QUALITATIVE AND QUANTITATIVE ANALYSIS OF FLAVONOIDS IN THE Euphorbia Milii PLANT

ABSTRACT

This article presents a comparative analysis of the types and amounts of flavonoids in the leaves of Euphorbia Milii, a representative of the succulent family, genus Euphorbia. Three regions with different climatic conditions—Tashkent, Fergana, and Khorezm—were selected for the analysis. The article's introduction primarily discusses the significance of the Euphorbia genus and the plant Euphorbia Milii in folk medicine. The methods and materials section outlines the process for determining the quantity and type of flavonoids in the plant. The flavonoids in the leaves of each sample were studied both qualitatively and quantitatively, compared, and concluded.

АННОТАЦИЯ

В данной статье представлен сравнительный анализ типов и количества флавоноидов в листьях Euphorbia Milii, представителя семейства суккулентных рода Euphorbia. Для анализа были выбраны три региона с различными климатическими условиями — Ташкент, Фергана и Хорезм. Во введении к статье в первую очередь обсуждается значение рода Euphorbia и растения Euphorbia Milii в народной медицине. В разделе «Методы и материалы» изложен процесс определения количества и типа флавоноидов в растении. Флавоноиды в листьях каждого образца были изучены как качественно, так и количественно, сравнены и сделаны выводы.

Keywords: Euphorbia, flavonoids, chromatography, stems, leaves, roots.

Ключевые слова: молочай, флавоноиды, хроматография, стебли, листья, корни.

Introduction. Euphorbia plants (Euphorbiaceae) form the third-largest flowering plant genus with nearly two thousand species in the world. They are distributed all over the world except in very cold areas. They have been used since ancient times as traditional medicinal herbs for treatment of different ailments as asthma, cough, tumors, and warts. The determination of the chemical constituents of herbal samples is an essential issue since plant materials have a variety of compounds with different chemical structures and complex matrices.

Euphorbia milii, also known as the crown of thorns, is an important medicinal plant species belonging to the family Euphorbiaceae. Euphorbia plants have been used as traditional medicine for treating scorpion and snake bites, liver diseases, respiratory disorders, asthma, rheumatism, tumors, and warts for hundreds of years. These plants are medicinally valuable due to their diverse secondary metabolites. Previous reports have shown that plant pigments, showing antitumor, antioxidant, antiaging, and anti-inflammatory activities, are associated with several health benefits; therefore, they are widely used in the modern food, cosmetic, and healthcare industries.

Among the phenolic compounds found in plants, the most important are flavonoids, which have high biological activity. The literature reviewed shows that all plants belonging to the Euphorbia genus contain flavonoids and that they can be used to treat various diseases. Therefore, one of the important tasks of the research was to determine the flavonoids and their content in this plant species.

Materials and methods.

Qualitative and quantitative analysis of biologically active substances in plants requires extraction first. Extraction is the first method to analyze the composition of all herbs of medicinal value and the results will also depend on this method.

For the research work, air-dried roots, stems, and leaves of plant samples of Euphorbia milii were used. Samples were brought from the Fergana, Tashkent, and Khorezm regions of the Republic of Uzbekistan.

The preparation of raw materials is carried out as follows:

• roots, stems and leaf parts of plant species are collected and washed with cold water and laid on paper in a thin layer to dry.
• drying was carried out where direct sunlight does not fall (humidity 78.8%).

Enough drying was determined by the fluff and color of the plant. The plant should not have a dark green color so that the chlorophylls do not mix in the extract.

Acetone extraction.

The dried plant stem was ground to a powder using a grinder; acetone was removed to remove impurities; the sample and solvent were taken in a ratio of 1:10.

One end of the reflux condenser was connected to a flask with a tube, the other end was connected to cold water, and the third end to wastewater; a stove was placed under the reflux condenser; the crushed plant stem and acetone were placed in the flask; the mixture was boiled for 3 hours, controlling the boiling temperature.

The process of extracting plant leaves in acetone was also performed in the above manner.

Ethanol extraction.

The dried plant stem was ground to a powder using a grinder; ethanol (96%) was removed to remove impurities; the sample and solvent were taken in a ratio of 1:10.

One end of the reflux condenser was connected to a flask with a tube, the other end was connected to cold water, and the third end to wastewater; a stove was placed under the reflux condenser; The crushed plant stem and ethanol were placed in a flask; the mixture was boiled for 3 hours while controlling the boiling temperature.

The process of extracting the plant leaf in ethanol was also performed in the above procedure.

Chloroform extraction.

The dried plant stem was ground into a powder using a grinder; the sample and solvent were taken in a ratio of 1:10.

One end of the reflux condenser was connected to the flask using a tube, the other end was connected to cold water, and the third end to wastewater; a stove was placed under the reflux condenser; the crushed plant stem and chloroform were placed in the flask; the mixture was boiled for 3 hours while controlling the boiling temperature.

Table 1.

Samples and solvents selected for extraction

After the presence of flavonoids in the Euphorbia milii plant was studied through qualitative reactions,  to determine the types and amounts of flavonoids, the vegetative organs of each of the three plant samples were extracted in chloroform and analyzed using high-performance liquid chromatography.

 The high-performance liquid chromatography method was performed using isocratic elution mode and a diode array detector (DAD). Acetonitrile and buffer solution were used as the mobile phase. Spectral data were studied in the spectral range from 200 to 400 nm.

Chromatography conditions:

Chromatograph - Agilent Technologies 1260

Mobile phase (isocratic mode) - acetonitrile - buffer solution (35:75)

pH = 2.92 15-20 min.

Injection volume - 5 μl.

Mobile phase speed - 0.75 ml / min.

Column - Eclipse XDB - C18. 5.0 μm, 4.6x250mm.

Detector – diode array detector, wavelength 254, 320, 381nm.

Results and discussions.

Table 2.

Results of the extraction of Euphorbia milii leaves using different solvents (Tashkent-1, Fergana-2, Khorezm-3)

According to the results of the extraction process, it was concluded that chloroform was the best solvent for extracting the leaves of Euphorbia milii. The amount of extract extracted from the Fergana species sample of E. milii was found to be higher than that of other samples in all selected solvents.

Table 3.

Results of the extraction of Euphorbia milii stem using different solvents (Tashkent-1, Fergana-2, Khorezm-3)

Extraction of the stem of Euphorbia milii plant samples yielded relatively high amounts of extract in chloroform extraction. The stem of the Fergana plant sample (72%) yielded 8% more extract than the stems of the Tashkent (64%) and Khorezm (64%) samples.

Table 4.

The number of flavonoids detected in Euphorbia milii leaves (mg\100)

Picture 1. Chromatogram of flavonoids identified in the leaves of Euphorbia milii (Fergana)

Picture 2. Chromatogram of flavonoids identified in the leaf of Euphorbia milii (Tashkent)

Picture 3. Chromatogram of flavonoids identified in the leaf of Euphorbia milii (Khorezm)

Conclusion. Based on the results compared in the table,  the leaves of the Euphorbia milii species growing in Tashkent and Khorezm regions contain relatively more flavonoids than the Fergana species. The leaves of Euphorbia milii mainly contain flavonoids such as apigenin, hyperazide, and hypoleucin. It was found that apigenin and hyperazide are relatively abundant in the leaves of the Tashkent and Khorezm species, and hypoleucin is relatively abundant in the Khorezm species.

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