Oʻzbekiston Biologiya Jurnali 1 2019 Узбекский Биологический Журнал
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OʻZBEKISTON RESPUBLIKASI FANLAR AKADEMIYASI АКАДЕМИЯ НАУК РЕСПУБЛИКИ УЗБЕКИСТАН OʻZBEKISTON BIOLOGIYA JURNALI 1 2019 УЗБЕКСКИЙ БИОЛОГИЧЕСКИЙ ЖУРНАЛ Издается с января 1957 г. по 6 номеров в год ТАШКЕНТ – 2019 РЕДАКЦИОННАЯ КОЛЛЕГИЯ Б.А. ТАШМУХАМЕДОВ (главный редактор) И.У. АТАБЕКОВ (ответственный секретарь) А.А. АБДУКАРИМОВ Дж.А. АЗИМОВ Т.Ф. АРИПОВ М.И. МАВЛОНИЙ И.М. МИРАБДУЛЛАЕВ В.П. ПЕЧЕНИЦЫН Т.С. СААТОВ Р. САБИРОВ Дж. С. САТТАРОВ П.Б. УСМАНОВ Адрес редакции: 100047, Ташкент, ул. Я. Гулямова, 70. Телефон (71)232-11-81 На обложке: Торғоқ Кречётка Chettusia gregaria (Pallas, 1771) Журнал зарегистрирован Агентством по печати и информации Республики Узбекистан 22.12.2006 Регистрационный номер 0052. 2 БИОХИМИЯ И БИОФИЗИКА S.N. GAYIBOVA, S.Z. OMONTURDIYEV, V.N. SYROV, T.F. ARIPOV VASORELAXANT EFFECT OF CYNAROSIDE ON RAT AORTIC RINGS [email protected] С.Н. Гайибова, С.З. Омонтурдиев, В.Н. Сыров, Т.Ф. Арипов КАЛАМУШ АОРТАСИДАН АЖРАТИБ ОЛИНГАН СЕГМЕНТЛАРИДА ЦИНАРОЗИДНИ ВАЗОРЕЛАКСАНТ ТАЪСИРИНИ ЎРГАНИШ Ўсимликлардан ажратиб олинган биологик фаол бирикмалар силлиқ мускул ҳужайралардаги Ca2+- транспорт тизими функцияларини ўзига ҳос модификация қилиш ҳусусиятига эга. Мазкур тадқиқот доира- сида цинарозидни ажратиб олинган каламуш аорта препаратидаги силлиқ мускул ҳужайраларининг қасқаришига таъсири ўрганилган. Цинарозид аорта препаратини, 50 мМ KCl билан дастлаб қисқартирилган ҳолатда, кальций ионлари таъсирида ҳамда кальций канал блокатори верапамил таъсирида, қисқартириш ҳусусияти кўрсатиб берилган. Шундай қилиб, цинарозидни релаксант таъсири потенциалга боғлиқ кальций каналларига таъсири туфайли бўлиши мумкинлиги таҳмин қилинди. Калит сўзлар: флавонлар, қисқариш, силлиқ мускул ҳужайралари, кальций канал блокаторлари. С.Н. Гайибова, С.З. Омонтурдиев, В.Н. Сыров, Т.Ф. Арипов ИЗУЧЕНИЕ ВАЗОРЕЛАКСАНТНОГО ДЕЙСТВИЯ ЦИНАРОЗИДА НА ИЗОЛИРОВАННЫХ СЕГМЕНТАХ АОРТЫ КРЫСЫ Биологически активные соединения растительного происхождения обладают уникальным свойством модифицировать функцию Ca2+-транспортных систем клеток гладкой мускулатуры. В представленной рабо- те изучалось влияние цинарозида на изометрическое сокращение гладкомышечных клеток на изолирован- ном препарате аорты крыс. Было показано, что цинарозид оказывает расслабляющее действие на препарат аорты, предварительно сокращенный 50 мМ KCl, в присутствие ионов кальция в среде, а также в присутст- вие блокатора кальциевых каналов – верапамила. В связи с этим, предполагается, что релаксантный эффект цинарозида связан с потенциал-зависимыми кальциевыми каналами. Ключевые слова: флавоны, сокращение, гладкомышечные клетки, блокаторы кальциевых каналов. S.N. Gayibova, S.Z. Omonturdiyev, V.N. Syros, T.F. Aripov VASORELAXANT EFFECT OF CYNAROSIDE ON RAT AORTIC RINGS Biologically active compounds of plant origin have the unique property of modifying the function of the Ca2+transport systems of smooth muscle cells. In the present study, the effect of cynaroside on isometric contraction of smooth muscle cells in an isolated rat aorta preparation was studied. Cynaroside has been shown to have a relaxing effect on the aortic preparation, previously contracted with 50 mM KCl, in the presence of calcium ions in the medium, as well as in the presence of a calcium channel blocker - verapamil. In this regard, it is assumed that the relaxant effect of cynaroside is associated with potential-dependent calcium channels. Key words: flavones, vasodilatation, smooth muscle cells, calcium channels blockers. Flavone-derived compounds are a large group of phytochemicals with a great number of properties present in vegetables, fruits, and aromatic plants [1]. Recent studies on the effects of flavonoid intake on cardiovascular mortality found that flavone consumption was associated with the greatest reduction in cardiovascular diseases in women (other flavonoids classes used in the study were anthocyanidins, flavan-3-ols, flavanones, and flavonols) and but with the lowest risk of fatal ischemic heart disease, both in men and women [2-4]. Cynaroside (luteolin-7-O-β-D-glucopyranoside or luteolin-7-O-β-D- glucopyranoside5,3`,4`-trihydroxyflavone) is a natural flavone, one of the bioactive compounds purified from aerial part of Ferula Varia (Schrenk) Trautv. (Apiaceae) [5]. In recent years, several lines of evidence suggested that cynaroside reduces hepatotoxicity [6], repairs antioxidant system [7], normalizes carbohydrate, energy and lipid metabolism as well as improves flow of bile [8]. Therefore, scientific interest in flavones as therapeutic agents is rapidly increasing. However, many studies often report on 3 biological effects of cynaroside without elucidating the underlying molecular, cellular, and physiological mechanisms. In the present study, the potential vasorelaxant activity of cynaroside was examined in the endothelium-intact aortic rings isolated from rat. The experiments were conducted on white outbred laboratory rats (150-200g) as described elsewhere [9, 10]. In these experiments endothelium was not removed. Cynaroside (2.5 mg) was dissolved in 1 ml of dimethyl sulfoxide (DMSO) and then the solution was added with 5 ml of double distilled water. Changes in tension were recorded by isometric Grass transducer (FT-03; Grass, USA). For statistical analysis Student’s t-test was used. Statistical significance as p<0.05 (Winter, 2013). Dose-dependent relaxation effect of cynaroside. In concentrations up to 50 µM cynaroside itself had no effect on muscular tonus of aorta indicating that the flavone does not cause contractility of smooth muscle cells. However, cynaroside exhibited a strong dose-dependent relaxing effect on the rat aorta rings pre-contracted by KCl. Cynaroside reduced vascular tension induced by KCl as a calcium sensitizing stimulus (50 mM), with EC50=28.2μM and EC90=3.52μM. Fig. 1. Concentration-response curve of aortic Fig. 2. Ca2+-dependent effect of cynaroside on rings pre-contracted by KCl in presence of aorta rings tonic contraction induced by KCl cynaroside with an EC50 of about 28.2µM (n=5). (50mM) (A - CaCl2, B - cynaroside, n=5). Effect of cynaroside on extracellular Ca2+-induced contraction. The aorta rings contraction induced by KCl (50 mM) is mainly due to the depolarization of smooth muscle cells (SMC) and the influx of extracellular Ca2+ through voltage-gated Ca2+channels [12, 13]. Our results show that in Ca2+- + free high-K medium, cumulative addition of CaCl2 (0-2.5 mM) induced a gradually increased tension of aortic rings. Pre-treatment of the rings with cynaroside at 15 μM noticeably depressed the maximal contraction to 84.8±4.2%. Fig. 3. Effect of cynaroside on KCl pre-contracted aorta rings in the presence of verapamil (A – control (KCl 50 mM); B – in presence of verapamil; C – in presence of verapamil and cynaroside) (n=5). Vasorelaxant effect of cynaroside in presence of verapamil. The effect of cynaroside was studied in the presence of verapamil, an L-type Ca2+-channels inhibitor. Verapamil itself did not significantly affect the resting tension. However verapamil reduced KCl-induced contractile response to 50.3±4.5%. Preincubation of aorta with cynaroside (28.2 µM) reduced the contractile response to 18.6±4.9%. 4 It is known that KCl-induced reduction of aorta SMC is associated with activation of potential- 2+ + dependent Ca -channels of plasma membranes of SMC. At the same time, the increase of (K )o changes the membrane potential and causes depolarization that activates potential-dependent Ca2+-channels and 2+ leads to an increase of (Ca )i, which in its turn causes a contraction of SMC [13]. Taking this into account and analyzing the data obtained, we can assume that the vasorelaxant mechanisms of cynaroside may be due to inhibition of Ca2+ flow into the cytosol of SMC by blocking potential-dependent Ca2+-channels of sarcolemma. To test this hypothesis, we performed a special series of experiments using a calcium-free Krebs solution. The results of these experiments demonstrated that in the absence of Ca2+ in the incubation medium cynaroside retain the ability to inhibit the contractile responses induced by KCl. Additional confirmation was obtained in experiments with verapamil (0.1 µM), a specific blocker of potential-dependent Ca2+-channels. In presence of verapamil the relaxant effect of cynaroside significantly increased. Our findings suggest that the relaxant effect of cynaroside in KCl-induced contraction is evoked by its interaction with the potential-gated Ca2+channels of plasma membranes of SMC. As a result Ca2+ entry is blocked and its concentration in the cytoplasm of SMC is reduced. Finally, the decrease in the concentration of Ca2+ in the SMC is known to be a cascade of reactions leading to the inhibition of the contractile apparatus and relaxation. Conclusion. Overall, on the basis of the data we can conclude that the relaxant effect of cynaroside mainly is based on blocking L-type channels (in KCl-induced contraction) with micromolar affinity. These data may bring new insights into the mechanisms by which flavones regulate vascular contraction in the human artery. Acknowledgment. The study was financially supported with grants from Academy of Sciences of Uzbekistan under project F7-ТО48 and project FA-F6-T083. The authors would like to recognize the contributions of Prof. Usmanov P.B. for useful discussions during the planning stages of the study and Mrs Mirzaeva Y. for her assistance with sample processing. REFERENCES 1. Kumar, S. and Pandey, A. K. (2013). Chemistry and biological activities of flavonoids: an overview, The Sci World J, 2013,