EVALUATION OF ANTIOXIDANT (FREE RADICAL SCAVENGING) ACTIVITY OF MINT (Mentha sp.) ALCOHOLIC EXTRACT BASED ON DPPH METHOD

ABSTRACT

This study investigated the antioxidant, i.e., free radical scavenging activity of mint (Mentha sp.) alcoholic extract using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical. Antioxidants play a vital role in reducing oxidative stress in living organisms, and their extraction from plant sources has been widely studied in recent years.

Mint samples were prepared using 96% ethanol via ultrasonic extraction, and the ability of the extract to inhibit DPPH• free radicals was measured spectrophotometrically at 517 nm. Experiments were performed based on the classic method proposed by Blois, with minor modifications. Different volumes of extract (25–100 µL) were reacted with DPPH solution, and changes in absorbance were monitored for 30 minutes.

From these results, the radical scavenging activity (RSA%) and IC50 value were determined. The mint alcoholic extract showed significant free radical scavenging activity, with an IC50 value of 391.33 µL. These findings highlight mint as a natural source of antioxidants, with potential applications in food, pharmaceutical, and traditional medicine.

АННОТАЦИЯ

В данном исследовании была оценена антиоксидантная, то есть способность к нейтрализации свободных радикалов, активность спиртового экстракта мяты (Mentha sp.) с использованием радикала DPPH (2,2-дифенил-1-пикрилгидразил). Антиоксиданты играют важную роль в снижении окислительного стресса у живых организмов, а их извлечение из растительных источников в последние годы широко изучается.

Образцы мяты были приготовлены с использованием 96% этанола и ультразвуковой экстракции, а способность экстракта ингибировать свободные радикалы DPPH• измерялась спектрофотометрически при 517 нм. Эксперименты проводились на основе классического метода Блуа, с небольшими модификациями. Разные объемы экстракта (25–100 µL) взаимодействовали с раствором DPPH, и изменения оптической плотности отслеживались в течение 30 минут.

На основании полученных результатов были рассчитаны антирадикальная активность (ARF%) и значение IC50. Спиртовой экстракт мяты продемонстрировал значительную антирадикальную активность с IC50 = 391,33 µL. Эти данные подчеркивают ценность мяты как натурального источника антиоксидантов, с перспективами применения в пищевой, фармацевтической и народной медицине.

Keywords: mint, antioxidant activity, DPPH method, free radical scavenging, IC50, alcoholic extract, spectrophotometry.

Ключевые слова: мята, антиоксидантная активность, метод DPPH, нейтрализация свободных радикалов, IC50, спиртовой экстракт, спектрофотометрия.

Introduction

In recent years, the importance of antioxidant compounds in maintaining human health and preventing various chronic diseases has been steadily increasing. Free radicals are generated in the body as a result of metabolic processes and can negatively affect cell membranes, proteins, lipids, and DNA structure. These processes lead to oxidative stress, which may contribute to cardiovascular diseases, oncological disorders, diabetes, and accelerated aging. Therefore, the identification of antioxidant compounds derived from natural sources and the evaluation of their activity represent an urgent scientific issue [1; 83-89].

Plants are rich in biologically active substances, particularly phenolic compounds, flavonoids, ascorbic acid, and essential oils, which possess strong antioxidant properties. Mint (Mentha sp.) is one of the widely used medicinal plants in folk and traditional medicine, known for its anti-inflammatory, antibacterial, antispasmodic, and sedative properties. In addition, the antioxidant potential of the biologically active compounds present in mint requires further scientific substantiation [2;113-121].

Various chemical and biochemical methods are available for determining antioxidant activity. Among them, the spectrophotometric method based on the DPPH free radical is considered one of the simplest, fastest, and most reliable techniques. DPPH• is a stable free radical characterized by a color change from purple to yellow when reduced by antioxidant substances. This color change can be measured using a spectrophotometer, allowing a quantitative assessment of the radical scavenging activity of the sample [3;203-209].

The aim of this study is to determine the free radical scavenging activity of the alcoholic extract of mint using the DPPH method, to evaluate its ability to inhibit free radicals at different concentrations, and to calculate the IC₅₀ value. The obtained results will contribute to the scientific validation of mint as a natural source of antioxidants [4;1118].

Materials and methods

The colour shift of the purple-colored 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution suggests that there are some pure antioxidant compounds having hydrogen atom/electrons donating ability. The stable DPPH• radical is widely used in spectrophotometrical analyses as a reagent. Here, the method established by Blois to measure the radical-scavenging activity of DPPH• was employed with some changes [5;1-6]. 

Analysis: Determination of antiradical activity. For the blank samples, 3 mL of DPPH solution and 100 μL of solvent (without extract) was put into a 4 mL quartz cuvette, and absorbance D1 was recorded every 5 minutes and at 517 nm at 30 minutes on a YOKE K7000 spectrophotometer (China). For the following four doses of extract to assess the antiradical activity of 25, 50, 75 and 100 μL of extracts, and 3 mL of DPPH solution. The absorbance values (D2) were further read under similar conditions. The remaining volume was filled with solvent to the total volume of 3.1 mL in the cuvette.

Results and discussion

The obtained results are presented in the following table:

Table 1.

Measured absorbance values and calculated radical scavenging activity (RSA%) of the blank and the tested alcoholic extract samples added to the DPPH solution

Figure 1. Graphical representation of the measured absorbance of the blank and tested alcoholic extract samples added to the DPPH solution

Figure 2. Graph showing the relationship between the AA% values determined at the 10th minute and the volumes of the alcoholic extract sample

To calculate the IC₅₀ value of the samples — the concentration required to inhibit 50% of the DPPH solution — a graph was constructed for each experiment based on the antiradical activity (ARA%) values measured at the 30th minute and the volume of the added alcoholic samples. The IC₅₀ value was then determined using the function of the trend line fitted to the graph. Using the equation of the trend line fitted to the graph, y = mx + b, the volume corresponding to 50% AA (IC₅₀) was calculated according to the formula x = (y − b) / m.

Discussion

In this study, the dried aerial parts of mint (Mentha sp.) were selected as the research object. The sample was prepared according to standard procedures, and the extraction process was carried out using 96% ethanol. To 1 g of plant material, 25 ml of ethanol was added, and the extraction was performed in an ultrasonic bath for 20 minutes. This method is advantageous as it ensures a more complete release of biologically active compounds.

The obtained extract was filtered through a 0.45 µm syringe filter, prepared for analysis, and diluted tenfold.

The DPPH• working solution was prepared in ethanol at a concentration of 7.92 mM and kept protected from light for 30 minutes. To determine antiradical activity, 3 ml of DPPH solution and different volumes (25, 50, 75, and 100 µl) of mint extract were added into a quartz cuvette. Ethanol was used as a blank sample. The total reaction mixture volume was adjusted to 3.1 ml.

Absorbance was measured using a YOKE (China) K7000 spectrophotometer at a wavelength of 517 nm at 5-minute intervals over a period of 0–30 minutes. Based on the obtained absorbance values, antiradical activity (ARA%) was calculated. The results showed that as the concentration of mint extract increased, the inhibition degree of DPPH• radicals also increased [6;126-129].

When 25 µl of extract was added, the ARA% value was approximately 3%, whereas at 100 µl it exceeded 12%. This confirms the presence of compounds in mint that possess electron- or hydrogen-donating properties. Based on graphical analysis, the IC₅₀ value was determined to be 391.33 µl. This result indicates that the alcoholic extract of mint exhibits a moderate level of antiradical activity.

Comparison of the obtained results with literature data confirms that mint, among other medicinal plants, is an effective natural source of antioxidants.

Conclusion

In conclusion, the alcoholic sample demonstrated antiradical activity. Specifically, the IC₅₀ value of the alcoholic extract was 391.33 µl, indicating its antiradical potential. The conducted study revealed that the alcoholic extract of mint (Mentha sp.) exhibits significant antiradical activity according to the DPPH method.

During the study, it was observed that the extract’s ability to inhibit DPPH• free radicals depended on time and concentration. As the sample volume increased, a decrease in absorbance values was recorded, indicating a reduction in the concentration of free radicals.

The IC₅₀ value of 391.33 µl demonstrates that the alcoholic extract of mint has a certain degree of effectiveness against free radicals. These results can be explained by the presence of phenolic compounds and other biologically active substances in mint.

In summary, mint can be considered a promising natural antioxidant source for application in the food industry, pharmaceuticals, and traditional medicine. Future studies should focus on identifying individual biologically active components of mint extract, conducting their quantitative analysis, and evaluating antioxidant efficacy under in vivo conditions. The findings of this research provide a scientific basis for the development of mint-based functional products and biologically active supplements.

References:

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