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. 2023 Apr 24;45(5):3733-3756.
doi: 10.3390/cimb45050241.

GC-MS Based Characterization, Antibacterial, Antifungal and Anti-Oncogenic Activity of Ethyl Acetate Extract of Aspergillus niger Strain AK-6 Isolated from Rhizospheric Soil

Shaik Kalimulla Niazi  1 , Dhanyakumara Shivapoojar Basavarajappa  2 , Sushma Hatti Kumaraswamy  3 , Asmatanzeem Bepari  4 , Halaswamy Hiremath  2 , Shashiraj Kariyellappa Nagaraja  2 , Muthuraj Rudrappa  2 , Anil Hugar  2 , Mary Anne Wong Cordero  4 , Sreenivasa Nayaka  2
Affiliations

GC-MS Based Characterization, Antibacterial, Antifungal and Anti-Oncogenic Activity of Ethyl Acetate Extract of Aspergillus niger Strain AK-6 Isolated from Rhizospheric Soil

Shaik Kalimulla Niazi et al. Curr Issues Mol Biol. .

Abstract

Rhizospheric soil is the richest niche of different microbes that produce biologically active metabolites. The current study investigated the antimicrobial, antifungal and anticancer activities of ethyl acetate extract of the potent rhizospheric fungus Aspergillus niger AK6 (AK-6). A total of six fungal isolates were isolated, and isolate AK-6 was selected based on primary screening. Further, it exhibited moderate antimicrobial activity against pathogens such as Klebsiella pneumonia, Candida albicans, Escherichia coli, Shigella flexneri, Bacillus subtilis and Staphylococcus aureus. The morphological and molecular characterization (18S rRNA) confirmed that the isolate AK-6 belonged to Aspergillus niger. Further, AK-6 showed potent antifungal activity with 47.2%, 59.4% and 64.1% of inhibition against Sclerotium rolfsii, Cercospora canescens and Fusarium sambucinum phytopathogens. FT-IR analysis displayed different biological functional groups. Consequently, the GC-MS analysis displayed bioactive compounds, namely, n-didehydrohexacarboxyl-2,4,5-trimethylpiperazine (23.82%), dibutyl phthalate (14.65%), e-5-heptadecanol (8.98%), and 2,4-ditert-butylphenol (8.60%), among the total of 15 compounds isolated. Further, the anticancer activity of AK-6 was exhibited against the MCF-7 cell line of human breast adenocarcinoma with an IC50 value of 102.01 μg/mL. Furthermore, flow cytometry depicted 17.3%, 26.43%, and 3.16% of early and late apoptosis and necrosis in the AK-6 extarct treated MCF-7 cell line, respectively. The results of the present analysis suggest that the isolated Aspergillus niger strain AK-6 extract has the potential to be explored as a promising antimicrobial, antifungal and anticancer drug for medical and agricultural applications.

Keywords: Aspergillus niger; GC-MS analysis; antifungal; antimicrobial; apoptosis; rhizospheric soil.

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Conflict of interest statement

The authors declare that there is no conflict of interest of this study.

Figures

Figure 1
Figure 1
Isolation of different fungi from the collected soil samples. (A) AK-1, (B) AK-2, (C) AK-3, (D) AK-4, (E) AK-6, and (F) AK-7.
Figure 2
Figure 2
Morphological characterization of isolate AK-6. (A) aerial mycelium, (B) substrate mycelium, (C) scanning electron microscope image showing conidiophores and (D) scanning electron microscope image showing spore morphology.
Figure 3
Figure 3
Antimicrobial activity of ethyl acetate extract of isolate AK-6. (A) K. pneumonia, (B) C. albicans, (C) E. coli, (D) S. flexneri, (E) B. subtilis and (F) S. aureus.
Figure 4
Figure 4
Graphical representation of the antimicrobial activity of ethyl acetate extract isolate AK-6 against pathogenic microorganisms. Two-way ANOVA with Tukey’s multiple comparisons was performed to identify group differences. p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****). ns: not significant.
Figure 5
Figure 5
Antifungal activity of isolate AK-6 assessed by dual culture assay against phytopathogens.
Figure 6
Figure 6
Graphical representations of antifungal activity of isolate AK-6 against phytopathogens assessed by dual culture assay. Two-way ANOVA with multiple comparisons was accomplished to identify group differences. p < 0.001 (***), and p < 0.001 (****).
Figure 7
Figure 7
Dendrogram showing the phylogenetic relationship of Aspergillus niger strain AK-6.
Figure 8
Figure 8
FTIR analysis of ethyl acetate extract of Aspergillus niger strain AK6.
Figure 9
Figure 9
GC-MS chromatogram of ethyl acetate extract of Aspergillus niger strain AK-6.
Figure 10
Figure 10
Mass spectra of individual metabolites found in ethyl acetate extract of the Aspergillus niger strain AK-6. (A) 1-Dodecene, (B) tetradecane, (C) cyclodecane, (D) 2,4-di-tert-butylphenol, (E) E-14-hexadecenal, (F) N-didehydrohexacarboxyl-2,4,5-trimethylpiperazine, (G) dibutyl phthalate and (H) E-15-heptadecenal.
Figure 11
Figure 11
Mass spectra of individual metabolites found in ethyl acetate extract of the Aspergillus niger strain AK-6. (A) 1-Heneicosanol, (B) trifluroacetoxy hexadecane, (C) cyclopentane, heneicosyl, (D) squalene, (E) tricosane, (F) nonadecane and (G) octadecane 2 methyl.
Figure 12
Figure 12
Anticancer activity (MTT assay) of ethyl acetate extract of the Aspergillus niger strain AK-6 against the MCF-7 cell line. (A) Untreated, (B) positive control treated, (C) 12.5 μg/mL, (D) 25 μg/mL, (E) 50 μg/mL, (F) 100 μg/mL, (G) 200 μg/mL, (H) graphical representation of anticancer activity of the Aspergillus niger strain AK-6 extract.
Figure 13
Figure 13
Flow cytometric analysis of ethyl acetate extract of the Aspergillus niger strain AK-6 against the MCF-7 cell line. Quadrangular plot of Annexin V/PI expression on MCF-7 cells: (A) untreated control group, (B) Cell cycle of MCF-7 cell line in untreated control group. Quadrangular plot of Annexin V/PI expression on MCF-7 cells: (C) positive control treated group. (D) Cell cycle of MCF-7 cell line positive control treated group. Quadrangular plot of Annexin V/PI expression on MCF-7 cells: (E) MCF-7 cell line treated with ethyl acetate extract of the Aspergillus niger strain AK-6. (F) Cell cycle of MCF-7 cell line treated with ethyl acetate extract of the Aspergillus niger strain AK-6.
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