STUDY OF ANTI-RADICAL ACTIVITY OF MIXTURES OF CHAMOMILE (Matricaria Chamomilla) AND OAK BARK (Corticis Quercus) IN VARIOUS RATIO

ABSTRACT

This article presents the results of an in vitro evaluation of the antiradical activity of extracts prepared in different proportions from the local medicinal plant chamomile (Matricaria chamomilla) and oak bark (Corticis Quercus) using the DPPH method. It discusses how the biologically active compounds in these extracts can neutralize radicals that contribute to inflammation, making them potentially beneficial for treating conditions in the oral cavity, such as candidiasis and inflammation caused by various microbes. Among the tested extracts, the one with the highest antiradical index was selected for further study on its effectiveness in treating oral cavity diseases, including gingivitis and candidiasis.

АННОТАЦИЯ

В данной статье представлены результаты in vitro оценки антирадикальной активности экстрактов, приготовленных в разных пропорциях из местного лекарственного растения ромашки (Matricaria chamomilla) и коры дуба (Corticis Quercus) с использованием метода DPPH. В ней обсуждается, как биологически активные соединения в этих экстрактах могут нейтрализовать радикалы, способствующие воспалению, что делает их потенциально полезными для лечения состояний полости рта, таких как кандидоз и воспаления, вызванные различными микробами. Среди протестированных экстрактов был выбран экстракт с самым высоким антирадикальным индексом для дальнейшего изучения его эффективности при лечении заболеваний полости рта, включая гингивит и кандидоз.

Keywords: Radical, spectrophotometer, optical density, inhibition, Matricaria Chamomilla, Oak bark, Corticis Quercus, Candidiasis, Gingivitis.

Ключевые слова: Радикал, спектрофотометр, оптическая плотность, ингибирование, Matricaria Chamomilla, кора дуба, Corticis Quercus, кандидоз, гингивит.

Introduction: Diseases of the oral cavity, such as gingivitis, periodontitis, and candidiasis, are primarily considered infectious diseases caused by pathogenic factors. Often, a decrease in immunity and the presence of viral infections can lead to the activation of pathogenic microorganisms, including staphylococci and fungi that are normally present in the body. This may result in an inflammatory process characterized by a mixed flora infection [1]. When we examine the mechanism of the inflammatory process,  radicals are produced in the body as a result of metabolism. These active particles can oxidize healthy cells, leading to inflammation. Consequently, the antioxidant status of the body serves as a key indicator of overall health. Many pathological processes, especially cardiovascular diseases, atherosclerosis, valvular disorders, and other heart-related conditions are associated with oxidative stress and the formation of free radicals [2].

The active form of oxygen (ROS) causes various free radical reactions in cells. Oxidation of cell membrane lipids, nucleic acids, proteins, enzymes and DNA molecules can lead to a wide range of pathogenetic effects. In this regard, reactive oxygen species (singlet oxygen atom (O), superoxide anion radical (O^-2), hydrogen peroxide (H2O2), hydroxyl radical (OH), peroxyl radical (RRCOO), nitric oxide (NO), peroxynitrite (ONOO)) are important biological factors [3].

Plants are an inexhaustible source of medicinal products with various therapeutic effects. The study of the molecular mechanisms of pathogenesis of many diseases occurring in plant, animal and human organisms has shown that all of them are to one degree or another associated with the reduction of free radicals. The activity of medicinal plants can be explained by the antiradical activity of biologically active compounds (for example, vitamins, beta-carotene and polyphenols) contained in them. It is known that polyphenols, unlike low molecular weight compounds (tocopherol, ascorbic acid, low molecular weight phenols, etc.), have fast and slow antioxidant properties [4]. Therefore, the search and study of the control of such processes based on natural and synthetic raw materials remains relevant.

Materials and methods: The AA of extracts of chamomile and oak bark of various concentrations was determined spectrophotometrically. The method of determining antiradical activity (using DPPH) is an indirect method, based on the interaction of antioxidants with the stable chromogenic radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and shows correlation with the results obtained by direct methods [5].

Chamomile (Matricaria Chamomilla) plant and Oak bark (Corticis Quercus) were ground to a size of 0.1-1 mm and 3 different samples were prepared based on them. The quantitative ratio of plant parts in the samples is given in the table below.

Table 1.

Proportions of plant parts in sample preparation

Extracts of the samples were prepared in two different ways.

1. Preparation of aqueous extract. 1 g of plant sample was boiled in 25 ml of water for 10 minutes in a flask equipped with a reflux condenser. The obtained extract was filtered through a 0.45 μm syringe filter and used for analysis.

2. Preparation of ethanolic extract. 1 g of plant sample was ultrasonically extracted in 25 ml of 96% ethanol for 20 minutes at 60 °C. The obtained extract was filtered through a 0.45 μm syringe filter and used for analysis.

The discolouration of the purple 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution allows the detection of the presence of some pure antioxidant compounds that have the properties of donating a hydrogen atom or an electron. Stable DPPH• is a reagent used in spectrophotometric analysis [6]. In this experiment, the method of evaluating the inhibition of DPPH• free radicals by the method developed by Blois [7] was used with minor modifications [8]. A 0.812 mM DPPH• solution in ethanol was prepared in a 100 ml volumetric flask, wrapped in aluminium foil and kept in the dark at room temperature for 30 minutes. A 4 ml quartz cuvette was filled with 3 ml of DPPH solution and 200 μl of ethanol (blank sample) and placed in a spectrophotometer, and the absorbance (D1) at a wavelength of 517 nm was measured every 30 seconds for 10 minutes using a K7000 spectrophotometer manufactured by YOKE (China). To evaluate the antiradical properties of the sample, 100, 150, 200 μl of the sample was mixed with 3 ml of DPPH solution and the absorbance (D2) at 517 nm was measured in the above order. Ethanol was added to the remaining volume to bring the total volume of the solution in the cuvette to 3.2 ml. The antiradical properties of the samples were calculated using the following formula:

The results obtained are presented in the following table:

Table 2.

Measured light absorption and antiradical activities of blank and tested alcoholic extracts of Chamomile plant and Oak bark added to DPPH solution

Table 3.

 Measured light absorption and antiradical activities of blank and test aqueous extracts of Chamomile plant and Oak bark added to DPPH solution

Figure 1. Graphical representation of the measured light absorption of blank and tested sample solutions added to DPPH solution

Figure 2. Graphical representation of the measured light absorption of blank and tested sample solutions added to DPPH solution

Results and discussions: To calculate the IC₅₀ of the sample, which represents the 50% inhibitory concentration of the DPPH solution, a graph was created using the absorbance (D2) values and the antiradical scavenging activity (AA%) values recorded after 30 minutes in each experiment. The IC₅₀ was then determined based on the trend line function applied to this graph.

Table 4.

IC50 values ​​of alcoholic extracts of all samples

Figure 3. Graph of the relationship between AA% and volumes determined at 10 minutes for sample A

The trend line plotted on the graph was calculated from the function formula y=mx+b, and the volume that exhibits 50% AA% (IC50) was calculated based on the formula x=(y-b)/m:

Table 5.

 IC50 values ​​of alcoholic extracts of all samples

Figure 4. Graph of the relationship between AA% and volumes determined at 10 minutes for sample A

The trend line plotted on the graph was calculated from the function formula y=mx+b, and the volume that exhibits 50% AA% (IC₅₀) was calculated based on the formula x=(y-b)/m:

Conclusion: The samples demonstrate antiradical activity, particularly the extract of Chamomile and Black Oak in a 1:3 ratio, which exhibits a higher antiradical effect compared to the other samples, with an IC₅₀ value of 92.02 μl. The aqueous extract from this combination also shows superior antiradical activity, with an IC₅₀ value of 114.19 μl, supporting the conclusion that it effectively scavenges free radicals, which are known to cause inflammation.

The aim of our study was to develop a food additive based on this ratio to help prevent and treat oral diseases, considering the well-documented antibiotic and antifungal properties of Chamomile (Matricaria chamomilla) and Oak Bark (Corticis quercus L.). We have named this biologically active compound AS-ZIYOD.

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