)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/omcl/2015/843237.pdf · by the following method. Brie y, volumes of . mM BHT in HPLC-grade methanol were mixed with

Research ArticleAntioxidant Capacity of lsquolsquoMexican ArnicarsquorsquoHeterotheca inuloides Cass Natural Products and SomeDerivatives Their Anti-Inflammatory Evaluation andEffect on C elegans Life Span

Joseacute Luis Rodriacuteguez-Chaacutevez1 Elvia Coballase-Urrutia2

Antonio Nieto-Camacho1 and Guillermo Delgado-Lamas1

1 Instituto deQuımica UniversidadNacional Autonoma deMexico Circuito Exterior CiudadUniversitaria 04510Mexico DFMexico2Laboratorio de Neuroquımica Instituto Nacional de Pediatrıa Insurgentes Sur 3700-C 04530 Mexico DF Mexico

Correspondence should be addressed to Elvia Coballase-Urrutia elcoballaseyahoocommx and Guillermo Delgado-Lamasdelgadounammx

Received 28 November 2014 Revised 16 January 2015 Accepted 26 January 2015

Academic Editor Vladimir Jakovljevic

Copyright copy 2015 Jose Luis Rodrıguez-Chavez et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

It has been suggested that the accumulation of biomolecular damage caused by reactive oxygen species (ROS) contributes to agingThe antioxidant activity is related to the ability of certain compounds to protect against the potentially harmful effect of processesor reactions involving ROS This ability is associated with the termination of free radical propagation in biological systems FromHeterotheca inuloides various compounds which have shown to possess antioxidant capacity and scavenging ROS The aim of thisstudywas to determine the antioxidant capacity of additional natural components isolated fromH inuloides and some semisyntheticderivatives their anti-inflammatory activity and the effect on Caenorhabditis elegans nematode life span Compounds showedability to inhibit various biological processes such as lipid peroxidation scavenge nonbiological important oxidants such as 1O

2

OH∙ H2O2 and HOCl and scavenge non biological stable free radicals (DPPH) Some cadinane type compounds showed possess

antioxidant ROS scavenging capacity anti-inflammatory activity and effect on the C elegans life span Flavonoid type compoundsincreased the life of the nematode and quercetin was identified as the compound with the greatest activity The modification ofchemical structure led to a change in the antioxidant capacity the anti-inflammatory activity and the survival of the worm

1 Introduction

Reactive oxygen species (ROS) exist as products of normalcellular physiology and play vital roles in the stimulationof signaling pathways in plant and animal cells [1] Aerobicorganisms produce ROS during the reduction of molecularoxygen by mitochondria [2] ROS include free radicals suchas superoxide anion (O

2

minus) hydroxyl radical (OH∙) andnonradical molecules like hydrogen peroxide (H

2O2) singlet

oxygen (1O2) and other species such as nitric oxide (NO)

hypochlorous acid (HOCl) and peroxynitrite (ONOOminus)When the level of ROS exceeds away cellular factors respon-sible for protecting cellular biomolecules against damagegenerated by oxidizing species is said to be in a state of

ldquooxidative stressrdquo Under these conditions ROS can damagebiomolecules like nucleic acids proteins lipids carbohy-drates and enzymes This condition has been implicated inthe pathogenesis of a number of multiple pathologies suchas senescence [3] ischemiareperfusion injury [4] neurode-generative diseases [5] infectious processes [6] rheumatoidarthritis [7] arterial diseases [8] obesity diabetes chronickidney disease [9] and other ailments

Heterotheca inuloides is commonly known as ldquoMexicanarnicardquo and it is known by other names in different regions ofMexico [10 11] InMexican traditionalmedicine the infusionsof this plant are mainly used for treatment of contusionsand bruises [12] Phytochemical studies of this plant allowedisolating different classes of compounds mainly cadinane

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 843237 11 pageshttpdxdoiorg1011552015843237

2 Oxidative Medicine and Cellular Longevity

type sesquiterpenes [13] flavonoids [14] and phytosterols[15] Previous studies have reported that metanolic extractand certain natural products isolated fromH inuloides driedflowers have antioxidant activity and ability to inhibit lipidperoxidation scavenging ROS and act as hepatoprotectiveagents [16ndash18] However the antioxidant activity of naturalproducts isolated from the acetone extract has not beenreported In the present study we evaluated the antioxidantand ROS scavenging capacity of H inuloides metabolitesisolated from the acetonic extract and prepared semisyntheticderivatives and also evaluated their anti-inflammatory effectsand effect on the C elegans life span Here we report theresults considering the structure-activity relationships

2 Materials and Methods

21 Reagents All reagents used were of analytical gradeSodium pyruvate dimethyl thiourea (DMTU) nordihydro-guaiaretic acid (NDGA) ascorbic acid histidine xylenolorange (FOX) 22-diphenyl-1-picrylhydrazyl (DPPH) dim-ethylsulfoxide (DMSO) NN-dimethyl-4-nitrosoaniline(DMNA) catalase xanthine xanthine oxidase nitroblue tet-razolium (NBT) dL-penicillamine 2-thiobarbituric acid(TBA) 120572-tocopherol Folin and Ciocalteursquos phenol reagent35-di-tert-4-butylhydroxytoluene (BHT) and 5-fluoro-21015840-deoxyuridine were purchased from Sigma-Aldrich (TolucaMexico or Sigma St Louis MO) Absolute ethanolhydrogen peroxide (H

2O2) sulfuric acid (H

2SO4) methanol

ethylenediaminetetraacetic acid disodium salt (EDTA)NaCl KH

2PO4 NaHPO

4sdot7H2O NaOH and H

2SO4were

purchased from Merck-Mexico Sodium hypochlorite(NaOCl) was purchased from Hycel (Mexico City Mexico)

22 Strains of C elegans Experimental C elegans strain waswild type N2 (Bristol) The strain was obtained from theCaenorhabditis Genetics Center (University of MinnesotaMinneapolis MN USA) and was maintained on nematodegrowth medium (NGM) at 20∘C as described previously byBrenner [19] Age-synchronized worms were generated inall experiments through the sodium hypochlorite methodWorms were allowed to hatch in Petri dishes in liquid S-medium with concentrated Escherichia coli OP50 as food[20]

23 Plant Material Flowers of H inuloides (provided byMIXIM Laboratories Naucalpan Mexico) were collected in2010 in the town of Mesas Altas de San Juan Xoconuscomunicipality of Donato Guerra (State of Mexico) andauthenticated by MS Abigail Aguilar-Contreras A voucherof plant material was deposited under code IMSSM-16064 inthe Medicinal Plant Herbarium of the Instituto Mexicano delSeguro Social (IMSS Mexico City)

24 Heterotheca inuloides Metabolites and Derivatives Com-pounds 1ndash12 were isolated from the acetone extract ofdried flowers of H inuloides in a previous study Semisyn-thetic compounds 7-acetoxy-34-dihydrocadalene (13) 7-benzoxy-34-dihydrocadalene (14) 7-acetoxycadalene (15)7-benzoxycadalene (16) quercetin pentaacetate (17) and

7-hydroxy calamenene (18) were obtained by conven-tional chemical procedures as was previously described [21](Figure 1) Due to the paucity of material compounds 10 and11 were not included in all the bioassays

25 Antioxidant Effects and ROS Scavenging ofH inuloides Metabolites

251 Estimation of Lipid Peroxidation Lipid peroxidationwas measured by TBARS assay using rat brain homogenates[22] with some modifications Adult male Wistar rats (200ndash250 g) were provided by the Instituto de Fisiologıa CelularUNAM and their use was approved by the Animal Careand Use Committee [23] The animals sacrifice was carriedout avoiding unnecessary pain with CO

2 cerebral tissue

(whole brain) was rapidly dissected and homogenized Thehomogenate was centrifuged for 10min at 3400 rpm to yielda pellet that was discarded protein content in the supernatantwas measured using the Folin and Ciocalteursquos phenol reagent[24] and adjusted to 266mg proteinmL with PBS Thesupernatant (375120583L) was incubated at 37∘C for 30min inpresence of test sample (25120583L) dissolved inDMSOor ethanoland 50 120583L of EDTA solution (20120583M) Lipid peroxidationwas started adding 50 120583L of a freshly prepared 100 120583MFeSO4solution (final concentration 10 120583M) and incubated

at 37∘C for 60min TBARS were determined as described byOhkawa et al [25]with somemodifications Concentration ofTBARSwas calculated by interpolation in a standard curve oftetrametoxipropane (TMP) [26] Final results were expressedas mmoles of TBARS per mg of protein The inhibition ratio() was calculated using the following formula

Inhibition ratio () = (119862 minus 119864)119862

times 100 (1)

where119862was the absorbance of control and 119864was the absorb-ance of the test sample

252 DPPH∙ Scavenging Capacity DPPH assay was assessedin 96-well microtiter plates according to the method ofBlois [27] with minor changes Briefly 005mL of test com-pounds in DMSO at different concentrations was added toan ethanolic solution of 22-diphenylpicrylhydrazyl (DPPH∙)(13333 120583M 0150mL)The control sample contained distilledwater Reactionmixtures were incubated at 37∘C for 30min inthe dark After incubation the absorbance was measured at515 nm in a microplate reader ELx808 (BioTek InstrumentsInc Vermont USA) The inhibition of each compoundwas determined by comparison with a DPPH ethanol blanksolution [28]The scavenging capacity is given as percent ()DPPH scavenged calculated as [(optical density of control minusoptical density of compound)(optical density of control) times100] 120572-tocopherol was used as standard

253 Superoxide Radical-Scavenging Activity The superox-ide radicals (O

2

minus) were generated through the system xan-thine oxidase [29] O

2

minus in the assay system and xanthineoxidase activity were measured as NBT reduction usinga DU-640 series Beckman spectrophotometer 800120583L of

Oxidative Medicine and Cellular Longevity 3

OHHO

H

12

11

HOOH

HO

HO

OHOH

10

HO

OHOOC

OHRO RO

3 5

OH

4

HO H

H H

R1 R2 R3 R4 R56 H H H H H7 H H8 H17 AcAc Ac Ac Ac

O

O

1 13 14R H Ac Bz

O

OOH

OH

9

HO

18

2 15 16R H Ac Bz

12

345

6

7 8 12

345

6

7 8

12

3456

78

A

B

CO OO

CH3CH3CH3

CH3CH3 CH3

CH3

R1O

OR4

OR5

OR3

OR2

H3CO

OCH3

Figure 1 Natural products isolated from H inuloides flowers and derivatives

the following reaction mixtures 90 120583M xanthine 16mMNa2CO3 228120583M NBT and 18mM phosphate buffer (pH

70) was mixed with 100 120583L of different concentrations ofH inuloides metabolites The reaction was started by theaddition of 100120583L of xanthine oxidase (168UL) Opticaldensity was registered both at 295 (for uric acid production)and at 560 nm (for O

2

minus in the assay system) The absorbancewas determined using a BeckmanDU640 Spectrophotometer(Beckman Coulter Inc California USA)

254 Peroxide Scavenging Activity Determination of scav-enging capacity was made by ferrous ion oxidation-xylenolorange (FOX) assay [30] Concentration of H

2O2was cal-

culated from a standard curve prepared with increasingH2O2concentrations A solution of 75120583M H

2O2was mixed

(1 1 vv) with water (0 scavenging tube) or with differentconcentrations of H inuloidesmetabolites and incubated for30min at room temperature After this H

2O2was measured

by the following method Briefly 9 volumes of 44mMBHT in HPLC-grade methanol were mixed with 1 volumeof 1mM xylenol orange and 256mM ammonium ferroussulphate in 025M H

2SO4to give the working FOX reagent

45 120583L of the metabolite solutions and 45 120583L of 75 120583M H2O2

were dispensed in 15mL Eppendorf tubes and mixed with10 120583L of HPLC-grade methanol immediately followed bythe addition of 09mL of FOX reagent mixed on a vortexmixer for 5 s and then incubated at room temperature for10min The tubes were centrifuged for 15000 g for 10minand absorbance at 560 nm was read against a methanol

blank using a Beckman DU640 Spectrophotometer (Beck-man Coulter Inc California USA) The concentration ofH2O2was calculated from a standard curve prepared with

increasing H2O2concentrations Sodium pyruvate was used

as standard for H2O2scavenging activity

255 Hydroxyl Radical-Scavenging Activity The malondi-aldehyde formed from the decay of deoxyribose was eval-uated in reaction with thiobarbituric acid and measuredat 532 nm [31] The reaction mixture containing deoxyri-bose (0056mM) H

2O2(1mM) potassium phosphate buffer

(10mM pH 74) FeCl3(02mM) EDTA (02mM) ascorbic

acid (02mM) and 100 120583L of different concentrations of Hinuloides was incubated in a water bath at 37 plusmn 05∘C for 1 hThe extent of the deoxyribose degradation by theOH∙ formedwas measured by the thiobarbituric acid test at 532 nm usinga Beckman DU640 Spectrophotometer (Beckman CoulterInc California USA) The ability of H inuloidesmetabolitesto scavenge OH∙ was compared with that of DMTU (0 12 10 20 and 31 120583gmL) The final results were expressed asinhibition percent in relation to a control test (without thesample)

256 Singlet Oxygen (1O2) Scavenging Activity The ability of

the compounds to scavenging 1O2was detected spectropho-

tometrically at 440 nm using the bleaching of NN-dimethylp-nitrosoaniline (DMNA) as a specific detector [32] Theassay mixture contained 45mM Na-phosphate buffer (pH


71) 10mM histidine 10mM NaOCl 10mM H2O2 50120583M

DMNA and 01mL of different concentration of compoundsThe total volume of reaction (20mL) was incubated at 30∘Cfor 40min The extent of 1O

2production was determined

by measuring the decrease in the absorbance of DMNA at440 nm using a Beckman DU640 Spectrophotometer (Beck-man Coulter Inc California USA) The relative scavengingefficiency ( inhibition production of 1O

2) of H inuloides

metabolites was estimated from the difference in absorbanceof DMNAwith andwithout the addition of compounds beingtested or reference compound Glutathione (0 092 153215 245 and 307mgmL) was used as standard for 1O

2

scavenging

257 HOCl Scavenging Assay TheHOCl scavenging activitywas evaluated by measuring the decrease in absorbance ofcatalase at 404 nm and was carried out as described byAruoma and Halliwell [33] with minor changes Briefly150 120583L of 498 120583M bovine liver catalase solution (166 120583Mfinal concentration) was mixed with 150120583L of 18mM HOCl(6mM final concentration) and 150 120583L of H inuloidesmetabolite solutions in increasing concentrations or the ref-erence compound ascorbic acid (0 22 44 88 and 176 120583gmL)Spectra (370ndash450 nm) of catalase alone catalase plus HOClor catalase plus HOCl and H inuloides metabolites or thereference compound were registered and the optical den-sities (OD) at 404 nm were determined using a BeckmanDU640 Spectrophotometer (Beckman Coulter Inc Califor-nia USA) The value of the OD of catalase alone minusthe OD of catalase plus HOCl was considered as 100 ofdegradation of catalase (or 0of scavenging activity) and thedifference of the catalase alone minus the OD of the catalaseplus HOCl in presence of either H inuloides metabolites orreference compound was compared against this value Theability of TPM to scavenge HOCl was compared with that ofascorbic acid

26 Evaluation of Anti-Inflammatory Effect The biologicalmodel employed was ear edema in mice induced by 13-ethyl-12-O-tetradecanoylphorbol (TPA) as described by Raoet al [34] with slight modifications CD1 male mice wereused Mice were administered with sodium pentobarbital(315mgkg ip) and TPA solution (25 120583gear) dissolved inethanol (10 120583L) This was done topically on the right earin both faces of the ear The left ear received only ethanol10min after the test substances (1 120583molear) or indomethacin(031 120583molear) as drug reference The tested substancesand indomethacin were dissolved in 20120583L of acetone andadministered on both sides of the ears (10 120583Lside) Thecontrol only received the vehicle (20120583L of acetone) Fourhours later the mice were killed with CO

2 A 7mm diam-

eter plug was removed from each ear The swelling wasassessed as the difference in weight between right and leftear plugs The anti-inflammatory activity was expressed asinhibition of edema (IE) in percent relative to the edemaformed in control animals according with the followingformula IE () = 100 minus [B times 100A] where A = edemainduced by TPA alone and B = edema induced by TPA plussample

27 Lifespan Assay Lifespan assays were assessed in liq-uid medium at 20∘C in 96-well plates (Corning NY)and were carried out according to the established pro-tocols [35] Briefly the nematodes were age-synchronizedand distributed in wells as L1 larvae (10ndash20 animals perwell) together with Escherichia coli OP50 To prevent self-fertilization 5-fluoro-21015840-deoxyuridine was added 36 h afterseeding (012mM final) Media were supplemented with dif-ferent doses of compounds (100 or 200120583M) DMSOethanol(5050 vv) was included as a solvent control The wormswere monitored daily to observe the number of live wormsin each treatment the fraction of animals alive was scored onthe basis of body movement Observations and worm countswere performed using a microscope Eclipse TS 100 (NikonInstruments Inc Tokyo Japan)

28 Statistical Analysis For lipid peroxidation and DPPHscavenging data were represented as mean plusmn standard errorof mean (SEM) Data were analyzed by one-way ANOVAfollowed by Dunnettrsquos test for comparisons against controlValues of 119875 le 005 (lowast) and 119875 le 001 (lowastlowast) were con-sidered statistically significant The inhibitory concentration50 (IC

50) was estimated by means of a linear regression

equation For scavenging capacity data were expressed asmean plusmn SEMThe data were compared against the blank tubewithoutH inuloidesmetabolites or the reference compoundsusing one-way analysis of variance (ANOVA) followed bythe Dunnettrsquos Multiple Comparison test (GraphPad Prism40 Software San Diego CA USA) 119875 lt 005 was con-sidered statistically significant The scavenging capacity wasexpressed as the 50 inhibitory concentration (IC

50) value

which denotes the concentration of H inuloides metabolitesor the reference compounds required to give a 50 reductionin oxidating effect relative to the blank tube The data of thelifespan assays were processed using the GraphPad Prism40 Software (GraphPad Software Inc San Diego CAUSA) Survival was plotted by the Kaplan-Meier methodand the curves compared for significance using the log-ranktest

3 Results

31 Antioxidant Effects and ROS Scavenging Our resultsshowed that only compounds 2 6 and 9 displayed the abilityto reduce DPPH radical with a IC

50of 3066 plusmn 814 120583M

1311 plusmn 12 120583M and 697 plusmn 014 120583M Compounds 1 and 7showed slight activity with IC

50higher than 100 120583M The

other compounds tested showed a rate of antioxidant capacityless than 25 during the preliminary screening and thereforewere not considered active (Table 1)

In peroxidation assay some compounds showed abilityto inhibit this process The inhibitory capacity of thesecompounds decreased in the following order 18 gt 13 gt 2 gt17 gt 6 gt 1 gt 7 gt 9 gt 11 Compound 18 had an IC

50of 058 plusmn

0008 120583Mand showed to possess greater ability to inhibit lipidperoxidation than 120572-tocopherol used as reference substancewhose IC

50was 678 plusmn 216 120583M The derivatives 14 15 and

16 showed little ability to inhibit lipid peroxidation with IC50

higher than 100 120583M


Table 1 Capacity to inhibit lipid peroxidation and to reduce theDPPH radical

Compound IC50 120583MLipid peroxidation Reduction of DPPH

1 472 plusmn 011 gt1002 309 plusmn 019 3066 plusmn 8143 ndlowast nd4 nd nd6 430 plusmn 027 1311 plusmn 127 616 plusmn 006 gt1008 nd nd9 1446 plusmn 061 697 plusmn 01411 1735 plusmn 140 nd12 nd mdash13 182 plusmn 012 nd14 gt100 nd15 gt100 nd16 gt100 nd17 367 plusmn 043 mdash18 058 plusmn 0008 nd120572-Tocopherol 678 plusmn 216 3174 plusmn 1lowastNot determined at the concentrations tested the activity of compoundswas low

The H inuloides natural products derivatives as well asthe reference compounds displayed O

2

minus HOCl H2O2 1O2

and OH∙ scavenging activity in a concentration-dependentway The IC

50values were calculated from the dose-response

curve (Table 2 Figure 2)The hypochlorous acid scavenging capability decreases

in the following order 9 gt 7 gt 2 gt 13 gt 15 gt 8 gt 3 gt 4Compound 9 showed to possess the higher scavenging abilityagainst HOCl Compounds 13 and 15 acetylated derivativesof compounds 1 and 2 displayed a reduction in radicalscavenging ability with an increase in the IC

50value The

standard ascorbic acid showed an IC50

value of 2786 plusmn167 120583M

In the case of hydroxyl radical the capacity to scavengingdecreased in the following order 2 gt 9 gt 7 gt 8 gt 15 gt 13 gt 3 gt4 The IC

50value for the dimethylthiourea used as reference

substance was 1315plusmn36 120583Mand was lower than those of thecompounds tested

H inuloides metabolites were capable of scavenginghydrogen peroxide in an amount-dependent manner Resultsshowed that the scavenging activity values decreased in theorder of 9 gt 7 gt 8 gt 2 gt 13 gt 3 gt 15 gt 4 Compound 9 showedthe best scavenging ability with an IC

50of 154 plusmn 63 120583M

however none of the compounds showed higher activity thanthe pyruvate used as reference substance whose IC

50was

322 plusmn 17 120583MThe superoxide scavenger capacity decreased in the fol-

lowing order 9 gt 7 gt 8 gt 3 gt 2 gt 4 gt 13 gt 15 Compound 9showed to possess the best ability to trap superoxide radicalwith IC

50value of 122plusmn022 120583Mhowever it is not statistically

different from NDGA reference compound whose IC50

was116 plusmn 013 120583M

With respect to the singlet oxygen scavenger capacity theability of the compounds to trap the singlet oxygen decreasedin the following order 4 gt 7 gt 8 gt 9 gt 15 gt 3 gt 13 gt 2The calamenoic acid (4) showed comparatively better activitythan the GHS The IC

50values (Table 2) of calamenoic acid

and GHS were 1124 plusmn 293 120583M y 471 plusmn 77 120583M

32 Evaluation of Anti-Inflammatory Effect The results showthat only the compounds 2 4 and 16 displayed a significantanti-inflammatory activity but this was less than that ofindomethacin used as reference drug The rest of the com-pounds showed a percentage of inhibition lower than 50(Table 3)

33 Life Span Assay Exposure of C elegans to compoundsgave the following results at 100 120583M flavonoids type com-pounds extended half-life compared to the control (Table 4)Significant differences were observed for treatments com-pared with the vehicle-treated control (119875 lt 0001) The sur-vival curve exhibited significant difference among worms fedwith flavonoid compounds There was not an increase inlifespan of the worm in the presence of cadinane compoundsand some of them decreased survival as shown in Figure 3

4 Discussion

H inuloides metabolites showed to possess the ability toinhibit lipid peroxidation and scavenging free radicals andROS In lipid peroxidation assaywe observed that the replace-ment of H at the hydroxyl in position 7 of sesquiterpenes1 and 2 led to a decrease in the activity It is further notedthat the increase in the unsaturation of cadinanes led to adecrease in their activity which is observed for compound18 whose activity is greater than 1 that is greater than 2In the case of flavonoid type compounds 6 (quercetin)had the best ability to inhibit lipid peroxidation naturalmethoxy derivatives 7 and 8 showed lower activity Theseresults are consistent with those reported that indicate thatmethoxy substituent perturbs the planarity due to sterichindrance imparted by the methyl group [36] It is reportedthat the antioxidant activity of flavonoids is proportional tothe number of hydroxyl groups present in the molecule [37]Compound 6 (quercetin) having a 3573101584041015840-pentahydroxygroup showedDPPHradical scavenging activity Compounds1 and 2 showed a statistically similar activity to 6 and higherthan that of 120572-tocopherol We noted that several testedcompounds had the ability to inhibit lipid peroxidationhowever few of them reduced the DPPH radical The aboveobservationsmay be explained because the DPPH radical hasa long life and no resemblance to the highly reactive peroxylradicals involved in lipid peroxidation and some antioxidantsthat react quickly with peroxyl radicals react slowly or areinert to DPPH [38]

Hypochlorous acid radicals have the ability to inactivatethe antioxidant enzyme catalase through degrade its hemegroup [33] Catalase inactivation is inhibited in the presenceof some H inuloides compounds The results showed thatcompounds are more efficient scavenger than the standardascorbic acid Among the flavonoid type compounds 9


Superoxide

2 3 4 7 8 9 13 15 C0

100

200

300

Compound2 3 4 7 8 9 13 15 C

Compound

2 3 4 7 8 9 13 15 CCompound

2 3 4 7 8 9 13 15 CCompound

2 3 4 7 8 9 13 15 CCompound

0

200

400

600

0

200

400

600

800

0

200

400

600

800

1000

HOCl

0

50

100

150

200

250

300

350

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

H2O2

OH∙minus1O2

CI50

(120583M

)

CI50

(120583M

)

CI50

(120583M

)CI

50

(120583M

)

CI50

(120583M

)

Figure 2 ROS scavenging capacity of H inuloides metabolites and semisynthetic derivatives C=NDGA (superoxide) pyruvate (H2O2)

DMTU (OHminus) GSH (1O2) ascorbic Ac acid (HOCl) The results were analyzed by ANOVA The Dunnettrsquos Multiple Comparison test was

used to compare outcomes between experimental and control group lowast119875 lt 005

showed the highest ability to scavenge hypochlorous acidwhile 7 and 8 showed lower activity In the last two com-pounds the hydroxyl at position 3 present in the quercetinmolecule has been replaced by a methoxy group It has beenreported that the hydroxyl at position 3 of the C ring ofquercetin plays an important role in the antioxidant activityincluding scavenging hypochlorite [39]

Hydroxyl free radicals cause damage to oxidative cellstrough damage DNA lipids and proteins [31]The flavonoidsscavenge hydroxyl (OH∙) radicals Hydroxyl radical (OH∙)generated by the Fenton reaction system was evaluated byTBARS assayWe observed that compound 9wasmore activethan 7 which in turn was more active than 8 with onlyone hydroxyl group The results obtained are consistent with


Table 2 Scavenging capacity of H inuloides metabolites on hypochlorous acid hydroxyl radical peroxide superoxide anion and singletoxygen (IC50)

Compound Scavenging capacity IC50 120583MHOCl OH∙ H2O2 O2

minus 1O2

2 691 plusmn 88lowast 1334 plusmn 326 270 plusmn 27lowast 1146 plusmn 139lowast 8804 plusmn 554lowast

3 2023 plusmn 219lowast 5454 plusmn 230lowast 422 plusmn 80lowast 105 plusmn 72lowast 5389 plusmn 5864 2035 plusmn 239lowast 6079 plusmn 335lowast 536 plusmn 14lowast 148 plusmn 130lowast 1124 plusmn 293lowast



9 600 plusmn 52lowast 1395 plusmn 11 154 plusmn 63lowast 122 plusmn 022 3144 plusmn 15813 1061 plusmn 44lowast 5238 plusmn 204lowast 322 plusmn 210lowast 1815 plusmn 298lowast 697 plusmn 1870lowast

15 1100 plusmn 158lowast 43578 plusmn 820lowast 4345 plusmn 233lowast 2441 plusmn 780lowast 4786 plusmn 447lowast

Reference compound Ascorbic acid DMTU Pyruvate NDGA GSH2786 plusmn 167 1315 plusmn 36 322 plusmn 17 116 plusmn 013 471 plusmn 77

Data represent the mean plusmn SE of the three independent assays The results were analyzed by ANOVA The Dunnetrsquos multiple comparison test was used tocompare outcomes between experimental and control group lowast119875 lt 005 versus reference compound

Table 3 Anti-inflammatory activities of additional H inuloidesmetabolites

Compound (1 120583molear) Edema (mg average SE) Inhibition

Negative control 1207 mdash1 1050 plusmn 099 5412 185 plusmn 052 8333lowastlowast

4 527 plusmn 070 6573lowastlowast

5 980 plusmn 036 7987 730 plusmn 115 3886lowastlowast


13 1187 plusmn 066 minus114214 1107 plusmn 115 110615 1360 plusmn 070 minus139016 373 plusmn 162 6644lowastlowast

17 907 plusmn 070 194618 1317 plusmn 051 1357Indomethacin (031 120583mol) 6190lowastlowast

The data represent the mean of three animals plusmn the standard error of themean All the compounds were tested at 1120583molear doses The results wereanalyzed byANOVA Studentrsquos 119905-test was used to compare outcomes betweenexperimental and control groups lowast119875 lt 005 versus reference drug

reports indicating that the scavenging activity of flavonoidsincreases with the number of free hydroxyl groups and it isindependent of the presence of double bond between C-2 andC-3 of ring C [40]

In biological systems H2O2produces hydroxyl ions and

hydroxyl radicals by reacting with Fe2+ and Cu2+ ions andthey have been related to the initiation of many toxic effects[41] It is therefore biologically advantageous for cells tocontrol the accumulation of H

2O2 H inuloides metabolites

showed ability to trap peroxide however they were no betterthan pyruvate

The superoxide radical is a highly reactive species andnot readily diffuses through the cell Because the mainproduction site of O

2

minus is the inner mitochondrial membrane

Table 4 Summary of Caenorhabditis elegans life span

Treatment Adult life span 20∘C (119899) 119875 versus controlMean CI 95 (Log-rank)

1 1 0171ndash1962 449 lt000012 1 0169ndash1964 479 lt000013 13661 1313ndash14209 513 lt000014 14391 13776ndash15006 557 011756 192 1841ndash1982 444 lt000017 16991 16387ndash17596 470 lt000018 16583 15926ndash17239 575 lt000019 1774 1704ndash1844 466 lt0000113 11012 10414ndash11609 434 0008014 13424 12845ndash14003 531 0000115 11659 11103ndash12215 499 0015016 13520 12917ndash14123 496 0000117 15216 14478ndash15955 499 01751Control 14943 14471ndash15414 1098

it has been proposed that mitochondrial DNA (mtDNA) isthe main target of damage [42] We noted that compounds9 and 7 have the capacity to scavenging superoxide It hasbeen reported that the superoxide scavenging capacity offlavonoids is dependent on the number of hydroxyl groupsin the B ring on the presence of a free hydroxyl group at C3on the presence of a saturated C2-C3 bond and the absenceof a C4 carbonyl group [43] Also it has been reported thatthe number of conjugated double bonds and the presence ofconjugated keto groups increase the quenching rate whilethe presence of a hydroxyl epoxy and methoxy group haslesser effects [44] Cadinane type compound 4 and flavonoid9 showed the best ability to scavenge the singlet oxygen 1O

2

other flavonoid type compounds showed lesser activity The1O2scavenger activity of flavonoids is associated with the

presence of a catechol moiety on ring B and with the presenceof a hydroxyl group activating the double bond onC ring [45]The cadinane type sesquiterpenes showed capacity to trap


0 4 8 12 16 20 24 28 320

20

40

60

80

100

9678

15Control

Time (days)

Surv

ival

()

(a)

Control

0 4 8 12 16 20 24 28 32Time (days)

Surv

ival

()

0

20

40

60

80

100

113

14

(b)

Control

Surv

ival

()

0 4 8 12 16 20 24 28 320

20

40

60

80

100

215

16

Time (days)

(c)

Surv

ival

()

Control

34

0 4 8 12 16 20 24 28 320

20

40

60

80

100

Time (days)

(d)

Figure 3 Lifespan survival curves in the presenceH inuloidesmetabolites Labels denote the compound used (100 120583M) Curve (a) flavonoidscurves (b) (c) and (d) natural and semisynthetic cadinane type compounds

singlet oxygen greater than glutathione In another study theability of 7-hydroxy-34-dihydrocadalene to trap this specieshas been reported [46]

Preparations from H inuloides are used as anti-inflammatory and analgesic agents in Mexican traditionalmedicine and these properties have been investigated inseveral studies [13 47 48] The results of anti-inflammatoryassay activity showed that acetylation of compounds 1 and2 to the corresponding derivatives 13 and 15 respectivelygenerated a change in biological response since thesederivatives exhibited proinflammatory activity Howeverthe benzoyl derivatives 14 and 16 (obtained from 1 and 2)

resulted in opposite effects Compound 14 increased the anti-inflammatory activity with respect to 1 while compound 16displayed less activity in comparison with 2 It has previouslybeen reported that quercetin (6) showed anti-inflammatorycapacity in the mice ear edema test [13] in this study itwas observed that naturally methylated derivative 7 andsemisynthetic acetylated derivative 17 displayed little activityin this assay (see Table 3)

We evaluated the effect of some natural products isolatedfrom H inuloides and derivatives in extension of life of Celegans nematode (Figure 3) Treatment of C elegans withsome H inuloides metabolites prolonged the lifespan of the


worm and the best effects were obtained with flavonoid typecompounds The results are consistent with other studiesconducted wherein it has been reported that certain com-pounds of flavonoid type increase lifespan of C eleganswhich protect against oxidative stress and cause an increasein the translocation of the transcription factor DAF-16 [49]In another study it was observed that blueberry polyphenolsincreased lifespan and slowed aging related decline in Celegans but these benefits did not just reflect antioxidantactivity [50] Similarly results ofWilson et al [51] showed thatthe prolongevity effect of myricetin is dependent on DAF-16 and not on direct antioxidative effects of the flavonoidBuchter et al [52] observed that quercetin significantlyincreased reproductive capacity ofC elegans and enlarged thebody size whereas no modification of these characteristicswas induced by their methylated derivatives isorhamnetinand tamarixetin Certain cadinane type compounds isolatedfrom H inuloides have shown to possess antioxidant activitybut its antioxidant capacity was not reflected in an increasein the life span of C elegans Although several compoundsisolated from H inuloides have antiantigiardial activity [21]and it has been found that structurally similar compoundsshowed activity against Leishmania chagasi promastigotes[53]

5 Conclusions

In the present paper we have shown that H inuloidesmetabolites in vitro scavenged to O

2

minus HOCl H2O2 OH∙

and 1O2in a concentration-dependent way The IC

50in

some cases were comparable to the reference compoundThese observations suggest that metabolites of H inuloideshave the ability to capture free radicals The results of theanti-inflammatory assay of the natural products and somederivatives showed both anti- and proinflammatory effectsdepending on the functionalization of the OHminus groups Wehave found that antioxidant capacities are not predictive ofC elegans lifespan benefits Even where a life span exten-sion and antioxidant effect were observed it appears thatother factors are also likely to be involved in modulatinglifespan

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

Jose Luis Rodrıguez-Chavez thanks the Consejo Nacionalde Ciencia y Tecnologıa (Mexico) for providing a graduatescholarship The authors thank B Quiroz R Patino M IChavez A Pena E Huerta R Gavino L Velasco J PerezInstituto de Quımica UNAM and D Ortega CuellarInstituto Nacional de Pediatrıa for technical assistance Theauthors also acknowledge UNAM-DGAPA-PAPIIT (ProjectIG200514) for support This paper is dedicated to DrBernardino Huerta-Gertrudis (In memoriam) InstitutoNacional de Pediatrıa Mexico

References

[1] T Jabs ldquoReactive oxygen intermediates as mediators of pro-grammed cell death in plants and animalsrdquo Biochemical Phar-macology vol 57 no 3 pp 231ndash245 1999

[2] P Venditti L Di Stefano and S Di Meo ldquoMitochondrial me-tabolism of reactive oxygen speciesrdquoMitochondrion vol 13 no2 pp 71ndash82 2013

[3] J Dasgupta S Kar R Liu et al ldquoReactive oxygen species controlsenescence-associated matrix metalloproteinase-1 through c-Jun-N-terminal kinaserdquo Journal of Cellular Physiology vol 225no 1 pp 52ndash62 2010

[4] T Koyama M Tawa N Yamagishi et al ldquoRole of super-oxide production in post-ischemic cardiac dysfunction andnorepinephrine overflow in rat heartsrdquo European Journal ofPharmacology vol 711 no 1ndash3 pp 36ndash41 2013

[5] H Xie S Hou J JiangM Sekutowicz J Kelly and B J BacskaildquoRapid cell death is preceded by amyloid plaque-mediatedoxidative stressrdquoProceedings of theNational Academy of Sciencesof the United States of America vol 110 no 19 pp 7904ndash79092013

[6] M Pohanka ldquoRole of oxidative stress in infectious diseases Areviewrdquo Folia Microbiologica vol 58 no 6 pp 503ndash513 2013

[7] S Kundu P Ghosh S Datta A Ghosh S Chattopadhyayand M Chatterjee ldquoOxidative stress as a potential biomarkerfor determining disease activity in patients with RheumatoidArthritisrdquo Free Radical Research vol 46 no 12 pp 1482ndash14892012

[8] C Marın E M Yubero-Serrano J Lopez-Miranda and FPerez-Jimenez ldquoEndothelial aging associated with oxidativestress can be modulated by a healthy Mediterranean dietrdquoInternational Journal of Molecular Sciences vol 14 no 5 pp8869ndash8889 2013

[9] S R Khan ldquoIs oxidative stress a link between nephrolithiasisand obesity hypertension diabetes chronic kidney diseasemetabolic syndromerdquo Urological Research vol 40 no 2 pp95ndash112 2012

[10] J L Dıaz Indice y Sinonimia de las Plantas Medicinales deMexico Monografıas Cientıficas I IMEPLAM Mexico CityMexico 1976

[11] A Argueta L Cano and M E Rodarte Atlas de las plantasmedicinales de la medicina tradicional mexicana vol 1ndash3Instituto Nacional Indigenista Mexico City Mexico 1994

[12] X Lozoya A Aguilar and R Camacho ldquoEncuesta sobre el usoactual de plantas en la Medicina Tradicional Mexicanardquo RevistaMedica del InstitutoMexicano del Seguro Social vol 25 pp 283ndash291 1987

[13] G Delgado M Del Socorro Olivares M I Chavez et alldquoAntiinflammatory constituents from Heterotheca inuloidesrdquoJournal of Natural Products vol 64 no 7 pp 861ndash864 2001

[14] C Jerga I Merfort and G Willuhn ldquoFlavonoidaglyka aus denbluten von Heterotheca inuloidesrdquo Planta Medica vol 56 no 1pp 122ndash123 1990

[15] G Willuhn and R Schneider ldquoSesquiterpene Triterpene undSterine aus Bluten von Heterotheca inuloides (lsquoMexikanischeArnikablutenrsquo)rdquo Archiv der Pharmazie vol 320 no 5 pp 393ndash396 1987

[16] I Kubo S K Chaudhuri Y Kubo et al ldquoCytotoxic and antiox-idative sesquiterpenoids from Heterotheca inuloidesrdquo PlantaMedica vol 62 no 5 pp 427ndash430 1996

[17] H Haraguchi T Saito H Ishikawa Y Sanchez T Ogura andI Kubo ldquoInhibition of lipid peroxidation by sesquiterpenoid in


Heterotheca inuloidesrdquo Journal of Pharmacy and Pharmacologyvol 48 no 4 pp 441ndash443 1996

[18] E Coballase-Urrutia J Pedraza-Chaverri N Cardenas-Rod-rıguez et al ldquoHepatoprotective effect of acetonic and methano-lic extracts of Heterotheca inuloides against CCl4-inducedtoxicity in ratsrdquo Experimental and Toxicologic Pathology vol 63no 4 pp 363ndash370 2011

[19] S Brenner ldquoThe genetics of Caenorhabditis elegansrdquo Geneticsvol 77 no 1 pp 71ndash94 1974

[20] J A Lewis and J T Fleming ldquoBasic culture methodsrdquo inMethods in Cell Biology H F Epstein and D C Shakes Edsvol 48 pp 3ndash29 Academic Press San Diego Calif USA 1995

[21] J L Rodrıguez-Chavez Y Rufino-Gonzalez M Ponce-Maco-tela and G Delgado ldquoIn vitro activity of lsquoMexican Arnicarsquo Het-erotheca inuloides Cass natural products and some derivativesagainst Giardia intestinalisrdquo Parasitology 2014

[22] T BNg F Liu andZ TWang ldquoAntioxidative activity of naturalproducts from plantsrdquo Life Sciences vol 66 no 8 pp 709ndash7232000

[23] Secretarıa de Agricultura ldquoNorma Oficial Mexicana NOM-062-ZOO-1999 Especificaciones tecnicas para la produccioncuidado y uso de los animales de laboratoriordquo

[24] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[25] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[26] H Esterbauer and K H Cheeseman ldquoDetermination of alde-hydic lipid peroxidation productsrdquoMethods in Enzymology vol186 pp 407ndash421 1990

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

[28] N Cotelle J-L Bernier J-P Catteau J Pommery J-C Wal-let and E M Gaydou ldquoAntioxidant properties of hydroxy-flavonesrdquo Free Radical Biology ampMedicine vol 20 no 1 pp 35ndash43 1996

[29] I Fridovich ldquoQuantitative aspects of the production of super-oxide anion radical by milk xanthine oxidaserdquo The Journal ofBiological Chemistry vol 245 no 16 pp 4053ndash4057 1970

[30] S P Wolff ldquoFerrous ion oxidation in presence of ferric ionindicator xylenol orange for measurement of hydroperoxidesrdquoMethods in Enzymology vol 233 pp 182ndash189 1994

[31] B Halliwell J M C Gutteridge and O I Aruoma ldquoThedeoxyribose method a simple lsquotest-tubersquo assay for determi-nation of rate constants for reactions of hydroxyl radicalsrdquoAnalytical Biochemistry vol 165 no 1 pp 215ndash219 1987

[32] N Chakraborty and B C Tripathy ldquoInvolvement of singlet oxy-gen in 5-aminolevulinic acid-induced photodynamic damage ofcucumber (Cucumis sativus L) chloroplastsrdquo Plant Physiologyvol 98 no 1 pp 7ndash11 1992

[33] O I Aruoma and B Halliwell ldquoAction of hypochlorous acidon the antioxidant protective enzymes superoxide dismutasecatalase and glutathione peroxidaserdquo Biochemical Journal vol248 no 3 pp 973ndash976 1987

[34] Y K Rao S-H Fang and Y-M Tzeng ldquoAnti-inflammatoryactivities of flavonoids isolated from Caesalpinia pulcherrimardquoJournal of Ethnopharmacology vol 100 no 3 pp 249ndash253 2005

[35] GM Solis andM Petrascheck ldquoMeasuringCaenorhabditis ele-gans life span in 96 well microtiter platesrdquo Journal of VisualizedExperiments JoVE no 49 Article ID e2496 2011

[36] A J Dugas Jr J Castaneda-Acosta G C Bonin K L PriceN H Fischer and G W Winston ldquoEvaluation of the totalperoxyl radical-scavenging capacity of flavonoids structure-activity relationshipsrdquo Journal of Natural Products vol 63 no3 pp 327ndash331 2000

[37] G Cao E Sofic and R L Prior ldquoAntioxidant and prooxidantbehavior of flavonoids structure-activity relationshipsrdquo FreeRadical Biology and Medicine vol 22 no 5 pp 749ndash760 1997

[38] R Amorati S Menichetti C Viglianisi and M C FotildquoProton-electron transfer pathways in the reactions of peroxyland dpph∙ radicals with hydrogen-bonded phenolsrdquo ChemicalCommunications vol 48 no 97 pp 11904ndash11906 2012

[39] Y Hirose M Kakita T Washizu and S Matsugo ldquoStructuraldetermination of oxidation products of flavonoids in alcoholicaqueous solution with reactive oxygen speciesrdquo Journal ofPhotoscience vol 9 no 2 pp 424ndash426 2002

[40] S R Husain J Cillard and P Cillard ldquoHydroxyl radicalscavenging activity of flavonoidsrdquo Phytochemistry vol 26 no9 pp 2489ndash2491 1987

[41] A J Nappi and E Vass ldquoHydroxyl radical production byascorbate and hydrogen peroxiderdquo Neurotoxicity Research vol2 no 4 pp 343ndash355 2000

[42] I G Kirkinezos and C TMoraes ldquoReactive oxygen species andmitochondrial diseasesrdquo Seminars in Cell and DevelopmentalBiology vol 12 no 6 pp 449ndash457 2001

[43] J P Hu M Calomme A Lasure et al ldquoStructure-activityrelationship of flavonoids with superoxide scavenging activityrdquoBiological Trace Element Research vol 47 no 1ndash3 pp 327ndash3311995

[44] P Di Mascio M E Murphy and H Sies ldquoAntioxidant defensesystems the role of carotenoids tocopherols and thiolsrdquo TheAmerican Journal of Clinical Nutrition vol 53 no 1 supple-ment pp 194Sndash200S 1991

[45] C Tournaire S CrouxM-TMaurette et al ldquoAntioxidant activ-ity of flavonoids efficiency of singlet oxygen (1Δg) quenchingrdquoJournal of Photochemistry and Photobiology B Biology vol 19no 3 pp 205ndash215 1993

[46] E Coballase-Urrutia J Pedraza-Chaverri R Camacho-Carranza et al ldquoAntioxidant activity of Heterotheca inuloidesextracts and of some of its metabolitesrdquo Toxicology vol 276no 1 pp 41ndash48 2010

[47] L Segura B Freixa T Ringbom et al ldquoAnti-inflammatoryactivity of dichloromethane extract of Heterotheca inuloides invivo and in vitrordquo Planta Medica vol 66 no 6 pp 553ndash5552000

[48] H I Rocha-Gonzalez E Blaisdell-Lopez V Granados-Sotoand A Navarrete ldquoAntinociceptive effect of 7-hydroxy-34-dihydrocadalin isolated from Heterotheca inuloides role ofperipheral 5-HT1 serotonergic receptorsrdquo European Journal ofPharmacology vol 649 no 1ndash3 pp 154ndash160 2010

[49] A Kampkotter C G Nkwonkam R F Zurawski et al ldquoInves-tigations of protective effects of the flavonoids quercetin andrutin on stress resistance in themodel organismCaenorhabditiselegansrdquo Toxicology vol 234 no 1-2 pp 113ndash123 2007

[50] N Saul K Pietsch R Menzel S R Sturzenbaum and C E WSteinberg ldquoCatechin induced longevity in C elegans from keyregulator genes to disposable somardquoMechanisms of Ageing andDevelopment vol 130 no 8 pp 477ndash486 2009

[51] M A Wilson B Shukitt-Hale W Kalt D K Ingram J AJoseph and C A Wolkow ldquoBlueberry polyphenols increaselifespan and thermotolerance in Caenorhabditis elegansrdquo AgingCell vol 5 no 1 pp 59ndash68 2006


[52] C Buchter D Ackermann S Havermann et al ldquoMyricetin-mediated lifespan extension in Caenorhabditis elegans is mod-ulated by DAF-16rdquo International Journal of Molecular Sciencesvol 14 no 6 pp 11895ndash11914 2013

[53] I A Rodrigues M M B Azevedo F C M Chaves et al ldquoInvitro cytocidal effects of the essential oil from Croton cajucara(red sacaca) and its major constituent 7-hydroxycalameneneagainst Leishmania chagasirdquo BMCComplementary and Alterna-tive Medicine vol 13 article 249 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


type sesquiterpenes [13] flavonoids [14] and phytosterols[15] Previous studies have reported that metanolic extractand certain natural products isolated fromH inuloides driedflowers have antioxidant activity and ability to inhibit lipidperoxidation scavenging ROS and act as hepatoprotectiveagents [16ndash18] However the antioxidant activity of naturalproducts isolated from the acetone extract has not beenreported In the present study we evaluated the antioxidantand ROS scavenging capacity of H inuloides metabolitesisolated from the acetonic extract and prepared semisyntheticderivatives and also evaluated their anti-inflammatory effectsand effect on the C elegans life span Here we report theresults considering the structure-activity relationships

2 Materials and Methods

21 Reagents All reagents used were of analytical gradeSodium pyruvate dimethyl thiourea (DMTU) nordihydro-guaiaretic acid (NDGA) ascorbic acid histidine xylenolorange (FOX) 22-diphenyl-1-picrylhydrazyl (DPPH) dim-ethylsulfoxide (DMSO) NN-dimethyl-4-nitrosoaniline(DMNA) catalase xanthine xanthine oxidase nitroblue tet-razolium (NBT) dL-penicillamine 2-thiobarbituric acid(TBA) 120572-tocopherol Folin and Ciocalteursquos phenol reagent35-di-tert-4-butylhydroxytoluene (BHT) and 5-fluoro-21015840-deoxyuridine were purchased from Sigma-Aldrich (TolucaMexico or Sigma St Louis MO) Absolute ethanolhydrogen peroxide (H

2O2) sulfuric acid (H

2SO4) methanol

ethylenediaminetetraacetic acid disodium salt (EDTA)NaCl KH

2PO4 NaHPO

4sdot7H2O NaOH and H

2SO4were

purchased from Merck-Mexico Sodium hypochlorite(NaOCl) was purchased from Hycel (Mexico City Mexico)

22 Strains of C elegans Experimental C elegans strain waswild type N2 (Bristol) The strain was obtained from theCaenorhabditis Genetics Center (University of MinnesotaMinneapolis MN USA) and was maintained on nematodegrowth medium (NGM) at 20∘C as described previously byBrenner [19] Age-synchronized worms were generated inall experiments through the sodium hypochlorite methodWorms were allowed to hatch in Petri dishes in liquid S-medium with concentrated Escherichia coli OP50 as food[20]

23 Plant Material Flowers of H inuloides (provided byMIXIM Laboratories Naucalpan Mexico) were collected in2010 in the town of Mesas Altas de San Juan Xoconuscomunicipality of Donato Guerra (State of Mexico) andauthenticated by MS Abigail Aguilar-Contreras A voucherof plant material was deposited under code IMSSM-16064 inthe Medicinal Plant Herbarium of the Instituto Mexicano delSeguro Social (IMSS Mexico City)

24 Heterotheca inuloides Metabolites and Derivatives Com-pounds 1ndash12 were isolated from the acetone extract ofdried flowers of H inuloides in a previous study Semisyn-thetic compounds 7-acetoxy-34-dihydrocadalene (13) 7-benzoxy-34-dihydrocadalene (14) 7-acetoxycadalene (15)7-benzoxycadalene (16) quercetin pentaacetate (17) and

7-hydroxy calamenene (18) were obtained by conven-tional chemical procedures as was previously described [21](Figure 1) Due to the paucity of material compounds 10 and11 were not included in all the bioassays

25 Antioxidant Effects and ROS Scavenging ofH inuloides Metabolites

251 Estimation of Lipid Peroxidation Lipid peroxidationwas measured by TBARS assay using rat brain homogenates[22] with some modifications Adult male Wistar rats (200ndash250 g) were provided by the Instituto de Fisiologıa CelularUNAM and their use was approved by the Animal Careand Use Committee [23] The animals sacrifice was carriedout avoiding unnecessary pain with CO

2 cerebral tissue

(whole brain) was rapidly dissected and homogenized Thehomogenate was centrifuged for 10min at 3400 rpm to yielda pellet that was discarded protein content in the supernatantwas measured using the Folin and Ciocalteursquos phenol reagent[24] and adjusted to 266mg proteinmL with PBS Thesupernatant (375120583L) was incubated at 37∘C for 30min inpresence of test sample (25120583L) dissolved inDMSOor ethanoland 50 120583L of EDTA solution (20120583M) Lipid peroxidationwas started adding 50 120583L of a freshly prepared 100 120583MFeSO4solution (final concentration 10 120583M) and incubated

at 37∘C for 60min TBARS were determined as described byOhkawa et al [25]with somemodifications Concentration ofTBARSwas calculated by interpolation in a standard curve oftetrametoxipropane (TMP) [26] Final results were expressedas mmoles of TBARS per mg of protein The inhibition ratio() was calculated using the following formula

Inhibition ratio () = (119862 minus 119864)119862

times 100 (1)

where119862was the absorbance of control and 119864was the absorb-ance of the test sample

252 DPPH∙ Scavenging Capacity DPPH assay was assessedin 96-well microtiter plates according to the method ofBlois [27] with minor changes Briefly 005mL of test com-pounds in DMSO at different concentrations was added toan ethanolic solution of 22-diphenylpicrylhydrazyl (DPPH∙)(13333 120583M 0150mL)The control sample contained distilledwater Reactionmixtures were incubated at 37∘C for 30min inthe dark After incubation the absorbance was measured at515 nm in a microplate reader ELx808 (BioTek InstrumentsInc Vermont USA) The inhibition of each compoundwas determined by comparison with a DPPH ethanol blanksolution [28]The scavenging capacity is given as percent ()DPPH scavenged calculated as [(optical density of control minusoptical density of compound)(optical density of control) times100] 120572-tocopherol was used as standard

253 Superoxide Radical-Scavenging Activity The superox-ide radicals (O

2

minus) were generated through the system xan-thine oxidase [29] O

2

minus in the assay system and xanthineoxidase activity were measured as NBT reduction usinga DU-640 series Beckman spectrophotometer 800120583L of


OHHO

H

12

11

HOOH

HO

HO

OHOH

10

HO

OHOOC

OHRO RO

3 5

OH

4

HO H

H H


O

O

1 13 14R H Ac Bz

O

OOH

OH

9

HO

18

2 15 16R H Ac Bz

12

345

6

7 8 12

345

6

7 8

12

3456

78

A

B

CO OO

CH3CH3CH3

CH3CH3 CH3

CH3

R1O

OR4

OR5

OR3

OR2

H3CO

OCH3




2



2O2was cal-


2O2was mixed


2O2was measured




















2) of H inuloides


2

scavenging



2 A 7mm diam-






50) value


3 Results


50of 3066 plusmn 814 120583M





50of 058 plusmn










2

minus HOCl H2O2 1O2





50value The







50of 154 plusmn 63 120583M


50was











4 Discussion




Superoxide

2 3 4 7 8 9 13 15 C0

100

200

300

Compound2 3 4 7 8 9 13 15 C

Compound

2 3 4 7 8 9 13 15 CCompound

2 3 4 7 8 9 13 15 CCompound

2 3 4 7 8 9 13 15 CCompound

0

200

400

600

0

200

400

600

800

0

200

400

600

800

1000

HOCl

0

50

100

150

200

250

300

350

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

H2O2

OH∙minus1O2

CI50

(120583M

)

CI50

(120583M

)

CI50

(120583M

)CI

50

(120583M

)

CI50

(120583M

)









minus 1O2
























2






2




0 4 8 12 16 20 24 28 320

20

40

60

80

100

9678

15Control

Time (days)

Surv

ival

()

(a)

Control

0 4 8 12 16 20 24 28 32Time (days)

Surv

ival

()

0

20

40

60

80

100

113

14

(b)

Control

Surv

ival

()

0 4 8 12 16 20 24 28 320

20

40

60

80

100

215

16

Time (days)

(c)

Surv

ival

()

Control

34

0 4 8 12 16 20 24 28 320

20

40

60

80

100

Time (days)

(d)








5 Conclusions


2



50in




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















OHHO

H

12

11

HOOH

HO

HO

OHOH

10

HO

OHOOC

OHRO RO

3 5

OH

4

HO H

H H


O

O

1 13 14R H Ac Bz

O

OOH

OH

9

HO

18

2 15 16R H Ac Bz

12

345

6

7 8 12

345

6

7 8

12

3456

78

A

B

CO OO

CH3CH3CH3

CH3CH3 CH3

CH3

R1O

OR4

OR5

OR3

OR2

H3CO

OCH3




2



2O2was cal-


2O2was mixed


2O2was measured




















2) of H inuloides


2

scavenging



2 A 7mm diam-






50) value


3 Results


50of 3066 plusmn 814 120583M





50of 058 plusmn










2

minus HOCl H2O2 1O2





50value The







50of 154 plusmn 63 120583M


50was











4 Discussion




Superoxide

2 3 4 7 8 9 13 15 C0

100

200

300

Compound2 3 4 7 8 9 13 15 C

Compound

2 3 4 7 8 9 13 15 CCompound

2 3 4 7 8 9 13 15 CCompound

2 3 4 7 8 9 13 15 CCompound

0

200

400

600

0

200

400

600

800

0

200

400

600

800

1000

HOCl

0

50

100

150

200

250

300

350

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

H2O2

OH∙minus1O2

CI50

(120583M

)

CI50

(120583M

)

CI50

(120583M

)CI

50

(120583M

)

CI50

(120583M

)









minus 1O2
























2






2




0 4 8 12 16 20 24 28 320

20

40

60

80

100

9678

15Control

Time (days)

Surv

ival

()

(a)

Control

0 4 8 12 16 20 24 28 32Time (days)

Surv

ival

()

0

20

40

60

80

100

113

14

(b)

Control

Surv

ival

()

0 4 8 12 16 20 24 28 320

20

40

60

80

100

215

16

Time (days)

(c)

Surv

ival

()

Control

34

0 4 8 12 16 20 24 28 320

20

40

60

80

100

Time (days)

(d)








5 Conclusions


2



50in




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2) of H inuloides


2

scavenging



2 A 7mm diam-






50) value


3 Results


50of 3066 plusmn 814 120583M





50of 058 plusmn










2

minus HOCl H2O2 1O2





50value The







50of 154 plusmn 63 120583M


50was











4 Discussion




Superoxide

2 3 4 7 8 9 13 15 C0

100

200

300

Compound2 3 4 7 8 9 13 15 C

Compound

2 3 4 7 8 9 13 15 CCompound

2 3 4 7 8 9 13 15 CCompound

2 3 4 7 8 9 13 15 CCompound

0

200

400

600

0

200

400

600

800

0

200

400

600

800

1000

HOCl

0

50

100

150

200

250

300

350

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

H2O2

OH∙minus1O2

CI50

(120583M

)

CI50

(120583M

)

CI50

(120583M

)CI

50

(120583M

)

CI50

(120583M

)









minus 1O2
























2






2




0 4 8 12 16 20 24 28 320

20

40

60

80

100

9678

15Control

Time (days)

Surv

ival

()

(a)

Control

0 4 8 12 16 20 24 28 32Time (days)

Surv

ival

()

0

20

40

60

80

100

113

14

(b)

Control

Surv

ival

()

0 4 8 12 16 20 24 28 320

20

40

60

80

100

215

16

Time (days)

(c)

Surv

ival

()

Control

34

0 4 8 12 16 20 24 28 320

20

40

60

80

100

Time (days)

(d)








5 Conclusions


2



50in




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2

minus HOCl H2O2 1O2





50value The







50of 154 plusmn 63 120583M


50was











4 Discussion




Superoxide

2 3 4 7 8 9 13 15 C0

100

200

300

Compound2 3 4 7 8 9 13 15 C

Compound

2 3 4 7 8 9 13 15 CCompound

2 3 4 7 8 9 13 15 CCompound

2 3 4 7 8 9 13 15 CCompound

0

200

400

600

0

200

400

600

800

0

200

400

600

800

1000

HOCl

0

50

100

150

200

250

300

350

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

H2O2

OH∙minus1O2

CI50

(120583M

)

CI50

(120583M

)

CI50

(120583M

)CI

50

(120583M

)

CI50

(120583M

)









minus 1O2
























2






2




0 4 8 12 16 20 24 28 320

20

40

60

80

100

9678

15Control

Time (days)

Surv

ival

()

(a)

Control

0 4 8 12 16 20 24 28 32Time (days)

Surv

ival

()

0

20

40

60

80

100

113

14

(b)

Control

Surv

ival

()

0 4 8 12 16 20 24 28 320

20

40

60

80

100

215

16

Time (days)

(c)

Surv

ival

()

Control

34

0 4 8 12 16 20 24 28 320

20

40

60

80

100

Time (days)

(d)








5 Conclusions


2



50in




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Superoxide

2 3 4 7 8 9 13 15 C0

100

200

300

Compound2 3 4 7 8 9 13 15 C

Compound

2 3 4 7 8 9 13 15 CCompound

2 3 4 7 8 9 13 15 CCompound

2 3 4 7 8 9 13 15 CCompound

0

200

400

600

0

200

400

600

800

0

200

400

600

800

1000

HOCl

0

50

100

150

200

250

300

350

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

lowast

H2O2

OH∙minus1O2

CI50

(120583M

)

CI50

(120583M

)

CI50

(120583M

)CI

50

(120583M

)

CI50

(120583M

)









minus 1O2
























2






2




0 4 8 12 16 20 24 28 320

20

40

60

80

100

9678

15Control

Time (days)

Surv

ival

()

(a)

Control

0 4 8 12 16 20 24 28 32Time (days)

Surv

ival

()

0

20

40

60

80

100

113

14

(b)

Control

Surv

ival

()

0 4 8 12 16 20 24 28 320

20

40

60

80

100

215

16

Time (days)

(c)

Surv

ival

()

Control

34

0 4 8 12 16 20 24 28 320

20

40

60

80

100

Time (days)

(d)








5 Conclusions


2



50in




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















minus 1O2
























2






2




0 4 8 12 16 20 24 28 320

20

40

60

80

100

9678

15Control

Time (days)

Surv

ival

()

(a)

Control

0 4 8 12 16 20 24 28 32Time (days)

Surv

ival

()

0

20

40

60

80

100

113

14

(b)

Control

Surv

ival

()

0 4 8 12 16 20 24 28 320

20

40

60

80

100

215

16

Time (days)

(c)

Surv

ival

()

Control

34

0 4 8 12 16 20 24 28 320

20

40

60

80

100

Time (days)

(d)








5 Conclusions


2



50in




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 4 8 12 16 20 24 28 320

20

40

60

80

100

9678

15Control

Time (days)

Surv

ival

()

(a)

Control

0 4 8 12 16 20 24 28 32Time (days)

Surv

ival

()

0

20

40

60

80

100

113

14

(b)

Control

Surv

ival

()

0 4 8 12 16 20 24 28 320

20

40

60

80

100

215

16

Time (days)

(c)

Surv

ival

()

Control

34

0 4 8 12 16 20 24 28 320

20

40

60

80

100

Time (days)

(d)








5 Conclusions


2



50in




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusions


2



50in




Acknowledgments


References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/omcl/2015/843237.pdf · by the following method. Brie y, volumes of . mM BHT in HPLC-grade methanol were mixed with