DETERMINATION OF THE CHEMICAL COMPOSITION AND BIOLOGICAL ACTIVITY OF FOOD ADDITIVES DEVELOPED FROM KNAPWEED AND YARROW PLANTS

ABSTRACT

This article presents the results of determining the antioxidant activity of yarrow (Achillea millefolium L.) and knapweed (Centaurea cyanus L.) plants and their mixtures in different mass ratios. Additionally, chromatographic analysis was performed to identify and quantify certain polyphenolic compounds in the food supplement “AS-KAMOL,” developed based on these plants. The results confirmed the presence of polyphenols, vitamins, and minerals in the supplement.

While these compounds are commonly associated in the scientific literature with potential health benefits, including anti-inflammatory properties, it is important to note that the present study did not include biological or clinical evaluations. Therefore, any claims regarding the therapeutic effects of “AS-KAMOL” require further experimental and clinical investigation.

АННОТАЦИЯ

В данной статье представлены результаты определения антиоксидантной активности растений тысячелистника (Achillea millefolium L.) и василька (Centaurea cyanus L.) и их смесей в различных массовых соотношениях. Также проведен хроматографический анализ для идентификации и количественного определения некоторых полифенольных соединений в пищевой добавке «AS-KAMOL», разработанной на основе этих растений. Результаты подтвердили наличие в добавке полифенолов, витаминов и минералов.

Хотя эти соединения в научной литературе часто связывают с потенциальной пользой для здоровья, включая противовоспалительные свойства, следует отметить, что в настоящем исследовании не проводилась биологическая или клиническая оценка. Следовательно, любые утверждения о терапевтических эффектах «AS-KAMOL» требуют дальнейших экспериментальных и клинических исследований.войствами для лечения и профилактики воспалительных заболеваний.

Keywords: Achillea millefolium L., Centaurea cyanus L., antioxidant, “AS-KAMOL” food supplement, HPLC, vitamins, polyphenols, macro- and microelements, inflammation.

Ключевые слова: Achillea millefolium L., Centaurea cyanus L., антиоксидант, пищевая добавка «AS-KAMOL», ВЭЖХ, витамины, полифенолы, макро- и микроэлементы, воспаление.

Introduction: Yarrow (Achillea millefolium L.) and knapweed (Centaurea cyanus L.) are plants belonging to the Asteraceae family and widely used in folk medicine and modern medicine for the treatment of various diseases. These plants are widely used to treat and prevent kidney stones, gastrointestinal diseases, liver and gallbladder diseases, cardiovascular diseases, various genital diseases, fever, inflammation, and injuries, and to stop internal and external bleeding [1].

Today, kidney stones and bladder inflammation are among the diseases that are increasingly developing and becoming more common in the world. In particular, kidney stones, which are called nephrolithiasis in modern medicine, have become one of the most common urological diseases due to the many factors that lead to the formation of crystal agglomerates in the urinary tract. Kidney stones and cystitis currently affect about 12% of the world's population, and by 2045, this figure is projected to reach 64.46%. If a kidney stone is larger than 5 millimetres, it can cause bladder obstruction and acute and severe abdominal pain, which increases the risk of kidney disease. The main factors that lead to the formation of calcium oxalate stones are hypercalciuria, hyperphosphatemia, hypocitraturia, hyperuricosuria, and hypomagnesuria [1,2].

In human health, various vitamins, such as vitamin B₆, with magnesium, can prevent the formation of oxalate stones. Magnesium and vitamin B₆ are used by the body to convert oxalate into other substances. Vitamin B₆ deficiency leads to an increase in kidney stones due to an increase in oxalate in the urine. 50 mg of vitamin B₆ per day and 200-400 mg of magnesium per day may prevent the formation of oxalate stones [3,4]. Researchers have found that low calcium and potassium intakes are associated with a higher risk of developing symptomatic kidney stones [5]. Drinking plenty of fluids, including calcium and potassium, can help prevent kidney stones. Potassium is an important mineral that acts as an electrolyte, helping to regulate fluid balance. The electrolytes that affect fluid balance are mainly potassium and sodium. The human body is made up of 60% water (in a healthy person) [6]. The movement of water in and out of cells is controlled by the elements Na and K. If the water balance is disrupted due to a deficiency of K or Na, salts can accumulate in the kidneys and form crystals [7].

Studies on the role of vitamins in the formation of kidney stones have focused mainly on water-soluble vitamins. Studies on the primary prevention of kidney stones show that low intake of water-soluble vitamins is associated with a higher risk of kidney stone formation. Water-soluble vitamins have high antioxidant properties and can prevent diseases by strengthening the immune system [8,9]. It is important to develop and implement natural, environmentally friendly, harmless, biologically active food additives based on medicinal plants for the prevention and treatment of kidney stones and bladder inflammation [10, 11].

Currently, scientific research focused on the development and practical application of natural medicinal products derived from bioactive compounds obtained from plants is rapidly advancing in many countries. In particular, in our country, studies have been conducted to develop medicinal products with potential therapeutic and preventive effects against kidney stones and bladder inflammation, based on medicinal plants and natural sources, yielding certain scientific results. However, it is recognised that the existing outcomes are insufficient to significantly reduce the incidence of kidney stones and bladder inflammation in the general population. Considering that the chemical composition of local yarrow and knapweed plants is rich in beneficial natural compounds, including polyphenols, vitamins, and minerals known to support kidney and bladder health, we undertook research to develop a new type of medicinal food supplement based on these plants. The following sections describe these studies and their results.

In the initial studies, we conducted research to determine the optimal composition of the medicinal composition to be developed on the basis of yarrow and yarrow. In this study, the antioxidant activities of yumadaron, botakoz, and their mixtures in different mass ratios (Mixture 1 (1:3), Mixture 2 (3:1), Mixture 3 (1:1)) were determined spectrophotometrically. The experiments are described below.

MATERIALS AND METHODS

Antioxidant determination experiment. The antioxidant activity of bilberry, bilberry and their mixtures in different mass ratios was determined by the method of inhibition of the autooxidation reaction of adrenaline in vitro. This method consists of determining the ability (activity) of the compounds in the extract under investigation to prevent the formation of free oxygen, and the activity indicator is expressed in %.

Work execution: 2.0 ml of 0.2 M sodium carbonate (Na₂CO₃-NaHCO₃) buffer with pH=10.65 and 56 μl of 0.18% adrenaline (epinephrine) hydrochloride solution were taken. 30 μl of antioxidant preparations were added and examined in a spectrophotometer (Cary 60 UV-Vis Agilent Technologies) at a wavelength of 347 nm for 30 seconds to 10 minutes. The amount of the test substance (concentration of 1 mg in 1 ml of solution) was used as a standard. 2.0 ml of 0.2 M buffer and 56 μl (5.46 mM) of 0.18% adrenaline were used as controls.

The antioxidant activity was calculated based on the inhibition of adrenaline autooxidation by the following formula.

Here, the optical density of the adrenaline hydrochloride solution is added to buffer D1, and the optical density of the extract under study, and adrenaline hydrochloride is added to buffer D2.

Table 1.

Antioxidant activities of plants and their compounds

The table above shows that the antioxidant activity of the plant is higher than that of the other mixtures, and among the mixtures, this indicator of Mixture 2 is higher than that of the other mixtures and plants. This means that the antioxidant activity, that is, the biological activity, of the mixture of the plant and the plant in a 3:1 mass ratio, respectively, in vitro, is high. This result served as a scientific basis for determining the optimal composition of a new medicinal food supplement, which we plan to develop from the plant and the plant. Taking this into account, we developed a natural food supplement called ‘AS-KAMOL’ based on a 3:1 mass ratio of the plant and the plant. Technological instructions to produce this food supplement have been developed, approved by the Department of Sanitary and Epidemiological Wellbeing and Public Health Service of the Ministry of Health of the Republic of Uzbekistan, and are being produced and put into practice at local enterprises.

Next, we conducted studies to determine the content of some polyphenols and water-soluble vitamins in the food supplement ‘AS-KAMOL.’ The conducted studies and their results are described below:

Determination of the content of phenolic compounds in the food additive ‘AS-KAMOL’ by the method of HPLC

Experimental part.

Reagents and equipment used. Gallic acid from ‘Macklin’ (China), Salicylic acid from ‘Rhydburg Pharmaceuticals’ (Germany), quercetin, apigenin, kaempferols from ‘Regal’ (China), and rutin were extracted from natural sources by extraction and column chromatography methods. Water, acetonitrile, acetic acid of chemically pure brand, and sodium hydroxide were used as reagents with HPLC purity.

The content of polyphenols in the food additive was determined on an LC-40 Nexera Lite high-performance liquid chromatograph manufactured by Shimadzu, Japan.

Preparation of standard solutions. Gallic acid (5.2 mg), salicylic acid (5.2 mg), rutin (5 mg), quercetin (5 mg), apigenin (5 mg), kaempferol (5 mg) were dissolved in 96% ethanol in an ultrasonic bath for 20 minutes and transferred to a 50 ml flask and made up to the mark with ethanol. 200 μl of each solution was taken and mixed and diluted to prepare a total of 4 different solutions. Each solution was poured into a vial and used for analysis.

Preparation of the extract of food additive ‘AS-KAMOL.’ For the extraction of phenolic compounds, 1 g of the test sample was weighed with an accuracy of 0.01 g on an NV222 scale manufactured by OHAUS (USA), placed in a 50 ml conical flask, and 25 ml of 96% ethanol was added. The mixture was extracted in an ultrasonic bath GT SONIC-D3 (China) for 20 minutes at a temperature of 60 oC. Then the mixture was cooled, filtered, and made up to 25 ml with ethanol in a volumetric flask. 1.5 ml of the extract was centrifuged at 7000 rpm in a Mini-7 centrifuge (BIOBASE, China) and filtered through a 0.45 μm syringe filter and used for analysis.

Chromatographic conditions.

Determination of phenolic compounds. Standard solution, sample extract Shim pack GIST C18 reversed-phase column (150 × 4.6 mm; 5 μm, Shimadzu, Japan) and a gradient mobile phase consisting of acetonitrile (A) and 0.5% acetic acid in water (B) (Table 1) were used. The injection volume was 10 μl, the flow rate was 0.5 ml/min, and the column thermostat was set to 40°C. The analytical signal (peak area) of phenolic compounds was recorded at 300 nm (Figure 1).

Table 2.

Mobile phase gradient program

Results obtained.

Determination of the  number of phenolic compounds in the food additive ‘AS-KAMOL.’ A chromatogram of a sample extract weighing 1 g was obtained, and based on the results, the number of phenolic compounds in 100 g of the sample was calculated using the following formula and presented in Tables 3-4.

Here, X is the amount of phenolic compounds in 100 grams of fruit, mg;

C_phen is the concentration of phenolic compounds in the extract determined by the HPLC method, mg/l;

V_extract is the volume of the sample extract, l;

m_example is the mass of the sample taken for extract preparation.

Table 3.

The number of polyphenols and retention times in the food supplement ‘AS-KAMOL.’

DISCUSSION OF THE OBTAINED RESULTS. The chromatographic analysis of the ‘AS-KAMOL’ food supplement revealed the presence of several polyphenolic compounds, including gallic acid, rutin, salicylic acid, quercetin, apigenin, and kaempferol. Among these, rutin and apigenin were found in relatively high concentrations, with kaempferol also present in moderate amounts.

The retention times and concentration values obtained during high-performance liquid chromatography (HPLC) confirmed that the polyphenols present in the supplement belong to biologically active classes of compounds, most notably flavonoids and phenolic acids. These compounds are widely studied in the scientific literature for their antioxidant and potential therapeutic properties. However, it should be noted that the presence of these compounds in the supplement alone does not provide sufficient evidence of any biological or health-promoting effects in humans.

The observed chemical composition suggests that the supplement may have antioxidant potential, given the known properties of rutin, apigenin, and kaempferol reported in previous studies. Nevertheless, verifying such bioactivity would require additional in vitro, in vivo, or clinical studies involving human subjects. Therefore, while the identified compounds are promising from a phytochemical perspective, their exact health effects remain to be determined through separate pharmacological or medical research.

CONCLUSION

The conducted chromatographic analysis of the ‘AS-KAMOL’ food supplement demonstrated the presence of several phenolic compounds, including rutin, apigenin, kaempferol, quercetin, gallic acid, and salicylic acid. Among these, rutin and apigenin were found in relatively high concentrations, suggesting a rich polyphenolic profile.

These findings confirm that the supplement contains natural compounds commonly associated with antioxidant and anti-inflammatory properties in the scientific literature. However, since the present study focused solely on chemical profiling, no direct conclusions regarding the supplement's biological efficacy or health benefits can be drawn.

Further experimental work, including in vitro, in vivo, and clinical investigations, is necessary to evaluate the potential functional effects of ‘AS-KAMOL’ on human health. Until such studies are conducted, any therapeutic claims should be considered hypothetical.

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