Chemical Composition, Antioxidant and Anticholinesterase Activity of Melissa

Industrial Crops and Products 53 (2014) 34 45

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Chemical composition, antioxidant and anticholiMelissa ofcinalis

Romaiana Picada Pereiraa,, Aline Augusti Boligonc, Andr StoRoselei Fachinetto f, Carla Speroni Cerond, Jos Eduardo TanusMargareth Linde Athaydec, Joo Batista Teixeira Rochab,e,

a Departament l de Pb Departament ederac Departamentd Departament o, SP, Be Abdus Salam f Departament dade F

a r t i c l

Article history:Received 12 August 2013Received in revised form27 November 2013Accepted 2 De

Keywords:Oxidative streMelissa ofcinaGallic acidQuercetinAlzheimers di

Oxidative stress is associated with various diseases, in particular those related with the central nervoussystem, such as Alzheimers disease. Based on the various benets of Melissa ofcinalis, we investigated thechemical composition and antioxidant activity of different fractions from M. ofcinalis extract. Further-more, the fraction with the highest antioxidant activity was tested as a potential acetylcholinesterase

1. Introdu

Reactiveclass of higcesses and ROS and RNan imbalanof the physand Gutteri

Abbreviatioxidative stresthiobarbituric

CorresponGraduac o emTel.: +55 21 55 CorresponGraduac o emTel.: +55 21 42

E-mail addjbtrocha@yaho

0926-6690/$ http://dx.doi.ocember 2013

sslis

sease

(AChE) inhibitor. Gallic acid, an important water soluble constituent of M. ofcinalis, was tested onthe matrix metalloproteinase-2 (MMP-2) activity. High performance liquid chromatography (HPLC), gaschromatography coupled with mass spectrometry (GCMS) and nuclear magnetic resonance (NMR) wereused to analyze the chemical composition of M. ofcinalis. TBARS, DPPH, epinephrine autoxidation wereused to verify antioxidant properties of M. ofcinalis or its constituents. Ethyl acetate fraction presentedthe highest avonoids content as well as the antioxidant activities when compared with other tested frac-tions. The ethyl acetate fraction was also a weak inhibitor of brain AChE. Moreover, gallic acid inhibitedMMP-2 activity. In conclusion, M. ofcinalis ethyl acetate fraction should be further investigated for itspossible use in the treatment of oxidative stress related diseases, such as Alzheimers disease.

2013 Elsevier B.V. All rights reserved.

ction

oxygen and nitrogen species (ROS and RNS) are ahly reactive molecules generated by metabolic pro-by some external factors. An excessive production ofS can lead to oxidative stress (OS), which is dened asce between generation of these species and the activityiologic antioxidant defenses (Aruoma, 1998; Halliwelldge, 1999). In OS conditions, the excessive presence of

ons: ROS, reactive oxygen species; RNS, reactive nitrogen species; OS,s; AD, Alzheimers disease; MMPs, matrix metalloproteinase; TBARS,

acid reactive substances; AChE, acetylcholinesterase.ding author at: Centro de Cincias Naturais e Exatas, Programa de Ps-

Bioqumica Toxicolgica, 97105-900 Santa Maria, RS, Brazil. 220 8140; fax: +55 21 55 220 8978.ding author at: Centro de Cincias Exatas e Naturais, Programa de Ps-

Qumica Aplicada, 84030-900 Ponta Grossa, PR, Brazil. 220 3062.resses: [email protected] (R.P. Pereira),o.com.br (J.B.T. Rocha).

reactive species can cause DNA, protein and lipid oxidation, whichcan cause cellular failure and neuronal death (Finkel and Holbrook,2000).

Of particular importance, the brain is an organ extremely sus-ceptible to free radical damage because of its high consumption ofoxygen and its relatively low concentration of antioxidant enzymesand free radicals scavengers. Consequently, OS has been directlyimplicated in the pathogenesis of a number of chronic neurode-generative diseases (Coyle and Puttfarcken, 1993; Aliev et al., 2008,2009). For instance, installation and progression of Alzheimersdisease (AD) has been linked to OS. AD is an age-related neurode-generative disease recognized as one of the most important medicalproblems affecting the elderly. Brain aging is known to be related toexcessive neuronal loss, decrease in ACh level, increase in inam-mation and OS (Nie et al., 2009).

The amyloid formation hypothesis postulates that the 4042amino acid peptide amyloid- (A) fragment from -amyloide pre-cursor protein triggers the deposition of the senile plaques in thebrain that are associated with AD development (Zhang-Nunes et al.,2006). Recent evidence suggests that cerebrovascular abnormali-ties associated with AD may have been underestimated (Stopa et al.,

see front matter 2013 Elsevier B.V. All rights reserved.rg/10.1016/j.indcrop.2013.12.007o de Qumica, Programa de Ps-Graduac o em Qumica Aplicada, Universidade Estaduao de Qumica, Programa de Ps-Graduac o em Bioqumica Toxicolgica, Universidade Fo de Farmcia Industrial, Universidade Federal de Santa Maria, RS, Brazilo de Farmacologia, Faculdade de Medicina de Ribeiro Preto, Universidade de So PaulInternational Centre for Theoretical Physics, Italyo de Fisiologia e Farmacologia, Programa de Ps-Graduac o em Farmacologia, Universi

e i n f o a b s t r a c t/ locate / indcrop

nesterase activity of

rti Appelb,-Santosd,

onta Grossa, PR, Brazill de Santa Maria, RS, Brazil

razil

ederal de Santa Maria, RS, Brazil

R.P. Pereira et al. / Industrial Crops and Products 53 (2014) 34 45 35

2008). However, how AD pathology can inuence or can be affectedby these changes is unclear. Furthermore, the factors that cause Adeposits in vessels forming plaques, as well as the molecular path-ways activated by vascular A causing breakdown of the vesselwall are poinduced act(MMPs) and

MMPs arthe remode2008). Prevand activitycells (Jung that ROS ca2007) and net al., 2000)plaques depactivation o

Based otreatment sinhibitors (Perry, 1986have been smodest bencurrent theidentify dru(Francis et a

Consequof new merally occurrattention asanti-amyloi(Singh et alhave demoa polyphenet al., 2010of cognitive2003). It haplants may (Dastmalchaceae), has b(Perry et al.2006). This its nerve ca(Kennedy egroup demosented a vepro-oxidan

Considereases, and tprevious stcompositioferent M. ochemical cofrom M. ofof gallic acithe MMP-2crude extrathe AChE ac

2. Method

2.1. Chemic

All chem60 F254 cocal procedu

n-butanol, acetonitrile, gallic acid, chlorogenic acid, ellagic acid,caffeic acid, catechin and epicatechin were purchased from Merck(Darmstadt, Germany). Iron sulfate (FeSO4), ascorbic acid, chlo-ridric and acetic acid were obtained from Merck (Rio de Janeiro,

zil).1-1

hyl a(St. LR speuipp

of 10 relere

chrom (Shequipted

detnalyomettt-Paplitleass satogrer an

metin).

g rat

ant c

plans mar a waportractichloactiocetatracted ju

imal

le Wis, frols eacontroith l

acco for /08.

alys

erse grad

withing adien%, 6folloodi

tion oentraorly understood. Among potential candidates are A-ivation of the extracellular matrix metalloproteinases

A-induced OS (Garcia-Alloza et al., 2009).e zinc-dependent endopeptidases with a major role inling of the extracellular matrix (Cao et al., 1995; Fu et al.,ious studies have shown that A induces the expression

of MMP-2 in human cerebrovascular smooth muscleet al., 2003). Furthermore, other studies have shownn also activate MMPs (Cao et al., 1995; Haorah et al.,atural antioxidants can reduce MMP activity (Demeule. Consequently, antioxidant compounds could block Aosits formation, which could blunt the subsequent OSf MMP.n cholinergic hypothesis of AD, the most commontrategy in AD has involved the use of cholinesteraseaiming to re-establish acetylcholine level in the brain). However, a number of these drugs used to treat AD

hown to produce several side effects and yield relativelyets (Van Marum, 2008). To reverse these limitations ofrapeutics for AD, extensive research are in progress togs that are effective and free of undesirable side effectsl., 1999; Van Marum, 2008; Dastmalchi et al., 2009).ently, there is still a great demand for discoverydical alternatives for AD treatment. Certain natu-ing dietary phytochemicals have received considerable

alternative candidates for AD therapy, because of theirdogenic, antioxidant and anti-inammatory properties., 2008; Sun et al., 2010). Furthermore, literature datanstrated MMP inhibition by epigallocatechin gallate,ol found in greentea, in a brain disease model (Park). Accordingly, plants have been used in the treatment

dysfunction (Kennedy et al., 2002; Akhondzadeh et al.,s been shown that ethnopharmacological screening ofbe useful in the discovery of new drugs for AD therapyi et al., 2007). For instance, Melissa ofcinalis L. (Lami-een assessed for its potential therapeutic efcacy in AD

, 1999; Akhondzadeh et al., 2003; dos Santos-Neto et al.,plant is used in traditional medicine to prepare tea forlming effect and to treat nervous disturbance of sleept al., 2004, 2006; Wheatley, 2005). A recent study of ournstrates that different extracts from M. ofcinalis pre-ry pronounced antioxidant property against differentts in brain homogenates (Pereira et al., 2009).ing the important role of OS in several neurological dis-he neurological benets described for M. ofcinalis inudies, it becomes interesting to further investigate itsn and to study the pharmacological properties of dif-fcinalis extracts. So, in this study, we determine themposition and antioxidant activity of different fractionscinalis crude extract. Furthermore, we tested the effectd, a phenolic compound found in this plant extract, on

activity. The potential inhibitory effect of M. ofcinalisct and its fraction with higher antioxidant activity ontivity was also determined.

s

als, apparatus and general procedures

icals were of analytical grade. Silica gel 60, silica gelated plates, solvents for the extractions and analyti-res, dichloromethane, ethyl acetate, ethanol, methanol,

RJ, Bra(TBA), (dimetSigma

NMeter eqwidth as ppmform wliquid system20A), connecUVvisSP1. ASpectrHewlesplit-sand mchromdiamet95% of2 mL/mheatin70 eV.

2.2. Pl

Therial wa70% fowas evThe exwith deach frethyl aous exprepar

2.3. An

Mamonthanimawith ccycle wused inciation11.794

2.4. An

Revunder packedcontaintion gr40%, 50tively, slight mcentraa conc Rutin and quercetin, TrisHCl, thiobarbituric aciddiphenyl-2 picrylhydrazyl (DPPH), malonaldehyde bis-cetal) (MDA) and all other reagents were obtained fromouis, MO, USA).ctra were carried out on a Bruker AMX 400 spectrom-ed with a broadband 5-mm probe, using a spectral

ppm (parts per million). Chemical shifts were expressedative to the TMS, deuterated methanol and chloro-

used as solvent for the samples. High performanceatography (HPLC-DAD) was performed with the HPLC

imadzu, Kyoto, Japan), Prominence Auto-Sampler (SIL-ped with Shimadzu LC-20 AT reciprocating pumps

to the degasser DGU 20A5 with integrator CBM 20A,ector DAD SPD-M20A and Software LC solution 1.22ses in the Gas Chromatography coupled with Massry (GCMS) were performed on gas chromatographckard 6890 Series Plus + equipped with an automaticss model HP 6890 Series GC Auto Sampler Controllerelective detector model HP 5973 MSD, using capillaryaphic fused silica HP-5 MS (30 m 0.32 mm internald thickness of the lm 0.25 mM) with 5% of phenyl andhylsiloxane. The carrier gas was helium (ow rate ofInjector temperature was 250 C programming with ae of 12 C min1 up to 280 C. Ionization potential was

ollection and extractions

t was obtained from commercial sources. This mate-cerated in the dark at room temperature with ethanoleek with daily shake-up. After ltration, the extractated under reduced pressure to remove the ethanol.

was suspended in water and partitioned successivelyromethane, ethyl acetate and n-butanol. The yield ofn was: 2.36% for dichloromethane fraction, 7.42% forte fraction and 7.95% for butanolic fraction. The aque-s were obtained by infusion in hot water and they werest before use.

s

star rats weighing 270320 g and with age from 3 to 3.5m our own breeding colony were kept in cages of 3 or 4h, with continuous access to foods and water in a roomlled temperature (22 3 C) and on a 12-h light/darkights on at 7:00 am. The animals were maintained andrdance to the guidelines of the Brazilian Society of Asso-Laboratory Animal Science (SBCAL) following the law

is of M. ofcinalis fractions composition by HPLC

phase chromatographic analyses were carried outient conditions using C18 column (4.6 mm 150 mm)

5 m diameter particles; the mobile phase was water2% acetic acid (A) and methanol (B), and the composi-t was: 5% of B until 2 min and changed to obtain 25%,

0%, 70% and 100% B at 10, 20, 30, 40 and 50 min, respec-wing the method described by Sabir et al. (2012) withcations. M. ofcinalis fractions were analyzed at a con-f 10 mg/mL, and M. ofcinalis infusion was analyzed attion of 20 mg/mL. The presence of eight antioxidants

36 R.P. Pereira et al. / Industrial Crops and Products 53 (2014) 34 45

compounds was investigated, namely, gallic acid, chlorogenic acid,caffeic acid, ellagic acid, catechin, epicatechin, quercetin and rutin.Identication of these compounds was performed by comparingtheir retention time and UV absorption spectrum with those ofthe commetion volume280 nm forand chlorogsamples anbrane lterto use. Stocchlorogenicmobile phaquercetin, chin and 0detection (Lbased on thusing threeculated as deviation ocurve.

2.5. Isolatio

The chrowas rst intion was spacetate:meand 366 nmstances wivisualized. and quantif

Thereformatography(50:50, v/v)in 10% of dused, startinincreasing started withof methano

The proctions of 5together onle. These fand II). Fracby repeatedwith dichlo(35 mg). Frarepeated co(30:70, v/v)ples were s

2.6. Analys

The dichfor CGMS,database (Ational litera

2.7. TBARS

Rats weand placed 10 mM Tristrifuged forand a low-

assay. An aliquot of 100 L of S1 was incubated for 1 h at 37 Cwith freshly prepared FeSO4 (10 M) in the presence or absenceof different fractions from crude extract of M. ofcinalis or isolatedcompounds. Then, TBARS production was determined as described

awaat 5;

dica

antfcin

theing Cavenir IC5

formcha,; 10M Dn w

afteed dted ns w

inhiuati

Abssextrae of

inep

potlis atin wline sam

buffitiated in

nm,

n vitr

examitro, ws, Ming t, humusing

Ther (exolecue or

100010)mm2055s pos

n vitr

s weaced rcial standards. The ow rate was 0.8 mL/min, injec- 50 L and the wavelength were 257 nm for gallic acid,

catechin and epicatechin, 325 nm for caffeic, ellagicenic acids, and 365 nm for rutin and quercitrin. Thed mobile phase were ltered through 0.45 m mem-

(Millipore) and then degassed by ultrasonic bath priork solutions of quercetin, rutin and gallic, caffeic and

acids standards references were prepared in the HPLCse at a concentration range of 0.1800.280 mg/mL for0.01250.200 mg/mL for rutin, catechin and epicate-.06250.250 mg/mL for phenolics acids. The limit ofOD) and limit of quantication (LOQ) were calculatede standard deviation of the responses and the slope

independent analytical curves. LOD and LOQ were cal-3.3 and 10 /S, respectively, where is the standardf the response and S is the slope of the calibration

n of dichloromethane fraction compounds

matographic prole of the dichloromethane fractionvestigated by TLC. A quantity of 2 L of the frac-otted in plate and eluted with dichloromethane:ethylthanol (5:3:2) and then observed under UV light (254) and sprayed with anisaldehyde-H2SO4/100 C. Sub-

th triterpenoids and avonoids characteristics wereThis preliminary TCL screening prompts us to identifyy the major compounds in this fraction.e, 2.5 g of dried fraction was submitted to column chro-

on silica gel 60 using initially dichloromethane:hexane as mobile phase and increasing the polarity by risingichloromethane until 100%. A new solvent system wasg with dichloromethane:ethyl acetate (90:10, v/v) and

in 10% of ethyl acetate until 100%. Finally the system ethyl acetate:methanol (90:10, v/v), increasing in 10%l until 100%.edure described above furnished ninety-eight (98) frac-0 mL each, which were analyzed by TLC and pooled

the basis of similarities in their chromatographic pro-ractions were combined to give two major fractions (Ition I (sub-fractions 130, 0.8 g), was further puried

chromatography column on silica gel 60 and elutedromethane:hexane (80:20, v/v), to yield compounds 1ction II (fractions 3587, 1.3 g), was further puried bylumn chromatography and eluted with CH2Cl2/EtOH, to yield compounds 2 (53 mg) and 3 (29 mg). The sam-ubmitted to NMR 1H and 13C analysis.

is of M. ofcinalis fractions composition by GCMS

loromethane and ethyl acetate fractions were analyzed all compounds were identied using the Adams MSdams, 2001) and the NIST library of spectra, and addi-ture data were also consulted.

re killed and the cerebral tissue was rapidly dissectedon ice. Tissues were immediately homogenized in coldHCl, pH 7.5 (1/10, w/v). The homogenate was cen-

10 min at 4000 g to yield a pellet that was discardedspeed supernatant (S1) that was used for the TBARS

by Ohktested

2.8. Ra

Theof M. oitoringaccordical scas theradicaland Rotion (5a 0.3 msolutio518 nmpreparcalculasolutioing orthe eqwhereferent absenc

2.9. Ep

Theofcinaquercean alkatested bonatethen inpreparat 480

2.10. Iactivity

To ity in vSystemacid usBrieysured buffer.rimeteEM; Mabsenc50 andet al., 2as recoKit (E1used a

2.11. I

Ratand pl et al. (1979) and Puntel et al. (2007). The fractions were 10; 25; 50 and 100 g/mL.

l-scavenging activity-DPPH assay

ioxidant activity of the fractions from crude extractalis and isolated compounds was evaluated by mon-

ir ability in quenching the stable free radical DPPH,hoi et al. (2002) with minor modications. Free rad-ging capacity (FRSC) of plant extracts was calculated0 values (the concentration necessary to inhibit 50%ation), using the method of Dixon and Web (Oboh

2007). Five different ethanol dilutions of each frac-; 50; 100 and 500 g/mL) were mixed with 100 L ofPPH ethanol solution. Ethanol (80 L) plus plant extractas used as a blank. The absorbance was measured atr 30 min of reaction at room temperature. DPPH wasaily and protected from light. Relative activities werefrom the calibration curve of l-ascorbic acid standardorking in the same experimental conditions. Scaveng-bitory capacity in percent (IC%) was calculated usingon: IC% = 100 [(Abssample Absblank) 100/Abscontrol)ample is the absorbance obtained in the presence of dif-ct concentrations and Abscontrol is that obtained in theextracts. Tests were carried out in triplicate.

hrine autoxidation

ential superoxide anion scavenging activity of the M.queous extract, ethyl acetate fraction, gallic acid andere determined using the epinephrine autoxidation inpH. Briey, 100 L of vehicle, ascorbic acid (100 M) orples were mixed with 2.8 mL of 50 mM of sodium car-er, pH 10.2 containing 0.1 mM of EDTA. The reaction wased by adding 100 L of epinephrine (epinephrine 10 mM

HCl 10 mM) and the kinetics readings were performedas described before (Misra and Fridovich, 1972).

o effect of gallic acid or ascorbic acid on MMP-2

ine whether gallic acid directly inhibits MMP-2 activ-e measured human recombinant MMP-2 activity (R&D

inneapolis, MN, USA) in the absence or presence of galliche Gelatinolytic Activity Kit (E12055; Molecular Probes).an recombinant MMP-2 (2.5 ng/L) activity was mea-

DQ gelatin (5 g/mL; Molecular Probes) in TrisCaCl2 activity was evaluated in a microplate spectrouo-citation at 495 nm and emission at 515 nm; Geminilar Devices) after 120 min of incubation at 37 C in thepresence of gallic acid or ascorbic acid (0, 0.1, 1.0, 10,

M), as previously described (Castro et al., 2009; Ceron. A standard curve of gelatinolytic activity was preparedended by the manufacturer of the Gelatinolytic Activity; Molecular Probes). Phenanthroline (0.1 mmol/L) wasitive control for MMP-2 activity inhibition.

o AChE activity

re killed and the cerebral tissue was rapidly dissectedon ice. Tissues were immediately homogenized in cold


Fig. 1. (A) High performance liquid chromatography phenolics and avonoids prole of dichloromethane (a), ethyl acetate (b) and butanolic (c) fractions, infusion (d) ofthe Melissa ofcinalis. Gallic acid (peak 1), catechin (peak 2), chlorogenic acid (peak 3), caffeic acid (peak 4), ellagic acid (peak 5), epicatechin (peak 6), rutin (peak 7), andquercetin (peak 8). (B) GCMS dichloromethane fraction of Melissa ofcinalis. (C) GCMS ethyl acetate fraction of Melissa ofcinalis.


Table 1Phenolics and avonoids composition of Melissa ofcinalis.

Compounds Ethyl acetate fraction (%) Dichloromethane fraction (%) Butanolic fraction (%) Aqueous extract (%)

Gallic acid 4.91 0.03a 8.66 0.02a 3.29 0.07a 3.15 0.01aCatechin 0.53 0.01b 0.51 0.03b 0.48 0.02bChlorogenic acid 2.64 0.05b 1.83 0.08b 1.72 0.05cCaffeic acid 5.32 0.01a 3.10 0.11c 1.35 0.01b 3.26 0.01aEllagic acid 11.06 0.03d 3.45 0.04d 5.17 0.09c 2.19 0.01dEpicatechin 2.48 0.02c 0.49 0.03b 0.56 0.08d 0.64 0.03bRutin 6.81 0.09c 0.52 0.01d 3.47 0.04a 1.81 0.01cQuercetin 9.22 0.15d 5.19 0.05e 2.09 0.10c 2.04 0.03d

Results are expressed as mean SEM of three determinations. Averages followed by different letters in the column differ by Tukey test at p < 0.05. Calibration curve forgallic acid: y = 53,985x + 1020.6 (r = 0.9981); caffeic acid: y = 87,846x + 1093.7 (r = 0.9997); chlorogenic acid: y = 52,548x + 1082.3 (r = 0.9992); ellagic acid: y = 53,782x + 1156.3(r = 0.9999); rutin: y = 19,217x + 1694.9 (r = 1.000); quercetin: y = 30,153x + 1151.3 (r = 0.9983), catechin: y = 25873x + 1275.8 (r = 0.9996) and epicatechin: y = 24961x + 1407.1(r = 0.9990).

50 mM TrisHCl, pH 7.5 (1/10, w/v). The homogenate was cen-trifuged for 10 min at 4000 g to yield a pellet that was discardedand a low-speed supernatant (S1) that was used for the AChE assay.AChE was determined according to Ellman et al. (1961) modiedby Rocha et al. (1993). The reaction mixture (2 mL nal volume)was composed of 100 mM phosphate buffer pH 7.5, 1 mM 5,5-dithio-bis-2-nitrobenzoic acid (DTNB). The method is based onthe formation of yellow anion, 4,4-dithio-bis-2-nitrobenzoic mea-sured by absorbance at 412 nm during 5 min at 25 C. An aliquotof 100 L of S1 was pre-incubated in the presence or absence ofplant extracts (0, 1, 10, 100 and 1000 g/mL) or isolated compounds(quercetin or gallic acid 0, 0.1, 1 and 10 g/mL) for 20 min. The reac-tion was initiated by adding 0.8 mM acetylthiocholine iodide. Theenzyme activity was estimated in terms of percentage change inabsorbance compared to the control. Physostigmine (Eserine) 1 Mwas used as

2.12. Statis

Data weby DuncansDPPH) or Bautoxidatiosidered stat

Table 2GCMS analys

Substances

Propanoic acPropanoic ac

butyl este2,5 pyrrolidiEthyl hydrogConhydrin Ethyl iso-allHeptatriacotAcetic acid 2

7-oxa-bicyGallic acidHexadecanoHexadecano

ester-Sitosterol9,12,15 Octa

methyl esChlorogenicLinoleic acidHexadecano

esterLupeol Caffeic acid Caffeine Octanal

3. Results and discussion

3.1. HPLC analysis

The HPLC prole of dichloromethane, ethyl acetate and butano-lic fractions from the leaves M. ofcinalis is depicted in arepresentative chromatogram of each extract in Fig. 1. The sam-ples of M. ofcinalis contains other minor compounds in additionto gallic acid (retention time-tR 12.13 min, peak 1), catechin(tR = 14.32 min, peak 2), chlorogenic acid (tR = 21.56 min, peak 3),caffeic acidpeak 5), eppeak 7) andof quercetiby HPLC-DA

in T

MS

dich GCic looic ac aci

(heploro

-si haveng p

analys

nces

oic aci acid brrolidiydrogted hy the reference standard.

tical analysis

re statistically analyzed by one-way ANOVA, followed multiple range tests when appropriated (for TBARS andonferronis multiple comparison tests (for epinephrinen, MMP-2 and AChE activities). The results were con-istically signicant for P < 0.05.

is of dichloromethane fraction.

Retention time(min)

Concentration(%)

Peak inFig. 1B

id 4.79 1.93 1id 2-hydroxyr

7.95 1.98 2

nedione 11.57 1.04 3en succinate 12.71 3.88 4

19.64 1.62 5ocholate 24.41 5.06 6onol 25.03 1.33 7

(2,2,6 trimethyl)clo hepteno

27.47 2.05 8

31.40 9.54 9ic acid 32.05 1.97 10

sented

3.2. GC

Theted toaliphatadecanlinoleitial oilsin dichsterolssterolsloweri

Table 3GCMS

Substa

ButanAcetic2,5 PyEthyl hButylaic acid methyl 33.55 1. 09 11

34.64 45.59 12decatrienoic acidter

34.78 2.11 13

acid 34.85 1.81 14 ethyl ester 35.10 0.99 15ic acid butyl 38.42 2.97 16

42.35 0.98 1745.66 3.07 1852.95 1.26 1953.15 2.10 20

1,6Anhydro-

Phenol, 2 am(1-1-dime

Imidazole 2 carboxy)-

Gallic acid HexadecanoPterin 6 carb-SitosterolChlorogenicLupeol Caffeic acid (tR = 25.78 min, peak 4), ellagic acid (tR = 31.49 min,icatechin (tR = 33.82 min, peak 6), rutin (tR = 37.15 min,

quercetin (tR = 44.93 min, peak 8). The quanticationn, rutin, gallic acid, caffeic acid and chlorogenic acidD (together with standards calibration curves) is pre-

able 1.

analysis

loromethane and ethylacetate fraction was submit-MS analysis (Fig. 2 and Tables 2 and 3). Numerousng-chain fatty acids such as hexadecanoic acid, hex-cid methyl ester, octadecatrienoic acid methyl ester,

d ethyl ester, and some compounds related to essen-tatriacotonol and ethyl iso-allocholate) were identied

methane fraction. Besides caffeic acid, gallic acid, thetosterol and lupeol were identied in both fractions. The

received much attention because of their cholesterol-roperties, and several studies have shown a protective

is of ethyl acetate fraction.

Retention time(min)

Concentration(%)

Peak inFig. 1C

d, ethyl ester 3.37 4.32 1utyl ester 3.57 3.78 2nedione 9.77 0.84 3en succinate 13.64 1.45 4droxytoluene 20.4 1.97 5-d-glucopyranose21.11 1.53 6

ino-6thyl ethyl)

21.54 8.96 7

amino 5 (2vinil

26.01 8.15 8

31.42 4.86 9ic acid 32.07 2.14 10oxylic acid 32.25 2.44 11

34.72 1.09 12 acid 34.87 2.61 13

41.27 0.91 1442.07 5.32 15


Fig. 2. (A) 13C NMR spectra (400 MHz, CDCl3): -sitosterol. (B) 1H NMR spectra (400 MHz, CDCl3): -sitosterol. (C) 13C NMR spectra (100 MHz, CD3OD): quercetin. (D) 1HNMR spectra (400 MHz, CD3OD): quercetin. (E) Chemical structures of isolated compounds from dichloromethane fraction. -sitosterol (1) and quercetin (2).


Fig. 2. (Continued )


effect againcancer deve

3.3. Isolatio

After thethe fractionsuccessive cisolation ofwere identicomparisonet al., 2007)carbon atomindicates thacteristic ofKvr, 2004signal at the compou-sitosterocompound (1H, m, H-6100 MHz): 140.74 (C-529.68 (C-1216); 51.23 ((C-23); 19.1

The isolaBriey, the6.12 (1H, dwith the mat: H 7.7260) and 6.8protons on

on a plac58.24.67tin (Btad, 2

vitroprod

side damlcohantlationnt fromerude cord

Table 4IC50 (g/mL) v

Ethyl acetate

30.41 0.56

Different alphFig. 2. (Continued )

st cardiovascular disease as well as colon and breastlopment (Ferretti et al., 2010).

n of compounds

detection and identication of specic compounds ins, dichloromethane fraction was submitted to furtherolumn chromatographic procedures, which led to the

two compounds. The structures of these compoundsed by 1H NMR and 13C NMR spectra (Fig. 2AD), and by

with literature data (Boligon et al., 2009a; Jayaprakasha. The 13C NMR spectra of subfraction 130 showed 29s, two sp2 signals at 121.67 and 140.74 ppm, which

e presence of one double bonds, this signals are char- steroids with a double bond at C5 and C6 (Forgo and; De-Eknankul and Potduang, 2003). The large singlet5.28 ppm in the 1H NMR is characteristic of H-6 innd. Therefore, the substance 1 was characterized as

l and this structure is shown in Fig. 2E. Some signs of

15 carbcarbon(C-5), 1(C-6), 9querceSlimes

3.4. InTBARS

ConradicalHydroasignicprepardifferedichlorthan cis in ac1: 1NMR (400 MHz, CDCl3): H 3.46 (1H, m, H-3); 5.30); 1.02 (3H, s, H-19); 0.87 (3H, t, H-29). 13C NMR (CDCl3,C 37.25 (C-1); 31.65 (C-2); 71.77 (C-3); 49.44 (C-4);); 121.67 (C-6); 50.14 (C-9); 33.94 (C-10); 21.22 (C-11);); 45.84 (C-13); 56.05 (C-14); 25.40 (C-15); 28.22 (C-C-17); 36.14 (C-20); 23.08 (C-21); 33.94 (C-22); 28.226 (C-25); 19.80 (C-26); 19.05 (C-27).ted compound 2 showed characteristics of avonoids.1H NMR spectrum of compound showed two peaks at, J = 2.0 Hz) and 6.39 ppm (1H, d, J = 2.0 Hz) consistenteta protons H-6 and H-8 on A-ring and an ABX system

(1H, d, J = 2.1 Hz, H-20), 7.62 (1H, dd, J = 8.4, 2.1 Hz, H-7 (1H, d, J = 8.4 Hz, H-50) corresponding to the catecholB-ring. The 13C NMR spectrum indicated the presence of

the fractioncompoundsthe literatuet al., 2006;where quertested compacid and ruinhibition o

3.5. DPPH rfractions or

The 1,1widely use

alues for DPPH inhibition by M. ofcinalis crude extract and fraction. Data show means

fraction Dichloromethane fraction Butanolic fractio

b 125.68 12.5b 384.77 62.37c

abets indicate statistical signicance among different fractions, extract and ascorbic acid.toms, the signal at: 177.3 was attributed to a carbonyled at C-4, the other signals were: 165.92 (C-7), 162.474 (C-9), 121.63 (C-6), 116.32 (C-5), 116.00 (C-2), 99.79

(C-8), the spectral data were compatible with those ofoligon et al., 2009b; Fossen et al., 1998; Liu et al., 2008;003), the structure is shown in Fig. 2E.

effects of M. ofcinalis on iron-induced cerebraluction

ring that the brain is particularly susceptible to freeage, we used cerebral tissue for the TBARS assay.olic extract and different fractions from M. ofcinalisy inhibited iron-induced TBARS production in brains (P < 0.001). However, the inhibitory potency of theactions and crude extract varied. Ethyl acetate andthane fraction presented better antioxidant propertiesextract and the butanolic fraction (Fig. 3A). This resultance with the phenolic and avonoids composition of

s measured by HPLC (Table 1). In effect, the detected

have strong antioxidant activity as demonstrated inre (Vinson et al., 1995; Wiseman et al., 1997; Wagner

Pereira et al., 2009). This can also be observed in Fig. 3Bcetin exhibited the highest antioxidant activity of theounds, followed by gallic acid, caffeic acid, chlorogenictin. In constrast, sitosterol did not exhibit signicantf TBARS production (Fig. 3B).

adical-scavenging activity of M. ofcinalis extract andisolated compounds

-diphenyl-2-picrylhydrazyl (DPPH) radical has beend to test the free radical scavenging ability of various

SEM values average from 3 independent experiments.

n Crude extract Ascorbic acid

379.98 50.44c 6.00 0.06a


Fig. 3. Effects (B and D) on Fascorbic acid w

natural promodel comet al., 1989different fraradical-scavan accentuDichloromeDPPH radicfraction (Fifound in thand Table 5inhibitory rutin. In conTable 5).

In accordfraction prebly due its h(Table 1).

Table 5IC50 (g/mL) v

Caffeic acid

8.45 0.736

Different alphof different concentrations of hydroalcoholic extract, ethyl acetate, dichloromethane and e(II) (10 M) induced TBARS production in brain homogenates (A and B) and on DPPHas used as a positive control). Data show means SEM values average from 3 to 6 indep

ducts (Brand et al., 1995) and has been accepted as apound for free radicals originating in lipids (Hatano; Yasuda et al., 2000). Hydroalcoholic extract andctions from M. ofcinalis presented a signicant DPPHenging activity (P < 0.001). Ethyl acetate exhibitedated DPPH radical inhibition (Fig. 3C and Table 4).thane, butanolic and crude fraction also quenched theal color, but with a lower potency than ethyl acetateg. 3C and Table 4). Isolated polyphenolic compoundse M. ofcinalis fractions inhibited DPPH radical (Fig. 3D). Caffeic acid and gallic acid displayed the strongesteffect, followed by quercitin, chlorogenic acid andtrast, sitosterol had no inhibitory activity (Fig. 3D and

ance with data obtained in TBARS assay, ethyl acetatesented the lowest IC50 value in DPPH assay, proba-igh avonoids content demonstrated in HPLC analysis

3.6. Effect o

Here wethe highest2009), ethyactivity detpounds, galpotential inous extract of epinepha signican(Fig. 4C). Hotion (Fig. 4Din a differeautoxidatiocated that ganion.

alues for DPPH inhibition by isolated compounds. Data show means SEM values averag

Chlorogenic acid Gallic acid

5a 12.7467 0.1503 b 17.61 0.4107d

abets indicate statistical signicance among different substances.butanolic fractions from M. ofcinalis (A and C) or isolated compounds test (C and D; the results are expressed as percentage of control andendent experiments performed in duplicate.

f M. ofcinalis on epinephrine autoxidation

tested M. ofcinalis aqueous extract (which presented antioxidant activity in our previous study; Pereira et al.,l acetate fraction (which present the highest antioxidantermined in the present study), and two phenolic com-lic acid and quercetin, present in M. ofcinalis extract ashibitors of epinephrine autoxidation. M. ofcinalis aque-and ethyl acetate fraction caused a signicant inhibitionrine autoxidation only at 1 mg/mL. Gallic acid causedt inhibition of epinephrine autoxidation at 100 Mwever, quercetin did not inhibit epinephrine autoxida-). The results indicated that these compounds interactnt way with superoxide anion, which is involved inn of epinephrine (Misra and Fridovich, 1972) and indi-allic acid had a modest capacity of scavenge superoxide

e from 3 independent experiments.

Quercetin Rutin

12.3167 0.04055b 15.2533 0.09821c


50

100

150

#

neph

rine

Aut

oxid

atio

n (%

)

Contr

ol

Etha

nol M

AA 10

01 10 10

010

000

50

100

150(A) (C)

(B) (D)M. officinalis Ethyl AcetateFraction ( g/mL)

#

#

Epin

ephr

ine

Aut

oxid

atio

n (%

)

Contr

ol M

AA 10

0 1 10 10

00

50

100

150

#

#

Gallic Acid ( M)

Epi

neph

rine

Aut

oxid

atio

n (%

)

Contr

o

50

100

150

inep

hrin

e A

utox

idat

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(%)

Fig. 4. (A) In v (110(D) on autoxid .05 ver

3.7. Effect oMMP-2 acti

Increasecause of MMRecent stud(Garcia-Allostrate a poantioxidant(Sen et al., uated only

y (Fighich

Fig. 5. (A) In vActivity Kit, inData are showContr

ol M

AA 10

01 10 10

010

000

M. officinalis Aqueous Extract ( g/mL)

#Epi

0Ep

itro effects of M. ofcinalis ethyl acetate fraction (11000 g/mL), aqueous extractation of epinephrine. Data are shown as means SEM of three experiments (#p < 0

f gallic acid or ascorbic acid on human recombinantvity

activitacid, wd concentrations of ROS have been implicated as aPs activation (Van Wart and Birkedal-Hansen, 1990).

ies indicated that MMPs plays an important role in ADza et al., 2009). Furthermore, literature data demon-lyphenol epigallocatechin-3-gallate, compound with

activity (Aldini et al., 2003) causing MMP inhibition2009). Here, due to solubility problems, we have eval-the effect of gallic acid on human recombinant MMP-2

in several mascorbic acity. Howeveand 100 Mthroline). Tgood bioavshould be fThese resulacid seems

Contr

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100

200

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itro effects of gallic acid and ascorbic acid (B) on human recombinant MMP-2 activity. Hum the absence or presence of ascorbic acid or gallic acid. Phenanthroline (Phe) was used as pn as means SEM of three experiments done in duplicate.l

Etha

nol M

AA 10

0 1 10 10

0

Quercetin ( M)

#

00 g/mL) (B), gallic acid (1100 M) (C) and quercetin (1100 M)sus control). Ascorbic acid was used as a positive control.

. 5A). We have also determined the effect of ascorbic is frequently used as a standard antioxidant compound

odels, on MMP-2 activity (Fig. 5B). As shown in Fig. 5,

id had no effect on human recombinant MMP-2 activ-r, gallic acid inhibited MMP-2 activity (P < 0.01), at 50

(Fig. 5A), similarly to the positive control (phenan-his result is very interesting because gallic acid has aailability from human diet (Shahrzad et al., 2001) andurther investigated as a possible modulator of MMP-2.ts also suggest that the MMP-2 inhibitory effect of gallic

not to be directly related with its antioxidant activity

trol

enan

tholin

e M

Asco

rbic

Ac 0.

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Ac 1

M

Asco

rbic

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M

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an recombinant MMP-2 activity was measured using a Gelatinolyticositive control for MMP-2 activity inhibition (#p < 0.01 versus control).


100

(A) (C)

# # #

rase

Co

0

50

100

tera

se

Co

0

50

100

Fig. 6. A In vit (C) anfor AChE activ depen

because ascMMP-2 actiexact mech

3.8. Effect o

M. ofci(Fig. 6B). Cothis enzymdependent acid (Fig. 6Cpolyphenolthe weak icould be attdifferent coand isolateddetermine tAD treatmedata that alM. ofcinali

4. Conclus

Here, wlic acid (anactivity. Wtion from Mand antioxiexhibited aM. ofcinalithe treatmeand antichocompound ing drug ininhibitory a

wled

nasoal

rac o CienparofullyContr

ol M

Eseri

ne 1

Etha

nol 1 10 10

010

000

50

(B) (D)M. officinalis Ethyl

Acetate Fraction ( g/mL)

#

#Ace

tylc

holin

este

Act

ivity

(%)

Contr

ol M

Eseri

ne 1

1 10 100

1000

0

50

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M. officinalis AqueousExtract ( g/mL)

#Ace

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este

rase

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ivity

(%)

Ace

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ity(%

)

Ace

tylc

holin

este

rase

Act

ivity

(%)

ro effects of Melissa ofcinalis ethyl acetate fraction, aqueous extract (B), gallic acid ity inhibition (#p < 0.05 versus control). Data are shown as means SEM of three in

orbic acid, a strong antioxidant compound, did not altervity. However, more studies are needed to elucidate theanism involved in this inhibition.

f M. ofcinalis on AChE activity

nalis aqueous extract did not change AChE activitynversely, ethyl acetate fraction signicantly inhibited

Ackno

Thede PesCoopementode Amis gratee when compared with control in a concentrationmanner (Fig. 6A). So, we evaluated the effect of gallic) and quercetin (Fig. 6D) on AChE activity. The isolated

compounds did not inhibit brain AChE. Consequently,nhibitory effect observed with ethyl acetate fractionributed to the synergistic effect or interaction betweennstituents of this fraction. Thus, M. ofcinalis fractions

compounds should be considered in future studies toheir feasibility as potential alternative medicine for thent. Moreover, this result is in accordance with literatureso demonstrate AChE inhibition by crude extracts froms (Ferreira et al., 2006; Dastmalchi et al., 2009).

ion

e demonstrated the in vitro inhibitory effect of gal- important constituent of M. ofcinalis) in the MMP-2e have also demonstrated that the ethyl acetate frac-. ofcinalis presented the highest avonoids content

dant properties. Furthermore, the ethyl acetate fraction moderate inhibition of cerebral AChE. In conclusion,s could be further investigated for its possible use innt of AD, due its very pronounced antioxidant activitylinesterase activity. In addition, gallic acid, a phenolicpresent in M. ofcinalis extract could be also a promis-

the treatment of AD due its antioxidant and MMP-2ctivities showed here.

Anglica daversity of S

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Chemical composition, antioxidant and anticholinesterase activity of Melissa officinalis1 Introduction2 Methods2.1 Chemicals, apparatus and general procedures2.2 Plant collection and extractions2.3 Animals2.4 Analysis of M. officinalis fractions composition by HPLC2.5 Isolation of dichloromethane fraction compounds2.6 Analysis of M. officinalis fractions composition by GCMS2.7 TBARS2.8 Radical-scavenging activity-DPPH assay2.9 Epinephrine autoxidation2.10 In vitro effect of gallic acid or ascorbic acid on MMP-2 activity2.11 In vitro AChE activity2.12 Statistical analysis

3 Results and discussion3.1 HPLC analysis3.2 GCMS analysis3.3 Isolation of compounds3.4 In vitro effects of M. officinalis on iron-induced cerebral TBARS production3.5 DPPH radical-scavenging activity of M. officinalis extract and fractions or isolated compounds3.6 Effect of M. officinalis on epinephrine autoxidation3.7 Effect of gallic acid or ascorbic acid on human recombinant MMP-2 activity3.8 Effect of M. officinalis on AChE activity

4 ConclusionAcknowledgementsReferences

Chemical Composition, Antioxidant and Anticholinesterase Activity of Melissa

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