Antioxidant and Antimicrobial Activities of Methanolic Extract of Sorghum Bicolor Seeds

Sorghum bicolor (L.) Moench, commonly known as sorghum, is a cereal crop extensively cultivated in tropical and subtropical regions worldwide. It serves as a staple food for millions of people, particularly in arid and semi-arid regions, due to its drought tolerance and nutritional value [1]. Sorghum seeds are rich in carbohydrates, proteins, and bioactive compounds, including phenolic acids, flavonoids, and tannins, which contribute to their health-promoting properties [2]. These phytochemicals exhibit potent antioxidant and antimicrobial activities, making sorghum a valuable candidate for nutraceutical and therapeutic applications [3]. Antioxidants play a crucial role in neutralizing free radicals, preventing oxidative stress-related diseases such as cancer, cardiovascular disorders, and neurodegenerative conditions [4]. Plant-derived antioxidants are particularly valued for their safety and efficacy. Similarly, antimicrobial agents are vital in combating infections caused by pathogenic microorganisms. With the rise of antimicrobial resistance, there is an increasing demand for natural antimicrobials derived from plants [5]. Sorghum seeds, with their rich phytochemical profile, offer a dual benefit of antioxidant and antimicrobial properties, addressing critical health challenges while supporting industrial applications in food preservation and pharmaceuticals [6]. Methanol is widely used as a solvent in phytochemical extraction due to its ability to efficiently solubilize both polar and non-polar compounds [7]. The methanolic extract of Sorghum bicolor seeds is particularly effective in isolating phenolic compounds and flavonoids, which are key contributors to antioxidant and antimicrobial activities [8]. Additionally, methanolic extraction ensures higher yields of bioactive compounds compared to aqueous or other organic solvents, enhancing the potential for biological efficacy [9].

LITERATURE REVIEW

Sorghum bicolor is a cereal crop widely recognized for its nutritional and medicinal properties. Studies have shown that sorghum seeds are rich in bioactive compounds, particularly phenolic acids, flavonoids, and tannins, which contribute significantly to its antioxidant and antimicrobial potential [10,11]. Beta and Rooney [12] reported that the polyphenolic content in sorghum varies among cultivars and is influenced by environmental conditions. These polyphenols exhibit strong free radical scavenging activity, which has implications for preventing oxidative stress-related diseases. Furthermore, sorghum extracts have been evaluated for their antimicrobial properties, with significant activity observed against Gram-positive and Gram-negative bacteria, making them a promising natural alternative to synthetic antimicrobials [13].

Phenolic compounds are secondary metabolites in plants that play a vital role in defense mechanisms against environmental stressors, including UV radiation, pathogens, and oxidative damage [14]. These compounds act as free radical scavengers, reducing agents, and metal chelators, thereby preventing lipid peroxidation and cellular damage [15]. Flavonoids, a subclass of phenolics, are particularly effective in reducing oxidative stress and are associated with various health benefits, such as reducing the risk of cardiovascular diseases, diabetes, and certain cancers [16]. Sorghum, due to its high phenolic content, is an excellent source of dietary antioxidants. Moreover, the unique structural properties of tannins in sorghum contribute to its antimicrobial efficacy by disrupting microbial cell membranes and inhibiting enzyme activity [17]. Plant extracts have been extensively studied for their antimicrobial properties due to their diverse bioactive constituents, including phenolics, alkaloids, terpenoids, and essential oils [18]. These natural compounds target multiple sites in microbial cells, making them effective against a broad spectrum of pathogens and reducing the likelihood of resistance development [19]. Methanolic extracts, in particular, are known to yield higher concentrations of bioactive compounds, enhancing their antimicrobial potency [20]. Research on sorghum methanolic extracts has demonstrated significant inhibitory effects on common pathogens such as Escherichia coli, Staphylococcus aureus, and Candida albicans [21].

MATERIALS AND METHODS

3.1. Materials

Mature seeds of Sorghum bicolor were collected from certified local suppliers. Solvents such as hexane, methanol, glacial acetic acid, ferric chloride, sulphuric acid, chloroform and ammonia used were analytical grade.

3.2. Microbial Species

The antimicrobial activity of Sorghum bicolor seed extract was tested against three microbial species: Bacillus megaterium, Bacillus thuringiensis, and Escherichia coli.

3.2. Methods

3.2.1. Sample Preparation

 The seeds were thoroughly cleaned to remove dirt and impurities, dried at room temperature in a well-ventilated area, and then ground into a fine powder using a laboratory mill. The powdered seeds were stored in airtight containers at 4°C to prevent degradation before extraction [22].

3.2.2. Extraction of Sorghum bicolor Seeds

The methanolic extraction of Sorghum bicolor seed powder was carried out using the Soxhlet apparatus. Approximately 100 g of the seed powder was extracted with 500 mL of 80% methanol at a temperature of 60°C for 6–8 hours [23]. Alternatively, a maceration technique was employed, where the powdered seeds were soaked in methanol for 72 hours with occasional shaking. The extract was filtered using Whatman No. 1 filter paper to remove solid residues. The filtrate was concentrated using a rotary evaporator under reduced pressure at 40°C, followed by storage in a desiccator until further analysis [24,25].

3.2.3. Phytochemical Screening

Phytochemical screening of methanolic extraction of Sorghum bicolor seed was performed by using keller-killiani, borntrager’s, foam and hemolytic tests.

1. Test for Cardiac Glycosides (Keller-Killiani Test)

The test was performed by taking 2 mL of the methanolic extract in a test tube, followed by the addition of glacial acetic acid and one drop of 5?rric chloride solution. Concentrated sulfuric acid was carefully added along the side of the test tube to form a separate layer. The formation of a brown ring at the interface indicated the presence of cardiac glycosides [26,27].

2. Test for Anthraquinone Glycosides (Borntrager’s Test)

For this test, 3 mL of the methanolic extract was mixed with dilute sulfuric acid and boiled for 5 minutes. The mixture was filtered, and the filtrate was cooled. An equal volume of chloroform was added to the filtrate and shaken vigorously. The organic layer was separated and mixed with an equal volume of ammonia. The development of a pink or red color in the ammoniacal layer confirmed the presence of anthraquinone glycosides [28-30].

3. Test for Saponins

Foam Test: The methanolic extract was shaken vigorously with distilled water. The presence of stable foam persisting for at least 10 minutes was considered a positive indicator for saponins.

Hemolytic Test: A drop of fresh blood was placed on a glass slide, and a small amount of the methanolic extract was added. Observation of hemolysis (rupture of red blood cells) indicated the presence of saponins [31].

3.2.4. Antimicrobial Activity Determination

The antimicrobial activity of Sorghum bicolor seed extract was evaluated using the disc diffusion method. The test organisms used were Bacillus megaterium, Bacillus thuringiensis, and Escherichia coli. The bacterial cultures were grown overnight at 37°C on appropriate media: nutrient agar for B. megaterium and B. thuringiensis, and MacConkey agar for E. coli. The Sorghum bicolor seed extract was prepared at concentrations of 20 mg/L, 100 mg/L, and 200 mg/L by dissolving the extract in sterile distilled water, while penicillin (20 mg/L) served as the positive control. Each concentration of the extract (20 µL) was applied to sterile filter paper discs, which were placed on the surface of the inoculated agar plates. After incubation at 37°C for 24 hours, the plates were examined for inhibition zones. The diameter of the inhibition zone was measured in millimeters to assess the antimicrobial activity. The results indicated a dose-dependent increase in antimicrobial activity, with larger inhibition zones observed at higher concentrations of the seed extract [32,33].

3.2.5. Antioxidant Activity Determination

To evaluate the antioxidant activity of Sorghum bicolor seed extract, the DPPH (2,2-diphenyl-1-picrylhydrazyl) method was employed using a 96-well plate assay. A stock solution of the Sorghum bicolor seed extract was prepared by dissolving the powdered seed extract in methanol to achieve a concentration of 1 mg/ml. Ascorbic acid, used as standard, was also prepared at 1 mg/ml concentration. The assay was conducted in triplicate for each sample. In sample wells, 1 mg/ml of the sample (Sorghum bicolor extract) and in standard wells, 1 mg/ml of Ascorbic acid (Standard) was added. The 100 µL of a 0.1 mM DPPH solution, freshly prepared in methanol was added in each well including control. The mixture was incubated at room temperature in the dark for 30 minutes to allow the reaction to occur. Absorbance was measured at 517 nm using a microplate reader. The percentage inhibition of the DPPH radical was calculated using the formula:

DPPH radical scavenging activity (%) = [(Absorbance of control - Absorbance of test sample) / Absorbance of control] × 100.

The results were expressed as the mean percentage inhibition of the DPPH radical for each sample. The control, containing no antioxidant, was used to measure the baseline absorbance, and the standard ascorbic acid was used for comparison to assess the antioxidant potential of the Sorghum bicolor seed extract [34,35].

RESULTS AND DISCUSSION

4.1. Extraction of of Sorghum bicolor Seeds

The methanolic extract of Sorghum bicolor seeds was obtained using Soxhlet extraction with 500 mL of 80% methanol at 60°C for 6–8 hours. The extraction yielded 68 percentage of the extract. Soxhlet extraction is efficient in obtaining bioactive compounds, especially phenolics and flavonoids, by allowing continuous extraction under heat, which helps break down plant cell walls and enhances solvent penetration. After filtration, the extract was concentrated using a rotary evaporator at 40°C and stored in a desiccator to preserve its integrity. The high yield and efficient extraction suggest that this method is effective for obtaining bioactive compounds with potential antioxidant and antimicrobial activities, consistent with findings in other studies [36-38].

4.2. Phytochemical Screening

Phytochemical analysis of Sorghum bicolor seed extract was performed using several tests to identify the presence of bioactive compounds, results were shown in Table 1. The Keller-Killiani test revealed the presence of cardiac glycosides, as indicated by the appearance of a reddish-brown color at the junction of the two liquid layers, with the upper layer turning bluish-green. Borntrager’s test for anthraquinone glycosides showed a red color in the ammoniacal layer after treatment with diluted sulfuric acid, boiling, and filtration, confirming the presence of anthraquinone glycosides. The foam test, which involved shaking the extract vigorously with water, resulted in the formation of a persistent, stable foam, indicating the presence of saponin glycosides. Additionally, the hemolytic test, where the extract was added to a drop of blood on a glass slide, produced a hemolytic zone, further confirming the presence of saponin glycosides. These results are consistent with previous studies and suggest that Sorghum bicolor seeds contain a variety of bioactive compounds with potential pharmacological applications

Table 1: Phytochemical Identification Tests for Sorghum bicolor Seeds Extract

4.3. Antimicrobial Activity Determination

The antimicrobial activity of Sorghum bicolor seed extract was assessed against three test organisms: Bacillus megaterium, Bacillus thuringiensis, and Escherichia coli at various concentrations (20 mg/L, 100 mg/L, and 200 mg/L). The results, as shown in Table 2, indicate a dose-dependent increase in antimicrobial activity. At the lowest concentration (20 mg/L), the extract demonstrated mild activity, with inhibition zones of 4 mm against B. megaterium and B. thuringiensis, and 5 mm against E. coli. At 100 mg/L, the inhibition zones increased significantly, with B. megaterium and B. thuringiensis showing inhibition zones of 12 mm, and E. coli showing a 14 mm zone. The highest concentration (200 mg/L) exhibited the strongest activity, with inhibition zones of 16 mm against B. megaterium, 15 mm against B. thuringiensis, and 18 mm against E. coli. The control, which used penicillin at 20 mg/L, showed inhibition zones of 6 mm against B. megaterium and B. thuringiensis, and 8 mm against E. coli, indicating that the Sorghum bicolor seed extract has comparable or slightly stronger antimicrobial activity than penicillin at higher concentrations.

Table 2: Determination of Antimicrobial Activity of Sorghum bicolor Seeds Extract

4.4. Antioxidant Activity

The DPPH assay was conducted to assess the antioxidant activity of Sorghum bicolor seeds extract. DPPH, characterized by its deep violet color with an absorption band at around 517 nm, is reduced upon interaction with a hydrogen atom donor, resulting in the loss of this color. Table 3 presents the results of the antioxidant activity of Sorghum bicolor seed extract using the DPPH (96 Well Method) assay, where the absorbance values were measured at a concentration of 1 mg/ml for both the standard (ascorbic acid) and the seed extract (Sample).

Table 3: Effect of Plant Extract by using Antioxidant Activity by DPPH (96 Well Method)

The antioxidant activity of Sorghum bicolor seed extract was evaluated using the DPPH method with a 96-well assay. The results, shown in Table 3, indicate that the Sorghum bicolor seed extract exhibited moderate antioxidant activity compared to the standard ascorbic acid. The control samples, which contained no antioxidant, showed an average absorbance of 1.253, indicating no inhibition of the DPPH radical. The standard ascorbic acid at 1 mg/ml concentration demonstrated significant antioxidant activity with a mean absorbance of 0.196, resulting in a % inhibition of 84.35%, confirming its strong ability to neutralize free radicals. Sorghum bicolor seed extract at 1 mg/ml concentration showed a mean absorbance of 0.660, resulting in a % inhibition of 47.33%. This indicates that the Sorghum extract has moderate antioxidant potential but is significantly weaker than the standard ascorbic acid. The observed lower % inhibition suggests that the antioxidant compounds present in the Sorghum bicolor seed extract are less efficient in scavenging free radicals compared to ascorbic acid.