Determination of xanthones in plants and the nutrient medium under in vitro cultivation conditions

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Determination of xantones in plants and the nutrient medium under in vitro cultivation conditions

А. Revutska, PhD stud., V. Belava, PhD, A. Golubenko, PhD, N. Taran, DSc, Prof.

ESC "Institute of Biology and Medicine", Taras Shevchenko National

In recent years, xanthones have received considerable attention from scientists due to their biological activity: anti- carcinogenic, antiviral, antibacterial, antioxidant, anti-inflammatory and other properties.Therefore they are useful for prevention and treatment of different diseases:cancer, Alzheimer's and Parkinson's disease, cardiovascular disorders, diabetes, etc. Extracts of different species of plants containing xanthones are components of chemotherapeutic and other medical drugs. In order to find the most sensitive and environmentally safe method of quantitative determination of xanthones in the plant material and the nutrient medium, known methods were tested and selected for the prototype Vyisochina G. I. et al., 2011 method, which uses ethanol as an extractor. As the plant material we used plants of different species that were grown under in vitro cultivation conditions on the agarized nutrient medium. This agarized nutrient medium was also used for the xanthone content analysis. Based on the performed research, modifications of the method for determining the content of xanthones were adapted to the in vitro conditions, which detail the specificity of extraction and quantitative calculation of the xanthone content in plant explants. Our own method of determination of these compounds in the agarized nutrient medium was developed as well. The method, that we proposed, will significantly speed up the process of xanthone detecting and will also increase their yield in biotechnological processes for obtaining the pharmacologically valuable secondary metabolites of phenolic nature.

Key words: xanthones, chromatography, spectrophotometry, in vitro, plants, nutrient medium.

В останні роки вторинні метаболіти, зокрема ксантони, отримали значну увагу науковців завдяки своїй біологічній активності: антиканцерогенним, противірусним, антибактеріальним, антиоксидантним, протизапальним та іншим властивостям. Тому вони використовуються для профілактики та лікування різних захворювань: раку, хвороб Альцгеймера та Паркінсона, порушень серцево- судинної системи, діабету тощо. Ектракти різних видів рослин, що містять ксантони, є компонентами хіміотерапевтичних та інших лікарських засобів. Щоб підібрати найбільш чутливий і екологічно безпечний метод якісного та кількісного визначення ксантонів у рослинному матеріалі та агаризованому живильному середовищі, було апробовано відомі методи та відібрано як прототип метод Vyisochina G. I. Et al., 2011, який застосовує етиловий спирт як екстрактор. Як рослинний матеріал ми використовували рослини різних видів, які вирощували в умовах культивування in vitro на агаризованому живильному середовищі. Це живильне середовище також було використане для аналізу вмісту ксантонів. На основі здійснених досліджень модифікації методу визначення вмісту ксантонів було адаптовано до умов in vitro, що деталізують специфічність екстракції та кількісний розрахунок вмісту цих сполук у рослинних експлантах. Було також розроблено власний метод визначення ксантонів у агаризованому живильному середовищі. Запропонований нами метод значно прискорить процес детекції ксантонів, а також збільшить їх вихід у біотехнологічних процесах отримання фармакологічно цінних вторинних метаболітів фенольної природи.

Ключові слова: ксантони, хроматографія, спектрофотометрія, in vitro, рослини, живильне середовище.

В последние годы вторичные метаболиты, особенно ксантоны, получили значительное внимание учених благодаря своей биологической активности: антиканцерогенным, противовирусным, антибактериальным, антиоксидантным, противовоспалительным и другим свойствам. Потому они используются для профилактики и лечения различных заболеваний: рака, болезней Альцгеймера и Паркинсона, сердечнососудистых заболеваний, диабета и т. д. Экстракты разных видов растений, содержащих ксантоны, входят в состав лекарственных препаратов. С целью подбора наиболее чувствительного и экологически безопасного метода качественного и количественного определения ксантонов в растительном материале и питательной среде были апробированы известные методы и отобран как прототип метод Vyisochina G. I. и др., 2011, который использует этанол как экстрактор. В качестве експериментального материала мы использовали растения разных видов, выращеные в условиях in vitro на агаризованной питательной среде, которая также была использована для анализа. Основываясь на проведеных исследованиях, модификации метода определения содержания ксантонов были адаптированы к условиям in vitro, которые подробно описывают специфичность экстракции и количественного определения содержания ксантонов в эксплантах растений. Также был разработан наш собственный метод определения этих соединений в питательной среде. Предложенный нами метод значительно ускорит процесс детекции ксантонов, а также увеличит их выход в биотехнологических процесах получения фармакологически ценных вторичных метаболитов фенольной природы.

Ключевые слова: ксантоны, хроматография, спектрофотометрия, in vitro, растения, питательная среда.

Introduction

Xanthones - heterocyclic polyphenolic compounds, which are particularly valuable secondary metabolites. Vascular plants produce 79 % of all known xanthones of natural origin, non-lichenized fungi - 16 %, lichens - the remaining 5 % [4, 9].In recent years, these polyphenolic compounds have received considerable attention from scientists due to their biological activity, namely: anti-carcinogenic, antiviral, antibacterial, antioxidant, antiinflammatory and other properties. Therefore they are used to prevent and treat cancer, Alzheimer's and Parkinson's disease, cardiovascular disorders, diabetes, etc. [1, 10]. Extracts of different species of plants containing xanthones are components of chemotherapeutic and other medical drugs.These compounds perform a protective function in plants: provide resistance to stress and pathogens, participate in the allelopathic interactions, as well as in the processes of general development of the plant organism [3].

The massive collection of plants as a medicinal material, which is the source of xanthones, destructively affects the species composition of natural phytocoenoses. Thus, the species of plants as sources of xanthones from the families Gentianaceae, Hypericaceae and others already have a rare and endangered status [2, 6, 7]. Consequently, it is important to search for alternative sources of xanthones, which can be, for example, high-yielding plant tissues cultures in vitro. In our opinion, an additional source of xanthones may also be the nutrient medium on which in vitro-plants were cultivated, whose roots are known to produce secondary metabolites, including a wide range of phenolic compounds.

Several methods for the extraction of xanthones are proposed in the scientific works, but most of them are labor-intensive, have low selectivity or practical yield (a large amount of materials is needed) and, moreover, require the use of toxic organic solvents (acetone, hexane, chloroform, methanol) [8]. Such methods are ineffective in the researching of the content of plant xanthones from in vitro tissue cultures and the nutrient medium.

Therefore, the aim of our study was to select the most sensitive and environmentally safe method for qualitative determination of xanthones in plant material, as well as to develop a method for the extraction of these compounds from an agarized nutrient medium as an additional source of pharmacologically valuable secondary metabolites. The main task in developing the modification of the method was to minimize the amount of material needed for the effective detection of xanthones.

Materials and methods

Chemicals. The following reagents were used in the experiment:

For growing plants under in vitro conditions: Murazige-Skuga nutrient medium (mix of macro- and microsoles) (Sigma, Germany), agar (Spain), mesoinozit (China), thiamine (LLC UKRCHIMEXPO, Ukraine), pyridox- ine (LlC UKRCHIMEXPO, Ukraine), nicotinic acid (LLC "UKRCHIMEXPO", Ukraine), ascorbic acid (LLC "UKRCHIMEXPO", Ukraine), Fe2SO4 (LLC "UKRCHIMEXPO", Ukraine), Na-EDTA (LLC "UKRCHIMEXPO", Ukraine), kinetin (Sigma, Germany), indolyl acetic acid (Sigma, Germany), casein hydrolyzate (Sigma, Germany), yeast extract (LLC "UKRCHIMEXPO", Ukraine), sucrose (LLC "UKRСHIMEXPO", Ukraine).

For extraction of xanthones, chromatography and spectrophotometryanalysis: 70 % ethanol (v/v) (DKP Pharmaceutical Factory LLC, Ukraine), 40 % acetic acid (v/v) (LLC "UKRСHIMEXPO", Ukraine).

Plant material. For approbation of known methods for the xanthone determination in plant material, the roots of plants of the genus Iris L. were used.

For developing our own modifications of the method for the xanthone determination in in vitro-plants we used plants of the genus Phalaenopsis sp. [18] and Acoruscala- mus L. (various variants of explants originating from different localities) [17]. The investigated plants were grown under in vitro conditions for 3-5 month sat a temperature of 24 °C, with a 16-hour photo period on the modified agarized Murassigu-Skooganutrient medium. In vitro cultivation of plants was performed in the research laboratory of "Introduced and natural phytodiversity" of O.V. Fomin Botanical Garden named of Taras Shevchenko National University of Kyiv.

Approbation of different extraction methods and quantitative determination of xanthones were carried out in the research laboratory "Physiological bases of plant productivity" of the Department of Plant Biology at the Educational and Scientific Centre "Institute of Biology and Medicine" of the Taras Shevchenko National University of Kyiv.

The agarized nutrient medium on which the plants under study were grown were also use das a material for extraction and quantitative determination of xanthonecontent.

Extraction and quantitative determination of xanthone content. In order to select the most sensitive method for the xan- thone determination in plant material, we have already tested the methods known in the scientific literature (Table 1 ).

Table 1. Extraction methods of xanthones from the plant material

Quantitative analysis of xanthone content was carried out using paper chromatography and spectrophotometrical- ly (SHIMADZU UV-1800, Japan) at X = 200-400 nm.

Statistical analysis. Statistical processing of the results was carried out using common methods using the standard program Microsoft Office Excel 2007. The biological repetition of the experiments was 3 times, the analytical repetition - 9 times. In preparation of the method, the average values of the indices were analyzed, the differences between which were considered significant at Р < 0.05 according to the Student's criterion [13].

Results and discussion. On the basis of the elaborated publications [5, 12, 14-16, 19, 20], we have developed our own modifications of methods for the xanthonedetermi- nation in plants grown under in vitro conditions. Since in literary sources we did not find methods for xanthone extraction from agarized nutrient medium, we have developed our own method of isolating these compounds and determining their content in the agarized nutrient medium.

Extraction of xanthones from plant material. The methods of extraction and determination of the xanthone content (see Table 1) presented in the literature are different in terms of the amount of dry plant material (from 0,2 g (№ 7) to 1,7 kg (№ 4)), by the composition of the extraction solution (methanol, ethanol, hexane, chloroform (№ 3), ethanol, chloroform, butanol (№ 4), ethanol, chloroform (№ 6)), as well as the duration extraction (from 1 hour (№ 7) to 360 hours (№ 2)). The presence of toxic solvents in solutions for extraction does not allow the use of extracts obtained without further purification.

Our analysis of the data of xanthone extraction methods has shown that method №5 is the most environmentally friendly and requires a small amount of materials. Therefore, this method became the basis for developing a modified version: xanthone in vitro nutrient medium

weigh 0,1-0,3 g of raw plant material gomogenise in a porcelain mortar with a small amount of 70 % ethanol (v/v);

add 70 % ethanol (v/v) and transfer quantitatively into heat resistant conical flasks (volume 100 ml) with reflux condensers so that the total volume of the extract does not exceed 4-5 ml;

extract in a boiling water bath for 3 hours;

the cooled extract filtere through a Shota-filter in a heat-resistant flask; add a 70 % ethanol (v/v) portionwise (0,3-0,5 ml) to the Shota-filter several times until the solution slips from the filter, does not become colorless; combine filtrate;

heat-resistant flasks with the resulting total filtrate place on a water bath and drop the contents to dryness (if necessary, cooled flasks with a dry residue hermetically sealed with rubber stoppers and left for 24 hours at a temperature of 3-7 °C);

add 0,5-1 ml of 70 % ethanol (v/v) to the dry residue (for better dissolution of the remainder of the flask can be heated to 60° C);

to carry out paper chromatography in the ascending method, take a chromatographic paper 8 x 11 cm, marking the starting line at a distance of 1 cm from the lower edge; obtain the extract step by step on the marked line (at the same time at each stage it should be completely dried); add thrice (0,1--0,2 ml) 70 % ethanol (v/v) in small portions to wash the remainder of the extract from the flask, apply it to the starting line;

upstream chromatography in a small chromatography chamber using 40 % acetic acid (v/v) as a mobile phase (the time required for chromatography is 15-20 minutes under normal conditions);

dry the chromatograms, place under the UV-irradiation, bright orange spots of xanthones circle with a pencil;

cut blotted spots, grind, place in test tubes and add 4 ml of 60 % ethanol (v/v), seal the tubes with rubber stoppers and leave them for 6 hours;

in the case of saturated bright orange color of the obtained eluates (which indicates the high concentration of xanthones in the experimental plant material), they must be diluted with 60 % ethanol (v/v),and then multiplicity of this dilution should be taken into account during calculations;

the eluates obtained should be used for spectropho- tometric analysis (the range of wavelength to be used for the analysis is X = 200-400 nm);

to calculate the xanthone content in 1 g of raw plant material, use the formula:

C - content of xanthone in the sample,%; D - optical density of the investigated solution; M - molecular weight of xanthone compound; l - path length of sample (cuvette thickness), cm; - specific absorption coefficient of xanthone compound; P - weight of plant material, g; z - multiplicity of dilution of the eluate used for spectrophotometric analysis.

Extraction of xanthones from the agarized nutrient medium. In the vast majority of cases, extraction of secondary metabolites is carried out from a liquid nutrient medium in the cultivation of suspension cultures [20]. For the effective use of in Wtro-cultures of plants grown on an agarized medium, along with the modification of the xanthone extraction from plant material, a method for extracting xanthones from agarized nutrient medium was developed. In the process of developing the method, the weight of the initial weight gain of the agarized nutrient medium was 0,1 g. However, we failed to establish the statistically significant values of the studied parameters. With an increase in cravings of the nutrient medium up to 0,5 g, the determination of the xan- thone content was successful.

Thus, the following sequence of analysis of agarized nutrient medium has been developed:

homogenize the nutrient medium (0,5 g) and place it in a 100 ml flask;

add 3 ml of 70 % ethanol (v/v) to the homogenate, mix thoroughly, close with rubber stoppers and withstand under the temperature of 5 oC for 24 hours;

transfer the samples quantitatively to centrifuge tubes and centrifuge for 10 min. at 2000 rpm;

take the supernatant carefully into the test tubes;

add 1 ml of 70 % ethanol (v/v) to the precipitate remaining in the centrifuge tube, mix and centrifuge for 10 min.;

take the supernatant obtained and combine with the previous extract (repeat the procedure 2 more times) and use for chromatographic analysis;

the quantitative determination of xanthones is carried out using the same method as in the plant material.

With the help of developed methods it was found that the xanthone content in in Wtro-plants remained stable or increased during cultivation, and in the nutrient medium - decreased. Consequently, for the biosynthesis the maximum amount of xanthonesin the in Wtro-plants, it is necessary to regulate the plant cultivation conditions, in particular the duration of cultivation [17, 18].

In conclusion, an ecologically safe and effective for a small amount of experimental material modification to the method for xanthone determination in plant material was selected and a method for their extraction from the agarized nutrient medium was developed. The method, that we have proposed, will significantly speed up the process of detecting xanthones and also increase their yield in biotechnological processes for obtaining a pharmacologically valuable substance.

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