ANTIRADICAL AND ANTIOXIDANT ACTIVITIES OF Acorus calamus L. AND Oryza sativa L. EXTRACTS USING THE DPPH• ASSAY

ABSTRACT

In this study, the antiradical and antioxidant activities of Acorus calamus L. (sweet flag) and Oryza sativa L. (rice) extracts were evaluated. Antioxidant activity was determined using a spectrophotometric method based on the scavenging of the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) free radical. This method enables the detection of biologically active compounds capable of donating hydrogen atoms or electrons. The DPPH• radical scavenging assay proposed by Blois was applied with minor modifications. The results demonstrated that the extracts of Acorus calamus L. and Oryza sativa L. possess significant antiradical and antioxidant activities.

АННОТАЦИЯ

В этом исследовании изучалась антирадикальная и антиоксидантная активность экстрактов Acorus calamus L. (аир обыкновенный) и Oryza sativa L. (рис). Антиоксидантная активность определялась методом спектрофотометрии с использованием ингибирования свободного радикала 2,2-дифенил-1-пикрилгидразила (DPPH•). Цель этого подхода — выявить биологически активные соединения, способные отдавать атом водорода или электрон. В этом исследовании использовался метод ингибирования радикала DPPH•, предложенный Блуа, с небольшими модификациями. Экстракты Acorus calamus L. и Oryza sativa L. демонстрируют значительную антирадикальную и антиоксидантную активность, что подтверждается полученными данными.

Keywords: Acorus calamus L., Oryza sativa L., antioxidant properties, antiradical effects, DPPH• assay, free radicals.

Ключевые слова: Acorus calamus L., Oryza sativa L., антиоксидантная активность, антирадикальная активность, метод DPPH•, свободные радикалы.

INTRODUCTION

Antioxidants in different chronic diseases and prophylaxis by others have been gaining attention for many years and they are used increasingly, at the level of human health care to relieve disease burden. Such the free radicals emitted by body metabolism may damage cellular membranes, proteins, lipids and DNA integrity. Such damage results in oxidative stress, a risk factor for a variety of other diseases, from cardiovascular diseases to cancer to diabetes to premature aging. Thus, one of the main lines of scientific inquiry has now been to explore natural sources of antioxidant compounds and their effectiveness investigation.

Essential oils, flavonoids, phenolic compounds, and other biologically active substances are contained in one of the medicinal plants, Acorus calamus. Moreover, these compounds could neutralize free radicals and decrease the activity of inflammatory mediators as well. Igir has been used for centuries in folk medicine for gastrointestinal diseases, colds, joint pain, and other inflammatory conditions. Its rhizome was used in the form of a decoction and infusion. Moreover, Oryza sativa (rice) is not just the primary food crop but also a plant that is rich in biologically active components. Phenolic compounds, gamma-orizanol, tocopherols, and other antioxidant substances are present and enriched in rice grain, especially its husk. These substances are key for reducing oxidative stress as well as suppressing inflammatory processes. In folk medicine, rice water and decoction were utilized for intestinal inflammation, gastritis, and general weakness. A range of chemical and biochemical methods are available in research aimed at determining antioxidant activity. The DPPH free radical-based spectrophotometric method is recognized as one of the easiest, fastest, and most trustworthy of these techniques. DPPH• is a stable free radical with a color change from purple to yellow as it is reduced by antioxidant compounds. A spectrophotometer can be used to allow quantification of the color-changing activity of the sample when its radicals are being scavenged. This study aims to elucidate the free radical scavenging activity of mint's alcoholic extract under the DPPH system, assess its ability to inhibit free radicals at different concentrations, and evaluate the IC₅₀ value.

MATERIALS AND METHODS

The discoloration of the purple 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution allows the detection 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 [1]. In this experiment, the method of Blois [2] was used to evaluate the inhibition of free radicals of DPPH• with minor modifications [3]. 

Preparation of DPPH• working solution. A 7.92 mM DPPH• solution in ethanol was prepared in a 100 ml volumetric flask, wrapped in aluminum foil and stored in the dark at room temperature for 30 minutes. 

Preparation of sample extracts. Samples were prepared in different proportions of alcohol extracts based on the yarrow plant and rice flour to test the antioxidant activity.

Figure 1. Composition of Igir and Rice Flour Samples

ANALYSIS OF THE RESULTS OBTAINED.

The antioxidant activity of alcoholic extracts of the Igir and rice flour mixtures was determined by DPPH radical scavenging methods. Results are shown as ARF (%) values and IC₅₀ concentrations, after 30 minutes of reaction. The antioxidant activity differed with the ratio of Igir and rice flour in the mixtures as shown in the data obtained. The combination (3:1 Igir to rice flour) with the highest antioxidant activity demonstrated an ARF of 28.59% (IC₅₀ of 171.15 µL) suggesting that when greater Igir percentage is employed in a mixture, the antioxidant potency increases on the extract. Igir and rice flour 1:1 mixture had a moderate antioxidant activity (ARF of 25.98% and IC₅₀ of 193.81 µL). Although more moderate than that of 3:1, it displayed a remarkable radical scavenging ability. By contrast, the 1:3 mixture (Igir : rice flour) exhibits the lowest antioxidant activity, with an ARF value of 14.16% and an IC₅₀ value of 347.67 µL. IC₅₀ value may reflect a weaker antioxidant effect with respect to reduction in bioactive phenolic compounds found in Igir. The overall results were suggestive that the antioxidant activity of the extracts are significantly impacted by the Igir components. Moreover, higher Igir content results to potent radical scavenging effect for the extracts, so the Igir could serve as a good source of natural antioxidant compounds.

Table 1.

Values of antioxidant activity (ARF%) of the samples’ alcoholic extracts at 30 minutes per 100 µL, and IC50 – the concentration (µL) required to inhibit 50% of the DPPH solution

DISCUSSION

In this study, extracts of the samples were prepared by extracting 1 g of plant material with 25 mL of an extractant (water or 96% ethanol) in an ultrasonic bath for 20 min. After the extract was filtered with a 0.45 µm syringe filter, it was analyzed. 3 mL of DPPH solution and 100 µL of ethanol (blank sample) were added to a 4 mL quartz cuvette for spectrophotometric assay and were placed first in the spectrophotometer. Absorbance (D₁) at 517 nm wavelength was recorded every 5 min for 30 min using a K7000 YOKE (China) spectrophotometer. For an assessment of the antiradical activity of the samples, 25, 50, 75, and 100 µL of the sample were combined with 3 mL of DPPH solution and absorbance at 517 nm (D₂) was recorded by the same procedure. Ethanol was added above to the residual volume adjusting total volume of solution at 3.1 mL in the cuvette.

CONCLUSION. A solution containing 2,2-diphenyl-1-picrylhydrazyl (DPPH) decolorizes, allowing for the detection of certain pure antioxidant compounds capable of donating hydrogen atoms or electrons. The stable DPPH• radical serves as a reagent in spectrophotometric assays. In our experimental study, a slightly modified version of the Blois method was exploited to analyze the DPPH• free radical scavenging activity. Importantly, the optimum ethanolic extraction profile was obtained in the extract of a 3:1 mixture of Igir and rice flour, which showed antiradical activity of 28.59% after 30 min. The IC₅₀ value, 171.15 µL, confirmed the antiradical activity of the extract to be significant.

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