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Phytomedicine 15 (2008) 1087–1092

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Endothelium-dependent induction of vasorelaxation by Melissa officinalisL. ssp. officinalis in rat isolated thoracic aorta

S. Ersoya, I. Orhana,�, N.N. Turanb, G. S-ahanb, M. Arkb, F. Tosuna

aDepartment of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, TurkeybDepartment of Pharmacology, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey

Abstract

In the current study, vasorelaxant effect produced by the aqueous extract of Melissa officinalis L. ssp. officinalis

(MOO) (Lamiaceae) and its possible mechanism in isolated rat aortic rings precontracted with phenylephrine wereexamined. In the first series of experiments, effect of MOO on the baseline and phenylephrine (10�5M) precontractedarteries was investigated, while in the second group of experiments, endothelium intact or endothelium denuded effectwas determined. The agents used were No-nitro-L-arginine (L-NAME), an irreversible inhibitor of nitric oxide (NO)synthase, indomethacin (10 mM), a cyclooxygenase (COX) inhibitor, and glibenclamide (10 mM), an ATP-sensitivepotassium channel blocker. The extract was found to exert a vasorelaxant effect and rosmarinic acid quantity, thecharacteristic compound of the plant, was analyzed by reversed-phase high-performance liquid chromatography(18.75%), and was further confirmed by LC–MS analysis giving a prominent [M+1] molecular ion peak at m/z 365.Total phenol amount in the extract was determined using Folin–Ciocalteau reagent (0.284mg/mg extract).Vasorelaxant effect of the extract was entirely dependent on the presence of endothelium and was abolished bypretreatment with L-NAME, whereas pretreatment with indomethacin and glibenclamide reduced the relaxation to aminor extent. Rosmarinic acid was also tested in the same manner as the extract and was found to exert vasorelaxanteffect. These results suggest that the aqueous extract of MOO vasodilates via nitric oxide pathway with the possibleinvolvement of prostacycline and endothelium-derived hyperpolarizing factor (EDHF) pathways as well.r 2008 Elsevier GmbH. All rights reserved.

Keywords: Melissa officinalis ssp. officinalis; Lamiaceae; Vasorelaxant effect; Endothelium; Rat aorta; Rosmarinic acid

Introduction

The last decade has witnessed a growing interest incomplementary therapies, particularly phytotherapy.Parallel to the public interest, researchers have alsopaid attention to herbal remedies in recent years.Therefore, many traditionally used herbs have beeninvestigated in many different in vitro and in vivo studies.

e front matter r 2008 Elsevier GmbH. All rights reserved.

ymed.2008.05.007

ing author. Tel.: +90312 2023186;

35018.

ess: iorhan@gazi.edu.tr (I. Orhan).

Melissa officinalis L. (Lamiaceae), also known as‘‘lemon balm’’, is an aromatic perennial herb native toouter parts of Anatolia and Mediterranean region ofTurkey where it has been traditionally used as tea for itssedative, carminative, and antiseptic properties (Baytop,1999). In early epochs, the plant was also reported to beused for psychosomatic cardiac disorders and heartfailure by Paracelsus and Avicenna (Wichtl, 1994;Babulka, 2005). The characteristic constituents in lemonbalm have been so far reported as hydroxycinnamic acidderivatives (rosmarinic and caffeic acids) (Caniova andBrandsteterova, 2001; Toth et al., 2003; Ziakova et al.,

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2003; Boyadzhiev and Dimitrova, 2006), essential oil(Enjalbert et al., 1983; Sadraei et al., 2003; Mimica-Dukic et al., 2004; Basta et al., 2005), flavonoids(Mulkens and Kapetanidis, 1987; Heitz et al., 2000) aswell as acidic triterpenes (Herodez et al., 2003).

In the current study, our objective was to examinepossible vasorelaxant effect of the aqueous extract ofMelissa officinalis L. ssp. officinalis (MOO) in ratisolated thoracic aorta. Besides, rosmarinic acid amountwas analyzed by reversed-phase high-performance liquidchromatography (RP-HPLC) as well as liquid chroma-tography–mass spectrometry (LC–MS) and total phenolcontent of the extract was determined using Folin–Ciocalteau reagent.

Materials and methods

Plant material

The plant was collected by one of us (S.E.) fromBiyikali village of Tekirdag province (Turkey) in June2006 and identified as Melissa officinalis L. ssp.officinalis by Prof. Dr. Hayri Duman from Departmentof Biology, Faculty of Art and Science, Gazi University,Ankara (Turkey). The voucher specimen is preserved atthe Herbarium of Faculty of Pharmacy of GaziUniversity, Ankara (Turkey).

Extract preparation

The leaves of M. officinalis L. ssp. officinalis was driedin shade, chopped, and extracted with boiling distilledwater. After filtration, the aqueous phase was collectedand lyophilized to give a crude aqueous extract.

Animals

Experiments on MOO were carried out using adultmale Wistar rats weighing between 250 and 350 g. Allanimals were received in accordance to the ‘‘Guide forthe Case and Use of Laboratory Animal Resources’’.The rats were acclimatized to a 12-h light:12-h darkcycle at 25 1C and supplied with standard laboratorydiet and tap water ad libidum. They were brought dailyto the laboratory for the experiments, which compliedwith the Guide for the Care and Use of LaboratoryAnimals (NIH Publication No. 85–23, revised 1996) andwere approved by the Ethics Committee of GaziUniversity.

Chemicals

All drugs were obtained from Sigma Chemical Co.(St. Louis, MO, USA). A stock solution of noradrena-

line was prepared in 0.001N HCl and ascorbic acid wasadded to prevent oxidation. Acetylcholine was dissolvedin 0.001N HCl, glibenclamide was dissolved in di-methylsulfoxide (DMSO), indomethacin was dissolvedin 150mM NaHCO3, whereas all other drugs weredissolved in saline (0.9%). All subsequent dilutions wereprepared in Krebs–Henseleit solution and kept in a coldand dark medium. The vehicles were found to have noeffect on the relaxation responses.

On the other hand, rosmarinic acid for HPLCanalysis was purchased from Sigma-Aldrich (Fluka-44699, Buchs, Switzerland). Chromatographic grade-double distilled water, HPLC grade methanol (Merck-1,06007), isopropyl alcohol (Merck-101040), and analy-tical grade ortho-phosphoric acid 85% (Merck-563)were used. Gallic acid and Folin–Ciocalteau’s reagentwere obtained from Sigma-Aldrich Chemie GmbH(Taufkirchen, Germany).

Determination of vasorelaxant effect

The rats were sacrificed after ether anesthetized andthe thoracic aorta was removed, cleaned off adherentconnective tissues, and cut into rings of 4–5mm long.Segments were suspended in a water-jacketed organbath of 10ml filled with Krebs–Heinseleit solution(37 1C) of the following composition in mM: NaCl119; NaHCO3 25; KCl 4.6; MgCl 21.2; KH2PO4 1.2;CaCl2 2.5; and glucose 11. The solution was aeratedwith a gas mixture containing 95% O2:5% CO2. Therings were suspended on a pair of stainless-steel hooks,one of which was fixed to an L-shaped rod inside thechamber and the other to an isometric transducer(PowerLab ML750) under optimum resting force. Thestainless-steel hook was connected to the force displace-ment transducer. Isometric contractions were measuredand recorded continuously in a computer by using theLabsys computer program w17x. Arterial rings wereequilibrated in Krebs–Heinseleit solution for 1 h at 1 goptimum resting force. In some rings, the endotheliumwas removed gently by rubbing the luminal surface ofthe ring with a roughened polyethylene tube. At the endof the equilibration period of 1 h, viabilities of thearteries segments with and without endothelium werechecked by KCl (60mM) and phenylephrine (10�5M).The effectiveness of endothelium removal was confirmedby the inability of acetylcholine (10�6M) to inducerelaxation on phenylephrine precontracted rubbed ringsand confirmed by a less than 10% relaxation. Theaqueous extract of MOO was dissolved in distilledwater (1mgml�1), and dilution (0.001–1mgml�1) wasmade by Krebs–Henseleit solution. In the first series ofexperiments, effect of the extract on the baseline andphenylephrine (10�5M) precontracted arteries wasinvestigated, while endothelium intact or endothelium

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denuded effect was determined in the second group ofexperiments. Finally, we investigated vasorelaxantmechanisms of MOO on endothelium intact arteries.In this series of experiments, endothelium intact arterieswere incubated for 30min with inhibitors of productionof diverse endothelium-derived relaxing factors beforeprecontraction with phenylephrine. An irreversible nitricoxide (NO) synthase inhibitor; L-NAME (No-nitro-L-arginine-2.5� 10�4M), a cyclooxygenase (COX)inhibitor; indomethacin (10 mM), and an ATP-sensitivepotassium channel blocker; glibenclamide (10 mM) weretested separately. Both control and treated ringsprecontracted and the MOO extract cumulative con-centration–response curve was made. Rosmarinic acidwas also tested for its vasorelaxant effect applying thesame experimental protocol.

HPLC apparatus

Rosmarinic acid analysis was performed with an LCsystem consisting of an HP Agilent 1100 seriesquaternary pump with a degasser and photodiode arraydetector. The samples were injected to an HP Agilent1100 Autosamplers with thermostatted column com-partment on a Phenomenex-Hyperclone ODS C18

column (5 m, 250mm; 4.6mm) at 30 1C. The systemwas controlled and data analysis was performed withAgilent ChemStation software. All the calculationsconcerning the quantitative analysis were performedwith external standardization by measurement of thepeak areas.

Chromatographic conditions

HPLC analysis was performed by a gradient elutionwith flow rate of 1.0mlmin�1 as described elsewhere(Kan et al., 2007). Briefly describing, the mobilephase was delivered from three separate containerswith gradient elution program. The first container waso-phosphoric acid 0.085% in water (solution A) andthe second container was o-phosphoric acid 0.085% inmethanol (solution B), while the third one waso-phosphoric acid 0.085% in 2-propanol (solution C).All solvents were filtered through a 0.45 mm Milliporefilter prior to use and degassed in an ultrasonic bath.A gradient system with o-phosphoric acid 0.085% inwater (A), o-phosphoric acid 0.085% in methanol (B), ando-phosphoric acid 0.085% in 2-propanol (C) were used.

Total phenol content

Phenolic compounds were assayed according to theFolin–Ciocalteau method (Singleton and Rossi, 1965).Briefly, the samples (150 ml) were put into test tubes;750 ml of Folin–Ciocalteau’s reagent and 600 ml of

sodium carbonate (7.5%) were added. The tubes werevortexed and incubated at 40 1C for 30min. Later,absorption was measured at 760 nm. The total phenoliccontent was expressed as gallic acid equivalents.

Statistical analysis

All biological activity data were expressed as themean7SEM for the number of experiments indicated.The relaxations to MOO and rosmarinic acid wereexpressed as the percent decreases of the precontractionto KCl. The sensitivity of arteries to MOO is expressedas the effective concentrations that elicited 50% of themaximal responses (EC50) and maximal decrease intension (Emax). EC50 values were expressed as negativelog M. The responses were quantified in terms of EC50

values, obtained from the concentration–response curvefor MOO by nonlinear curve fitting, using the Prism 3Graph Pad program. Statistical analysis was performedby Student’s-t test in order to evaluate the differencebetween two groups at the same time and the one-wayANOVA. Values were considered to be significantlydifferent when the P value was less than 0.05.

Results

The aqueous extract of MOO (1–1000 mg/ml) pro-duced concentration-dependent relaxation in pheny-lephrine-precontracted endothelium intact thoracicaorta rings (Emax: 9171.5%, �logEC50: 1.6470.02,n ¼ 6), while abolished in de-endothelised rings in alldoses (Emax: 1.570.3%, �logEC50: 1.6170.038, n ¼ 8).The extract caused concentration-dependent relaxationsin endothelium-intact aortic rings precontracted withphenylephrine (Fig. 1). The relaxation effects of MOOwas significantly abolished by L-NAME (2.4� 10–4M)(Emax: 7.3270.34%, �logEC50: 1.470.34; n ¼ 8)(Fig. 1). Glibenclamide and indomethacin significantlyreduced the vasorelaxant effect of MOO in 1 and0.3mgml�1 doses (Emax: 53.470.12%, �logEC50:2.070.12 n ¼ 8, and Emax: 5070.44%; �logEC50:1.9570.02, n ¼ 8, respectively). Rosmarinic acid alsoproduced a dose-dependent vasorelaxant effect in thesame experiment as seen in Fig. 1.

Quantity of rosmarinic acid analyzed by RP-HPLCwas found to be 18.75% in the aqueous extract of MOO.The extract possessed 0.284mg/mg extract of totalphenol amount as gallic acid equivalent using Folin–Ciocalteau reagent.

We herein studied the aqueous extract of MOO inthoracic aorta strips and observed a concentration-dependent vasorelaxant effect in arteries precontractedwith phenylephrine. Damaging the intima of aortastrips or pretreatment with L-NAME almost abolished

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0.0 0.5 1.0 1.5 2.0 2.5 3.0

0

50

100

Control E(+)L-NAMEIndomethacinGlibenclamide

* * * * *

* *R

elax

atio

n (%

)

-6 -5 -4 -3

0

50

100

Concentration of rosmarinic acid log [M]

Rel

axat

ion

(%)

Concentration of M. officinalis log µg/ml

Fig. 1. Relaxations to cumulative-concentration of Melissa officinalis L. ssp. officinalis (MOO) in rat isolated aorta rings with (+E)

and without (�E) endothelium (A) and rosmarinic acid (B). The effects of L-NAME (2.5� 10�4M); indomethacin (10 mM), and

glibenclamide (10 mM) on the concentration–relaxation curves to MOO with endothelium intact rat aorta rings. The relaxations to

MOO were expressed as the percent decreases of the precontraction to KCl. *Po0.05 vs control. Data are represented as

mean7SEM (n ¼ 6–12).

S. Ersoy et al. / Phytomedicine 15 (2008) 1087–10921090

MOO-induced relaxation, whereas pretreatment withindomethacin and glibenclamide only decreased therelaxation at high concentration of the extract.

Although M. officinalis, as a medicinal plant, has beenrecorded for its utilization against some cardiacdisorders, there is a restricted knowledge about itscardiovascular activity. If the biochemical study per-formed on hyperlipidemic rats in which the M. officinalis

extract exerted a hypolipidemic effect is excluded(Bolkent et al., 2005), there are limited reports inliterature investigating its cardiac activity. In one studywhich was performed on the isolated hearts of rats,M. officinalis extract provoked significant cardiac ratereduction but did not alter the contractile force (Gazolaet al., 2004).

The vascular endothelium performs a wide array ofhomeostatic functions within normal blood vessels.Located between the vascular lumen and the smoothmuscle cells of the vessel wall, the monolayer ofendothelial cells is able to transduce blood-bornesignals, sense mechanical forces within the lumen, andregulate vascular tone through the production of avariety of factors. Endothelium produces potent vaso-dilators, such as endothelium-derived relaxing factor(EDRF, NO), prostacyclin and endothelium-derivedhyperpolarizing factor (EDHF). Perhaps, the mostimportant vasodilator substance produced by endothe-lial cells is endothelium-derived relaxing factor (EDRF),which has been identified as the nitric oxide (NO) radical(Ganz and Ganz, 2001). NO is the predominantmediator in large conduit arteries, whereas EDHF andprostacyclin are more prominent in smaller vessels suchas the mesenteric vessels, coronary arteries, andperipheral resistance vessels (Shimokawa et al., 1996).

Up to date, innumerable studies have been reported inherbal extracts and some of them made a conclusionabout the active component(s) responsible for the

vasorelaxant effect of those extracts. In those studieswhere the herbal extracts showed vasodilator effect onisolated aorta strips, as a result of investigatingthe underlying mechanism, some were suggested to beeffective by releasing NO through endothelium, whileseveral others were suggested to show their vasorelaxanteffect not only via NO pathway, but also EDHF and/orprostacyclin pathways which play a significant role inthe process. In the present study, we also suggest thatthe aqueous extract of MOO has an endothelium-dependent vasorelaxant effect via NO pathway, invol-ving EDHF and prostacyclin pathways as well.

Many earlier studies with plant extracts which haveused similar experimental models proved that phenolicconstituents such as flavonoids, glucose derivatives,coumarines, phenylethanoids, and other polyphenolsare possibly accountable for vasorelaxant activity (Koet al., 1991; Goto et al., 1996; Lee et al., 2002; Legssyeret al., 2004; Brixius et al., 2006; Yoshikawa et al., 2006;Rocha et al., 2007; Anselm et al., 2007).

These studies have indicated that in addition to andindependently from their antioxidant effects, polyphe-nols enhance the productions of vasodilating factors;NO, endothelium-derived factor (EDHF), together withprostacyclin and inhibit the synthesis of vasoconstrictorendothelin-1 in endothelial cells. Besides, other probablemechanisms are the increased level of Ca2+ and redox-sensitive activation of the phosphoinositide-3 (PI3)-kinase/Akt pathway (leading to rapid and sustainedactivation of NOS and formation of EDHF) andenhanced expression of NOS (Stoclet et al., 2004).

In our study in which the aqueous extract of MOOwas revealed to exert an endothelium-dependent vasor-elaxation in rat thoracic aorta, the extract was furtherexamined by different analytical methods. Total phe-nolic content of the extract (0.284mg/mg extract)emphasized that the extract is quite rich in phenolics

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Fig. 2. LC–MS spectrum of the aqueous extract of MOO (Rt: 3.47, rosmarinic acid).

S. Ersoy et al. / Phytomedicine 15 (2008) 1087–1092 1091

and HPLC analysis supported as well that rosmarinicacid was the dominant phenolic acid (18.75%). Inaddition, LC–MS data indicated the presence ofrosmarinic acid again in the extract as the majorcomponent giving a single abundant peak in total ionchromatogram (TIC) that emerged at 3.47min (Fig. 2).Mass spectrum of the peak scanned in positive modewith electron-spray mass detector gave a prominent[M+1] molecular ion peak at m/z 365 which clearlydefined the molecular weight of rosmarinic acid, whichis consistent with a previous report (Almela et al., 2006).As rosmarinic acid was the most abundant in the MOOextract, we also tested the compound for its possiblevasorelaxant effect and observed that it exerted a dose-dependent vasorelaxative effect.

In conclusion, our results demonstrated that theaqueous extract of MOO possessed the concentration-dependent vasorelaxant activity in phenylephrine-pre-contracted thoracic aorta rings with endothelium. Thisvasorelaxant activity is caused by the stimulation ofendothelial nitric oxide formation with the possibleinvolvements of prostacyclin and EDHF activation aswell. The phytochemical analysis of the extract clearlyshowed the existence of phenolic compounds withrosmarinic acid as the main compound. Since purerosmarinic acid also produced vasorelaxation in the

same experimental model, we suggest that the vasor-elaxation produced by the MOO extract might probablybe due to phenolic compounds, with rosmarinic acid asthe major contributor to the mentioned effect. To thebest of our knowledge, we herein report the first studyon vasorelaxant effect of MOO and rosmarinic acid inisolated rat thoracic aorta. Finally, we would like to addthat under the illuminations of the findings above,M. officinalis ssp. officinalis could be considered as aphytotherapeutic co-medication for the treatment ofcardiovascular diseases. Supplementary studies shouldbe done to investigate its cardioprotective potency inmore details.

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