EKSPERIMENTINIAI TYRIMAI

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1 406 EKSPERIMENTINIAI TYRIMAI Inotropic and lusitropic effects of Perilla frutescens (L.) Britton extract on the rabbit myocardium Igor Korotkich, Žibuoklė Senikienė, Genovaitė Šimonienė, Robertas Lažauskas, Alė Laukevičienė, Egidijus Kėvelaitis Department of Physiology, Kaunas University of Medicine, Lithuania Key words: Perilla frutescens, myocardial contractility, inotropic effect, lusitropic effect. Summary. Background. Common perilla (Perilla frutescens (L.) Britton) is a plant cultivated in many countries around the world. Although its immunomodulating and antioxidative properties are well known, there is a lack of data about the cardiotropic activity of the plant. The objective of this study was to determine the influence of Perilla frutescens extract on the myocardial contractility in vitro and as a food supplement in vivo. Material and methods. Rabbits of the experimental group were fed with a supplement of 100 mg/kg of Perilla frutescens extract for 14 days. Rabbits of control group were fed with ordinary food. The maximal mechanical activity of isolated myocardial preparations, obtained from the rabbits of both groups, was tested during the perfusion with Ringer s solution containing 5 µm of adrenaline and 4.5 mm of CaCl 2. For the assessment of the direct influence of Perilla frutescens extract on the myocardial contractility in vitro isolated heart preparations were perfused with 0.01, 0.1, and 1.0 mg/ml of Perilla frutescens extract. Results. The maximum force of isometric contraction, maximum velocity of force development, and maximum velocity of relaxation were higher among the atrial and ventricular preparations from the experimental group, as compared with the control group. Perfusion of the myocardial preparations with different concentrations of Perilla frutescens extract revealed slight dosedependent increase in the parameters of contraction and relaxation. Conclusions. The consumption of Perilla frutescens extract as a food supplement leads to an increase in the contractility of the rabbit myocardium. Perilla frutescens extract in vitro had a dose-dependent positive inotropic and lusitropic effect on the rabbit myocardium. Introduction Common perilla, Perilla frutescens (L.) Britton, plant of the Laminaceae family native to South-East Asia, which is successfully cultivated all over the world, is becoming very promising herb in Lithuania. Its well-known antimicrobial, antiallergic, antitumor, and antioxidative properties are used extensively and constantly investigated (1 4). In spite of widespread occurrence there are only few references regarding the influence of P. frutescens preparations on cardiovascular system. Some authors describe cytoprotective effects, using cardiotoxic drugs, which are related to the antioxidative properties of the substances isolated from P. frutescens (5, 6). O. Ezaki et al. report longterm influence of perilla oil-containing diet on the cardiovascular system, which is related to the increase of serum omega-3 polyunsaturated fatty acids (7). Although the recent investigations refer to the vasoactive properties of certain flavonoids (8, 9), influence of P. frutescens preparations on contractile function of myocardium still remains undisclosed. The aim of the present study was to determine the influence of P. frutescens extract on the mechanical properties of the contractile myocardium in vitro and effect of P. frutescens extract used as a food supplement on contractility of the myocardium. Material and methods Frozen-dried P. frutescens extract was produced by Department of Food Technology of Kaunas University of Technology. Chinchilla rabbits of both sexes (3 3.5 kg, 6 10 Correspondence to I. Korotkich, Department of Physiology, Kaunas University of Medicine, A. Mickevičiaus 9, Kaunas, Lithuania. igor@kmu.lt

2 Inotropic and lusitropic effects of Perilla frutescens (L.) Britton extract on the rabbit myocardium 407 months old) were divided into two groups: control and experimental. Rabbits of the control group were fed with ordinary food. Rabbits of the experimental group were fed with an addition of 100 mg/kg of P. frutescens extract during 14 days before the experiments. The experiments were carried out in vitro on the isolated preparations of rabbits left atria and right ventricle. Animals were killed in accordance with ethical guidelines approved by Lithuanian State Food and Veterinary Service (License No. 0112). The chest was opened; the heart promptly excised and immersed in a modified Ringer s solution consisting of mm NaCl, 3.5 mm KCl, 0.58 mm NaH 2 PO 4, 2.1 mm Na 2 HPO 4, 2.5 mm CaCl 2, 1.3 mm MgCl 2, 11.1 mm glucose; ph was adjusted to with NaHCO 3. The preparation ready for experiment, stripe of the auricle from the left atria or papillary muscle from the right ventricle, was transferred into the organ bath perfused with the oxygenated Ringer s solution. The bath temperature was maintained at 25±0.5 o C by means of thermostatically controlled water jacket. Mechanical activity of the preparations was registered by micromechanographic device Mioton- CAO12 under isometric conditions. Preparation was stimulated by suprathreshold rectangular electrical pulses of 0.3 Hz, V, 3 10 ms, delivered by a pair of Ag/AgCl electrodes placed inside the organ bath. Electrodes were arranged along the papillary muscle or stripe of the atria in order to provide simultaneous excitation of the entire preparation. The preparation was stretched to an optimal length, until the amplitude of isometric contraction reached its maximum. Before starting the experimental procedures, the preparation was allowed to stabilize for about 1.5 hours. The preparation was considered to be well adapted when its force amplitude remained unchanged. To determine the possible differences in the mechanical properties of myocardium obtained from experimental and control groups of rabbits, preparations were superfused with Ringer s solution containing 5 µm of adrenaline and 4.5 mm of CaCl 2. Previous studies have shown that in general the combination of adrenaline and calcium chloride is optimal in order to reach the state of maximum contraction comparing the contractility of the myocardium preparations obtained from the different groups of rabbits (10). The direct influence of P. frutescens extract on the mechanical properties of contractile myocardium was also examined. Preparations from the part of rabbits of control group were perfused with the Ringer s solution containing three different concentrations of extract: 0.01 mg/ml, 0.1 mg/ml, and 1.0 mg/ml. In all cases mechanical activity of preparations was registered during the sixth minute after the change of the solution in the organ bath. Data were recorded by the polygraph and stored in a computer for off-line processing using the original software. To evaluate mechanical activity of preparations, the following parameters were measured: maximum force of isometric contraction, ; maximum velocity of force development, +dp/dt; maximum velocity of relaxation, dp/dt. Data are reported as mean±sem. Each parameter is expressed in percent, comparing to its baseline value. Data obtained during investigation of the direct influence of P. frutescens extract on the mechanical properties of contractile myocardium are also expressed in absolute values (mn) and standardized per square unit of section of the preparation (mm 2 ). The Student s t-test was used to determine differences between the means. Results were considered to be statistically different when p<0.05. Results All parameters of mechanical activity registered under the influence of 5 µm of adrenaline with higher concentration of CaCl 2 on the myocardial preparations obtained from the rabbits of experimental group were increased, as compared with the preparations obtained from the control group. Maximum force of isometric contraction and maximum velocity of force development were significantly higher testing the preparations of left atria (p<0.01) and right ventricle (p<0.05) from the rabbits of experimental group, than the same parameters of preparations obtained from the control group (Fig. 1). Maximum velocity of relaxation was also increased among the preparations of left atria from the experimental group (p<0.001), while the increase in this parameter during the test of the right ventricle from the experimental group was not statistically significant (Fig. 1). Superfusion of myocardial preparations with different concentrations of P. frutescens extract enhanced their mechanical activity even when lowest concentration of extract (0.01 mg/ml) was used. At this concentration the increase of maximum force of isometric contraction and maximum velocity of force development was statistically significant (p<0.05) on the preparations of right ventricle (Fig. 2B, Table), in contrast to the preparations of left atria (Fig. 2A, Table). Higher concentrations of extract (0.1 and 1.0 mg/ml) caused further increase of these parameters of both left atria and right ventricle.

3 408 Igor Korotkich, Žibuoklė Senikienė, Genovaitė Šimonienė et al. control group (n=18) experimental group (n=5) control group (n=9) experimental group (n=6) Fig. 1. Parameters of isometric contraction registered on the preparations of left atria (A) and right ventricle (B) from the rabbits of experimental and control group during the perfusion with Ringer s solution containing 5 µm of adrenaline and 4.5 mm of CaCl 2 maximum force of isometric contraction; +dp/dt maximum velocity of force development; dp/dt maximum velocity of relaxation. * p<0.05; ** p<0.01; *** p<0.001 as compared to control group. A statistically significant increase (p<0.05) in maximum velocity of relaxation of the preparation from right ventricle was observed only at concentrations of extract, 0.1 and 1.0 mg/ml (Fig. 2B, Table), whereas none of the concentrations could cause a statistically significant increase in the maximum velocity of relaxation among the preparations of left atria (Fig. 2A, Table).

4 Inotropic and lusitropic effects of Perilla frutescens (L.) Britton extract on the rabbit myocardium 409 baseline PFE 0.01 mg/ml PFE 0.1 mg/ml PFE 1 mg/ml n=5 baseline PFE 0.01 mg/ml PFE 0.1 mg/ml PFE 1 mg/ml n=6 Fig. 2. Effect of different concentrations of Perilla frutescens extract on the parameters of isometric contraction of the preparations from left atria (A) and right ventricle (B) of the rabbits maximum force of isometric contraction; +dp/dt maximum velocity of force development; dp/dt maximum velocity of relaxation; PFE Perilla frutescens extract. Baseline parameters were registered during the perfusion with Ringer s solution. * p<0.05; ** p<0.01 as compared to baseline. Discussion In the present study we investigated the influence of P. frutescens extract on the mechanical properties of mammalian myocardium. The preparations from the left atria and right ventricle of the rabbits, which were fed with an addition of P. frutescens extract during two weeks before the experiment, demonstrated an increase in the maximum mechanical response

5 410 Igor Korotkich, Žibuoklė Senikienė, Genovaitė Šimonienė et al. Table. Effect of different concentrations of Perilla frutescens extract on the parameters of isometric contraction of the preparations from left atria and right ventricle of the rabbits (absolute values) Concentration of PFE Left atria (n=5) Right ventricle (n=6) mn/mm 2 mn/s/mm 2 mn/s/mm 2 mn/mm 2 mn/s/mm 2 mn/s/mm 2 Baseline 8.15± ± ± ± ± ± mg/ml 9.01± ± ± ±5.36* 68.35±23.19* 34.48± mg/ml 9.14± ±16.19* 46.62± ±5.13* 72.71±23.30* 37.26±11.25* 1.0 mg/ml 10.30±1.48** ±22.06* 53.35± ±5.12** 76.16±22.85** 37.56±11.10* maximum force of isometric contraction; +dp/dt maximum velocity of force development; dp/dt maximum velocity of relaxation; PFE Perilla frutescens extract. Baseline parameters were registered during the perfusion with Ringer s solution. * p<0.05; ** p<0.01 as compared to baseline. during the perfusion with Ringer s solution containing 5 µm of adrenaline and 4.5 mm of CaCl 2, as compared with the preparations obtained from the control group. These data conform to the results obtained in another part of the study, where perfusion of the preparations with the Ringer s solution, containing different concentrations of P. frutescens extract, revealed dosedependent positive inotropic and positive lusitropic effects on the myocardium of left atria and right ventricle of the rabbits. Such effects can probably be explained by the presence of certain substances in the extract which affect several aspects of calcium metabolism in the cardiomyocyte: 1) increase of inward calcium current through the L-type calcium membrane channels; and 2) accelerate an uptake of calcium into the sarcoplasmic reticulum. P. frutescens leaves and fruits are known to be rich of flavonoids and polyphenolics. Among them, catechin, ferulic acid, apigenin, luteolin, rosmarinic acid, and caffeic acid are mentioned as the most important active ingredients of the plant (11). Although polyphenolics, rosmarinic, caffeic acids and their derivatives are intensively tested due to their cytoprotective antioxidative properties using cardiotoxic drugs (6, 12) or during ischemia-reperfusion injury (13 15), there are no references regarding their direct influence on the myocardial contractility. Some flavonoids, such as ferulic acid, apigenin, luteolin, besides their antioxidative characteristics also possess vasorelaxing properties (8, 9, 16, 17). In addition, as it is reported by M. Itoigawa et al., apigenin and luteolin produced a moderate positive inotropic effect on guinea pig papillary muscle, while catechins did not produce any positive inotropic effect. Authors suppose the presence of cyclic AMP-dependent mechanism for the positive inotropic effect of flavonoids (18). During another study, luteolin derivative, luteolin-7-glucoside, was tested on isolated Langendorff-perfused guinea pig heart. Observed slight positive inotropic and positive lusitropic effects were explained by the possible inhibition of phosphodiesterase (19). Thus, on the supposition that P. frutescens contains substances which increase the amount of cyclic AMP and/or inhibit cyclic AMP splitting enzyme in cardiomyocytes, it is possible to explain contractile effects of P. frutescens extract. Cyclic AMP via protein kinase A increases the entry of calcium ions through the L-type calcium channels and enhances calcium-induced calcium release from sarcoplasmic reticulum. As a consequence, peak force and velocity of contraction increase (positive inotropic effect). Due to phosphorylation of phospholamban the rate of uptake of calcium into the sarcoplasmic reticulum rises, causing an increase in velocity of relaxation (positive lusitropic effect). Although some flavonoids seem to be able to affect contractility of the myocardium, it is important to emphasize that since P. frutescens extract is a mixture of several substances, it cannot be excluded that we are observing the result of several simultaneous influences. That is why additional studies of composition of P. frutescens extract and its influence on the myocardium are required. Conclusions The consumption of Perilla frutescens extract as a food supplement leads to an increase in the contractility of the rabbit myocardium. Perilla frutescens extract in vitro has a dose-dependent positive inotropic and positive lusitropic effects on the rabbit myocardium. Acknowledgement This study was supported by Lithuanian State Science and Studies Foundation (contract No. C-03/ 2005).

6 Inotropic and lusitropic effects of Perilla frutescens (L.) Britton extract on the rabbit myocardium 411 Krūminės perilės (Perilla frutescens (L.) Britton) ekstrakto inotropinis ir luzitropinis poveikis triušių miokardui Igor Korotkich, Žibuoklė Senikienė, Genovaitė Šimonienė, Robertas Lažauskas, Alė Laukevičienė, Egidijus Kėvelaitis Kauno medicinos universiteto Fiziologijos katedra Raktažodžiai: krūminė perilė, miokardo kontraktiliškumas, inotropinis poveikis, luzitropinis poveikis. Santrauka. Krūminė perilė, Perilla frutescens (L. Britton) tai vaistinis augalas nuo seno auginamas daugelyje šalių. Nors antimikrobinės, priešalerginės, priešnavikinės, antioksidacinės šio augalo savybės gerai žinomos, tačiau duomenų apie Perilla frutescens preparatų įtaką širdies mechaniniam aktyvumui yra mažai. Tyrimo tikslas. Nustatyti Perilla frutescens ekstrakto poveikį miokardo kontraktiliškumui in vitro ir jo kaip maisto papildo vartojimą in vivo. Medžiaga ir metodai. Eksperimentinės grupės triušiai prieš tyrimą kasdien (14 dienų) kartu su maistu gaudavo 100 mg/kg Perilla frutescens ekstrakto. Kontrolinės grupės triušiai buvo maitinami įprastu maistu. Po 14 dienų atlikti abiejų grupių triušių izoliuotų miokardo preparatų maksimalaus mechaninio aktyvumo tyrimai perfuzuojant juos Ringerio tirpalu, turinčiu 5 µm adrenalino ir 4,5 mm CaCl 2. Tiriant tiesioginį Perilla frutescens ekstrakto poveikį in vitro miokardo preparatai buvo perfuzuojami Ringerio tirpalu su trimis skirtingomis krūminės perilės ekstrakto koncentracijomis: 0,01 mg/ml; 0,1 mg/ml ir 1,0 mg/ml. Rezultatai. Eksperimentinės grupės triušių kairiojo prieširdžio ir dešiniojo skilvelio preparatų maksimali izometrinio susitraukimo jėga, maksimalus izometrinio susitraukimo greitis ir maksimalus izometrinio atsipalaidavimo greitis buvo didesni nei kontrolinės grupės triušių. Perfuzuojant skirtingomis Perilla frutescens ekstrakto koncentracijomis, nustatytas priklausomas miokardo preparatų susitraukimo ir atsipalaidavimo rodmenų padidėjimas, priklausomas nuo dozės. Išvados. Perilla frutescens ekstraktas, vartojamas su maistu, didina triušių miokardo kontraktiliškumą. Perilla frutescens ekstraktas in vitro sukelia nuo dozės priklausomą teigiamą inotropinį ir luzitropinį poveikį triušių miokardui. Adresas susirašinėti: I. Korotkich, KMU Fiziologijos katedra, A. Mickevičiaus 9, Kaunas El. paštas: igor@kmu.lt References 1. Sanbongi C, Takano H, Osakabe N, Sasa N, Natsume M, Yanagisawa R, et al. Rosmarinic acid in perilla extract inhibits allergic inflammation induced by mite allergen, in a mouse model. Clin Exp Allergy 2004;34(6): Ragažinskienė O, Gailys V, Jankauskienė K, Šimonienė G, Jurkštienė V. Krūminė perilė (Perilla frutescens (L.) Britton) perspektyvus imunomoduliatorius. (Common perilla (Perilla frutescens (L.) Britton.) as a perspective immunomodulator). Medicina (Kaunas) 2004;40(3): Osakabe N, Yasuda A, Natsume M, Yoshikawa T. Rosmarinic acid inhibits epidermal inflammatory responses: anticarcinogenic effect of Perilla frutescens extract in the murine twostage skin model. Carcinogenesis 2004;25(4): Takano H, Osakabe N, Sanbongi C, Yanagisawa R, Inoue K, Yasuda A, et al. Extract of Perilla frutescens enriched for rosmarinic acid, a polyphenolic phytochemical, inhibits seasonal allergic rhinoconjunctivitis in humans. Exp Biol Med (Maywood) 2004;229(3): Psotova J, Chlopcikova S, Miketova P, Hrbac J, Simanek V. Chemoprotective effect of plant phenolics against anthracycline-induced toxicity on rat cardiomyocytes. Part III. Apigenin, baicalelin, kaempherol, luteolin and quercetin. Phytother Res 2004;18(7): Kim DS, Kim HR, Woo ER, Hong ST, Chae HJ, Chae SW. Inhibitory effects of rosmarinic acid on adriamycin-induced apoptosis in H9c2 cardiac muscle cells by inhibiting reactive oxygen species and the activations of c-jun N-terminal kinase and extracellular signal-regulated kinase. Biochem Pharmacol 2005;70(7): Ezaki O, Takahashi M, Shigematsu T, Shimamura K, Kimura J, Ezaki H, et al. Long-term effects of dietary alpha-linolenic acid from perilla oil on serum fatty acids composition and on the risk factors of coronary heart disease in Japanese elderly subjects. J Nutr Sci Vitaminol (Tokyo) 1999;45(6): Woodman OL, Chan E. Vascular and anti-oxidant actions of flavonols and flavones. Clin Exp Pharmacol Physiol 2004; 31(11): Calderone V, Chericoni S, Martinelli C, Testai L, Nardi A, Morelli I, et al. Vasorelaxing effects of flavonoids: investigation on the possible involvement of potassium channels. Naunyn Schmiedebergs Arch Pharmacol 2004;370(4): Senikienė Ž, Abraitis R, Laukevičienė A, Kėvelaitis E, Lažauskas R. Anafilaksinio šoko ir amžiaus įtaka miokardo kontraktiliškumui. (Influence of anaphylactic shock and age on the myocardial contractility.) Medicina (Kaunas) 1999; 35(4):

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