Antimicrobial and Antifungal Activity

The antimicrobial activity of plant species is normally determined using the disk diffusion method which measures the zone of inhibition of the plant extracts against gram-positive and gram-negative bacteria (Haque et al., 2014). The factors that influence the size of the inhibition zone include the capability of the substances in the plant extract to diffuse through the medium as well as the metabolic activity and growth of microorganisms in the medium (Haque et al., 2014). In addition, the lipid content of the membranes of the different bacterial groups and the permeability of various constituents of the plant extracts would influence the size of the inhibition zone (Roy et al., 2013). The ability of plant extracts to demonstrate antimicrobial activity against both gram-positive and gram-negative could indicate the presence of a broad-spectrum of antibiotic compounds (Haque et al., 2014).

An antimicrobial study on different extracts of A. indica leaves, including petroleum ether, chloroform, acetone, ethanol, and water against several microorganism exhibited significant antimicrobial activities against gram-positive and gram-negative bacterial as well as fungal strains. In addition, the Minimum Inhibitory Concentration (MIC) values of all extracts were reported between 5 and 20 mg/ml. The lowest and highest MIC values observed from the ethanol and chloroform extracts of the plant ranged between 10.23 and 13.18 mg/ml and 14.30–15.42 mg/ml, respectively. The inhibition zone of all extracts increased with increasing concentrations, ranging between 9 and 23 mm (Mulla Wahid et al., 2010a).

The study also showed that ethanol extract (10 mg/ml) recorded the most significant inhibitory activity against B. subtilis with an inhibition zone of 22 mm, followed by E. coli (19 mm), S. cerevisiae (18 mm), K. pneumonia (17 mm), S. aureus (16 mm), C. albicans (16 mm), and A. niger (15 mm). The extract inhibited the growth of bacteria compared to that of antibacterial standard (Gentamicin 0.5 mg/ml) but exhibited less inhibitory activity against fungi strains compared to the standard antifungal (fluconazole 0.5 mg/ml). This study suggested that bioactive compounds found in A. indica leave extracts such as polyphenolics compounds such as tannin played a major role in antimicrobial activity. Tannin in the form of crude extract is mostly tested compared to individual compound against microorganism. Tannin acts as antimicrobial by interacting with bacterial enzymes and precipitating them (Puljula et al., 2020).

In another study, Islam et al. (2013) used the ethanol extract of A. indica Schott tuber to evaluate its antimicrobial activity against 2 g-positive bacteria (S. aureus and Staphylococcus epidermidis), 6 g-negative bacteria (S. typhi, E. coli, S. flexneri, Shigella sonnei, Shigella dysenteriae, and K. pneumonia), and three fungal species (A. niger, C. albicans, and S. cerevisiae). The results revealed that the plant extracts demonstrated moderate antimicrobial activity with an inhibition zone ranging between 5.8–9.8 mm and 12.1–18 mm for gram-positive and gram-negative bacteria, respectively when the concentration was set at 250 and 500 µg/disc.

The evaluation of antimicrobial activity of A. macrorrhizos extracts (petroleum ether, carbon tetrachloride, chloroform, and aqueous fraction) at 400 µg concentration/disk against B. subtilis, S. aureus, Pseudomonas aeruginosa, S. typhi, E. coli, C. albicans, and A. niger showed that the methanol crude extract was effective against all tested microorganisms while the chloroform soluble fractions were selectively effective against all tested gram-negative bacteria only. In addition, the carbon tetrachloride soluble fractions were effective against all gram-positive and gram-negative bacteria, but not against fungi. In contrast, petroleum ether fraction was effective against all bacteria except S. areus and P. aeruginosa while the aqueous soluble fraction effective except against S. typhi. This study suggested that certain chemical constituents found in different parts of plant extracts may be responsible for their antimicrobial activity against certain types of microorganisms. Thus, further study is needed to detect the chemical compounds that exert the highest antimicrobial and antifungal activities (Banik et al., 2014).

Furthermore, Haque et al. (2014) examined the antimicrobial activity of A. fornicata leaf, stolon, and root extracts using ethanol and other soluble partitions (petroleum ether, chloroform, and ethyl acetate) against Bacillus megaterium, B. subtilis, Bacillus cereus, S. aureus, Sarcina lutea, Salmonella paratyphi, Vibrio parahaemolyticus, Vibrio mimicus, E. coli, S. dysenteriae, P. aeruginosa, and Shigella boydii. The results showed that all extracts (500 µg/disk) exhibited good antibacterial effect except for the petroleum ether extracts which did not record any antimicrobial activity. The ethanol extract of plant root was the most active against all the bacteria with a zone of inhibition ranging between 10 and 18 mm. Meanwhile, the chloroform and ethyl acetate leaves extracts were more active against most of the tested bacteria compared to the respective stolon extracts even though the chloroform extract of the stolon showed the highest zone of inhibition (20 mm) against S. lutea. Besides, both ethyl acetate and chloroform extracts of the plant leaves showed better MIC against B. subtilis at 64 μg/ml while ethanol extract of roots recorded a MIC of 64 μg/ml against P. aeruginosa.

The antimicrobial properties of methanol extract of A. decipiens Schott rhizome were investigated in-Vitro by Roy et al. (2013) against 2 g-positive bacteria (S. aureus and B. subtilis) and 2 g-negative bacteria (E. coli and Klebsiella sp.). The results showed a significant zone of inhibition against S. aureus, B. subtilis, E. coli, and Klebsiella sp. at 16 mm, 12 mm, 11 mm, and 10 mm, respectively when the extract was at 100% concentration. The MIC value of the extract was varied between 2 and 16 μg/ml with S. aureus displayed higher sensitivity while Klebsiella sp. was the most resistant bacteria towards the plant extracts. All the organisms were inhibited with the concentration of plant extracts at 25% except for S. aureus which was inhibited at 10% concentration. The methanol plant extracts were believed to have broad-spectrum activity against gram-positive bacteria.

A less effective antimicrobial effect was demonstrated by A. sanderiana Bull. leaves through three different solvent extracts (methanol crude extract, dichloromethane fraction of methanol extract, and hexane fraction of methanol extract) (Ongpoy Jr et al., 2015). The results showed that the antimicrobial activities of the plant leave extracts against 8 g-positive bacteria, 8 g-negative bacteria, and three fungi using at least an 8 mm inhibition zone was mostly non-active. However, some areas were observed below the 8 mm criteria, with the dichloromethane fraction displaying an inhibition zone of 4 mm, 3 mm, 1 mm, and 1 mm for Proteus mirabilis, P. aeruginosa, Pectrobacterium carotovorum, and C. albicans, respectively while methanol fraction showed an inhibition zone of 1 mm against P. aeruginosa. The study suggested that the polyphenolic compounds found in the plant extracts may not be an effective antimicrobial agent against all the microorganisms tested. Furthermore, the small zone of inhibition (less than 8 mm) exhibited by some of the plant extracts may be due to the presence of protease inhibitors, trypsin inhibitors or lectins in which their roles in antimicrobial activity still requires further analysis (Ongpoy Jr, 2015; 2017).

The antimicrobial study on 80% ethanol extract of A. denudata stem against selected gram-positive oral bacteria which include S. mutans, S. aureus, and E. faecalis as well as the non-oral pathogen Streptococcus pyogenes indicated the presence of the antimicrobial steroid compound β-sitosterol trimethylsilyl ether, the results recorded no antimicrobial effects by the plant extracts at any concentration even up to 32 μg/ml. In addition, compounds such as phenols, flavonoids, and alkaloids, which had been proven to possess antimicrobial effects, were not detected in the extract. It was believed that either the antimicrobial compounds were degraded before the susceptibility testing was performed or that the selected bacteria were already resistant to the compounds (Mohd Yusoff et al., 2020).

So far, seven Alocasia species had been studied for their antimicrobial and antifungal activities. Based on previous studies, A. indica syn. and A. macrorrhizos are the most studied species that showed significant antimicrobial effects comparable to that of standards. Meanwhile, A. fornicata and A. decipiens Schott showed moderate-to-good antimicrobial activities. In contrast, A. sanderiana Bull., A. denudata, and A. brisbanensis extracts exhibited no antimicrobial activities. Thus, it was believed that different extracts of the Alocasia species may contain different bioactive molecules that are responsible for antimicrobial and antifungal activity.