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Lipid peroxidation inhibition by ethanolic extract and fractions from Rhamnus sphaerosperma var. pubescens (Reissek) M.C. Johnst. (Rhamnaceae)


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5 (2013) 1



d fraction

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native to Brazil, cal activities of t y and determin s hexane, chloro phosphomolybd ted, in vitro a ormed from pero d from egg yolk , using stomach f Rhamnus sph e phosphomolyb (96.2%) showe thechloroform ( of lipid oxidation verification of inh showed better e ossible identify t rma var. pubesc

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bles are known oxidant capacity rapeutic potentia us exhibit a pro enger and inhib s and R. nakara amnus sphaeros

native in Brazil, n as „Cangica‰ dies that descri at potential ch in the genus phytochemical

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, Beatriz

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, found in the so this plant. There ne the ability t oform and ethyl denum complex nd in vivo exp oxidation induced k. And, for in vi

hs of female ra haerosperma va bdenum comple

d the best activ 63.3%) and he n. The crude ex hibition of lipid p effectiveness in the stigmasterol cens stem poss vivo methods. dation, antioxid

n to produce a y. The family Rh al of their specie onounced antiox ition lipid perox ai, species widel sperma var. pu found in the so and „Fruto-de-be their biologic emical and ph Rhamnus. In t composition o s, as well as, th




, Obdulio

outhern region, efore, the aims to inhibit lipid

acetate of the x method. And periments, by d. The in vitro vo assay, the ats injured by ar. pubescens x method, the vity than crude exane (59.7%) xtract showed peroxidation in inhibiting lipid and sitosterol sess potential

ant, ethanolic

a diversity of hamnaceae is es. Specimens xidant activity, idation, as for ly used in folk bescens is a outhern region.

-pombo‰. And cal properties, harmacological

his study we of Rhamnus he antioxidant



et al

. International Journal of Phytomedicine 5 (2) 136-140 [2013]




activity in vitro and the inhibition of lipid peroxidation in animal model of tissue injury caused by ethanol.

Material and methods

Plant Material

The stems of Rhamnus sphaerosperma var. pubescens were collected in Curitiba-PR, in May 2011. The botanical identification was performed in Municipal Botanical Museum of Curitiba, Paraná. The voucher specimen was deposited under the number 243989.

Extraction and isolation

The dried plant material (2Kg) was submitted to ethanol extraction in Soxhlet apparatus modified. The crude ethanolic extract obtained was concentrated and subjected to liquid-liquid partitioning with hexane, chloroform and ethyl acetate. The hexane fraction was subjected to purification by chromatography on silica column (silica 60 Merck 0.063-0.200mm) with hexane, ethyl acetate and methanol. A crystalline sample was obtained. This sample was analyzed by nuclear magnetic resonance spectroscopy (NMR) of1H and13C.

Phosphomolibdenum spectrophotometric method

This analysis evaluates the antioxidant capacity of both Lipophilic and hydrophilic components. The fractions and ethanol extracts of stems were submitted to the Phosphomolibdenum spectrophotometric method, at a concentration of 200μg/mL. The antioxidant capacity of the samples was expressed in relative antioxidant activity (RAA) regarding rutin[6,7].

Determination of the inhibition of lipid peroxidation



This system evaluates the potential of different samples in inhibiting lipid peroxidation process. As source of lipids was used the gem of an egg, diluted to a concentration of 5% (w/v), in sodium dodecyl sulfate solution(0.55%). Then 0.5 mL of this solution of lipids was placed in contact with samples or standard butyl hydroxyl toluene (0.1mL, final concentration of 70μg/mL), the oxidizing agent 2,2Ê-azo-bis-2-amidinopropanochloride (50μL, 0.035M), 20% acetic acid (1.5mL, pH 3.5), distilled water (0.4mL) and After, the thiobarbituric acid (1.5mL, 0.4%).The reaction was carried out at 95oC for 60 minutes. The organic phase was

extracted with butanol (1.5mL) and the absorbance was read at 532nm in UV-Spectrophotometer Shimadzu® 1601[8].

Determination of the inhibition of lipid peroxidation



The determination of lipid peroxidation index was held in stomachs injured by the administration of absolute ethanol, of animals previously treated with crude ethanolic extract through the quantification of thiobarbituric acid reactive species.

Were used rats (Rattusnorvegicus, Wistar) adults, females, weighing between 150g and 300g (Protocol approved by the Ethics Committee of PUCPR,CEUA number 700).The animals (n=8) were fasted for 6 hours, with free access to water. The ethanolic extract of the stem was administered orally (gavage) in doses of 20, 100 and 500 mg/Kg, absolute ethanol (0.5ml) was also administered orally one hour after extract administration. The control group was treated with sodium chloride 0.9% (0.1mL/100 g).After an hour of administration of ethanol, the animals were killed with ketamine and xylasina and stomachs removed, weighed and homogenized with potassium phosphate buffer (200mM, pH 6.5). This homogenate (1500øl) was diluted in potassium chloride (500øl,0.15M). After that, sodium dodecyl sulfate (0.2mL, 8.1% w/v); acetic acid (1,5ml, 20%, pH 3.5) and thiobarbituric acid (1.5mL, 0.8% w/v) were added. The reaction was carried out at 95oC for 60 minutes. And the organic phase was extracted with

butanol (2.0mL) and the absorbance was read at 532nm in UV-Spectrophotometer Shimadzu® 1601. The results were expressed

in respect of the amount of protein present in the sample[9,10].

Statistical analyses

Statistical analysis of the results were carried out through the evaluation of variance ANOVA followed by analysis of difference by Tukey test (using the software Statística 5.1).


Identification of phytosterols

The identification of the substances was accomplished through the comparison of NMR data of literature[11].

NMR analysis of the sample, in CDCl3, 200 MHz, showed the

mixture of stigmasterol and sitosterol, both classified as phytosterols. The signs in Ē140.75 and 121.72 indicate the double bond between carbons C-5 and C-6 of both steroids. The double bond between C-22 and C-23, characteristic of stigmasterol, was identified by the signs Ē138.32 and 129,26. There was even a sign in Ē71.79, attributed to carbon C-3 of both sterols.

Stigmasterol: 13C NMR (200 MHz, CDCl

3)Ē in ppm: 37.23(Că

1);31.88 (Că2);71.79 (Că3); 42.28 (Că4);140.75 (Că5);121.72 (Că

6);31.88 (Că7);31.88 (Că8);50.11 (Că9);36.49 (Că10);21.06 (Că

11);39.75 (Că12);42.28 (Că13);56.75 (Că14);24.29 (Că15);29.23 (Că16);56.03 (Că17);12.23 (Că18);19.38 (Că19);40.48(Că

20);21.06(Că21);138.32(Că22);129.26(Că23);51.22 (Că24);31.63 (Că25);21.20 (Că26);19.01(Că27);25.39(Că28);12.23(Că29). Sitosterol: 13C NMR (200 MHz, CDCl

3) Ē in ppm: 37.23 (Că1);

31.88 (Că2); 71.79(Că3);42.28(Că4); 140.75(Că5); 121.72(Că6); 31.63(Că7); 31.63(Că8); 50.11(Că9); 36.49 (Că10); 21.06 (Că11); 39.75 (Că12); 42.28 (Că13); 56.75 (Că14); 24.29 (Că15); 28.23(Că16);56.03 (Că17); 11.84 (Că18); 19.38 (Că19); 36.13 (Că


The vege respo


Evalu form an o (RAA rutin(


the s fractio

The of 68 chlor show purifi comp other than crude by th

identification of etable, and still

onsible for their

oxidant activi

uation of antioxi ation determine oxidation-reductio

A) was determ (figure2).

re 2.Antioxidant

tem; FH: hexane on). Different lette

crude ethanolic 8.7% in relation roform fraction wed the same ication of the pounds with the r hand, the activ the EE, indicat e extract compo his method.


Figure 1.Phy

substances is ne aims to estab biological prope

ty - Phosphom

idant activity by s the total antio on reaction[7]. T mined by com

activity relative

fraction, FC: chlo ers represent statis

extract of the ste to the activity o (86.4%) and e activity of the extract with best capacity re vity of hexane fra ting that the ext onents that contr



ytosterols of Rh

ecessary for cha lish component erties.


phosphomolybd oxidant capacity The relative ant mparison with

to rutin. (EE: eth oroform fraction; F stically considered

em has total ant of rutin, conside ethyl acetate fr standard, indic these solvent eduction in this m action was 53.2% traction with hex

ribute to the ant



et al

. Inte

Rhamnus sphaero

aracterization of s that may be


denum complex of a sample to ioxidant activity the standard

hanolic extract of FA: ethyl acetate d for p ª 0.01.

tioxidant activity ered 100%. The raction (96.2%)

cating that the s selects the method. On the %, slightly lower xane eliminates ioxidant activity

a a

ernational Jou

osperma var. pu


This t inhibit results and 4 fractio lipid o althou lower this ex


hidroxi fraction fraction consid

As the used this measu stoma

rnal of Phytom

ubescens.1. Stigm

rmination of li

test is used to ing lipid peroxi s of in vitro and 4, respectively. ons of the stem oxidation, 63.3% ugh the rate of

(46.3%), both s xperiment.

e 3. Inhibition

butiltolueno; EE: n of the stalk, FC: n of the stem).

ered for p ª 0.05.

e crude extract in the verificatio assay, the ra urement of thio ach injured by eth

a a

medicine 5 (2


masterol, 2. Sito

ipid peroxidat

o check the ca dation induced d in vivo experim In testing in v demonstrated h % and 59.7%, hig inhibition of cru samples tested s

n of lipid pe

crude ethanolic e : chloroform fracti

Different letters

showed effectiv on of inhibition ate of peroxid obarbituric acid

hanol administra


2) 136-140 [2





tion inhibition

apacity of samp by an oxidizin ments are show vitro, chloroform high capacity in gher standard r de extract have showed satisfac

eroxidation in

extract of the ste on of the stem; FA s indicate signific

veness in both of lipoperoxidat dation was de

reactive speci ation.

c c



ples tested at ng agent. The wn in figures 3 m and hexane prevention of esult (51.1%), e been slightly ctory results in

vitro. (BHT: em; FH: hexane

A: ethyl acetate cant difference

methods was ion in vivo. In etermined by

es formed in


Figur treate mg/K consi The The inhib contr effec vivo, 16.1% with


The demo used genu demo of an Evalu phos hydro capa base subs antio a co signi prop exam desc found preve



re 4. Inhibition

ed with sodium ch Kg). Equal letters idered for p ª 0.05)

ethanolic extrac lesser dose tes biting lipid oxidat rol. The extract ctive, but also sh both doses, 10 %, respectively control, conside


samples of onstrated an im d, consistent wit us, as Rham onstrating its sig nalysis used [3-5 uation of anti sphomolybdenum

ophilic compon acity of a sample ed on the red stances with ant oxidant activity, t ompound belong

ficant antioxida erties, such as mple[12]. Some cribed for Rham d in in Rhamnu ent oxidative da


Schafer FQ, environment o

of lipid peroxid

hloride 0.9%; EE = s indicate equalit


ct was tested in t ted, 20 mg/Kg, tion, with reduct t in the highes howed ability in 0 and 500 mg/K , showed a sta ered as 100% ox

Rhamnus sph mportant antioxi th studies condu mnus alaternus

gnificant antioxid 5].

oxidant activity m complex all nents and dete e to an oxidation

uction of moly tioxidant capacit the samples sho ging to the cla ant activity as anti-inflammato e flavonoids w mnus species, li us nakaharai [13 amage can be

Buettner GR of the cell as




dation in vivo. ( = ethanolic extract ty statistics (stat

three concentrat showed better e tion of 56.9% co st concentration

preventing lipid Kg, with a reduct atistically signifi xidation.

haerosperma va dant capacity i ucted for other and Rhamn dant activity, in v

y by the form lows evaluate ermines the to n reduction react ybdenum in the

ty [6]. In determ owed a similar p ass of flavonoid ssociated with

ry and antihype with antioxidan ike quercetin a 3]. Thus, the abi associated with

. Redox s viewed



et al

. Inte

(Vehicle = group 20; 100 and 500 istical difference

tions (Figure 4). effectiveness in ompared to the ns was not as peroxidation in tion of 19.2 and cant difference

ar. pubescens n the methods

species of this nus nakaharai, arious methods

mation of the lipophilic and otal antioxidant tion. This test is e presence of mining the total

rofile with rutin, ds, which have

several other erglycaemic, for nt activity are

nd kaempferol, ility to inhibit or h the treatment

through the glutathione d


ernational Jou


and physio The i quanti peroxi on the lipids (TBA) In pre better ethano vivo te In the showe experi the lipope tested activity Acute ROS a stress which role o oxidat The a on fac involve factors absorp conce reactio There antiox doses


There Rham results extrac prope


The a Paran provid

e redox stat disulfide/glutathio

rnal of Phytom

prevention of opathology asso

inhibition of lip ification of thiob idation induced e reaction of ma due to attack by , demonstrating liminary testing, activity, with hig olic extract also est.

e animal model ed greater effec

iment, the respo highest doses eroxidation. This d in this study y of this extract. or chronic expo and RNS, lower s increases in va alcohol cause c f lipid peroxidati tive stress and a ntioxidant activit ctors determine ed. However, in s influence in th ption, distributio entration and mo on and interactio fore, further st xidant mechanism s for this use.


is no data in t mnus sphaerospe

s stimulate the ct's antioxidant

rties of this plant


authors would ná, Pontifical Ca ding necessary s

e of the one couple.

medicine 5 (2


diseases, es ociated with oxida pid peroxidation

barbituric acid r by an oxidizing alondialdehyde ( y oxidizing agen , indirectly, the a the chloroform gher inhibition ra

has activity nea

of lipid peroxi ctiveness at the onse was not d s were not s may indicate t already have m

osure to ethano rs levels of cellu arious cells and cell injury is not y ion has been ex alcohol toxicity [1 ty of a sample a ed by the chem n reactions invo he antioxidant a on and drug rete obility of the sam on with other ant tudies are still m of action for t

he literature abo erma var. pubes e continuity of mechanism o t.


like to acknow atholic Universit support.

Free Radic 212.

2) 136-140 [2




specially those ative stress. n was evaluate

reactive species agent. The me MDA), formed b nts, with the thio

amount of oxidiz and hexane frac ate than BHT, in ar the standard,

idation, the eth e lowest dose dependent on th as effective that the lowest maximum in viv

ol increases the ular antioxidants tissues. The m yet fully defined xplored in the de


about ROS and mical structure o olving living orga

activity, as meta ention in tissues mple in the envir

tioxidants [2]. l needed to d this extract and

out the biologic escens, therefore

this study, aim of action and

wledge Federal y of Paraná an

cBiol Med. 2001


e who own

ed by testing s formed from ethod is based by oxidation of barbituric acid zed lipids [14]. ctions showed n addition, the used in the in

hanolic extract tested in this he dose, since in reducing concentration ivo antioxidant production of and oxidative mechanisms by , however, the evelopment of

RNS depends of the species

anisms, many abolic rates of s, such as the ronment of the

determine the establish safe

al activities of e the obtained m identify the other related

University of nd CAPES for



et al

. International Journal of Phytomedicine 5 (2) 136-140 [2013]





[2]. Niki E. Assessment of antioxidant capacity in vitro and in vivo. Free RadicBiol Med. 2010; 49(4):503-515. URL:

http://dx.doi.org/10.1016/j.freeradbiomed .2010.04.016.

[3]. Ammar RB, Sghaier MB, Boubaker J, Bhouri W, Naffeti A, Skandrani I, Bouhlel I, Kilani S, Ghedira K, Chekir-Ghedira L.Antioxidant activity and inhibition of aflatoxin B1-, nifuroxazide-, and sodium azide-induced mutagenicity by extracts from Rhamnus alaternus L. ChemBiol Interact. 2008;174(1):1-10. URL: http://dx.doi.org/10.1016/j.cbi.2008.04.0 06.

[4]. Ammar RB, Neffati A, Skandrani I, Sghaier MB, Bhouri W, Ghedira K, Chekir-Ghedira L. Anti-lipid peroxidation and induction of apoptosis in the erythroleukaemic cell line K562 by extracts from (Tunisian) Rhamnus alaternus L. (Rhamnaceae). Nat Prod Res. 2011; 25(11):1047-58.

[5]. Ng LT, Lin CC, Lu CM. Antioxidative effects of 6-Methoxysorigenin and Its Derivatives from Rhamnus nakaharai. Chem Pharm Bull. 2007;55(3):382-4. URL:


[6]. Prieto P, Pineda M, Aguilar M. Spectrophotometric quantitation of

antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem. 1999; 269(2):337-41. URL: http://dx.doi.org/10.1006/abio.1999.4019 .

[7]. Kalegari M, Miguel MD, Philippsen AF, Dias JFG, Zanin SMW, Lima CP, Miguel OG. Antibacterial, allelopathic and antioxidant activity of extracts and compounds from Roureainduta Planch. (Connaraceae).Journal of Applied Pharmaceutical Science. 2012; 2 (09):061-066.

[8]. Morais SM, Catunda-Junior FEA, Silva ARA, Martins-Neto JS, Rondina D, Cardoso JHL. Antioxidant activity of essential oils from Northeastern Brazilian Croton species. Quim Nova. 2006; 29(05):907-910.

[9]. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.Anal Biochem. 1979; 95(2):351-8.

[10].Maróstica-Junior MR, Silva TAAR, Franchi GC, Nowill A, Pastore GM, Hyslop S. Antioxidant potential of aroma compounds obtained by limonene biotransformation of orange essential oil. Food Chemistry.2009; 116:8ă12.

[11].Kongduang D, Wungsintaweekul J, De-Eknamkul W. Biosynthesis of b-sitosterol and stigmasterol proceeds exclusively

via the mevalonate pathway in cell suspensioncultures of Croton stellatopilosus. Tetrahedron Lett. 2008;

49(18):4067ă4072. URL:

http://dx.doi.org/10.1016/j.tetlet.2008.04. 049.

[12].Kamalakkannan N, Prince PS.

Antihyperglycaemic and antioxidant effect of rutin, a polyphenolic flavonoid, in streptozotocin-induced diabetic wistar rats. Basic ClinPharmacolToxicol. 2006; 98(1):97-103.

[13].Wei BL, Lu CM, Tsao LT, Wang JP, Lin CN. In Vitro Anti-Inflammatory Effects of Quercetin 3-O-Methyl Ether and Other Constituents from Rhamnus Species.Planta Med. 2001; 67(8):745-7.

[14].Liu J, Yeo HC, Doniger SJ, Ames BN. Assay of aldehydes from lipid peroxidation: gas chromatography-mass spectrometry compared with thiobarbituric acid. Anal Biochem. 1997;

245(2):161-6. URL: http://dx.doi.org/10.1006/abio.1996.9990


[15].Pan JS, He SZ, Xu HZ, Zhan XJ, Yang XN, Xiao HM, Shi HX, Ren JL. Oxidative stress disturbs energy metabolism of mitochondria in ethanol-induced gastric mucosa injury. World J Gastroenterol. 2008; 14(38):5857-67. URL:


Figure 1.Phyytosterols of RhRhamnus sphaeroosperma var. pu ubescens.1. Stigmmasterol, 2


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