Fresh fruit bodies of Ganoderma enigmaticum and G. mbrekobenum were collected from dead palm tree substrates in Lagos, Nigeria, at two separate locations ( Figure 1 ) (GPS: 006˚30'48''N 003˚23'47''E and 006˚51'61''N 003˚38'92''E, respectively).

Macroscopic and microscopic characteristics were used for identification with standard mycological manuals based on key morphological traits [12] [13] . Representative voucher specimens were deposited at the University of Lagos Herbarium, Lagos, Nigeria (with Herbarium Voucher Specimen number—LUH8947 for Ganoderma mbrekobenum, and LUH8961 for Ganoderma enigmaticum). Morphological identification of polypore fungi/mushrooms was confirmed by Professor Erute M. Adongbede, and specimen curation was supervised by Dr. Akeem B. Kadiri at the University of Lagos Herbarium.

Genomic DNA was isolated from approximately 100 mg of dried fungal tissue using the Norgen Biotek Plant/Fungi DNA Isolation Kit (Ontario, Canada), following the manufacturer’s protocol. Samples were lysed with 500 µL Lysis Buffer and 1 µL RNase A, then incubated at 65˚C for 10 minutes. DNA was purified using filter columns, eluted with the supplied buffer, and its quality and concentration were assessed using a NanoDrop spectrophotometer. The internal transcribed spacer (ITS) region was amplified by polymerase chain reaction (PCR) with primers ITS1-Forward (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4-Reverse (5'-TCC TCC GCT TAT TGA TAT GC-3'), sourced from IDT (Coralville, IA, USA).

PCR was performed with Solis BioDyne 5X Master Mix in a Prime thermal cycler (PRIMEX/02, Cole-Parmer Ltd, UK) using the following program: 95˚C for 5 min; 35 cycles of 95˚C for 40 s, 55˚C for 1 min, 72˚C for 1 min; and 72˚C for 10 min. Amplicons were detected on 1.5% agarose gels stained with ethidium bromide using a Bio-Rad Gel Doc EZ imager.

Sanger sequencing of purified PCR products was conducted by Eurofins Genomics (Ebersberg, Germany) utilizing ITS primers. Consensus sequences were assembled and curated using Geneious Prime (Version 2025.1.2). Sequence similarity searches were performed against the NCBI GenBank and UNITE databases using BLAST. Phylogenetic analyses were conducted employing the Maximum Likelihood method in MEGA X, with robustness evaluated via bootstrap analysis comprising five hundred replicates. Herbarium voucher specimens were deposited at the University of Lagos Herbarium, and corresponding sequence data were submitted to NCBI GenBank under their designated accession numbers.

A total of ten grams of powdered tissue were stirred in 200 mL of 70% methanol containing 1% formic acid at 250 rpm overnight, following the protocols outlined by Xiang et al. (2024) [14] , with modifications. The mixture underwent additional ultrasonication for 10 minutes at 40˚C, followed by centrifugation at 12,000 rpm to remove debris. The resulting filtrates were collected and concentrated at 40˚C under reduced pressure, after which the concentrate was lyophilized. The final powdered extract was weighed and reconstituted in sterile deionized water to prepare a 100 mg/mL stock solution, which was stored at 4˚C.

Polysaccharides were isolated from 10 g of lyophilized and pulverized fruiting bodies through extraction in 300 mL of distilled water at 100˚C for 2 hours, followed by two additional extractions of 1 hour each. The combined aqueous extracts were filtered, concentrated under reduced pressure at 60˚C, frozen, and subjected to lyophilization. The resulting material was redissolved, and polysaccharides were precipitated using 80% ethanol at 4˚C overnight. After centrifugation at 10,000 rpm for 10 minutes, the precipitate was freeze-dried and weighed.

Oligosaccharides were isolated from 10 g of lyophilized, pulverized mushroom tissue using 85% ethanol at 50˚C for one hour, with continuous magnetic stirring at 150 rpm. The resulting extracts were centrifuged at 14,000 rpm for 30 minutes and evaporated to dryness under reduced pressure via rotary evaporation, then redissolved in sterile deionized water. Low molecular weight compounds (oligosaccharides) were purified and separated from high molecular weight substances by 80% ethanol precipitation at 4˚C overnight. The recovered oligosaccharides were concentrated, subjected to lyophilization, redissolved in distilled water, and sequentially filtered through 0.45 µm and 0.22 µm membranes prior to use [15] .

TPC was determined using the Folin-Ciocalteu assay kit (Bioquochem), with all assays conducted in triplicate on 96-well plates and gallic acid employed as the standard following the manufacturer’s instructions. Absorbance was recorded at 700 nm. Results were reported as mg gallic acid equivalent (GAE) per gram of dry extract.

Total carbohydrate content was determined using the phenol-sulfuric acid method with the Abcam Total Carbohydrate Quantification Kit (ab155891) in 96-well microtiter plates. Glucose standard curves were prepared, and samples consisting of polysaccharides and oligosaccharides from G. enigmaticum and G. mbrekobenum (30 µL), at concentrations of 25, 50, 75, and 100 mg/mL, were incubated with 150 µL concentrated H₂SO₄ and 30 µL developer solution. Absorbance values were measured at 490 nm, and carbohydrate concentrations were quantified as glucose equivalents.

Antioxidant activity was evaluated utilizing the DPPH assay kit (Abcam ab289847), following the protocol established by Zangeneh et al. (2025) [16] . Extracts were evaluated at concentrations of 25, 50, 75, and 100 mg/mL in triplicate. Trolox was employed as the standard reference compound. Absorbance was recorded at 517 nm, and IC50 values were determined from the generated dose-response curves.

Antibacterial activity was assessed following CLSI guidelines (2012) against Escherichia coli O157:H7 (ATCC BAA-3162) and MRSA Staphylococcus aureus (ATCC 700798). Bacteria were diluted to 5 × 10⁵ CFU/well and incubated with 25, 50, 75, and 100 mg/mL concentrations of each extract, positive (Ciprofloxacin and Ceftazidime (2.5, 5.0, 7.5, and 1.00 mg/mL)) and negative controls in 96-well microtiter plates in three replications/wells. Absorbance was measured at 600 nm using a BioTek Instruments microplate reader. Percentage inhibition and IC₅₀ values were calculated using GraphPad Prism 10.6.0.

Cell lines—HepG2 (ATCC-HB-8065; hepatocellular carcinoma), HCT116 (ATCC-SLC25A16-KO-c7; colorectal adenocarcinoma), and MDA-MB231 (ATCC-HTB-26)—were obtained from the American Type Culture Collection (ATCC) and cultured in their respective media: Eagle’s Minimum Essential Medium, McCoy’s 5A, and RPMI 1640, each supplemented with 10% FBS. Cell viability and cytotoxicity were assessed using the 2,3-Bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) Cell Proliferation Assay Kit (ATCC), with absorbance measured at 475 nm and 660 nm using a microplate reader (BioTek). Cells were seeded at a density of 5000 per well in 96-well plates and exposed to varying concentrations of extract (25 - 100 µg/mL) as well as the standard anticancer drug cisplatin for 24 and 48 hours. Percentage inhibition and IC₅₀ values were calculated from dose-response curves using GraphPad Prism Software.

Data were expressed as mean ± SD, and differences between groups were analyzed by ordinary two-way ANOVA (P < 0.05). Half maximal inhibitory concentrations/effective doses (IC₅₀ values) were derived from non-linear regression using GraphPad Prism 10.6.0 (CA, USA).

The specimens ULSH/M137 and ULSC/M006 were identified as Ganoderma enigmaticum and Ganoderma mbrekobenum, respectively, based on morphological and molecular features. G. enigmaticum exhibited a fan-shaped, laccate pileus with concentric reddish-brown zones, while G. mbrekobenum displayed a thicker, circular, and velvety pileus ( Figure 1 ). Both species showed lateral stipes and brown pore surfaces typical of Ganoderma species. Herbarium voucher specimens were deposited at the University of Lagos Herbariums as LUH8961 (G. enigmaticum) and LUH8947 (G. mbrekobenum), with ITS sequences submitted to GenBank with accession numbers OK324048 and OK324049, respectively.

NCBI Mega-Blast analysis confirmed species identity: LUH8961 shared 99.68% similarity with Ganoderma enigmaticum voucher Ghana 1a/938398 (GenBank accession KR150678.1), while LUH8947 showed a 100% identity match with Ganoderma mbrekobenum voucher UMN7-4 GHA (GenBank accession KX000898.1).

The ITS sequence for specimen ULSH/M137 (voucher LUH8961) showed a 99.68% similarity to the GenBank record for Ganoderma enigmaticum voucher Ghana 1a/938398 18S ribosomal RNA gene and internal transcribed spacer 2, complete sequence (GenBank accession KR150678.1). The ITS sequence blast of specimen ULSH/M137 (voucher LUH8947) indicated a 100% identity with the NCBI repository record for Ganoderma mbrekobenum voucher UMN7-4 GHA 18S ribosomal RNA gene and ITS 2, complete sequence (GenBank accession KX000898.1). A maximum likelihood phylogenetic tree constructed using ITS sequences from GenBank clustered both species within their respective clades with strong bootstrap support (99% - 100%; Figure 2 and Figure 3 ).

Polyphenol fractions yielded the highest dry weight for both Ganoderma species, followed by oligosaccharides and polysaccharides ( Figure 4 ). Two-way ANOVA indicated significant effects of species (84.93% variance, P < 0.0001), extract type (7.01%, P < 0.0001), and their interaction (6.95%, P < 0.0001) on yield ( Table S1 ).

Oligosaccharide fraction contained the highest carbohydrate concentrations in both species, while G. mbrekobenum polysaccharides showed the least ( Figure 5 ). Total phenolic content was significantly higher in G. enigmaticum (~300 mg GAE/g dry extract) than in G. mbrekobenum ( Figure 5 ; P < 0.0001; Table S2 ).

All extract types demonstrated dose-dependent DPPH radical scavenging activity ( Figure 6 ). Oligosaccharide fractions exhibited the strongest effects (80% - 90%), followed by polysaccharides (70% - 88%) and polyphenols (50% - 60%). The two-way ANOVA revealed significant contributions of concentration (81.57% variation, P < 0.0001), extract type (14.54%, P < 0.0001), and the interaction was significant (2.87%, P < 0.05) ( Table S3 ).

G. mbrekobenum extracts consistently outperformed those of G. enigmaticum. Its oligosaccharides showed lower half maximal inhibitory concentration (IC₅₀) values ( Table 1 ), and its polyphenol fractions reached ~65% activity at 100 mg/mL compared to ~55% for G. enigmaticum. Interestingly, G. enigmaticum polysaccharides demonstrated higher activity than its polyphenols ( Figure 6 ). Overall, G. mbrekobenum exhibited greater antioxidant potential.

Extracts inhibited Escherichia coli and Staphylococcus aureus in a dose-dependent manner ( Figure 7 and Figure 8 ). Polyphenol and oligosaccharide fractions were the most effective, particularly from G. mbrekobenum against E. coli (~95% inhibition at 100 mg/mL). Two-way ANOVA showed significant effects of concentration and extract/antibiotic type (P < 0.0001), with no interactions (P = 0.4186) ( Table S4 ).

Oligosaccharide fractions, especially from G. mbrekobenum, exhibited the highest dose-dependent inhibition of S. aureus, while polysaccharides showed comparatively lower activity ( Figure 8 ). There was no significant difference between the two species for the polyphenol fraction, but a slight variation was observed in the polysaccharide and oligosaccharide fractions, with G. mbrekobenum outperforming G. enigmaticum ( Figure 8 ).

Ciprofloxacin showed strong, dose-dependent inhibition of E. coli and S. aureus, whereas Ceftazidime was moderately effective against E. coli but displayed markedly weaker activity against S. aureus ( Figure 9 ). Compared to the test extracts, ciprofloxacin exhibited strong inhibition of both E. coli and S. aureus, while Ceftazidime was less effective, particularly against S. aureus, where inhibition was markedly lower than that observed with Ganoderma oligosaccharide fractions ( Figures 7-9 ).

The two-way ANOVA also showed a consistent dose-dependent effect of extracts from the two test mushrooms and control antibiotics on the growth of S. aureus (P < 0.0001). There was also no significant interaction across treatments (P = 0.7748) ( Table S5 ).

All extracts showed strong anti-Staphylococcus activity (>80%), with G. enigmaticum oligosaccharide having the lowest IC₅₀ (6.96 mg/mL), followed by G. mbrekobenum polysaccharide (8.70 mg/mL) ( Table 1 ; Figure 8 ). These values were comparable to the IC₅₀ of ciprofloxacin (~7.50 - 8.80 mg/mL), a standard broad-spectrum antibiotic ( Table 1 ). S. aureus was marginally inhibited by ceftazidime in vitro and had the highest IC₅₀ values 18.04 (95% CI: 15.25 - 22.91) ( Table 1 ).

Values are IC₅₀ estimates from nonlinear regression analysis, expressed with 95% confidence intervals (CI), computed in GraphPad Prism (Version 10.5.0).

The half maximal inhibitory or effective concentrations (IC₅₀ values) were consistent with these inhibition patterns, with the polyphenol extracts of G. enigmaticum displaying the lowest IC₅₀ values against E. coli (7.70 mg/mL) with a 95% confidence interval (CI) ( Table 1 ).

Polyphenol fractions significantly inhibited the proliferation of HepG2 and HCT116 cells, while polysaccharide fractions were most effective against MDA-MB-231 cells ( Figures 10-13 ). Overall, G. mbrekobenum extracts were more potent than those from G. enigmaticum, and comparable to cisplatin.

After 24 hours of treatment, polyphenols inhibited HepG2 cell proliferation by 60% - 90%, and polysaccharides by 60% - 80% at all concentrations assessed. These effects surpassed those of cisplatin, which exhibited a 42% inhibition at the highest dose. After 48 hours, the inhibitory effects increased to 95%, 86%, and 70% for polyphenols and polysaccharides, respectively ( Figure 10 ). Two-way ANOVA showed significant effects of extract/drug type and time (77.75% variance, P < 0.0001) and concentration (17.75%, P < 0.0001) ( Table S6 ). The interaction effect was significant but minor (2.26%; ***, P < 0.003), indicating consistent concentration effects across treatments and time periods.

Polyphenol extracts from both G. mbrekobenum and G. enigmaticum inhibited HCT116 cell proliferation in a concentration-dependent manner, reaching >90% after 48 h ( Figure 11 ). Two-way ANOVA revealed that extract/anticancer drug type and time (74.12%, P < 0.0001), as well as their concentrations (20.03%, P < 0.0001), significantly affected HCT116 cell proliferation. No significant interaction between concentration and treatment/time was observed (1.46%, P = 0.146), indicating consistent concentration effects (see Table S7 ).

After 24-hour treatments, both polyphenol extracts strongly and dose-dependently inhibited cell proliferation: G. enigmaticum reduced MDA-MB231 cell growth by 86%, and G. mbrekobenum by 81.33% at 100 µg/mL ( Figure 12 ). The most potent effect was seen with the polysaccharide extract of G. mbrekobenum, which inhibited 93% of cells after 48 hours; the G. enigmaticum polysaccharide also significantly decreased proliferation by 84%.

Two-way ANOVA revealed that extract type, anticancer drug, time (78.28% variation, P < 0.0001), and their concentrations (20.61% variation, P < 0.0001) significantly affected MDA-MB231 cell proliferation. No significant interaction was found between concentration and extract/drug type/time (1.46% variation, P = 0.146), indicating stable concentration effects. See Table S8 for details.

Cisplatin inhibited the proliferation of HepG2, HCT116, and MDA-MB-231 cells in a dose-dependent manner, with the strongest effects observed at 10 µg/mL, where inhibition exceeded 60% in HepG2 and ~50% in the other cell lines ( Figure 13 ).

The IC₅₀ values were consistent with these findings; polyphenol and polysaccharide extracts from G. mbrekobenum showed lower values across all cell lines and time points at the 95% confidence interval (CI) ( Table 2(a) and Table 2(b) ).

The polyphenol extract of G. mbrekobenum had the lowest IC₅₀ value against HepG2 cells (9.33 µg/mL), while its polysaccharide fraction recorded a value of 9.47 µg/mL against MDA-MB231 cells at the 24-hour time point ( Table 2(a) ). At 48 hours, the lowest IC₅₀ values were observed for the polysaccharide extract of G. enigmaticum with MDA-MB231 cells (7.75 µg/mL), and the polyphenol extract of G. mbrekobenum (7.70 µg/mL) (95% CI) ( Table 2(b) ).

Values are IC₅₀ estimates from nonlinear regression analysis, expressed with 95% confidence intervals (CI), computed in GraphPad Prism (version 10.5.0).

These results suggest promising anticancer potential for both extract types, especially from G. mbrekobenum. Collectively, these findings indicate that both mushroom species possess significant, time-dependent antiproliferative activities, with G. mbrekobenum demonstrating potential comparable to that of cisplatin.