3. Extraction and preliminary phytochemical analyses 1 Introduction
4.3 Results
4.3.1 Qualitative antioxidant activity assay
The antioxidant activity of the plant was screened using DPPH on the TLC plates which were developed in different separation systems. The yellow spots on the purple
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background indicated the presence of antioxidant compounds. The plant extracts have antioxidant compounds but separated well in the EMW mobile system (Figure 4.1).
Most of the antioxidant compounds were visible in the EMW mobile system.
Figure 4. 1: Chromatograms of Dombeya rotundifolia developed in BEA, CEF and EMW and sprayed with 0.2% DPPH.
Key: H- Hexane, C- Chloroform, D- Dichloromethane, EA- Ethyl acetate, A- Acetone, E- Ethanol, M- Methanol, B- Butanol, W- Water, EMW- Ethyl acetate: Methanol: Water, CEF- Chloroform: Ethyl acetate: Formic acid and BEA- Benzene: Ethanol: Ammonium hydroxide.
4.3.2 Quantitative antioxidant activity assay
The antioxidant activity of the plant was quantified using the DPPH method. The graph below shows the quantitative antioxidant activity, which is represented by the percentage scavenging activity of the plant extracts. The increases in scavenging activity increase with the increase in the concentration of the plant extracts. Ascorbic acid (control) had the highest scavenging activity compared to the plant extracts. The dichloromethane extract had the highest scavenging activity while acetone and methanol extracts had the lowest scavenging activity (Figure 4.2). The antioxidant activity of the plant was quantified using the ferric reducing power method. The increase in the absorbance increases with the concentration in the plant extracts.
Ascorbic acid (control) had the highest reducing capacity compared to the plant extracts. In the plant extract, hexane had the highest reducing capacity while acetone had the least (Figure 4.3).
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Figure 4. 2: Percentage free radical (DPPH) scavenging of the plant extracts compared with the ascorbic acid as a standard at 517 nm.
Figure 4. 3: The ferric reducing power of the plant extracts compared with the ascorbic acid as a standard at 700 nm.
62 4.4 Discussion
The aim was to investigate the antioxidant activity of Dombeya rotundifolia. Plants are a key source of natural antioxidants and produce a variety of secondary metabolites with antioxidative properties that may be used therapeutically. Polyphenols are the most common antioxidant chemical found in plants. The antioxidant activity is based on the redox characteristics that allow them to work as reducing agents, hydrogen donors, singlet oxygen quencher and metal chelators (Stanković et al., 2016). The antioxidant activity of the plant was evaluated using DPPH and the ferric reducing power since one cannot conclude based on one method, because antioxidant activity occurs through different mechanisms and is influenced by numerous factors (El Jemli et al., 2016). The yellow spots against the purple background indicated the presence of antioxidant compounds (Figure 4.1). This means that the plant extracts have hydrogen donating antioxidant compounds because they were able to reduce violet DPPH to a yellow DPPPH-H. All the plant extracts had the antioxidant compounds represented by a yellow spot, but they did not separate in all the systems due to their polarity to the solvent in the system. There was a clear separation in the EMW mobile system and most of the antioxidant compounds were visible in the EMW mobile system. The chromatograms (Figure 4.1) are similar to the study of Kudumela and Masoko (2017), who noted a separation in one mobile system (EMW) whereas in other systems the compounds were found at the bottom.
The quantification of antioxidants was done using the DPPH assay and the ascorbic acid was used as control. The method is based on the reduction of violet DPPH into a yellow DPPH molecule, which represents the free radical reducing activity of antioxidant. Ascorbic acid had the highest scavenging activity compared to the plant extracts (Figure 4.2). Vitamins, phenolic and carotenoids are the important types of natural antioxidants that provide protection (Bajalan et al., 2016); hence, the ascorbic acid had the highest activity. In plant extracts, dichloromethane had the highest scavenging activity while acetone and methanol extracts had the least activity.
Analysing the results for each concentration, dichloromethane had the highest scavenging activity for all the concentrations except at 250 µg/mL where the methanol extract had the highest activity. The acetone extract had the least activity at all the concentrations except at 31 and 15 µg/mL, where methanol had the lowest activity at those concentrations. When relating the total content and the antioxidant activity,
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acetone and methanol as extracts both had lower content in phenolic, tannin and flavonoid; hence, they have the lower antioxidant activity. The absorbance of DPPH radical decreases as the concentration increases. This is because of the reaction between antioxidant and radical molecules, which results in the radical being scavenged by hydrogen donation (Bhalodia et al., 2013). Antioxidants offer an alternative method for both treating and preventing chronic disorders. Due to their oxidative activity, phenolic compounds have the potential to prevent and treat a wide range of oxidative stress-related disorders (Sofowora et al., 2013). Phytochemicals with antioxidant properties are needed so that plant products can be effective in treating human diseases such TB (Kumar et al., 2021).
The quantification of antioxidants was done using the ferric reducing power assay and the ascorbic acid was used as control. The assay was based on the reduction of Fe3+/ ferricyanide complex to the Fe2+/ ferrous form, which acts as a potential indicator of antioxidant activity. In the plant extract, hexane had the highest reducing capacity while acetone had the least due to their hydrogen donating ability (Figure 4.3). The hexane extract acts as an electron donor which reacts with free radicals to convert them into more stable molecules and end the free radical chain reactions (Irshad et al., 2012). The absorbance increases with an increase in concentrations which indicate the antioxidant activity; so, the reducing power of the plant extracts and ascorbic acid increase as the concentration increases. There is a relationship between the phenolic content and the antioxidant activity; hence, the extract that had the highest phenolic content had the highest reducing capacity, which indicated the antioxidant activity. Kudumela and Masoko (2018) evaluated antioxidant activity using the same method, where D rotundifolia had the highest reducing power when compared to other plants. The antioxidant activity was due to the flavonoids and phenolics that had been identified from the leaves.