emodin from rhubarb (Rheum rhabarbarum) inhibitslipopolysaccharide-induced inflammatory responses inRAW2647 macrophagesrdquo Journal of Ethnopharmacologyvol 153 no 3 pp 846ndash853 2014
[13] L Lu and H Yin ldquoEffects of Dahuang (Rhubarb) retentionenema on leukocyte interleukin-6 high sensitive C reactiveprotein and endotoxin in patients with acute pancreatitisrdquoMedicinal Plants vol 9 pp 60ndash62 2018
6 Journal of Food Quality
[14] H Matsuda N Tomohiro K Hiraba et al ldquoStudy on Anti-Oketsu Activity of Rhubarb II Anti-allergic effects of stilbenecomponents from Rhei undulati Rhizoma (dried rhizome ofRheum undulatum cultivated in Korea)rdquo Biological andPharmaceutical Bulletin vol 24 no 3 pp 264ndash267 2001a
[15] H Matsuda T Morikawa I Toguchida J Y Park S Harimaand M Yoshikawa ldquoAntioxidant constituents from rhubarbstructural requirements of stilbenes for the activity andstructures of two new anthraquinone glucosidesrdquo Bioorganicand Medicinal Chemistry vol 9 no 1 pp 41ndash50 2001b
[16] T M Ngoc T M Hung P T -uong et al ldquoInhibition ofhuman low density lipoprotein and high density lipoproteinoxidation by oligostilbenes from rhubarbrdquo Biological andPharmaceutical Bulletin vol 31 no 9 pp 1809ndash1812 2008
[17] M Ozturk F Aydogmus-Ozturk M E Duru and G TopccediluldquoAntioxidant activity of stem and root extracts of Rhubarb(Rheum ribes) An edible medicinal plantrdquo Food Chemistryvol 103 no 2 pp 623ndash630 2007
[18] P Raudsepp D Anton M Roasto et al ldquo-e antioxidativeand antimicrobial properties of the blue honeysuckle (Loni-cera caerulea L) Siberian rhubarb (Rheum rhaponticum L)and some other plants compared to ascorbic acid and sodiumnitriterdquo Food Control vol 31 no 1 pp 129ndash135 2013
[19] C S Shia S H Juang S Y Tsai et al ldquoMetabolism andpharmacokinetics of anthraquinones in Rheum palmatum inrats and ex vivo antioxidant activityrdquo Planta Medica vol 75no 13 pp 1386ndash1392 2009
[20] J P Silveira L N Seito S Eberlin et al ldquoPhotoprotective andantioxidant effects of Rhubarb inhibitory action on tyrosinaseand tyrosine kinase activities and TNF-alpha IL-1alpha andalpha-MSH production in human melanocytesrdquo BMCComplementary and Alternative Medicine vol 13 no 1 p 492013
[21] T Venkatesan M J Jeong Y W Choi E J ParkS K El-Desouky and Y K Kim ldquoDeoxyrhapontigenina natural stilbene derivative isolated from Rheum undulatumL induces endoplasmic reticulum stress-mediated apoptosisin human breast cancer cellsrdquo Integrative Cancer Berapiesvol 15 no 4 pp NP44ndashNP52 2016
[22] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquoMethods inEnzymology vol 299 pp 152ndash178 1999
[23] R Re N Pellegrini A Proteggente A Pannala M Yang andC Rice-Evans ldquoAntioxidant activity applying an improvedABTS radical cation decolorization assayrdquo Free Radical Bi-ology and Medicine vol 26 no 9-10 pp 1231ndash1237 1999
[24] I F Benzie and J J Strain ldquo-e ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo theFRAP assayrdquo Analytical Biochemistry vol 239 no 1pp 70ndash76 1996
[25] C Megıas E Pastor-Cavada C Torres-Fuentes et alldquoChelating antioxidant and antiproliferative activity of Viciasativa polyphenol extractsrdquo European Food Research andTechnology vol 230 no 2 pp 353ndash359 2009
[26] P Ke E Cervantes and C Robles-Martinez ldquoDeterminationof thiobarbituric acid reactive substances (TBARS) in fishtissue by an improved distillationndashspectrophotometricmethodrdquo Journal of the Science of Food and Agriculturevol 35 no 11 pp 1248ndash1254 1984
[27] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983
[28] E Borenfreund and J A Puerner ldquoToxicity determined invitro by morphological alterations and neutral red absorp-tionrdquo Toxicology Letters vol 24 no 2-3 pp 119ndash124 1985
[29] K Nishio M Horie Y Akazawa et al ldquoAttenuation of li-popolysaccharide (LPS)-induced cytotoxicity by tocopherolsand tocotrienolsrdquo Redox Biology vol 1 no 1 pp 97ndash1032013
[30] L Bravo ldquoPolyphenols chemistry dietary sources meta-bolism and nutritional significancerdquo Nutrition Reviewsvol 56 no 11 pp 317ndash333 1998
[31] Y Cai Q Luo M Sun and H Corke ldquoAntioxidant activityand phenolic compounds of 112 traditional Chinese medic-inal plants associated with anticancerrdquo Life Sciences vol 74no 17 pp 2157ndash2184 2004
[32] G Di Carlo N Mascolo A A Izzo and F Capasso ldquoFla-vonoids old and new aspects of a class of natural therapeuticdrugsrdquo Life Sciences vol 65 no 4 pp 337ndash353 1999
[33] F Shahidi and P Ambigaipalan ldquoPhenolics and polyphenolicsin foods beverages and spices antioxidant activity and healtheffectsmdasha reviewrdquo Journal of Functional Foods vol 18pp 820ndash897 2015
[34] M S Brewer ldquoNatural antioxidants sources compoundsmechanisms of action and potential applicationsrdquo Compre-hensive Reviews in Food Science and Food Safety vol 10 no 4pp 221ndash247 2011
[35] B Ou D Huang M Hampsch-Woodill J A Flanagan andE K Deemer ldquoAnalysis of antioxidant activities of commonvegetables employing oxygen radical absorbance capacity(ORAC) and ferric reducing antioxidant power (FRAP) as-says a comparative studyrdquo Journal of Agricultural and FoodChemistry vol 50 no 11 pp 3122ndash3128 2002
[36] R Apak K Guclu B Demirata et al ldquoComparative evalu-ation of various total antioxidant capacity assays applied tophenolic compounds with the CUPRAC assayrdquo Moleculesvol 12 no 7 pp 1496ndash1547 2007
capacity of Korean rhubarb Rheum officinale Baillon hasnever been investigated in spite of the rapid growing interestin using natural antioxidants and functional ingredients infoods and dietary supplements Given the close associationbetween food quality oxidative stress and health outcomesthe demand for applying natural antioxidants either in theform of raw extracts or their chemical constituents in foodshas been fueled by a growing consumer preference forhealthy foods -erefore the specific purpose of the studywas to investigate the inhibitory effect of aqueous extractfrom Rheum officinale Baillon on lipid oxidation and oxi-dative stress
2 Materials and Methods
21 Chemicals Potassium persulfate was purchased fromJunsei Chemical (Tokyo Japan) Pyrocatechol violet (PV) and22prime-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)were purchased from TCI (Tokyo Japan) Dimethyl sulfoxide(DMSO) was purchased from Amresco Inc (Solon OHUSA) Fetal bovine serum (FBS) was obtained from GibcoInvitrogen (Grand Island NY USA) Dulbeccorsquos modifiedEaglersquos medium (DMEM) and penicillin-streptomycin (PS)were obtained fromWelgene (Gyeongsan Gyeongsangbuk-doRepublic of Korea) Gum Arabic 1133-tetramethoxypropane(TEP) sodium dodecyl sulfate (SDS) phosphotungstic acidand 2-thiobarbituric acid (TBA) were purchased from Sigma-Aldrich (St Louis MO USA) Perilla oil was food grade andprocured from a local supplier All other chemicals unlessotherwise noted were purchased from Sigma-Aldrich(St Louis MO USA) All chemical reagents used in thisstudy were of the highest purity commercially available
22 Sample Preparation Dried Rheum officinale Baillonrhizome was obtained from a local market (GyeongsanGyeongsangbuk-do Republic of Korea) Rhizome waspulverized using a grinder Hot water extraction was carriedout in a 5 L round-bottomed flask fitted with a coolingcondenser at 121plusmn 1degC for 20min -e cooled extract wasfiltered through double layers of filter paper (No 1Whatman International Ltd UK) -e filtered extract wascentrifuged at 11000timesg for 20min and this process wasrepeated three times Supernatant was collected and stored at4degC for further analysis
23 Measurement of Total Phenolic Contents Total phenolconcentration of Rheum rhizome extract was determinedusing the FolinndashCiocalteu method [22] In brief 10 μL ofdifferent concentrations of Rheum extract sample (025 0510 25 and 50mgmL) was added to a 96-well platecontaining 10 FolinndashCiocalteu reagent (20 μL) After5min 80 μL of 700mM sodium carbonate was added to themixture and incubated for 1 h at room temperature -eabsorbance was measured by a microplate spectropho-tometer (Epoch BioTek Winooski VT USA) at a wave-length of 765 nm-e concentration of phenolic compoundswas calculated according to the following equation obtainedfrom a standard curve of gallic acid Total phenolic content
was expressed as mg of gallic acid equivalent (GAE) per mLof aqueous Rheum rhizome extract
24 Measurement of Total Flavonoid Contents Total flavo-noid content was measured by the aluminum chloridecolorimetric method 25 μL of Rheum rhizome extract in thefinal concentration range of 025ndash50mgmL was added toa 96-well plate containing 125 μL of distilled water followedby the addition of 10 μL of 5 sodium nitrite solution After6min the reaction was carried out by addition of 15 μL of10 (wv) aluminum chloride After 5min 50 μL of 1mMsodium hydroxide and 275 μL of distilled water were addedand mixed thoroughly -e absorbance was measured ata wavelength of 510 nm-e concentration of total flavonoidcompounds was determined as compared to a standardcurve of catechin as earlier described and expressed as mg ofcatechin per mL of aqueous Rheum rhizome extract
25 DPPH Radical Scavenging Activity Assay Free radicalscavenging activity of Rheum extract was measured by DPPHassay DPPH stock solution was freshly prepared in methanolat a concentration of 002 An aliquot of 50 μL samplesolution at a wide dose range from 025 to 50mgmL wasmixed with 100 μL of DPPH reagent solution After 30min ofincubation at room temperature in the dark the absorbanceof the mixture was measured at 510 nm against a blankScavenging activity was expressed as mg of ascorbic acid (AA)per mL of aqueous Rheum rhizome extract
DPPH radical scavenging activity()
1minusΔABS(sampleminus blank)
ABS(control)1113896 1113897 times 1001113890 1113891
(1)
26 ABTS Radical Scavenging Activity Assay As describedpreviously [23] ABTS stock solution (74mM) was added to26mM potassium persulfate solution in equal quantities andkept for 16 h at room temperature in the dark to yield a darkcolored solution containing ABTS radical cation Before useABTS radical cation was diluted to an initial absorbance ofabout 07plusmn 002 at 734 nm Free radical scavenging activitywas determined by the addition of 10 μL of Rheum rhizomeextract from 5 different final concentrations (025 05 10 25and 50mgmL) to 290 μL of ABTS working solution After6min the absorbance was determined at 734 nm ABSTradical scavenging activity was expressed as mg of troloxequivalent (TE) per mL of aqueous Rheum rhizome extract
ABTS radical scavenging activity()
1minusΔABS(sampleminus blank)
ABS(control)1113896 1113897 times 1001113890 1113891
(2)
27 Ferric Reducing Antioxidant Power (FRAP) AssayReducing power was measured based on conversion ofFe(III) to Fe(II) using the method described by Benzie andStrain [24] Briefly FRAP working reagent was prepared by
2 Journal of Food Quality
addition of 300mM acetate buffer (pH 36) 10mM 246-tripyridyl-s-triazine (TPTZ) solution in 40mM HCl and20mM ferric chloride (FeSO4middot6H2O) solution at a 10 1 1 ratioA wide dose range of 10μL Rheum rhizome aqueous extract(025 05 10 25 and 50mgmL) was mixed with 10μL ofdistilled water followed by the addition of prewarmed FRAPworking reagent (100μL) Optical density of the mixture wasmeasured at a wavelength of 593nm Reducing power wasdescribed as the use of ferrous sulfate (FeSO4middot7H2O)
28 Determination of Copper Chelating Activity Cupricreducing antioxidant capacity of Rheum extract was de-termined according to the method of Megıas [25] In eachwell of a 96-well plate 30 μL hot water extract sample whichfinal concentration was different from 025 to 50mgmLwasmixed with 6 μL of 4mM PV 290 μL of 50mM sodiumacetate buffer (pH 60) and 10 μL of 2mM copper sulfate Inorder to measure copper chelating activity absorbance wasdetermined at 632 nm according to the following equation
Copper chelating activity ()
1minusΔABS(sampleminus blank)
ABS(control)1113896 1113897 times 1001113890 1113891
(3)
29 Measurement of Lipid Peroxidation Oil emulsion wasprepared by mixing raw perilla oil and rhubarb aqueousextract at the ratio of 3 to 1 -en raw perilla oil emulsionswere mixed with 10 gum arabic as an emulsifier andhomogenized for 16min by homogenizer (AM-8 NisseiJapan) Lipid oxidation of the oil emulsion was induced at30degC or 70degC using incubator (JISICO Korea) and waterbath (Shaking Water Bath JEIO Tech Korea) respectivelyHeated raw perilla oil emulsions were collected at 0 7 and10 days of incubation -e lipid oxidation of raw perilla oilwas analyzed by thiobarbituric acid reactive substance(TBARS) assay [26] In brief 10 μL of samples (rhubarb) atthree different concentrations of 0 025 and 5mgmL orstandard solution (1133-tetramethoxypropane TEP) and40 μL of 20mM phosphate buffer (pH 70) were added to anEppendorf tube on ice In each tube 50 μL of 3 sodiumdodecyl sulfate (SDS) 200 μL of 01N HCl 30 μL of 10phosphotungstic acid and 100 μL of 07 of 2-thiobarbituricacid (TBA) were added -e tubes were firmly closed andboiled at 100degC for 30min in water bath -e reactionmixture was mixed with 400 μL of n-butanol and thencentrifuged at 3000 rpm for 10min Supernatants werecollected and loaded in a 96-well plate Fluorescence in-tensity was read at the excitationemission wavelengths of515 nm555 nm using microplate reader (VICTOR Multi-label Plate Reader PerkinElmer Korea)
210 In Vitro RAW2647 Macrophage Study
2101 Measurement of Cell Viability RAW2647 macro-phage cells were cultured at 37degC and 5 CO2 in DMEM with10FBS and 1PS Cell viability was determined by amodified
3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) assay [27] RAW2647 cells were seeded on a 24-wellplate at a density of 1times 105 cellswell and cultured overnightAfter 24h treatment of Rheum extract with different concen-trations (0 01 05 and 1mgmL) RAW2647 cells were in-cubated 05mM tert-butyl hydroperoxide (t-BHP) for 1hFurther incubation with MTT dye solution (05mgmL MTTsolution and medium at a ratio of 1 5) was executed for 1hAfter dissolving purple formazan intoDMSO the absorbance ofthe product was measured at 540nm -e percentage of cellviability was calculated by the following equation
Cell viability(of control) ODsampleminusblank
ODControlminusblanktimes 100 (4)
2102 Determination of Cell Toxicity Effect of aqueousRheum rhizome extract on cell toxicity was measured byneutral red assay [28] RAW2647 cells (1times 105 cellswell ina 24-well plate) were treated with different concentrations ofaqueous Rheum extract (0 01 05 and 1mgmL) for 24 hfollowed by treatment of 05mM t-BHP for an additional 1 hCulture medium was changed to 0004 neutral red solutiondissolved in medium and incubated for 3 h Lysis buffercontaining distilled water ethanol and acetic acid at a ratio of50 49 1 was added to each well and the plate was rocking for15min -e optical density at 540 nm was measured
2103 Measurement of Intracellular Reactive Oxygen Species(ROS) Levels -e level of intracellular ROS was determinedusing 2prime7prime-dichlorofluorescin diacetate (DCFH-DA) [29]RAW2647 cells were planted on a 96-well plate at a density of5times104 cellswell and incubated at 37degC and 5 CO2 for 24 hAfter 24 h of treatment of Rheum extract (0 01 05 and1mgmL) themediumwas changed to PBS containing 120μMDCFH-DA with or without 05mM t-BHP and incubated for60min at 37degC -e absorbance was determined at 488nmexcitation and 525nm emission by using a fluorescence platereader (VICTOR X3 PerkinElmer Turku Singapore)
2104 Measurements of Superoxide Anion ProductionPreincubated RAW2647 cells with Rheum extract (0 0105 and 1mgmL) for 24 h were incubated with or without05mM t-BHP for 1 h NBT solution (05mgmL NBT so-lution and medium at a ratio of 1 5) was added and furtherincubated at 37degC and 5 CO2 for 7 h Medium was re-moved and lysis buffer including DMSO and 2M potassiumhydroxide at a ratio of 1 1 was added -e absorbance wasmeasured at 570 nm
211 Statistical Analysis -e data are presented as themeanplusmn standard deviation (SD) of at least three independenttriplicate experiments Data were analyzed by the ANOVAprocedure using the Statistical Analysis System (SAS 94)software Differences among groups were determined usingBonferroni procedure at the 5 level
Journal of Food Quality 3
3 Results and Discussion
31 Total Phenolic and Flavonoid Contents Polyphenoliccompounds including flavonoids are known as powerfulantioxidants due to their hydroxyl groups and radical scav-enging activities -ese compounds may contribute directly toantioxidant capacity thus having protective functions againstoxidative damage and health benefits [30ndash33] In order toinvestigate antioxidant activity of Rheum aqueous extract wefirst examined concentrations of total phenols and flavonoidsAs shown in Figure 1(a) total phenolic contents of rhizomeextract significantly increased in a dose-dependent mannerwith a significant increase at 05mgmL Total flavonoidconcentration reached a maximum at 5mgmL about 30 foldshigher than 025mgmL (Figure 1(b)) -ese findings suggestthat flavonoids and phenols may be important components ofRheum officinale Baillon and its radical scavenging activitycould be attributed to the presence of these constituents
32 Radical Scavenging and Metal Chelating ActivitiesExcess generation of free radicals or ROS causes oxidativestress and disease [1ndash5] Increased use of naturally occurringantioxidants is considerably regarded as an effective and safe
way to prevent ROS-induced diseases -erefore we deter-mined the antioxidant effect of Rheum extract based on radicalscavenging -e electron donation ability of rhizome extractfrom Rheum was determined by DPPH purple-colored so-lution bleaching assay Aqueous Rheum extract significantlyincreased the degree of color change in a dose-dependentmanner indicating significant free radical scavenging activity(Table 1) In addition the extent of decolorizationmeasured asthe percentage inhibition of ABTS radical cation was assessedafter addition of Rheum rhizome extract (Table 1) Aqueousextract of Rheum rhizome significantly increased both DPPHand ABTS radical scavenging activities in a dose-dependentmanner from a concentration of 025 to 5mgmL indicatingstrong free radical scavenging activity
Copper(I) and iron(II) are regarded as catalysts for thegeneration of highly reactive hydroxyl radicals which causecell or tissue damages and consequently diseases [6 7]-erefore copper chelating capacity and ferric reducingactivity are important markers for antioxidant activity ofnatural resources and functional ingredients [34ndash36] Asshown in Table 1 both ferric reducing capacity and copperchelating activity were significantly increased by Rheumextract in a dose-dependent manner in ranges of 002ndash032mM ferric levels and 3ndash35 copper chelating compared
00
01
02
03
04
05
025 05 1 25 5
Tota
l phe
nol c
once
ntra
tion
(mg G
AE
mL)
Concentration (mgmL)
c
b
a a
d
(a)
00
01
02
03
04
025 05 1 25 5
Flav
onoi
d co
ncen
trat
ion
(mg
mL)
Concentration (mgmL)
d
e
c
b
a
(b)
Figure 1 Effect of aqueous Rheum rhizome extracts on total phenol (a) and flavonoid (b) contents -e concentrations of total phenoliccompounds and flavonoids are calculated according to standard curves of gallic acid and catechin and expressed as milligrams of gallic acidequivalent (GAE) and catechin per milliliter of aqueous Rheum rhizome extract Values are expressed as the meanplusmn standard deviation (SD)(n 9) Bars with different letters indicate significant differences (plt 005)
Table 1 Radical scavenging and metal chelating activities of aqueous Rheum extracts
Values are expressed as the meanplusmn standard deviation (SD) from at least three experiments (n 9) Different letters indicate a significant difference amonggroups according to Bonferroni procedure (plt 005) AA ascorbic acid ABTS 22prime-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid DPPH 22-diphenyl-1-picrylhydrazyl) FRAP ferric reducing antioxidant power TE Trolox equivalent
4 Journal of Food Quality
Table 2 Inhibitory esectect of rhubarb aqueous extract on lipid oxidation over storage time at 30degC and 70degC
Storage time (days) Storage temperature (degC)Concentration of rhubarb (mgmL)
Data are expressed as the meanplusmn standard deviation (SD) from at least three experiments (n 9) Disecterent letters (A B C) within same row indicatea signicant disecterence groups with the same storage time and temperature (plt 005) Small letters (andashe) demonstrate signicant disecterence at a givenconcentration of rhubarb aqueous extract (plt 005)
00 01 05 10 Concentration of Rheum (mgmL)
Controlt-BHP
0
20
40
60
80
100
120
140
Cel
l via
bilit
y (
of c
ontr
ol)
(a)
00 01 05 10 Concentration of Rheum (mgmL)
Controlt-BHP
0
20
40
60
80
100
120
Cell
toxi
city
( o
f con
trol
)
(b)
Figure 2 Esectects of Rheum rhizome extract on cell viability (a) and cell toxicity (b) in 05mM tert-butyl hydroperoxide- (t-BHP-) inducedRAW2647 macrophages Each value represents the meanplusmn standard deviation (SD) from at least three experiments (n 9) Bars withdisecterent letters disecter among aqueous Rheum rhizome extract treatment groups (plt 005)
0
50
100
150
200
250
300
350
00 01 05 10
ROS
gene
ratio
n(
of c
ontr
ol)
Concentration of Rheum (mgmL)
Controlt-BHP
cd
a
e
b
e e
bc
de
(a)
Controlt-BHP
0
20
40
60
80
100
120
140
160
00 01 05 10
Supe
roxi
de an
ion
prod
uctio
n (
of c
ontr
ol)
Concentration of Rheum (mgmL)
abc
a
bc
ab
bc bcbc
c
(b)
Figure 3 Esectects of Rheum rhizome extract on production of intracellular reactive oxygen species (a) and superoxide anion (b) in 05mMtert-butyl hydroperoxide- (t-BHP-) induced RAW2647 macrophages e value of each bar represents the meanplusmn standard deviation (SD)(n 9) Means sharing the same letter are not signicantly disecterent at the 5 level
Journal of Food Quality 5
to control respectively -ese results demonstrated thatRheum rhizome extract has antioxidant capacity mediatedby ferric reducing and copper chelating activities
33 LipidOxidation Increased oxidation process in the foodscontributes to food quality deterioration by increasing oxi-dative rancidity and deleterious food product as well as losingcolor and nutrient value [9 10] -ereby the ways in whichantioxidants inhibit oxidation of food and increase the anti-oxidant efficacy have attracted much attention In the presentstudy inhibitory effect of aqueous Rheum extract on lipidoxidation was measured using thiobarbituric acid reactivesubstances assay Malondialdehyde the end product of lipidoxidation was significantly generated as storage time andtemperature were increased (Table 2) Even though lipidperoxidation in oil emulsion mixture was significantly in-creased according to storage temperature and times treatmentof aqueous extract of Rheum rhizome significantly suppressedmalondialdehyde levels in the raw perilla oil emulsions ina dose-dependent manner -is result supported that aqueousextract of Rheum officinale Baillon inhibits lipid oxidation
34 Antioxidant Properties of Rheum Aqueous Extract int-BHP-Treated RAW2647 Murine Macrophages Antioxidanteffect of Rheum officinale Baillon was determined in in vitrocell culture model t-BHP treated RAW2647 macrophagecells First we measured the cytotoxic effect of Rheumofficinale Baillon rhizome extract After 24 h exposure to 0105 and 1mgmL of Rheum extract viabilities of RAW2647cells were not statistically different from control regardlessof t-BHP treatment (Figure 2(a)) In addition cell toxicitywas not altered by either Rheum extract or t-BHP treatment(Figure 2(b)) -ese results indicate that Rheum extract atany concentration ranging from 01 to 1mgmL had nosignificant cytotoxicity on RAW2647 cell viability
As an indicator of oxidative stress increased productionof ROS promotes the pathogenesis of multiple diseases [1ndash5]In order to evaluate the effect of Rheum extract on oxidativeprocess measurement of ROS and superoxide anion pro-duction were carried out in t-BHP-treated RAW2647 cells Inthe presence of Rheum extract t-BHP-induced ROS (Figure3(a)) and superoxide anion production (Figure 3(b)) weresignificantly diminished in a dose-dependent manner com-pared to t-BHP only-treated cells
4 Conclusions
In the present study analysis of radical scavenging abilitiesmetal chelating activities and total phenolic and flavonoidcontents showed that aqueous extract from Rheum officinaleBaillon rhizome could be a potent source of natural anti-oxidants In addition Rheum aqueous extract significantlyinhibited lipid oxidation in a dose-dependent manner Tothe best of our knowledge this is the first study to suggestthat aqueous extract of Korean Rhubarb (Rheum officinaleBaillon) rhizome may be useful as a natural antioxidant dueto its antioxidant capacities to prevent ROS generation anddelay oxidant degradation of lipids
Data Availability
-e data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
-e authors declare no conflicts of interest
Acknowledgments
-e authors greatly appreciate the technical assistance fromDarae Lee and Naeun Jeon Yeungnam University -iswork was supported by the 2014 Yeungnam UniversityResearch Grant
References
[1] R L Auten and J M Davis ldquoOxygen toxicity and reactiveoxygen species the devil is in the detailsrdquo Pediatric Researchvol 66 no 2 pp 121ndash127 2009
[2] A H Bhat K B Dar S Anees et al ldquoOxidative stressmitochondrial dysfunction and neurodegenerative diseasesa mechanistic insightrdquo Biomedicine and Pharmacotherapyvol 74 pp 101ndash110 2015
[3] J S Bhatti G K Bhatti and P H Reddy ldquoMitochondrialdysfunction and oxidative stress in metabolic disordersmdashastep towards mitochondria based therapeutic strategiesrdquoBiochimica et Biophysica Acta (BBA)-Molecular Basis ofDisease vol 1863 no 5 pp 1066ndash1077 2017
[4] L A Pham-Huy H He and C Pham-Huy ldquoFree radicalsantioxidants in disease and healthrdquo International Journal ofBiomedical Science vol 4 no 2 pp 89ndash96 2008
[5] B P Yu ldquoCellular defenses against damage from reactiveoxygen speciesrdquo Physiological Reviews vol 74 no 1pp 139ndash162 1994
[6] M Ott V Gogvadze S Orrenius and B ZhivotovskyldquoMitochondria oxidative stress and cell deathrdquo Apoptosisvol 12 no 5 pp 913ndash922 2007
[7] G Stark ldquoFunctional consequences of oxidative membranedamagerdquo Journal of Membrane Biology vol 205 no 1pp 1ndash16 2005
[8] E Birben U M Sahiner C Sackesen S Erzurum andO Kalayci ldquoOxidative stress and antioxidant defenserdquoWorldAllergy Organization Journal vol 5 no 1 pp 9ndash19 2012
[9] A J St Angelo J Vercellotti T Jacks and M LegendreldquoLipid oxidation on foodsrdquo Critical Reviews in Food Scienceand Nutrition vol 36 no 3 pp 175ndash224 1996
[10] I Gulccedilin ldquoAntioxidant activity of food constituents anoverviewrdquo Archives of Toxicology vol 86 no 3 pp 345ndash3912012
[11] Z H He R Zhou M F He et al ldquoAnti-angiogenic effect andmechanism of rhein from Rhizoma Rheirdquo Phytomedicinevol 18 no 6 pp 470ndash478 2011
[12] B Hu H Zhang X Meng F Wang and P Wang ldquoAloe-emodin from rhubarb (Rheum rhabarbarum) inhibitslipopolysaccharide-induced inflammatory responses inRAW2647 macrophagesrdquo Journal of Ethnopharmacologyvol 153 no 3 pp 846ndash853 2014
[13] L Lu and H Yin ldquoEffects of Dahuang (Rhubarb) retentionenema on leukocyte interleukin-6 high sensitive C reactiveprotein and endotoxin in patients with acute pancreatitisrdquoMedicinal Plants vol 9 pp 60ndash62 2018
6 Journal of Food Quality
[14] H Matsuda N Tomohiro K Hiraba et al ldquoStudy on Anti-Oketsu Activity of Rhubarb II Anti-allergic effects of stilbenecomponents from Rhei undulati Rhizoma (dried rhizome ofRheum undulatum cultivated in Korea)rdquo Biological andPharmaceutical Bulletin vol 24 no 3 pp 264ndash267 2001a
[15] H Matsuda T Morikawa I Toguchida J Y Park S Harimaand M Yoshikawa ldquoAntioxidant constituents from rhubarbstructural requirements of stilbenes for the activity andstructures of two new anthraquinone glucosidesrdquo Bioorganicand Medicinal Chemistry vol 9 no 1 pp 41ndash50 2001b
[16] T M Ngoc T M Hung P T -uong et al ldquoInhibition ofhuman low density lipoprotein and high density lipoproteinoxidation by oligostilbenes from rhubarbrdquo Biological andPharmaceutical Bulletin vol 31 no 9 pp 1809ndash1812 2008
[17] M Ozturk F Aydogmus-Ozturk M E Duru and G TopccediluldquoAntioxidant activity of stem and root extracts of Rhubarb(Rheum ribes) An edible medicinal plantrdquo Food Chemistryvol 103 no 2 pp 623ndash630 2007
[18] P Raudsepp D Anton M Roasto et al ldquo-e antioxidativeand antimicrobial properties of the blue honeysuckle (Loni-cera caerulea L) Siberian rhubarb (Rheum rhaponticum L)and some other plants compared to ascorbic acid and sodiumnitriterdquo Food Control vol 31 no 1 pp 129ndash135 2013
[19] C S Shia S H Juang S Y Tsai et al ldquoMetabolism andpharmacokinetics of anthraquinones in Rheum palmatum inrats and ex vivo antioxidant activityrdquo Planta Medica vol 75no 13 pp 1386ndash1392 2009
[20] J P Silveira L N Seito S Eberlin et al ldquoPhotoprotective andantioxidant effects of Rhubarb inhibitory action on tyrosinaseand tyrosine kinase activities and TNF-alpha IL-1alpha andalpha-MSH production in human melanocytesrdquo BMCComplementary and Alternative Medicine vol 13 no 1 p 492013
[21] T Venkatesan M J Jeong Y W Choi E J ParkS K El-Desouky and Y K Kim ldquoDeoxyrhapontigenina natural stilbene derivative isolated from Rheum undulatumL induces endoplasmic reticulum stress-mediated apoptosisin human breast cancer cellsrdquo Integrative Cancer Berapiesvol 15 no 4 pp NP44ndashNP52 2016
[22] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquoMethods inEnzymology vol 299 pp 152ndash178 1999
[23] R Re N Pellegrini A Proteggente A Pannala M Yang andC Rice-Evans ldquoAntioxidant activity applying an improvedABTS radical cation decolorization assayrdquo Free Radical Bi-ology and Medicine vol 26 no 9-10 pp 1231ndash1237 1999
[24] I F Benzie and J J Strain ldquo-e ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo theFRAP assayrdquo Analytical Biochemistry vol 239 no 1pp 70ndash76 1996
[25] C Megıas E Pastor-Cavada C Torres-Fuentes et alldquoChelating antioxidant and antiproliferative activity of Viciasativa polyphenol extractsrdquo European Food Research andTechnology vol 230 no 2 pp 353ndash359 2009
[26] P Ke E Cervantes and C Robles-Martinez ldquoDeterminationof thiobarbituric acid reactive substances (TBARS) in fishtissue by an improved distillationndashspectrophotometricmethodrdquo Journal of the Science of Food and Agriculturevol 35 no 11 pp 1248ndash1254 1984
[27] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983
[28] E Borenfreund and J A Puerner ldquoToxicity determined invitro by morphological alterations and neutral red absorp-tionrdquo Toxicology Letters vol 24 no 2-3 pp 119ndash124 1985
[29] K Nishio M Horie Y Akazawa et al ldquoAttenuation of li-popolysaccharide (LPS)-induced cytotoxicity by tocopherolsand tocotrienolsrdquo Redox Biology vol 1 no 1 pp 97ndash1032013
[30] L Bravo ldquoPolyphenols chemistry dietary sources meta-bolism and nutritional significancerdquo Nutrition Reviewsvol 56 no 11 pp 317ndash333 1998
[31] Y Cai Q Luo M Sun and H Corke ldquoAntioxidant activityand phenolic compounds of 112 traditional Chinese medic-inal plants associated with anticancerrdquo Life Sciences vol 74no 17 pp 2157ndash2184 2004
[32] G Di Carlo N Mascolo A A Izzo and F Capasso ldquoFla-vonoids old and new aspects of a class of natural therapeuticdrugsrdquo Life Sciences vol 65 no 4 pp 337ndash353 1999
[33] F Shahidi and P Ambigaipalan ldquoPhenolics and polyphenolicsin foods beverages and spices antioxidant activity and healtheffectsmdasha reviewrdquo Journal of Functional Foods vol 18pp 820ndash897 2015
[34] M S Brewer ldquoNatural antioxidants sources compoundsmechanisms of action and potential applicationsrdquo Compre-hensive Reviews in Food Science and Food Safety vol 10 no 4pp 221ndash247 2011
[35] B Ou D Huang M Hampsch-Woodill J A Flanagan andE K Deemer ldquoAnalysis of antioxidant activities of commonvegetables employing oxygen radical absorbance capacity(ORAC) and ferric reducing antioxidant power (FRAP) as-says a comparative studyrdquo Journal of Agricultural and FoodChemistry vol 50 no 11 pp 3122ndash3128 2002
[36] R Apak K Guclu B Demirata et al ldquoComparative evalu-ation of various total antioxidant capacity assays applied tophenolic compounds with the CUPRAC assayrdquo Moleculesvol 12 no 7 pp 1496ndash1547 2007
addition of 300mM acetate buffer (pH 36) 10mM 246-tripyridyl-s-triazine (TPTZ) solution in 40mM HCl and20mM ferric chloride (FeSO4middot6H2O) solution at a 10 1 1 ratioA wide dose range of 10μL Rheum rhizome aqueous extract(025 05 10 25 and 50mgmL) was mixed with 10μL ofdistilled water followed by the addition of prewarmed FRAPworking reagent (100μL) Optical density of the mixture wasmeasured at a wavelength of 593nm Reducing power wasdescribed as the use of ferrous sulfate (FeSO4middot7H2O)
28 Determination of Copper Chelating Activity Cupricreducing antioxidant capacity of Rheum extract was de-termined according to the method of Megıas [25] In eachwell of a 96-well plate 30 μL hot water extract sample whichfinal concentration was different from 025 to 50mgmLwasmixed with 6 μL of 4mM PV 290 μL of 50mM sodiumacetate buffer (pH 60) and 10 μL of 2mM copper sulfate Inorder to measure copper chelating activity absorbance wasdetermined at 632 nm according to the following equation
Copper chelating activity ()
1minusΔABS(sampleminus blank)
ABS(control)1113896 1113897 times 1001113890 1113891
(3)
29 Measurement of Lipid Peroxidation Oil emulsion wasprepared by mixing raw perilla oil and rhubarb aqueousextract at the ratio of 3 to 1 -en raw perilla oil emulsionswere mixed with 10 gum arabic as an emulsifier andhomogenized for 16min by homogenizer (AM-8 NisseiJapan) Lipid oxidation of the oil emulsion was induced at30degC or 70degC using incubator (JISICO Korea) and waterbath (Shaking Water Bath JEIO Tech Korea) respectivelyHeated raw perilla oil emulsions were collected at 0 7 and10 days of incubation -e lipid oxidation of raw perilla oilwas analyzed by thiobarbituric acid reactive substance(TBARS) assay [26] In brief 10 μL of samples (rhubarb) atthree different concentrations of 0 025 and 5mgmL orstandard solution (1133-tetramethoxypropane TEP) and40 μL of 20mM phosphate buffer (pH 70) were added to anEppendorf tube on ice In each tube 50 μL of 3 sodiumdodecyl sulfate (SDS) 200 μL of 01N HCl 30 μL of 10phosphotungstic acid and 100 μL of 07 of 2-thiobarbituricacid (TBA) were added -e tubes were firmly closed andboiled at 100degC for 30min in water bath -e reactionmixture was mixed with 400 μL of n-butanol and thencentrifuged at 3000 rpm for 10min Supernatants werecollected and loaded in a 96-well plate Fluorescence in-tensity was read at the excitationemission wavelengths of515 nm555 nm using microplate reader (VICTOR Multi-label Plate Reader PerkinElmer Korea)
210 In Vitro RAW2647 Macrophage Study
2101 Measurement of Cell Viability RAW2647 macro-phage cells were cultured at 37degC and 5 CO2 in DMEM with10FBS and 1PS Cell viability was determined by amodified
3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) assay [27] RAW2647 cells were seeded on a 24-wellplate at a density of 1times 105 cellswell and cultured overnightAfter 24h treatment of Rheum extract with different concen-trations (0 01 05 and 1mgmL) RAW2647 cells were in-cubated 05mM tert-butyl hydroperoxide (t-BHP) for 1hFurther incubation with MTT dye solution (05mgmL MTTsolution and medium at a ratio of 1 5) was executed for 1hAfter dissolving purple formazan intoDMSO the absorbance ofthe product was measured at 540nm -e percentage of cellviability was calculated by the following equation
Cell viability(of control) ODsampleminusblank
ODControlminusblanktimes 100 (4)
2102 Determination of Cell Toxicity Effect of aqueousRheum rhizome extract on cell toxicity was measured byneutral red assay [28] RAW2647 cells (1times 105 cellswell ina 24-well plate) were treated with different concentrations ofaqueous Rheum extract (0 01 05 and 1mgmL) for 24 hfollowed by treatment of 05mM t-BHP for an additional 1 hCulture medium was changed to 0004 neutral red solutiondissolved in medium and incubated for 3 h Lysis buffercontaining distilled water ethanol and acetic acid at a ratio of50 49 1 was added to each well and the plate was rocking for15min -e optical density at 540 nm was measured
2103 Measurement of Intracellular Reactive Oxygen Species(ROS) Levels -e level of intracellular ROS was determinedusing 2prime7prime-dichlorofluorescin diacetate (DCFH-DA) [29]RAW2647 cells were planted on a 96-well plate at a density of5times104 cellswell and incubated at 37degC and 5 CO2 for 24 hAfter 24 h of treatment of Rheum extract (0 01 05 and1mgmL) themediumwas changed to PBS containing 120μMDCFH-DA with or without 05mM t-BHP and incubated for60min at 37degC -e absorbance was determined at 488nmexcitation and 525nm emission by using a fluorescence platereader (VICTOR X3 PerkinElmer Turku Singapore)
2104 Measurements of Superoxide Anion ProductionPreincubated RAW2647 cells with Rheum extract (0 0105 and 1mgmL) for 24 h were incubated with or without05mM t-BHP for 1 h NBT solution (05mgmL NBT so-lution and medium at a ratio of 1 5) was added and furtherincubated at 37degC and 5 CO2 for 7 h Medium was re-moved and lysis buffer including DMSO and 2M potassiumhydroxide at a ratio of 1 1 was added -e absorbance wasmeasured at 570 nm
211 Statistical Analysis -e data are presented as themeanplusmn standard deviation (SD) of at least three independenttriplicate experiments Data were analyzed by the ANOVAprocedure using the Statistical Analysis System (SAS 94)software Differences among groups were determined usingBonferroni procedure at the 5 level
Journal of Food Quality 3
3 Results and Discussion
31 Total Phenolic and Flavonoid Contents Polyphenoliccompounds including flavonoids are known as powerfulantioxidants due to their hydroxyl groups and radical scav-enging activities -ese compounds may contribute directly toantioxidant capacity thus having protective functions againstoxidative damage and health benefits [30ndash33] In order toinvestigate antioxidant activity of Rheum aqueous extract wefirst examined concentrations of total phenols and flavonoidsAs shown in Figure 1(a) total phenolic contents of rhizomeextract significantly increased in a dose-dependent mannerwith a significant increase at 05mgmL Total flavonoidconcentration reached a maximum at 5mgmL about 30 foldshigher than 025mgmL (Figure 1(b)) -ese findings suggestthat flavonoids and phenols may be important components ofRheum officinale Baillon and its radical scavenging activitycould be attributed to the presence of these constituents
32 Radical Scavenging and Metal Chelating ActivitiesExcess generation of free radicals or ROS causes oxidativestress and disease [1ndash5] Increased use of naturally occurringantioxidants is considerably regarded as an effective and safe
way to prevent ROS-induced diseases -erefore we deter-mined the antioxidant effect of Rheum extract based on radicalscavenging -e electron donation ability of rhizome extractfrom Rheum was determined by DPPH purple-colored so-lution bleaching assay Aqueous Rheum extract significantlyincreased the degree of color change in a dose-dependentmanner indicating significant free radical scavenging activity(Table 1) In addition the extent of decolorizationmeasured asthe percentage inhibition of ABTS radical cation was assessedafter addition of Rheum rhizome extract (Table 1) Aqueousextract of Rheum rhizome significantly increased both DPPHand ABTS radical scavenging activities in a dose-dependentmanner from a concentration of 025 to 5mgmL indicatingstrong free radical scavenging activity
Copper(I) and iron(II) are regarded as catalysts for thegeneration of highly reactive hydroxyl radicals which causecell or tissue damages and consequently diseases [6 7]-erefore copper chelating capacity and ferric reducingactivity are important markers for antioxidant activity ofnatural resources and functional ingredients [34ndash36] Asshown in Table 1 both ferric reducing capacity and copperchelating activity were significantly increased by Rheumextract in a dose-dependent manner in ranges of 002ndash032mM ferric levels and 3ndash35 copper chelating compared
00
01
02
03
04
05
025 05 1 25 5
Tota
l phe
nol c
once
ntra
tion
(mg G
AE
mL)
Concentration (mgmL)
c
b
a a
d
(a)
00
01
02
03
04
025 05 1 25 5
Flav
onoi
d co
ncen
trat
ion
(mg
mL)
Concentration (mgmL)
d
e
c
b
a
(b)
Figure 1 Effect of aqueous Rheum rhizome extracts on total phenol (a) and flavonoid (b) contents -e concentrations of total phenoliccompounds and flavonoids are calculated according to standard curves of gallic acid and catechin and expressed as milligrams of gallic acidequivalent (GAE) and catechin per milliliter of aqueous Rheum rhizome extract Values are expressed as the meanplusmn standard deviation (SD)(n 9) Bars with different letters indicate significant differences (plt 005)
Table 1 Radical scavenging and metal chelating activities of aqueous Rheum extracts
Values are expressed as the meanplusmn standard deviation (SD) from at least three experiments (n 9) Different letters indicate a significant difference amonggroups according to Bonferroni procedure (plt 005) AA ascorbic acid ABTS 22prime-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid DPPH 22-diphenyl-1-picrylhydrazyl) FRAP ferric reducing antioxidant power TE Trolox equivalent
4 Journal of Food Quality
Table 2 Inhibitory esectect of rhubarb aqueous extract on lipid oxidation over storage time at 30degC and 70degC
Storage time (days) Storage temperature (degC)Concentration of rhubarb (mgmL)
Data are expressed as the meanplusmn standard deviation (SD) from at least three experiments (n 9) Disecterent letters (A B C) within same row indicatea signicant disecterence groups with the same storage time and temperature (plt 005) Small letters (andashe) demonstrate signicant disecterence at a givenconcentration of rhubarb aqueous extract (plt 005)
00 01 05 10 Concentration of Rheum (mgmL)
Controlt-BHP
0
20
40
60
80
100
120
140
Cel
l via
bilit
y (
of c
ontr
ol)
(a)
00 01 05 10 Concentration of Rheum (mgmL)
Controlt-BHP
0
20
40
60
80
100
120
Cell
toxi
city
( o
f con
trol
)
(b)
Figure 2 Esectects of Rheum rhizome extract on cell viability (a) and cell toxicity (b) in 05mM tert-butyl hydroperoxide- (t-BHP-) inducedRAW2647 macrophages Each value represents the meanplusmn standard deviation (SD) from at least three experiments (n 9) Bars withdisecterent letters disecter among aqueous Rheum rhizome extract treatment groups (plt 005)
0
50
100
150
200
250
300
350
00 01 05 10
ROS
gene
ratio
n(
of c
ontr
ol)
Concentration of Rheum (mgmL)
Controlt-BHP
cd
a
e
b
e e
bc
de
(a)
Controlt-BHP
0
20
40
60
80
100
120
140
160
00 01 05 10
Supe
roxi
de an
ion
prod
uctio
n (
of c
ontr
ol)
Concentration of Rheum (mgmL)
abc
a
bc
ab
bc bcbc
c
(b)
Figure 3 Esectects of Rheum rhizome extract on production of intracellular reactive oxygen species (a) and superoxide anion (b) in 05mMtert-butyl hydroperoxide- (t-BHP-) induced RAW2647 macrophages e value of each bar represents the meanplusmn standard deviation (SD)(n 9) Means sharing the same letter are not signicantly disecterent at the 5 level
Journal of Food Quality 5
to control respectively -ese results demonstrated thatRheum rhizome extract has antioxidant capacity mediatedby ferric reducing and copper chelating activities
33 LipidOxidation Increased oxidation process in the foodscontributes to food quality deterioration by increasing oxi-dative rancidity and deleterious food product as well as losingcolor and nutrient value [9 10] -ereby the ways in whichantioxidants inhibit oxidation of food and increase the anti-oxidant efficacy have attracted much attention In the presentstudy inhibitory effect of aqueous Rheum extract on lipidoxidation was measured using thiobarbituric acid reactivesubstances assay Malondialdehyde the end product of lipidoxidation was significantly generated as storage time andtemperature were increased (Table 2) Even though lipidperoxidation in oil emulsion mixture was significantly in-creased according to storage temperature and times treatmentof aqueous extract of Rheum rhizome significantly suppressedmalondialdehyde levels in the raw perilla oil emulsions ina dose-dependent manner -is result supported that aqueousextract of Rheum officinale Baillon inhibits lipid oxidation
34 Antioxidant Properties of Rheum Aqueous Extract int-BHP-Treated RAW2647 Murine Macrophages Antioxidanteffect of Rheum officinale Baillon was determined in in vitrocell culture model t-BHP treated RAW2647 macrophagecells First we measured the cytotoxic effect of Rheumofficinale Baillon rhizome extract After 24 h exposure to 0105 and 1mgmL of Rheum extract viabilities of RAW2647cells were not statistically different from control regardlessof t-BHP treatment (Figure 2(a)) In addition cell toxicitywas not altered by either Rheum extract or t-BHP treatment(Figure 2(b)) -ese results indicate that Rheum extract atany concentration ranging from 01 to 1mgmL had nosignificant cytotoxicity on RAW2647 cell viability
As an indicator of oxidative stress increased productionof ROS promotes the pathogenesis of multiple diseases [1ndash5]In order to evaluate the effect of Rheum extract on oxidativeprocess measurement of ROS and superoxide anion pro-duction were carried out in t-BHP-treated RAW2647 cells Inthe presence of Rheum extract t-BHP-induced ROS (Figure3(a)) and superoxide anion production (Figure 3(b)) weresignificantly diminished in a dose-dependent manner com-pared to t-BHP only-treated cells
4 Conclusions
In the present study analysis of radical scavenging abilitiesmetal chelating activities and total phenolic and flavonoidcontents showed that aqueous extract from Rheum officinaleBaillon rhizome could be a potent source of natural anti-oxidants In addition Rheum aqueous extract significantlyinhibited lipid oxidation in a dose-dependent manner Tothe best of our knowledge this is the first study to suggestthat aqueous extract of Korean Rhubarb (Rheum officinaleBaillon) rhizome may be useful as a natural antioxidant dueto its antioxidant capacities to prevent ROS generation anddelay oxidant degradation of lipids
Data Availability
-e data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
-e authors declare no conflicts of interest
Acknowledgments
-e authors greatly appreciate the technical assistance fromDarae Lee and Naeun Jeon Yeungnam University -iswork was supported by the 2014 Yeungnam UniversityResearch Grant
References
[1] R L Auten and J M Davis ldquoOxygen toxicity and reactiveoxygen species the devil is in the detailsrdquo Pediatric Researchvol 66 no 2 pp 121ndash127 2009
[2] A H Bhat K B Dar S Anees et al ldquoOxidative stressmitochondrial dysfunction and neurodegenerative diseasesa mechanistic insightrdquo Biomedicine and Pharmacotherapyvol 74 pp 101ndash110 2015
[3] J S Bhatti G K Bhatti and P H Reddy ldquoMitochondrialdysfunction and oxidative stress in metabolic disordersmdashastep towards mitochondria based therapeutic strategiesrdquoBiochimica et Biophysica Acta (BBA)-Molecular Basis ofDisease vol 1863 no 5 pp 1066ndash1077 2017
[4] L A Pham-Huy H He and C Pham-Huy ldquoFree radicalsantioxidants in disease and healthrdquo International Journal ofBiomedical Science vol 4 no 2 pp 89ndash96 2008
[5] B P Yu ldquoCellular defenses against damage from reactiveoxygen speciesrdquo Physiological Reviews vol 74 no 1pp 139ndash162 1994
[6] M Ott V Gogvadze S Orrenius and B ZhivotovskyldquoMitochondria oxidative stress and cell deathrdquo Apoptosisvol 12 no 5 pp 913ndash922 2007
[7] G Stark ldquoFunctional consequences of oxidative membranedamagerdquo Journal of Membrane Biology vol 205 no 1pp 1ndash16 2005
[8] E Birben U M Sahiner C Sackesen S Erzurum andO Kalayci ldquoOxidative stress and antioxidant defenserdquoWorldAllergy Organization Journal vol 5 no 1 pp 9ndash19 2012
[9] A J St Angelo J Vercellotti T Jacks and M LegendreldquoLipid oxidation on foodsrdquo Critical Reviews in Food Scienceand Nutrition vol 36 no 3 pp 175ndash224 1996
[10] I Gulccedilin ldquoAntioxidant activity of food constituents anoverviewrdquo Archives of Toxicology vol 86 no 3 pp 345ndash3912012
[11] Z H He R Zhou M F He et al ldquoAnti-angiogenic effect andmechanism of rhein from Rhizoma Rheirdquo Phytomedicinevol 18 no 6 pp 470ndash478 2011
[12] B Hu H Zhang X Meng F Wang and P Wang ldquoAloe-emodin from rhubarb (Rheum rhabarbarum) inhibitslipopolysaccharide-induced inflammatory responses inRAW2647 macrophagesrdquo Journal of Ethnopharmacologyvol 153 no 3 pp 846ndash853 2014
[13] L Lu and H Yin ldquoEffects of Dahuang (Rhubarb) retentionenema on leukocyte interleukin-6 high sensitive C reactiveprotein and endotoxin in patients with acute pancreatitisrdquoMedicinal Plants vol 9 pp 60ndash62 2018
6 Journal of Food Quality
[14] H Matsuda N Tomohiro K Hiraba et al ldquoStudy on Anti-Oketsu Activity of Rhubarb II Anti-allergic effects of stilbenecomponents from Rhei undulati Rhizoma (dried rhizome ofRheum undulatum cultivated in Korea)rdquo Biological andPharmaceutical Bulletin vol 24 no 3 pp 264ndash267 2001a
[15] H Matsuda T Morikawa I Toguchida J Y Park S Harimaand M Yoshikawa ldquoAntioxidant constituents from rhubarbstructural requirements of stilbenes for the activity andstructures of two new anthraquinone glucosidesrdquo Bioorganicand Medicinal Chemistry vol 9 no 1 pp 41ndash50 2001b
[16] T M Ngoc T M Hung P T -uong et al ldquoInhibition ofhuman low density lipoprotein and high density lipoproteinoxidation by oligostilbenes from rhubarbrdquo Biological andPharmaceutical Bulletin vol 31 no 9 pp 1809ndash1812 2008
[17] M Ozturk F Aydogmus-Ozturk M E Duru and G TopccediluldquoAntioxidant activity of stem and root extracts of Rhubarb(Rheum ribes) An edible medicinal plantrdquo Food Chemistryvol 103 no 2 pp 623ndash630 2007
[18] P Raudsepp D Anton M Roasto et al ldquo-e antioxidativeand antimicrobial properties of the blue honeysuckle (Loni-cera caerulea L) Siberian rhubarb (Rheum rhaponticum L)and some other plants compared to ascorbic acid and sodiumnitriterdquo Food Control vol 31 no 1 pp 129ndash135 2013
[19] C S Shia S H Juang S Y Tsai et al ldquoMetabolism andpharmacokinetics of anthraquinones in Rheum palmatum inrats and ex vivo antioxidant activityrdquo Planta Medica vol 75no 13 pp 1386ndash1392 2009
[20] J P Silveira L N Seito S Eberlin et al ldquoPhotoprotective andantioxidant effects of Rhubarb inhibitory action on tyrosinaseand tyrosine kinase activities and TNF-alpha IL-1alpha andalpha-MSH production in human melanocytesrdquo BMCComplementary and Alternative Medicine vol 13 no 1 p 492013
[21] T Venkatesan M J Jeong Y W Choi E J ParkS K El-Desouky and Y K Kim ldquoDeoxyrhapontigenina natural stilbene derivative isolated from Rheum undulatumL induces endoplasmic reticulum stress-mediated apoptosisin human breast cancer cellsrdquo Integrative Cancer Berapiesvol 15 no 4 pp NP44ndashNP52 2016
[22] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquoMethods inEnzymology vol 299 pp 152ndash178 1999
[23] R Re N Pellegrini A Proteggente A Pannala M Yang andC Rice-Evans ldquoAntioxidant activity applying an improvedABTS radical cation decolorization assayrdquo Free Radical Bi-ology and Medicine vol 26 no 9-10 pp 1231ndash1237 1999
[24] I F Benzie and J J Strain ldquo-e ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo theFRAP assayrdquo Analytical Biochemistry vol 239 no 1pp 70ndash76 1996
[25] C Megıas E Pastor-Cavada C Torres-Fuentes et alldquoChelating antioxidant and antiproliferative activity of Viciasativa polyphenol extractsrdquo European Food Research andTechnology vol 230 no 2 pp 353ndash359 2009
[26] P Ke E Cervantes and C Robles-Martinez ldquoDeterminationof thiobarbituric acid reactive substances (TBARS) in fishtissue by an improved distillationndashspectrophotometricmethodrdquo Journal of the Science of Food and Agriculturevol 35 no 11 pp 1248ndash1254 1984
[27] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983
[28] E Borenfreund and J A Puerner ldquoToxicity determined invitro by morphological alterations and neutral red absorp-tionrdquo Toxicology Letters vol 24 no 2-3 pp 119ndash124 1985
[29] K Nishio M Horie Y Akazawa et al ldquoAttenuation of li-popolysaccharide (LPS)-induced cytotoxicity by tocopherolsand tocotrienolsrdquo Redox Biology vol 1 no 1 pp 97ndash1032013
[30] L Bravo ldquoPolyphenols chemistry dietary sources meta-bolism and nutritional significancerdquo Nutrition Reviewsvol 56 no 11 pp 317ndash333 1998
[31] Y Cai Q Luo M Sun and H Corke ldquoAntioxidant activityand phenolic compounds of 112 traditional Chinese medic-inal plants associated with anticancerrdquo Life Sciences vol 74no 17 pp 2157ndash2184 2004
[32] G Di Carlo N Mascolo A A Izzo and F Capasso ldquoFla-vonoids old and new aspects of a class of natural therapeuticdrugsrdquo Life Sciences vol 65 no 4 pp 337ndash353 1999
[33] F Shahidi and P Ambigaipalan ldquoPhenolics and polyphenolicsin foods beverages and spices antioxidant activity and healtheffectsmdasha reviewrdquo Journal of Functional Foods vol 18pp 820ndash897 2015
[34] M S Brewer ldquoNatural antioxidants sources compoundsmechanisms of action and potential applicationsrdquo Compre-hensive Reviews in Food Science and Food Safety vol 10 no 4pp 221ndash247 2011
[35] B Ou D Huang M Hampsch-Woodill J A Flanagan andE K Deemer ldquoAnalysis of antioxidant activities of commonvegetables employing oxygen radical absorbance capacity(ORAC) and ferric reducing antioxidant power (FRAP) as-says a comparative studyrdquo Journal of Agricultural and FoodChemistry vol 50 no 11 pp 3122ndash3128 2002
[36] R Apak K Guclu B Demirata et al ldquoComparative evalu-ation of various total antioxidant capacity assays applied tophenolic compounds with the CUPRAC assayrdquo Moleculesvol 12 no 7 pp 1496ndash1547 2007
31 Total Phenolic and Flavonoid Contents Polyphenoliccompounds including flavonoids are known as powerfulantioxidants due to their hydroxyl groups and radical scav-enging activities -ese compounds may contribute directly toantioxidant capacity thus having protective functions againstoxidative damage and health benefits [30ndash33] In order toinvestigate antioxidant activity of Rheum aqueous extract wefirst examined concentrations of total phenols and flavonoidsAs shown in Figure 1(a) total phenolic contents of rhizomeextract significantly increased in a dose-dependent mannerwith a significant increase at 05mgmL Total flavonoidconcentration reached a maximum at 5mgmL about 30 foldshigher than 025mgmL (Figure 1(b)) -ese findings suggestthat flavonoids and phenols may be important components ofRheum officinale Baillon and its radical scavenging activitycould be attributed to the presence of these constituents
32 Radical Scavenging and Metal Chelating ActivitiesExcess generation of free radicals or ROS causes oxidativestress and disease [1ndash5] Increased use of naturally occurringantioxidants is considerably regarded as an effective and safe
way to prevent ROS-induced diseases -erefore we deter-mined the antioxidant effect of Rheum extract based on radicalscavenging -e electron donation ability of rhizome extractfrom Rheum was determined by DPPH purple-colored so-lution bleaching assay Aqueous Rheum extract significantlyincreased the degree of color change in a dose-dependentmanner indicating significant free radical scavenging activity(Table 1) In addition the extent of decolorizationmeasured asthe percentage inhibition of ABTS radical cation was assessedafter addition of Rheum rhizome extract (Table 1) Aqueousextract of Rheum rhizome significantly increased both DPPHand ABTS radical scavenging activities in a dose-dependentmanner from a concentration of 025 to 5mgmL indicatingstrong free radical scavenging activity
Copper(I) and iron(II) are regarded as catalysts for thegeneration of highly reactive hydroxyl radicals which causecell or tissue damages and consequently diseases [6 7]-erefore copper chelating capacity and ferric reducingactivity are important markers for antioxidant activity ofnatural resources and functional ingredients [34ndash36] Asshown in Table 1 both ferric reducing capacity and copperchelating activity were significantly increased by Rheumextract in a dose-dependent manner in ranges of 002ndash032mM ferric levels and 3ndash35 copper chelating compared
00
01
02
03
04
05
025 05 1 25 5
Tota
l phe
nol c
once
ntra
tion
(mg G
AE
mL)
Concentration (mgmL)
c
b
a a
d
(a)
00
01
02
03
04
025 05 1 25 5
Flav
onoi
d co
ncen
trat
ion
(mg
mL)
Concentration (mgmL)
d
e
c
b
a
(b)
Figure 1 Effect of aqueous Rheum rhizome extracts on total phenol (a) and flavonoid (b) contents -e concentrations of total phenoliccompounds and flavonoids are calculated according to standard curves of gallic acid and catechin and expressed as milligrams of gallic acidequivalent (GAE) and catechin per milliliter of aqueous Rheum rhizome extract Values are expressed as the meanplusmn standard deviation (SD)(n 9) Bars with different letters indicate significant differences (plt 005)
Table 1 Radical scavenging and metal chelating activities of aqueous Rheum extracts
Values are expressed as the meanplusmn standard deviation (SD) from at least three experiments (n 9) Different letters indicate a significant difference amonggroups according to Bonferroni procedure (plt 005) AA ascorbic acid ABTS 22prime-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid DPPH 22-diphenyl-1-picrylhydrazyl) FRAP ferric reducing antioxidant power TE Trolox equivalent
4 Journal of Food Quality
Table 2 Inhibitory esectect of rhubarb aqueous extract on lipid oxidation over storage time at 30degC and 70degC
Storage time (days) Storage temperature (degC)Concentration of rhubarb (mgmL)
Data are expressed as the meanplusmn standard deviation (SD) from at least three experiments (n 9) Disecterent letters (A B C) within same row indicatea signicant disecterence groups with the same storage time and temperature (plt 005) Small letters (andashe) demonstrate signicant disecterence at a givenconcentration of rhubarb aqueous extract (plt 005)
00 01 05 10 Concentration of Rheum (mgmL)
Controlt-BHP
0
20
40
60
80
100
120
140
Cel
l via
bilit
y (
of c
ontr
ol)
(a)
00 01 05 10 Concentration of Rheum (mgmL)
Controlt-BHP
0
20
40
60
80
100
120
Cell
toxi
city
( o
f con
trol
)
(b)
Figure 2 Esectects of Rheum rhizome extract on cell viability (a) and cell toxicity (b) in 05mM tert-butyl hydroperoxide- (t-BHP-) inducedRAW2647 macrophages Each value represents the meanplusmn standard deviation (SD) from at least three experiments (n 9) Bars withdisecterent letters disecter among aqueous Rheum rhizome extract treatment groups (plt 005)
0
50
100
150
200
250
300
350
00 01 05 10
ROS
gene
ratio
n(
of c
ontr
ol)
Concentration of Rheum (mgmL)
Controlt-BHP
cd
a
e
b
e e
bc
de
(a)
Controlt-BHP
0
20
40
60
80
100
120
140
160
00 01 05 10
Supe
roxi
de an
ion
prod
uctio
n (
of c
ontr
ol)
Concentration of Rheum (mgmL)
abc
a
bc
ab
bc bcbc
c
(b)
Figure 3 Esectects of Rheum rhizome extract on production of intracellular reactive oxygen species (a) and superoxide anion (b) in 05mMtert-butyl hydroperoxide- (t-BHP-) induced RAW2647 macrophages e value of each bar represents the meanplusmn standard deviation (SD)(n 9) Means sharing the same letter are not signicantly disecterent at the 5 level
Journal of Food Quality 5
to control respectively -ese results demonstrated thatRheum rhizome extract has antioxidant capacity mediatedby ferric reducing and copper chelating activities
33 LipidOxidation Increased oxidation process in the foodscontributes to food quality deterioration by increasing oxi-dative rancidity and deleterious food product as well as losingcolor and nutrient value [9 10] -ereby the ways in whichantioxidants inhibit oxidation of food and increase the anti-oxidant efficacy have attracted much attention In the presentstudy inhibitory effect of aqueous Rheum extract on lipidoxidation was measured using thiobarbituric acid reactivesubstances assay Malondialdehyde the end product of lipidoxidation was significantly generated as storage time andtemperature were increased (Table 2) Even though lipidperoxidation in oil emulsion mixture was significantly in-creased according to storage temperature and times treatmentof aqueous extract of Rheum rhizome significantly suppressedmalondialdehyde levels in the raw perilla oil emulsions ina dose-dependent manner -is result supported that aqueousextract of Rheum officinale Baillon inhibits lipid oxidation
34 Antioxidant Properties of Rheum Aqueous Extract int-BHP-Treated RAW2647 Murine Macrophages Antioxidanteffect of Rheum officinale Baillon was determined in in vitrocell culture model t-BHP treated RAW2647 macrophagecells First we measured the cytotoxic effect of Rheumofficinale Baillon rhizome extract After 24 h exposure to 0105 and 1mgmL of Rheum extract viabilities of RAW2647cells were not statistically different from control regardlessof t-BHP treatment (Figure 2(a)) In addition cell toxicitywas not altered by either Rheum extract or t-BHP treatment(Figure 2(b)) -ese results indicate that Rheum extract atany concentration ranging from 01 to 1mgmL had nosignificant cytotoxicity on RAW2647 cell viability
As an indicator of oxidative stress increased productionof ROS promotes the pathogenesis of multiple diseases [1ndash5]In order to evaluate the effect of Rheum extract on oxidativeprocess measurement of ROS and superoxide anion pro-duction were carried out in t-BHP-treated RAW2647 cells Inthe presence of Rheum extract t-BHP-induced ROS (Figure3(a)) and superoxide anion production (Figure 3(b)) weresignificantly diminished in a dose-dependent manner com-pared to t-BHP only-treated cells
4 Conclusions
In the present study analysis of radical scavenging abilitiesmetal chelating activities and total phenolic and flavonoidcontents showed that aqueous extract from Rheum officinaleBaillon rhizome could be a potent source of natural anti-oxidants In addition Rheum aqueous extract significantlyinhibited lipid oxidation in a dose-dependent manner Tothe best of our knowledge this is the first study to suggestthat aqueous extract of Korean Rhubarb (Rheum officinaleBaillon) rhizome may be useful as a natural antioxidant dueto its antioxidant capacities to prevent ROS generation anddelay oxidant degradation of lipids
Data Availability
-e data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
-e authors declare no conflicts of interest
Acknowledgments
-e authors greatly appreciate the technical assistance fromDarae Lee and Naeun Jeon Yeungnam University -iswork was supported by the 2014 Yeungnam UniversityResearch Grant
References
[1] R L Auten and J M Davis ldquoOxygen toxicity and reactiveoxygen species the devil is in the detailsrdquo Pediatric Researchvol 66 no 2 pp 121ndash127 2009
[2] A H Bhat K B Dar S Anees et al ldquoOxidative stressmitochondrial dysfunction and neurodegenerative diseasesa mechanistic insightrdquo Biomedicine and Pharmacotherapyvol 74 pp 101ndash110 2015
[3] J S Bhatti G K Bhatti and P H Reddy ldquoMitochondrialdysfunction and oxidative stress in metabolic disordersmdashastep towards mitochondria based therapeutic strategiesrdquoBiochimica et Biophysica Acta (BBA)-Molecular Basis ofDisease vol 1863 no 5 pp 1066ndash1077 2017
[4] L A Pham-Huy H He and C Pham-Huy ldquoFree radicalsantioxidants in disease and healthrdquo International Journal ofBiomedical Science vol 4 no 2 pp 89ndash96 2008
[5] B P Yu ldquoCellular defenses against damage from reactiveoxygen speciesrdquo Physiological Reviews vol 74 no 1pp 139ndash162 1994
[6] M Ott V Gogvadze S Orrenius and B ZhivotovskyldquoMitochondria oxidative stress and cell deathrdquo Apoptosisvol 12 no 5 pp 913ndash922 2007
[7] G Stark ldquoFunctional consequences of oxidative membranedamagerdquo Journal of Membrane Biology vol 205 no 1pp 1ndash16 2005
[8] E Birben U M Sahiner C Sackesen S Erzurum andO Kalayci ldquoOxidative stress and antioxidant defenserdquoWorldAllergy Organization Journal vol 5 no 1 pp 9ndash19 2012
[9] A J St Angelo J Vercellotti T Jacks and M LegendreldquoLipid oxidation on foodsrdquo Critical Reviews in Food Scienceand Nutrition vol 36 no 3 pp 175ndash224 1996
[10] I Gulccedilin ldquoAntioxidant activity of food constituents anoverviewrdquo Archives of Toxicology vol 86 no 3 pp 345ndash3912012
[11] Z H He R Zhou M F He et al ldquoAnti-angiogenic effect andmechanism of rhein from Rhizoma Rheirdquo Phytomedicinevol 18 no 6 pp 470ndash478 2011
[12] B Hu H Zhang X Meng F Wang and P Wang ldquoAloe-emodin from rhubarb (Rheum rhabarbarum) inhibitslipopolysaccharide-induced inflammatory responses inRAW2647 macrophagesrdquo Journal of Ethnopharmacologyvol 153 no 3 pp 846ndash853 2014
[13] L Lu and H Yin ldquoEffects of Dahuang (Rhubarb) retentionenema on leukocyte interleukin-6 high sensitive C reactiveprotein and endotoxin in patients with acute pancreatitisrdquoMedicinal Plants vol 9 pp 60ndash62 2018
6 Journal of Food Quality
[14] H Matsuda N Tomohiro K Hiraba et al ldquoStudy on Anti-Oketsu Activity of Rhubarb II Anti-allergic effects of stilbenecomponents from Rhei undulati Rhizoma (dried rhizome ofRheum undulatum cultivated in Korea)rdquo Biological andPharmaceutical Bulletin vol 24 no 3 pp 264ndash267 2001a
[15] H Matsuda T Morikawa I Toguchida J Y Park S Harimaand M Yoshikawa ldquoAntioxidant constituents from rhubarbstructural requirements of stilbenes for the activity andstructures of two new anthraquinone glucosidesrdquo Bioorganicand Medicinal Chemistry vol 9 no 1 pp 41ndash50 2001b
[16] T M Ngoc T M Hung P T -uong et al ldquoInhibition ofhuman low density lipoprotein and high density lipoproteinoxidation by oligostilbenes from rhubarbrdquo Biological andPharmaceutical Bulletin vol 31 no 9 pp 1809ndash1812 2008
[17] M Ozturk F Aydogmus-Ozturk M E Duru and G TopccediluldquoAntioxidant activity of stem and root extracts of Rhubarb(Rheum ribes) An edible medicinal plantrdquo Food Chemistryvol 103 no 2 pp 623ndash630 2007
[18] P Raudsepp D Anton M Roasto et al ldquo-e antioxidativeand antimicrobial properties of the blue honeysuckle (Loni-cera caerulea L) Siberian rhubarb (Rheum rhaponticum L)and some other plants compared to ascorbic acid and sodiumnitriterdquo Food Control vol 31 no 1 pp 129ndash135 2013
[19] C S Shia S H Juang S Y Tsai et al ldquoMetabolism andpharmacokinetics of anthraquinones in Rheum palmatum inrats and ex vivo antioxidant activityrdquo Planta Medica vol 75no 13 pp 1386ndash1392 2009
[20] J P Silveira L N Seito S Eberlin et al ldquoPhotoprotective andantioxidant effects of Rhubarb inhibitory action on tyrosinaseand tyrosine kinase activities and TNF-alpha IL-1alpha andalpha-MSH production in human melanocytesrdquo BMCComplementary and Alternative Medicine vol 13 no 1 p 492013
[21] T Venkatesan M J Jeong Y W Choi E J ParkS K El-Desouky and Y K Kim ldquoDeoxyrhapontigenina natural stilbene derivative isolated from Rheum undulatumL induces endoplasmic reticulum stress-mediated apoptosisin human breast cancer cellsrdquo Integrative Cancer Berapiesvol 15 no 4 pp NP44ndashNP52 2016
[22] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquoMethods inEnzymology vol 299 pp 152ndash178 1999
[23] R Re N Pellegrini A Proteggente A Pannala M Yang andC Rice-Evans ldquoAntioxidant activity applying an improvedABTS radical cation decolorization assayrdquo Free Radical Bi-ology and Medicine vol 26 no 9-10 pp 1231ndash1237 1999
[24] I F Benzie and J J Strain ldquo-e ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo theFRAP assayrdquo Analytical Biochemistry vol 239 no 1pp 70ndash76 1996
[25] C Megıas E Pastor-Cavada C Torres-Fuentes et alldquoChelating antioxidant and antiproliferative activity of Viciasativa polyphenol extractsrdquo European Food Research andTechnology vol 230 no 2 pp 353ndash359 2009
[26] P Ke E Cervantes and C Robles-Martinez ldquoDeterminationof thiobarbituric acid reactive substances (TBARS) in fishtissue by an improved distillationndashspectrophotometricmethodrdquo Journal of the Science of Food and Agriculturevol 35 no 11 pp 1248ndash1254 1984
[27] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983
[28] E Borenfreund and J A Puerner ldquoToxicity determined invitro by morphological alterations and neutral red absorp-tionrdquo Toxicology Letters vol 24 no 2-3 pp 119ndash124 1985
[29] K Nishio M Horie Y Akazawa et al ldquoAttenuation of li-popolysaccharide (LPS)-induced cytotoxicity by tocopherolsand tocotrienolsrdquo Redox Biology vol 1 no 1 pp 97ndash1032013
[30] L Bravo ldquoPolyphenols chemistry dietary sources meta-bolism and nutritional significancerdquo Nutrition Reviewsvol 56 no 11 pp 317ndash333 1998
[31] Y Cai Q Luo M Sun and H Corke ldquoAntioxidant activityand phenolic compounds of 112 traditional Chinese medic-inal plants associated with anticancerrdquo Life Sciences vol 74no 17 pp 2157ndash2184 2004
[32] G Di Carlo N Mascolo A A Izzo and F Capasso ldquoFla-vonoids old and new aspects of a class of natural therapeuticdrugsrdquo Life Sciences vol 65 no 4 pp 337ndash353 1999
[33] F Shahidi and P Ambigaipalan ldquoPhenolics and polyphenolicsin foods beverages and spices antioxidant activity and healtheffectsmdasha reviewrdquo Journal of Functional Foods vol 18pp 820ndash897 2015
[34] M S Brewer ldquoNatural antioxidants sources compoundsmechanisms of action and potential applicationsrdquo Compre-hensive Reviews in Food Science and Food Safety vol 10 no 4pp 221ndash247 2011
[35] B Ou D Huang M Hampsch-Woodill J A Flanagan andE K Deemer ldquoAnalysis of antioxidant activities of commonvegetables employing oxygen radical absorbance capacity(ORAC) and ferric reducing antioxidant power (FRAP) as-says a comparative studyrdquo Journal of Agricultural and FoodChemistry vol 50 no 11 pp 3122ndash3128 2002
[36] R Apak K Guclu B Demirata et al ldquoComparative evalu-ation of various total antioxidant capacity assays applied tophenolic compounds with the CUPRAC assayrdquo Moleculesvol 12 no 7 pp 1496ndash1547 2007
Data are expressed as the meanplusmn standard deviation (SD) from at least three experiments (n 9) Disecterent letters (A B C) within same row indicatea signicant disecterence groups with the same storage time and temperature (plt 005) Small letters (andashe) demonstrate signicant disecterence at a givenconcentration of rhubarb aqueous extract (plt 005)
00 01 05 10 Concentration of Rheum (mgmL)
Controlt-BHP
0
20
40
60
80
100
120
140
Cel
l via
bilit
y (
of c
ontr
ol)
(a)
00 01 05 10 Concentration of Rheum (mgmL)
Controlt-BHP
0
20
40
60
80
100
120
Cell
toxi
city
( o
f con
trol
)
(b)
Figure 2 Esectects of Rheum rhizome extract on cell viability (a) and cell toxicity (b) in 05mM tert-butyl hydroperoxide- (t-BHP-) inducedRAW2647 macrophages Each value represents the meanplusmn standard deviation (SD) from at least three experiments (n 9) Bars withdisecterent letters disecter among aqueous Rheum rhizome extract treatment groups (plt 005)
0
50
100
150
200
250
300
350
00 01 05 10
ROS
gene
ratio
n(
of c
ontr
ol)
Concentration of Rheum (mgmL)
Controlt-BHP
cd
a
e
b
e e
bc
de
(a)
Controlt-BHP
0
20
40
60
80
100
120
140
160
00 01 05 10
Supe
roxi
de an
ion
prod
uctio
n (
of c
ontr
ol)
Concentration of Rheum (mgmL)
abc
a
bc
ab
bc bcbc
c
(b)
Figure 3 Esectects of Rheum rhizome extract on production of intracellular reactive oxygen species (a) and superoxide anion (b) in 05mMtert-butyl hydroperoxide- (t-BHP-) induced RAW2647 macrophages e value of each bar represents the meanplusmn standard deviation (SD)(n 9) Means sharing the same letter are not signicantly disecterent at the 5 level
Journal of Food Quality 5
to control respectively -ese results demonstrated thatRheum rhizome extract has antioxidant capacity mediatedby ferric reducing and copper chelating activities
33 LipidOxidation Increased oxidation process in the foodscontributes to food quality deterioration by increasing oxi-dative rancidity and deleterious food product as well as losingcolor and nutrient value [9 10] -ereby the ways in whichantioxidants inhibit oxidation of food and increase the anti-oxidant efficacy have attracted much attention In the presentstudy inhibitory effect of aqueous Rheum extract on lipidoxidation was measured using thiobarbituric acid reactivesubstances assay Malondialdehyde the end product of lipidoxidation was significantly generated as storage time andtemperature were increased (Table 2) Even though lipidperoxidation in oil emulsion mixture was significantly in-creased according to storage temperature and times treatmentof aqueous extract of Rheum rhizome significantly suppressedmalondialdehyde levels in the raw perilla oil emulsions ina dose-dependent manner -is result supported that aqueousextract of Rheum officinale Baillon inhibits lipid oxidation
34 Antioxidant Properties of Rheum Aqueous Extract int-BHP-Treated RAW2647 Murine Macrophages Antioxidanteffect of Rheum officinale Baillon was determined in in vitrocell culture model t-BHP treated RAW2647 macrophagecells First we measured the cytotoxic effect of Rheumofficinale Baillon rhizome extract After 24 h exposure to 0105 and 1mgmL of Rheum extract viabilities of RAW2647cells were not statistically different from control regardlessof t-BHP treatment (Figure 2(a)) In addition cell toxicitywas not altered by either Rheum extract or t-BHP treatment(Figure 2(b)) -ese results indicate that Rheum extract atany concentration ranging from 01 to 1mgmL had nosignificant cytotoxicity on RAW2647 cell viability
As an indicator of oxidative stress increased productionof ROS promotes the pathogenesis of multiple diseases [1ndash5]In order to evaluate the effect of Rheum extract on oxidativeprocess measurement of ROS and superoxide anion pro-duction were carried out in t-BHP-treated RAW2647 cells Inthe presence of Rheum extract t-BHP-induced ROS (Figure3(a)) and superoxide anion production (Figure 3(b)) weresignificantly diminished in a dose-dependent manner com-pared to t-BHP only-treated cells
4 Conclusions
In the present study analysis of radical scavenging abilitiesmetal chelating activities and total phenolic and flavonoidcontents showed that aqueous extract from Rheum officinaleBaillon rhizome could be a potent source of natural anti-oxidants In addition Rheum aqueous extract significantlyinhibited lipid oxidation in a dose-dependent manner Tothe best of our knowledge this is the first study to suggestthat aqueous extract of Korean Rhubarb (Rheum officinaleBaillon) rhizome may be useful as a natural antioxidant dueto its antioxidant capacities to prevent ROS generation anddelay oxidant degradation of lipids
Data Availability
-e data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
-e authors declare no conflicts of interest
Acknowledgments
-e authors greatly appreciate the technical assistance fromDarae Lee and Naeun Jeon Yeungnam University -iswork was supported by the 2014 Yeungnam UniversityResearch Grant
References
[1] R L Auten and J M Davis ldquoOxygen toxicity and reactiveoxygen species the devil is in the detailsrdquo Pediatric Researchvol 66 no 2 pp 121ndash127 2009
[2] A H Bhat K B Dar S Anees et al ldquoOxidative stressmitochondrial dysfunction and neurodegenerative diseasesa mechanistic insightrdquo Biomedicine and Pharmacotherapyvol 74 pp 101ndash110 2015
[3] J S Bhatti G K Bhatti and P H Reddy ldquoMitochondrialdysfunction and oxidative stress in metabolic disordersmdashastep towards mitochondria based therapeutic strategiesrdquoBiochimica et Biophysica Acta (BBA)-Molecular Basis ofDisease vol 1863 no 5 pp 1066ndash1077 2017
[4] L A Pham-Huy H He and C Pham-Huy ldquoFree radicalsantioxidants in disease and healthrdquo International Journal ofBiomedical Science vol 4 no 2 pp 89ndash96 2008
[5] B P Yu ldquoCellular defenses against damage from reactiveoxygen speciesrdquo Physiological Reviews vol 74 no 1pp 139ndash162 1994
[6] M Ott V Gogvadze S Orrenius and B ZhivotovskyldquoMitochondria oxidative stress and cell deathrdquo Apoptosisvol 12 no 5 pp 913ndash922 2007
[7] G Stark ldquoFunctional consequences of oxidative membranedamagerdquo Journal of Membrane Biology vol 205 no 1pp 1ndash16 2005
[8] E Birben U M Sahiner C Sackesen S Erzurum andO Kalayci ldquoOxidative stress and antioxidant defenserdquoWorldAllergy Organization Journal vol 5 no 1 pp 9ndash19 2012
[9] A J St Angelo J Vercellotti T Jacks and M LegendreldquoLipid oxidation on foodsrdquo Critical Reviews in Food Scienceand Nutrition vol 36 no 3 pp 175ndash224 1996
[10] I Gulccedilin ldquoAntioxidant activity of food constituents anoverviewrdquo Archives of Toxicology vol 86 no 3 pp 345ndash3912012
[11] Z H He R Zhou M F He et al ldquoAnti-angiogenic effect andmechanism of rhein from Rhizoma Rheirdquo Phytomedicinevol 18 no 6 pp 470ndash478 2011
[12] B Hu H Zhang X Meng F Wang and P Wang ldquoAloe-emodin from rhubarb (Rheum rhabarbarum) inhibitslipopolysaccharide-induced inflammatory responses inRAW2647 macrophagesrdquo Journal of Ethnopharmacologyvol 153 no 3 pp 846ndash853 2014
[13] L Lu and H Yin ldquoEffects of Dahuang (Rhubarb) retentionenema on leukocyte interleukin-6 high sensitive C reactiveprotein and endotoxin in patients with acute pancreatitisrdquoMedicinal Plants vol 9 pp 60ndash62 2018
6 Journal of Food Quality
[14] H Matsuda N Tomohiro K Hiraba et al ldquoStudy on Anti-Oketsu Activity of Rhubarb II Anti-allergic effects of stilbenecomponents from Rhei undulati Rhizoma (dried rhizome ofRheum undulatum cultivated in Korea)rdquo Biological andPharmaceutical Bulletin vol 24 no 3 pp 264ndash267 2001a
[15] H Matsuda T Morikawa I Toguchida J Y Park S Harimaand M Yoshikawa ldquoAntioxidant constituents from rhubarbstructural requirements of stilbenes for the activity andstructures of two new anthraquinone glucosidesrdquo Bioorganicand Medicinal Chemistry vol 9 no 1 pp 41ndash50 2001b
[16] T M Ngoc T M Hung P T -uong et al ldquoInhibition ofhuman low density lipoprotein and high density lipoproteinoxidation by oligostilbenes from rhubarbrdquo Biological andPharmaceutical Bulletin vol 31 no 9 pp 1809ndash1812 2008
[17] M Ozturk F Aydogmus-Ozturk M E Duru and G TopccediluldquoAntioxidant activity of stem and root extracts of Rhubarb(Rheum ribes) An edible medicinal plantrdquo Food Chemistryvol 103 no 2 pp 623ndash630 2007
[18] P Raudsepp D Anton M Roasto et al ldquo-e antioxidativeand antimicrobial properties of the blue honeysuckle (Loni-cera caerulea L) Siberian rhubarb (Rheum rhaponticum L)and some other plants compared to ascorbic acid and sodiumnitriterdquo Food Control vol 31 no 1 pp 129ndash135 2013
[19] C S Shia S H Juang S Y Tsai et al ldquoMetabolism andpharmacokinetics of anthraquinones in Rheum palmatum inrats and ex vivo antioxidant activityrdquo Planta Medica vol 75no 13 pp 1386ndash1392 2009
[20] J P Silveira L N Seito S Eberlin et al ldquoPhotoprotective andantioxidant effects of Rhubarb inhibitory action on tyrosinaseand tyrosine kinase activities and TNF-alpha IL-1alpha andalpha-MSH production in human melanocytesrdquo BMCComplementary and Alternative Medicine vol 13 no 1 p 492013
[21] T Venkatesan M J Jeong Y W Choi E J ParkS K El-Desouky and Y K Kim ldquoDeoxyrhapontigenina natural stilbene derivative isolated from Rheum undulatumL induces endoplasmic reticulum stress-mediated apoptosisin human breast cancer cellsrdquo Integrative Cancer Berapiesvol 15 no 4 pp NP44ndashNP52 2016
[22] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquoMethods inEnzymology vol 299 pp 152ndash178 1999
[23] R Re N Pellegrini A Proteggente A Pannala M Yang andC Rice-Evans ldquoAntioxidant activity applying an improvedABTS radical cation decolorization assayrdquo Free Radical Bi-ology and Medicine vol 26 no 9-10 pp 1231ndash1237 1999
[24] I F Benzie and J J Strain ldquo-e ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo theFRAP assayrdquo Analytical Biochemistry vol 239 no 1pp 70ndash76 1996
[25] C Megıas E Pastor-Cavada C Torres-Fuentes et alldquoChelating antioxidant and antiproliferative activity of Viciasativa polyphenol extractsrdquo European Food Research andTechnology vol 230 no 2 pp 353ndash359 2009
[26] P Ke E Cervantes and C Robles-Martinez ldquoDeterminationof thiobarbituric acid reactive substances (TBARS) in fishtissue by an improved distillationndashspectrophotometricmethodrdquo Journal of the Science of Food and Agriculturevol 35 no 11 pp 1248ndash1254 1984
[27] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983
[28] E Borenfreund and J A Puerner ldquoToxicity determined invitro by morphological alterations and neutral red absorp-tionrdquo Toxicology Letters vol 24 no 2-3 pp 119ndash124 1985
[29] K Nishio M Horie Y Akazawa et al ldquoAttenuation of li-popolysaccharide (LPS)-induced cytotoxicity by tocopherolsand tocotrienolsrdquo Redox Biology vol 1 no 1 pp 97ndash1032013
[30] L Bravo ldquoPolyphenols chemistry dietary sources meta-bolism and nutritional significancerdquo Nutrition Reviewsvol 56 no 11 pp 317ndash333 1998
[31] Y Cai Q Luo M Sun and H Corke ldquoAntioxidant activityand phenolic compounds of 112 traditional Chinese medic-inal plants associated with anticancerrdquo Life Sciences vol 74no 17 pp 2157ndash2184 2004
[32] G Di Carlo N Mascolo A A Izzo and F Capasso ldquoFla-vonoids old and new aspects of a class of natural therapeuticdrugsrdquo Life Sciences vol 65 no 4 pp 337ndash353 1999
[33] F Shahidi and P Ambigaipalan ldquoPhenolics and polyphenolicsin foods beverages and spices antioxidant activity and healtheffectsmdasha reviewrdquo Journal of Functional Foods vol 18pp 820ndash897 2015
[34] M S Brewer ldquoNatural antioxidants sources compoundsmechanisms of action and potential applicationsrdquo Compre-hensive Reviews in Food Science and Food Safety vol 10 no 4pp 221ndash247 2011
[35] B Ou D Huang M Hampsch-Woodill J A Flanagan andE K Deemer ldquoAnalysis of antioxidant activities of commonvegetables employing oxygen radical absorbance capacity(ORAC) and ferric reducing antioxidant power (FRAP) as-says a comparative studyrdquo Journal of Agricultural and FoodChemistry vol 50 no 11 pp 3122ndash3128 2002
[36] R Apak K Guclu B Demirata et al ldquoComparative evalu-ation of various total antioxidant capacity assays applied tophenolic compounds with the CUPRAC assayrdquo Moleculesvol 12 no 7 pp 1496ndash1547 2007
to control respectively -ese results demonstrated thatRheum rhizome extract has antioxidant capacity mediatedby ferric reducing and copper chelating activities
33 LipidOxidation Increased oxidation process in the foodscontributes to food quality deterioration by increasing oxi-dative rancidity and deleterious food product as well as losingcolor and nutrient value [9 10] -ereby the ways in whichantioxidants inhibit oxidation of food and increase the anti-oxidant efficacy have attracted much attention In the presentstudy inhibitory effect of aqueous Rheum extract on lipidoxidation was measured using thiobarbituric acid reactivesubstances assay Malondialdehyde the end product of lipidoxidation was significantly generated as storage time andtemperature were increased (Table 2) Even though lipidperoxidation in oil emulsion mixture was significantly in-creased according to storage temperature and times treatmentof aqueous extract of Rheum rhizome significantly suppressedmalondialdehyde levels in the raw perilla oil emulsions ina dose-dependent manner -is result supported that aqueousextract of Rheum officinale Baillon inhibits lipid oxidation
34 Antioxidant Properties of Rheum Aqueous Extract int-BHP-Treated RAW2647 Murine Macrophages Antioxidanteffect of Rheum officinale Baillon was determined in in vitrocell culture model t-BHP treated RAW2647 macrophagecells First we measured the cytotoxic effect of Rheumofficinale Baillon rhizome extract After 24 h exposure to 0105 and 1mgmL of Rheum extract viabilities of RAW2647cells were not statistically different from control regardlessof t-BHP treatment (Figure 2(a)) In addition cell toxicitywas not altered by either Rheum extract or t-BHP treatment(Figure 2(b)) -ese results indicate that Rheum extract atany concentration ranging from 01 to 1mgmL had nosignificant cytotoxicity on RAW2647 cell viability
As an indicator of oxidative stress increased productionof ROS promotes the pathogenesis of multiple diseases [1ndash5]In order to evaluate the effect of Rheum extract on oxidativeprocess measurement of ROS and superoxide anion pro-duction were carried out in t-BHP-treated RAW2647 cells Inthe presence of Rheum extract t-BHP-induced ROS (Figure3(a)) and superoxide anion production (Figure 3(b)) weresignificantly diminished in a dose-dependent manner com-pared to t-BHP only-treated cells
4 Conclusions
In the present study analysis of radical scavenging abilitiesmetal chelating activities and total phenolic and flavonoidcontents showed that aqueous extract from Rheum officinaleBaillon rhizome could be a potent source of natural anti-oxidants In addition Rheum aqueous extract significantlyinhibited lipid oxidation in a dose-dependent manner Tothe best of our knowledge this is the first study to suggestthat aqueous extract of Korean Rhubarb (Rheum officinaleBaillon) rhizome may be useful as a natural antioxidant dueto its antioxidant capacities to prevent ROS generation anddelay oxidant degradation of lipids
Data Availability
-e data used to support the findings of this study areavailable from the corresponding author upon request
Conflicts of Interest
-e authors declare no conflicts of interest
Acknowledgments
-e authors greatly appreciate the technical assistance fromDarae Lee and Naeun Jeon Yeungnam University -iswork was supported by the 2014 Yeungnam UniversityResearch Grant
References
[1] R L Auten and J M Davis ldquoOxygen toxicity and reactiveoxygen species the devil is in the detailsrdquo Pediatric Researchvol 66 no 2 pp 121ndash127 2009
[2] A H Bhat K B Dar S Anees et al ldquoOxidative stressmitochondrial dysfunction and neurodegenerative diseasesa mechanistic insightrdquo Biomedicine and Pharmacotherapyvol 74 pp 101ndash110 2015
[3] J S Bhatti G K Bhatti and P H Reddy ldquoMitochondrialdysfunction and oxidative stress in metabolic disordersmdashastep towards mitochondria based therapeutic strategiesrdquoBiochimica et Biophysica Acta (BBA)-Molecular Basis ofDisease vol 1863 no 5 pp 1066ndash1077 2017
[4] L A Pham-Huy H He and C Pham-Huy ldquoFree radicalsantioxidants in disease and healthrdquo International Journal ofBiomedical Science vol 4 no 2 pp 89ndash96 2008
[5] B P Yu ldquoCellular defenses against damage from reactiveoxygen speciesrdquo Physiological Reviews vol 74 no 1pp 139ndash162 1994
[6] M Ott V Gogvadze S Orrenius and B ZhivotovskyldquoMitochondria oxidative stress and cell deathrdquo Apoptosisvol 12 no 5 pp 913ndash922 2007
[7] G Stark ldquoFunctional consequences of oxidative membranedamagerdquo Journal of Membrane Biology vol 205 no 1pp 1ndash16 2005
[8] E Birben U M Sahiner C Sackesen S Erzurum andO Kalayci ldquoOxidative stress and antioxidant defenserdquoWorldAllergy Organization Journal vol 5 no 1 pp 9ndash19 2012
[9] A J St Angelo J Vercellotti T Jacks and M LegendreldquoLipid oxidation on foodsrdquo Critical Reviews in Food Scienceand Nutrition vol 36 no 3 pp 175ndash224 1996
[10] I Gulccedilin ldquoAntioxidant activity of food constituents anoverviewrdquo Archives of Toxicology vol 86 no 3 pp 345ndash3912012
[11] Z H He R Zhou M F He et al ldquoAnti-angiogenic effect andmechanism of rhein from Rhizoma Rheirdquo Phytomedicinevol 18 no 6 pp 470ndash478 2011
[12] B Hu H Zhang X Meng F Wang and P Wang ldquoAloe-emodin from rhubarb (Rheum rhabarbarum) inhibitslipopolysaccharide-induced inflammatory responses inRAW2647 macrophagesrdquo Journal of Ethnopharmacologyvol 153 no 3 pp 846ndash853 2014
[13] L Lu and H Yin ldquoEffects of Dahuang (Rhubarb) retentionenema on leukocyte interleukin-6 high sensitive C reactiveprotein and endotoxin in patients with acute pancreatitisrdquoMedicinal Plants vol 9 pp 60ndash62 2018
6 Journal of Food Quality
[14] H Matsuda N Tomohiro K Hiraba et al ldquoStudy on Anti-Oketsu Activity of Rhubarb II Anti-allergic effects of stilbenecomponents from Rhei undulati Rhizoma (dried rhizome ofRheum undulatum cultivated in Korea)rdquo Biological andPharmaceutical Bulletin vol 24 no 3 pp 264ndash267 2001a
[15] H Matsuda T Morikawa I Toguchida J Y Park S Harimaand M Yoshikawa ldquoAntioxidant constituents from rhubarbstructural requirements of stilbenes for the activity andstructures of two new anthraquinone glucosidesrdquo Bioorganicand Medicinal Chemistry vol 9 no 1 pp 41ndash50 2001b
[16] T M Ngoc T M Hung P T -uong et al ldquoInhibition ofhuman low density lipoprotein and high density lipoproteinoxidation by oligostilbenes from rhubarbrdquo Biological andPharmaceutical Bulletin vol 31 no 9 pp 1809ndash1812 2008
[17] M Ozturk F Aydogmus-Ozturk M E Duru and G TopccediluldquoAntioxidant activity of stem and root extracts of Rhubarb(Rheum ribes) An edible medicinal plantrdquo Food Chemistryvol 103 no 2 pp 623ndash630 2007
[18] P Raudsepp D Anton M Roasto et al ldquo-e antioxidativeand antimicrobial properties of the blue honeysuckle (Loni-cera caerulea L) Siberian rhubarb (Rheum rhaponticum L)and some other plants compared to ascorbic acid and sodiumnitriterdquo Food Control vol 31 no 1 pp 129ndash135 2013
[19] C S Shia S H Juang S Y Tsai et al ldquoMetabolism andpharmacokinetics of anthraquinones in Rheum palmatum inrats and ex vivo antioxidant activityrdquo Planta Medica vol 75no 13 pp 1386ndash1392 2009
[20] J P Silveira L N Seito S Eberlin et al ldquoPhotoprotective andantioxidant effects of Rhubarb inhibitory action on tyrosinaseand tyrosine kinase activities and TNF-alpha IL-1alpha andalpha-MSH production in human melanocytesrdquo BMCComplementary and Alternative Medicine vol 13 no 1 p 492013
[21] T Venkatesan M J Jeong Y W Choi E J ParkS K El-Desouky and Y K Kim ldquoDeoxyrhapontigenina natural stilbene derivative isolated from Rheum undulatumL induces endoplasmic reticulum stress-mediated apoptosisin human breast cancer cellsrdquo Integrative Cancer Berapiesvol 15 no 4 pp NP44ndashNP52 2016
[22] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquoMethods inEnzymology vol 299 pp 152ndash178 1999
[23] R Re N Pellegrini A Proteggente A Pannala M Yang andC Rice-Evans ldquoAntioxidant activity applying an improvedABTS radical cation decolorization assayrdquo Free Radical Bi-ology and Medicine vol 26 no 9-10 pp 1231ndash1237 1999
[24] I F Benzie and J J Strain ldquo-e ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo theFRAP assayrdquo Analytical Biochemistry vol 239 no 1pp 70ndash76 1996
[25] C Megıas E Pastor-Cavada C Torres-Fuentes et alldquoChelating antioxidant and antiproliferative activity of Viciasativa polyphenol extractsrdquo European Food Research andTechnology vol 230 no 2 pp 353ndash359 2009
[26] P Ke E Cervantes and C Robles-Martinez ldquoDeterminationof thiobarbituric acid reactive substances (TBARS) in fishtissue by an improved distillationndashspectrophotometricmethodrdquo Journal of the Science of Food and Agriculturevol 35 no 11 pp 1248ndash1254 1984
[27] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983
[28] E Borenfreund and J A Puerner ldquoToxicity determined invitro by morphological alterations and neutral red absorp-tionrdquo Toxicology Letters vol 24 no 2-3 pp 119ndash124 1985
[29] K Nishio M Horie Y Akazawa et al ldquoAttenuation of li-popolysaccharide (LPS)-induced cytotoxicity by tocopherolsand tocotrienolsrdquo Redox Biology vol 1 no 1 pp 97ndash1032013
[30] L Bravo ldquoPolyphenols chemistry dietary sources meta-bolism and nutritional significancerdquo Nutrition Reviewsvol 56 no 11 pp 317ndash333 1998
[31] Y Cai Q Luo M Sun and H Corke ldquoAntioxidant activityand phenolic compounds of 112 traditional Chinese medic-inal plants associated with anticancerrdquo Life Sciences vol 74no 17 pp 2157ndash2184 2004
[32] G Di Carlo N Mascolo A A Izzo and F Capasso ldquoFla-vonoids old and new aspects of a class of natural therapeuticdrugsrdquo Life Sciences vol 65 no 4 pp 337ndash353 1999
[33] F Shahidi and P Ambigaipalan ldquoPhenolics and polyphenolicsin foods beverages and spices antioxidant activity and healtheffectsmdasha reviewrdquo Journal of Functional Foods vol 18pp 820ndash897 2015
[34] M S Brewer ldquoNatural antioxidants sources compoundsmechanisms of action and potential applicationsrdquo Compre-hensive Reviews in Food Science and Food Safety vol 10 no 4pp 221ndash247 2011
[35] B Ou D Huang M Hampsch-Woodill J A Flanagan andE K Deemer ldquoAnalysis of antioxidant activities of commonvegetables employing oxygen radical absorbance capacity(ORAC) and ferric reducing antioxidant power (FRAP) as-says a comparative studyrdquo Journal of Agricultural and FoodChemistry vol 50 no 11 pp 3122ndash3128 2002
[36] R Apak K Guclu B Demirata et al ldquoComparative evalu-ation of various total antioxidant capacity assays applied tophenolic compounds with the CUPRAC assayrdquo Moleculesvol 12 no 7 pp 1496ndash1547 2007
[14] H Matsuda N Tomohiro K Hiraba et al ldquoStudy on Anti-Oketsu Activity of Rhubarb II Anti-allergic effects of stilbenecomponents from Rhei undulati Rhizoma (dried rhizome ofRheum undulatum cultivated in Korea)rdquo Biological andPharmaceutical Bulletin vol 24 no 3 pp 264ndash267 2001a
[15] H Matsuda T Morikawa I Toguchida J Y Park S Harimaand M Yoshikawa ldquoAntioxidant constituents from rhubarbstructural requirements of stilbenes for the activity andstructures of two new anthraquinone glucosidesrdquo Bioorganicand Medicinal Chemistry vol 9 no 1 pp 41ndash50 2001b
[16] T M Ngoc T M Hung P T -uong et al ldquoInhibition ofhuman low density lipoprotein and high density lipoproteinoxidation by oligostilbenes from rhubarbrdquo Biological andPharmaceutical Bulletin vol 31 no 9 pp 1809ndash1812 2008
[17] M Ozturk F Aydogmus-Ozturk M E Duru and G TopccediluldquoAntioxidant activity of stem and root extracts of Rhubarb(Rheum ribes) An edible medicinal plantrdquo Food Chemistryvol 103 no 2 pp 623ndash630 2007
[18] P Raudsepp D Anton M Roasto et al ldquo-e antioxidativeand antimicrobial properties of the blue honeysuckle (Loni-cera caerulea L) Siberian rhubarb (Rheum rhaponticum L)and some other plants compared to ascorbic acid and sodiumnitriterdquo Food Control vol 31 no 1 pp 129ndash135 2013
[19] C S Shia S H Juang S Y Tsai et al ldquoMetabolism andpharmacokinetics of anthraquinones in Rheum palmatum inrats and ex vivo antioxidant activityrdquo Planta Medica vol 75no 13 pp 1386ndash1392 2009
[20] J P Silveira L N Seito S Eberlin et al ldquoPhotoprotective andantioxidant effects of Rhubarb inhibitory action on tyrosinaseand tyrosine kinase activities and TNF-alpha IL-1alpha andalpha-MSH production in human melanocytesrdquo BMCComplementary and Alternative Medicine vol 13 no 1 p 492013
[21] T Venkatesan M J Jeong Y W Choi E J ParkS K El-Desouky and Y K Kim ldquoDeoxyrhapontigenina natural stilbene derivative isolated from Rheum undulatumL induces endoplasmic reticulum stress-mediated apoptosisin human breast cancer cellsrdquo Integrative Cancer Berapiesvol 15 no 4 pp NP44ndashNP52 2016
[22] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquoMethods inEnzymology vol 299 pp 152ndash178 1999
[23] R Re N Pellegrini A Proteggente A Pannala M Yang andC Rice-Evans ldquoAntioxidant activity applying an improvedABTS radical cation decolorization assayrdquo Free Radical Bi-ology and Medicine vol 26 no 9-10 pp 1231ndash1237 1999
[24] I F Benzie and J J Strain ldquo-e ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo theFRAP assayrdquo Analytical Biochemistry vol 239 no 1pp 70ndash76 1996
[25] C Megıas E Pastor-Cavada C Torres-Fuentes et alldquoChelating antioxidant and antiproliferative activity of Viciasativa polyphenol extractsrdquo European Food Research andTechnology vol 230 no 2 pp 353ndash359 2009
[26] P Ke E Cervantes and C Robles-Martinez ldquoDeterminationof thiobarbituric acid reactive substances (TBARS) in fishtissue by an improved distillationndashspectrophotometricmethodrdquo Journal of the Science of Food and Agriculturevol 35 no 11 pp 1248ndash1254 1984
[27] T Mosmann ldquoRapid colorimetric assay for cellular growthand survival application to proliferation and cytotoxicityassaysrdquo Journal of Immunological Methods vol 65 no 1-2pp 55ndash63 1983
[28] E Borenfreund and J A Puerner ldquoToxicity determined invitro by morphological alterations and neutral red absorp-tionrdquo Toxicology Letters vol 24 no 2-3 pp 119ndash124 1985
[29] K Nishio M Horie Y Akazawa et al ldquoAttenuation of li-popolysaccharide (LPS)-induced cytotoxicity by tocopherolsand tocotrienolsrdquo Redox Biology vol 1 no 1 pp 97ndash1032013
[30] L Bravo ldquoPolyphenols chemistry dietary sources meta-bolism and nutritional significancerdquo Nutrition Reviewsvol 56 no 11 pp 317ndash333 1998
[31] Y Cai Q Luo M Sun and H Corke ldquoAntioxidant activityand phenolic compounds of 112 traditional Chinese medic-inal plants associated with anticancerrdquo Life Sciences vol 74no 17 pp 2157ndash2184 2004
[32] G Di Carlo N Mascolo A A Izzo and F Capasso ldquoFla-vonoids old and new aspects of a class of natural therapeuticdrugsrdquo Life Sciences vol 65 no 4 pp 337ndash353 1999
[33] F Shahidi and P Ambigaipalan ldquoPhenolics and polyphenolicsin foods beverages and spices antioxidant activity and healtheffectsmdasha reviewrdquo Journal of Functional Foods vol 18pp 820ndash897 2015
[34] M S Brewer ldquoNatural antioxidants sources compoundsmechanisms of action and potential applicationsrdquo Compre-hensive Reviews in Food Science and Food Safety vol 10 no 4pp 221ndash247 2011
[35] B Ou D Huang M Hampsch-Woodill J A Flanagan andE K Deemer ldquoAnalysis of antioxidant activities of commonvegetables employing oxygen radical absorbance capacity(ORAC) and ferric reducing antioxidant power (FRAP) as-says a comparative studyrdquo Journal of Agricultural and FoodChemistry vol 50 no 11 pp 3122ndash3128 2002
[36] R Apak K Guclu B Demirata et al ldquoComparative evalu-ation of various total antioxidant capacity assays applied tophenolic compounds with the CUPRAC assayrdquo Moleculesvol 12 no 7 pp 1496ndash1547 2007
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