STUDY OF THE CONTENT OF FLAVONOIDS AND BIOELEMENTS IN THE ABOVE-GROUND PARTS OF Atraphaxis spinosa L.

АННОТАЦИЯ

Впервые с помощью методов ВЭЖХ и оптической эмиссионной спектрометрии AVIO-200 (ICP-OES) было изучено содержание 5 флавоноидов и 30 макро- и микроэлементов в надземной части растения Atraphaxis spinosa L. произрастающего на территории Ташкентской области. Установлено, что растение накапливает больше всего рутина (28.71мг/г), розавина (21.61мг/г) и дигидрокверцетина (10.63мг/г). Кроме того, выявлено что в листьях растения содержание элементов таких как Са (536.259 мг/кг), К (102.265 мг/кг), Р (12.251 мг/кг), Мg (15.068мг/кг), Na (17.608мг/кг), S (2.899 мг/кг), Fe (3.744 мг/кг) и Mn (2.344 мг/кг) было значительно выше чем стеблях растения.

ABSTRACT

For the first time, using HPLC and AVIO-200 (ICP-OES) optical emission spectrometric methods, the amounts of 5 flavonoids and 30 macro-and microelements were studied in the aerial part of Atraphaxis spinosa L. growing in the Tashkent region. It was determined that the plant accumulates higher amounts of rutin (28.71mg/gr), rosavin (21.61mg/gr), and dihydroquercetin (10.63mg/gr). Additionally, it was found that the leaves contain higher levels of Ca (536.259 mg/kg), K (102.265 mg/kg), P (12.251 mg/kg), Mg (15.068 mg/kg), Na (17.608 mg/kg), S (2.899 mg/kg), Fe (3.744 mg/kg), and Mn (2.344 mg/kg) compared to the stem of plant.

Ключевые слова: Atraphaxis spinosa, экстракция, ВЭЖХ, флавоноиды, макроэлементы, микроэлементы, оптический эмиссионный  спектрометрический метод.

Keywords: Atraphaxis spinosa, extraction, HPLC, flavonoids, macroelements, microelements, optical emission spectrometric method

Introduction

In recent years, consistent reforms have been carried out in the Republic aimed at protecting medicinal plants, ensuring the rational use of natural resources, establishing plantations for the cultivation of medicinal species, and developing systems for their processing. The local flora comprises more than 4.3 thousand plant species, of which about 750 are classified as medicinal. Among them, 112 species are officially registered for use in scientific medicine, and approximately 70 are actively utilized in the pharmaceutical industry. At the same time, analytical studies indicate the need to strengthen efforts toward the conservation of medicinal plants, the development of their plantations, and the creation of value-added chains through processing. It is well known that many plant species in nature possess therapeutic properties. The quantity and quality of biologically active compounds present in plants constitute the most important factors in determining their medicinal potential. In the preparation of pharmaceuticals from plant-derived raw materials, biologically active substances including flavonoids, lipids, proteins, essential oils, saponins, and various other compounds with pharmacological value play a crucial role.

The mountainous forest ecosystems of the Tashkent region are particularly rich in medicinal plants. These species serve as an important source of raw materials not only for traditional medicine but also for the pharmaceutical industry. Among the medicinal plants rich in biologically active compounds is Atraphaxis spinosa L., a representative of the Polygonaceae family. Plants belonging to this family are known to contain carbohydrates, macro- and microelements, vitamins, hydrolyzable tannins, flavonoids, phenolic acids, and various other classes of biologically active substances. These compounds form the basis for the development of highly effective medicinal preparations widely used in medical practice. Therefore, conducting scientific research on the above-ground parts of Atraphaxis spinosa L.including the analysis of its chemical composition, the identification of macro- and microelements, the extraction of flavonoids and hydrolyzable tannins, the elucidation of their chemical structures, and the study of their biological activity is of significant scientific and practical importance [1].

Atraphaxis spinosa L. belongs to the family Polygonaceae. It is a low-growing, highly branched, woody shrub, typically reaching a height of 40–100 cm and forming bushy or semi-bushy structures. Its branches are slightly whitish, and the apical parts are leafless, ending either in a spine or without one. The leaves are usually thick, smooth, and glabrous [2–3].

Species of the genus Atraphaxis are known for containing polyphenolic compounds such as flavonoids and tannins, which are highly valued for their strong antioxidant properties. These compounds possess the ability to combat oxidative stress, inflammation, and cellular damage, making them particularly valuable in medicine and healthcare applications [4].     

Figure 1. The plant Atraphaxis spinosa L.

The aim of this study is to investigate the content of flavonoids and bioelements in Atraphaxis spinosa L. growing in the Tashkent region.

Experimental Part

Plant samples were collected from the mountainous areas of the Tashkent region specifically from the slopes surrounding the village of Suqoq during the flowering period of Atraphaxis spinosa L. The above-ground parts of the plant were used for analysis. The plant material was dried in a cool, shaded place protected from direct sunlight and then ground. From 500 g of the dried raw material, a portion was taken and extracted with chloroform in a 1:4 ratio (45–50°C, 2 hours, three repetitions) to remove pigments and lipophilic compounds. The plant residue was then dried at room temperature for 24 hours (until the smell of chloroform completely disappeared) and subsequently extracted with 70% aqueous acetone in a 1:5 ratio (45–50°C, 2 hours, three repetitions). The combined aqueous-acetone extracts were concentrated using a rotary evaporator under vacuum to remove the organic solvent, yielding an aqueous fraction.

The obtained aqueous fraction was repeatedly extracted with ethyl acetate to isolate the ethyl acetate fraction. This fraction was dried over anhydrous Na₂SO₄ and concentrated under vacuum using a rotary evaporator to obtain an ethyl acetate concentrate. The concentrate was mixed with chloroform to precipitate the compounds, and the precipitate was collected by filtration. The collected precipitate was dried at room temperature in a vacuum drying oven, yielding a polyphenolic fraction.

To study the chemical composition of the obtained polyphenolic fraction, High-Performance Liquid Chromatography (HPLC) was used.

 Chromatographic conditions: erituvchilar:

Solvents: A – acetonitrile; B – 0.1% trifluoroacetic acid buffer (pH = 3).

Gradient program for acetonitrile:
0–15 min: 15% (v/v) acetonitrile
15–27 min: 30% (v/v) acetonitrile
27–42 min: 95% (v/v) acetonitrile
42–45 min: 15% (v/v) acetonitrile
Flow rate: 0.8 mL/min
Detection wavelengths: 254 and 276 nm
Column thermostat temperature: 30°C
Standard flavonoids were used as reference substances.

The macro- and microelement contents of the plant samples were analyzed using an Avio 200 (ICP-OES) optical emission spectrometer. For mineralization, an accurately weighed 0.1000 g portion of sample was transferred into Teflon autoclaves. Then, 3 mL of purified concentrated nitric acid (HNO₃) and 2 mL of purified hydrogen peroxide (H₂O₂) were added. The autoclaves were sealed and placed into a Berghof SpeedWave Xpert (or similar) microwave digestion system. The system automatically controlled and monitored the temperature and pressure inside each autoclave according to a predefined program. Throughout the process, operational parameters were tracked via a liquid crystal display. The digestion was carried out for 35–45 minutes under conditions where the internal temperature ranged between a minimum of 50°C and a maximum of 230°C, and the maximum pressure reached up to 40 bar. After cooling to room temperature, the digested solution was quantitatively transferred to a 50 or 100 mL volumetric flask. The autoclaves were rinsed 2–3 times, and the rinse solutions were combined with the main solution, followed by dilution to the mark with distilled water. The solution was mixed thoroughly, transferred into autosampler vials, and placed in designated positions within the autosampler. The sample position, weighed mass, and dilution factor were entered into the analytical program [5].  The mineralized solutions were analyzed using a Perkin Elmer Avio-200 ICP-OES instrument to quantify macro- and microelements, heavy metal salts, and rare metals relative to certified standard reference materials. Upon completion of the analysis, the instrument automatically calculated the elemental concentrations based on sample mass and dilution factors, along with measurement precision and relative standard deviation (RSD) values.

Results and Conclusions

Comparison of the obtained results with the chromatograms of standard flavonoids showed that the polyphenolic fraction contains more than five phenolic-type compounds (Figure 1). 

Figure 1. Results obtained from the HPLC analysis of the polyphenolic fraction isolated from the plant

The presence of flavonoids such as rutin, quercetin, dihydroquercetin, rosavin, and salidroside in the above-ground part of Atraphaxis spinosa L. has been established. The results and quantitative analysis of flavonoids from the above-ground part of Atraphaxis spinosa L. are presented below (Table 1).

Table 1.

Content of flavonoids in the above-ground part of Atraphaxis spinosa L.

As shown in Figure 1 and Table 1, the above-ground part of Atraphaxis spinosa L. contains primarily rutin (28.71 mg/g) and rosavin (21.61 mg/g), and it was determined that these accumulate in significantly higher quantities compared to the other flavonoids present in the plant.

The content of macro- and microelements in the plant raw material was determined using the optical emission spectrometry method with an ICP-OES instrument (AVIO 200). The results obtained from the investigation of the macro- and microelement content in Atraphaxis spinosa L. are presented in the following table.

 Table 1.

Content of Macro- and Microelements in the Samples

Analysis of the macro- and microelement composition of Atraphaxis spinosa L. aerial parts revealed that elements such as potassium (K), calcium (Ca), and magnesium (Mg) are present in the highest concentrations across the plant's floral, stem, and leaf tissues. Furthermore, the plant's composition is characterized by substantial levels of aluminum (Al), iron (Fe), sulfur (S), phosphorus (P), and sodium (Na). The study also determined that Atraphaxis spinosa L. lacks detectable concentrations of elements harmful to human health, such as lead (Pb), cadmium (Cd), arsenic (As), and mercury (Hg). Additionally, the levels of other heavy metals did not exceed established regulatory limits. Conversely, the relatively high accumulation of biogenic elements like Mg, K, and Ca in the plant organs indicates promising prospects for the research object as a potential source of beneficial minerals.

Conclusion.

1. The HPLC results indicate that bioactive compounds such as rutin, rosavin, and dihydroquercetin possess significant antioxidant properties. Consequently, the leaves of Atraphaxis spinosa L. show potential for prophylactic and therapeutic application in conditions requiring oxidative stress reduction, anti-inflammatory action, and vascular fortification.

2. The high content of bioelements such as potassium (K), calcium (Ca), sodium (Na), and magnesium (Mg) in Atraphaxis spinosa L. suggests its potential for use in the prophylaxis and treatment of cardiovascular diseases, osteoporosis, muscle cramps, nervousness, and chronic fatigue.

References:

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3. Dini,I; Grumetto,L. Recent advances in natural polyphenol research. Molecules 2022, 27,8777
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