Получение препарата, обогащенного Zn-феофитином, из листьев пшеницы Triticum aestivum L.

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Abstract

Целью работы было получение препарата, обогащенного цинковым производным феофитина, из срезанных листьев озимой пшеницы Triticum aestivum L. Сначала проводили феофитинизацию хлорофилла и вымывание из листьев фенольных веществ, Mg2+ при рН 3.5–4.0 с добавкой ЭДТА-Na, затем инкубацию с ZnCl2, листья высушивали и хранили в темноте. Экстракцию цинковых производных проводили 2-кратной обработкой этанолом. Для увеличения выхода проводили повторную металлизацию. Полученный препарат соответствовал Zn-феофитину а и b по данным спектрофлуориметрии и тонкослойной хроматографии. Обсуждаются перспективы использования препарата Zn-феофитина, обладающего антиоксидантной и антивирусной активностью.

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М. С. Христин

Институт фундаментальных проблем биологии РАН ФИЦ “Пущинский научный центр биологических исследований Российской академии наук”

Author for correspondence.
Email: khristin_@rambler.ru
Russian Federation, Пущино

Т. Н. Смолова

Институт фундаментальных проблем биологии РАН ФИЦ “Пущинский научный центр биологических исследований Российской академии наук”

Email: khristin_@rambler.ru
Russian Federation, Пущино

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Supplementary files

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2. Fig. 1. Scheme of Zn-chlorophyll synthesis in wheat leaves before (I, II) and after remetallization with Zn2+ (III) without (a) and with the addition of EDTA–Na (b). I – Pheophytinization of chlorophylls by incubation of leaves at pH 3.5–4.0 with stirring until the color changes from green to brown, washing with distilled water until the release of colored substances stops and the pH in the medium increases to 5.5–6.5. II – Metallization of pheophytin by incubation of leaves with 100 mM ZnCl2 at pH 5.5 until the color changes from brown-green to blue-green stops, washing with distilled water, drying, storage in the dark, grinding in a blender, extraction with ethanol, centrifugation at 5000 g for 5 min. 1, 4 – ethanol extract of wheat leaves; 2, 5 – supernatant and sediment after metallization; 3, 6 – repeated ethanol extraction of the sediment after metallization (2, 5). III – Remetallization by adding 10 mM ZnCl2 to the ethanol extract, incubation for 2 days, dilution with distilled water 10 times and precipitation of Zn-pheophytin by centrifugation at 5000 g, 5 min. 1, 4 – ethanol extract after remetallization; 2, 5 – supernatant and sediment after remetallization; 3, 6 – repeated ethanol extraction of the sediment after remetallization (2, 5).

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3. Fig. 2. Fluorescence spectra at 77 K. (a) – wheat leaves after pheophytinization (1) and metallization with Zn2+ ions (2); (b) – ethanol extract of dry wheat leaves before (1) and after repeated metallization (2). 10 mM ZnCl2 in ethanol was added to the extract (1), and the mixture was incubated for 2 days at 23°C (2); a – maximum fluorescence of pheophytin a; b – maximum fluorescence of pheophytin b [19].

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4. Fig. 3. TLC of the Zn-pheophytin preparation (4) obtained by metallization with ZnCl2. Pheophytin was obtained by treating dry wheat leaves (1) at pH 3.5–4.0. 1 – dry leaf extract; 2, 3 – leaf extract after pheophytinization; 4 – leaf extract after substitution of Mg2+ by Zn2+ and repeated metallization with Zn2+ (Fig. 1, III 5).

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5. Fig. 4. Incorporation of Zn2+ ions into pheophytin on a silica gel TLC plate. (a) 1 – zone of Chl a + b, immediately after TLC, 2 – the same zone of Chl a + b after long-term aerobically storage at room temperature in the dark. 10 μl of 1 M ZnCl2 [17] in alcohol were applied to the surface of the zone (marked with a circle). A change in color is visible, indicating the incorporation of Zn2+ into pheophytin. (b) – zone of pheophytin on a TLC plate after chromatography of an ethanol extract of wheat leaves incubated at pH 4.0 and washed with EDTA-Na from Mg2+ ions and phenolic substances. 1 – 10 µl of 1 M ZnCl2 in alcohol was applied to the area of ​​the zone (marked with a circle), 2 – 10 µl of 1 M ZnCl2 in water was applied to the area of ​​the zone (marked with a circle).

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