Submerged fermentation conditions and bioactivity profiling of mycelial extract of the Philippine Oudemansiella canarii

Mushrooms are renowned as valuable sources of nutritional and pharmaceutical compounds, highlighting their potential in functional foods and therapeutic development. Edible mushrooms are particularly rich in protein, carbohydrates, dietary fibers, and ergosterols, while containing relatively low levels of fat^[1]. In addition, edible mushrooms provide essential vitamins, including vitamin C and members of the B-complex such as riboflavin, thiamine, and niacin, along with minerals such as zinc, magnesium, calcium, phosphorus, copper, potassium, manganese, selenium, and iron^[2,3]. Beyond their nutritional profile, mushrooms contain diverse bioactive compounds, including phenolic acids, terpenoids, polysaccharides, β-glucans, and lectins, that contribute to a wide range of functional activities, such as antioxidant, anti-inflammatory, and metabolic regulatory effects^[4]. Notably, the consumption of edible mushrooms has been associated with reductions in total cholesterol, low-density lipoprotein, plasma glucose, and plasma triglyceride levels^[5].

With the growing interest surrounding mushrooms, there is a pressing need for innovative technologies to harness their full potential. Numerous wild species remain insufficiently studied and underutilized, making advances in cultivation methods, optimization of growth conditions, and extraction of bioactive compounds crucial for unlocking their nutritional, medicinal, and industrial value.

One common approach to mushroom cultivation is solid-state fermentation, which makes use of a substrate made from agro-industrial wastes such as wheat straw, hardwood sawdust, rice bran, etc.^[6]. Although cost-effective and highly efficient for fruiting body production through the use of readily available natural materials, solid-state fermentation is often characterized by prolonged incubation periods that fluctuate depending on the specific mushroom species and substrate composition^[7]. This limitation in solid-state fermentation has prompted increasing interest in submerged fermentation, a method that allows greater control over culture conditions, thereby improving the yield and quality of target products, particularly bioactive compounds and mycelial biomass, within reduced timeframes^[8]. Despite these advantages, the application of submerged fermentation remains largely unexplored for many wild tropical species. This research gap presents an opportunity to investigate understudied mushrooms such as Oudemansiella canarii.

O. canarii is an edible macrofungus characterized by a cream-colored, mucilaginous cap with surface scales, free lamellar attachments, and club-shaped basidia^[9]. It is also the most common and widely distributed pantropical member of the genus, with records spanning 14 countries, including the Philippines^[10]. The first report of cultivation of this mushroom employed sugarcane bagasse and eucalyptus sawdust substrates supplemented with wheat bran^[11]. On the other hand, the first successful cultivation of O. canarii in the Philippines was with malt extract agar, enhancing mycelial growth, and cracked corn supporting robust colonization^[12]. The same study further demonstrated that a substrate mixture of rice straw (80%) and sawdust (20%) produced high biological efficiency in fruiting body development.

Despite the advances, research on O. canarii in the Philippines remains scarce, especially regarding its cultivation via submerged fermentation. Accordingly, the present study was carried out to optimize submerged fermentation conditions using fruit-based substrates for mycelial biomass production of O. canarii and to evaluate its anti-diabetic, cholinesterase inhibitory, anti-inflammatory, antioxidant, teratogenic, and cytotoxic properties, as these bioactivities have been previously reported in related taxa and are known to be associated with diverse secondary metabolites in this genus^[10]. These properties offer ideas for the sustainable development of new functional foods and potential therapeutic agents; however, such applications remain preliminary and require further investigation to establish safety, efficacy, and practical utility.

Pure culture of O. canarii, identified by laboratory code OcBs, was sourced from the culture collections of the Center for Tropical Mushroom Research and Development at Central Luzon State University, located in the Science City of Muñoz, Nueva Ecija, Philippines. Following standard mycological protocols, an agar block containing actively growing mycelium from the pure culture of O. canarii was aseptically transferred onto sterilized potato dextrose agar (PDA) plates and incubated at 28 °C to promote mycelial growth. After a 7-d incubation period, mycelial discs were obtained using a flame-sterilized cork borer (10 mm diameter) and were used as inoculants in subsequent experiments to evaluate optimum growth conditions.

Optimization studies for mycelial biomass production

Following the methods of Dulay et al.^[13] with minor modifications, optimization of liquid culture conditions was performed. The parameters of the optimization study were evaluated one at a time, and all experiments were conducted in triplicate.

The effect of low-cost and locally sourced liquid culture media, namely, 25% mango purée (MP), mature coconut water (CW), and their combination, on the mycelial production of the mushrooms was assessed under submerged fermentation. Each medium was prepared at a working volume of 100 mL in Erlenmeyer flasks. These media were obtained from local markets to ensure accessibility and practical relevance. Mango purée was selected as a carbohydrate-rich substrate, while CW was chosen for its mineral, vitamin, and natural growth factor content. Ripe Indian mangoes were washed, peeled, and blended into a purée, which was then diluted to 25% (v/v) with distilled water, determined to balance nutrient availability with manageable viscosity, ensuring proper mixing and oxygen transfer. Mature coconut water was filtered to remove particulates and was used at full strength to reflect its natural composition. The combination of 25% mango purée and CW was prepared in equal proportions to assess potential synergistic effects between carbohydrate and mineral sources.

In the screening of optimal pH, the optimal liquid culture medium was used and adjusted to pH 5.0 to 9.0 in increments of 1 using 1 M sodium hydroxide (NaOH) or hydrochloric acid (HCl). A 100 mL volume of each medium was transferred into 250 mL Erlenmeyer flasks, sterilized at 121 °C and 15 psi for 30 mi, and aseptically inoculated with a single mycelial disc. Cultures were incubated at 30 °C for 10 d in a rotary shaker at 100 rpm. After incubation, mycelia were collected by filtration, rinsed with distilled water, and lyophilized. Biomass yield was expressed as milligrams dry weight per milliliter (mg DW/mL).

The effect of temperature was evaluated by incubating cultures at 10, 20, and 30 °C using the optimal liquid medium and pH level. These values were selected to represent a broad range of environmental conditions: 10 and 20 °C to assess tolerance under cooler settings, and 30 °C to examine the potential upper limits of growth. Moreover, to determine the optimal agitation condition, optimal culture media adjusted to the previously identified optimal pH were inoculated with mycelial discs and incubated at the optimal temperature in a rotary shaker at agitation speeds of 100 and 150 rpm for 10 d. A separate static (0 rpm) condition was also included.

Mass production of mycelia

Using the optimized culture conditions, mushroom mycelia were mass-produced for subsequent applications, including the extraction of bioactive compounds from the mycelia and culture (mycelia + spent culture liquid).

Ethanolic extraction

Ethanol extraction of mycelial biomass was performed, following the protocol of Boukes et al.^[14]. Five grams of lyophilized mushroom mycelia were finely ground and soaked in 200 mL of 95% ethanol for 48 h at room temperature. The mixture was then filtered through Whatman No. 1 filter paper, and the filtrate was freeze-dried. The dried extract was stored at −8 °C until further analysis. Extraction yield was calculated and recorded.

α-Glucosidase inhibition assay for anti-diabetic activity screening

An α-glucosidase inhibition assay was performed in accordance with the method established by Naing et al.^[15]. A stock solution (10,000 µg/mL) and a working solution (300 µg/mL) of each sample were prepared in dimethyl sulfoxide (DMSO). The solutions were vortexed, sonicated, and centrifuged to ensure complete dissolution. Each reaction well in a 96-well quartz microplate contained 190 μL of 50 mmol/L sodium phosphate buffer solution (PBS) supplemented with 100 mmol/L sodium chloride (NaCl) (pH 6.8), 50 μL of α-glucosidase enzyme solution (120 mU/mL), and 10 μL of the sample working solution. All samples were tested in two independent trials with two replicates each (n = 2, t = 2). For the positive control, 200 μL of acarbose (final well concentration: 1,000 µg/mL) was used. The negative control consisted of 200 μL of 5% DMSO in PBS (final solvent concentration: 3.33%). After incubation at 37 °C for 10 min, 50 μL of 1.86 mmol/L p-nitrophenyl-α-D-glucopyranoside was added to initiate the reaction. The release of p-nitrophenol was monitored by measuring absorbance at 405 nm every 30 s for 30 min using a Multiskan Go® UV/Vis Spectrophotometer.

Cholinesterase inhibitory activity

The acetylcholinesterase (AChE) inhibition assay was conducted according to the established procedure of Ellman et al.^[16], with some modifications. A stock solution (10,000 µg/mL) and a working solution (2,000 µg/mL) of each sample were prepared. Solutions were vortexed, sonicated, and centrifuged to ensure complete dissolution. The reaction mixture in each well of a 96-well microplate consists of 180 μL of 100 mmol/L phosphate buffer (pH 8), 10 μL of acetylcholinesterase (AChE) solution (1.0 U/mL, Electrophorus electricus), 80 μL of buffered Ellman's reagent (10.0 mmol/L 5,5′-dithiobis-[2-nitrobenzoic acid] [DTNB] and 17.9 mmol/L sodium bicarbonate [NaHCO₃], pH 7.0), and 15 μL of each sample working solution, positive (100 µg/mL), or negative control (methanol). Each test sample solution has two trials and two replicates (n = 2, t = 2). The sample, positive, and negative controls have a solvent well concentration of 5% methanol. After incubation at 25 °C for 15 min, 15 μL of 2.87 mmol/L acetylthiocholine iodide (AChI) was added to initiate the reaction. The changes in absorbance were measured at 420 nm using a Multiskan (Thermo Scientific) microplate reader, with 31 readings taken at 25-s intervals.

COX-1 and COX-2 inhibition assays for anti-pain activity screening

Cyclooxygenase (COX-1 and COX-2) inhibition was assessed as previously described by Nacpil et al.^[17]. A stock solution of 10,000 µg/mL and a working solution of 200 µg/mL were prepared using DMSO. Samples were homogenized using a vortex mixer, sonicator, and centrifuge. Using a clean scintillation vial, 5,184 μL of 100 mmol/L Tris buffer (pH 8) was added. Then, 96 μL of 250 U/mL COX enzyme (human recombinant enzyme for COX-2 and sheep enzyme for COX-1) and 480 μL of 20 μM Hematin were mixed and subsequently dispensed into the vial with the buffer. This formed the enzyme cofactor solution. To each test well that had already been dispensed with 50 μL of the same buffer, 120 μL of the enzyme-cofactor mixture was added. Then, 10 μL of either the sample working solution, the 160 mmol/L positive control (indomethacin), or the negative control (DMSO) was added to the specified wells. Each working solution has two trials and two replicates (n = 2, t = 2). The sample, positive control, and negative control have a solvent well concentration of 5% DMSO. The mixture was incubated at 25 °C for 15 min. After incubation, 10 μL of 200 μM Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) was added to each well, followed by 10 μL of 2,000 μM arachidonic acid. The reaction mixture was briefly mixed and purged with nitrogen (N₂). The reaction was monitored for 2 min using the CLARIOstar plate reader at an excitation wavelength of 535 nm and an emission wavelength of 590 nm. Fluorescence intensity was measured at 12-s intervals.

Radical scavenging activity

The radical scavenging activity of the extracts at a concentration of 500 mg/L was determined using the stable 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical following the standard method of Shimada et al.^[18] with modifications. One hundred microliters (100 µL) of the test sample in ethanol was added to 5 µL of DPPH solution (prepared by dissolving 5 mg DPPH powder in 2 mL of ethanol) in 96-well microtiter plates. The mixture was shaken vigorously and left to stand in the dark for 30 min, after which the absorbance was measured at 517 nm. The inhibition of DPPH free radicals was calculated.

Danio rerio embryotoxicity and teratogenicity assay

The embryotoxic and teratogenic potential of the extract was evaluated using zebrafish (Danio rerio) embryos, following the method described by Dulay et al.^[19] with modifications. Test concentrations were prepared at 0.1, 1, 10, 100, 1,000, 10,000, and 100,000 µg/mL using embryo water as the diluent. This broad concentration range was intended to capture both environmentally relevant exposures and extreme levels to establish the upper threshold of the embryotoxic response, ensuring a comprehensive dose–response profile. A negative control group containing only embryo water and a positive control group treated with 95% ethanol were included. For each treatment, 200 µL of the prepared solution was dispensed into individual wells of a sterile, flat-bottom 96-well ELISA plate. Four embryos at the segmentation stage were transferred into each well, with three replicate wells per treatment (r = 3, n = 4). Hatchability and mortality rates were recorded, and the resulting data were subjected to probit analysis via SPSS Statistics software to determine the median lethal concentration (LC₅₀).

Following LC₅₀ determination, a second experimental setup was conducted to assess sublethal and teratogenic effects using concentrations based on the LC₅₀ value. Three working doses were prepared: a high dose (HD), a medium dose (MD), and a low dose (LD). In this setup, each treatment group, including the negative control, was tested in duplicate, with three embryos allocated per well (r = 2, n = 3). After incubation, embryos were examined under a compound microscope to assess morphological development. The embryos were classified based on the criteria of Nagel into three categories: lethal (coagulation, absent heartbeat), teratogenic (spinal curvature, pericardial edema, yolk deformities, head and tail malformations), and normal development (no abnormalities)^[20]. In addition to morphological observations, heartbeat rate was measured by manually counting cardiac contractions under the microscope for 10 s and multiplying by 6 to obtain beats per minute (bpm). The assay was deemed valid only if 100% of the embryos in the negative control group developed normally without mortality or malformation.

Artemia salina lethality assay

The cytotoxic potential of the mushroom extract was evaluated using the brine shrimp (Artemia salina) lethality assay, following the modified method of Olowa & Nuñeza^[21]. Brine shrimp larvae (nauplii) were obtained by hatching eggs in artificial seawater, prepared by dissolving 25 g of salt in 1 L of distilled water. Test solutions were prepared at concentrations of 1, 10, 100, 1,000, 10,000, and 100,000 µg/mL using artificial seawater as the diluent. Ten nauplii were introduced into each well of a 96-well ELISA plate containing the respective treatment solutions, with three replicate wells utilized per treatment (r = 3, n = 10). Artificial seawater and 95% ethanol were used as the negative and positive controls, respectively. After 12 h of exposure, the number of dead nauplii was recorded, and the percentage mortality was calculated. The median lethal concentration (LC₅₀) was determined using probit analysis via SPSS Statistics software. Toxicity levels were interpreted using the criteria established by Nguta et al.^[22]. Specifically, LC₅₀ values were classified as non-toxic (> 1,000 µg/mL), weakly toxic (500–1,000 µg/mL), toxic (100–500 µg/mL), or very toxic (< 100 µg/mL).

Statistical analysis

Data were analyzed using one-way analysis of variance (ANOVA) in GraphPad Prism v8.0.2 (Dotmatics, San Diego, CA, USA). Means were assessed and compared using Tukey's honestly significant difference (HSD) test at a 5% level of significance. Additionally, Dunnett's t post hoc test was performed using SPSS Statistics Software v25.0 (IBM Corp., Armonk, NY, USA) to compare each treatment group with the negative control.

The influence of liquid culture media, pH, temperature, and agitation on the mycelial biomass production of O. canarii is summarized in Table 1. Among the three locally available fruit-based media tested (25% mango purée, mature coconut water, and their combination), O. canarii grows best in coconut water, yielding a mean of 180.00 mg DW/100 mL, which was significantly greater than the other media (p < 0.05).

pH also exerted a significant effect, with the range of 6.0–7.0 proving most suitable, yielding a mean of 180.67 and 189.00 mg DW/100 mL, respectively. Likewise, temperature strongly influenced biomass production of O. canarii, with the highest yield observed at 30 °C (223.67 mg DW/100 mL), significantly higher than the other temperatures tested (p < 0.05). Agitation further enhanced growth, with O. canarii attaining its maximum biomass yield at 150 rpm (1,847.00 mg DW/100 mL), which was significantly greater than yields at 100 rpm and under static conditions (p < 0.05).

Inhibitory assays

The inhibitory activity against α-glucosidase, acetylcholinesterase, cyclooxygenase, and DPPH observed in this study for O. canarii extracts is presented in Table 2.

The α-glucosidase inhibitory activity of O. canarii extract at 10 µg/mL was evaluated to assess its antidiabetic potential. This study sought to evaluate the extract that might achieve inhibition above 50%, which is considered necessary for therapeutic relevance; however, the results revealed minimal activity at 10 µg/mL, with 1.42% inhibition, suggesting that the extract is not highly effective at this concentration.

The O. canarii extract also underwent preliminary screening for AChE inhibition, conducted at a concentration of 10 µg/mL. Results presented in Table 2 showed negative values, having −4.35% inhibition, which suggests that the extract has no inhibitory activity against AChE at this concentration.

A cyclooxygenase inhibition assay was performed to assess the anti-inflammatory potential of O. canarii extract. As shown in Table 2, the extract yielded 26.37% and 9.80% inhibition against COX-1 and COX-2, respectively. These values suggest the low effectiveness of the extract to inhibit cyclooxygenase at this concentration.

The antioxidant potential of O. canarii extract at 1,000 µg/mL was evaluated via DPPH radical scavenging assay. The extract exhibited a moderate DPPH radical scavenging activity with 51.56% inhibition, as presented in Table 2. This is significantly lower than the value observed from ascorbic acid, which showed a notably high scavenging activity (86.88% inhibition), confirming the assay's reliability.

Danio rerio embryotoxicity and teratogenicity

Zebrafish (Danio rerio) embryos were employed to evaluate the embryotoxic and teratogenic potential of the extract. The results, summarized in Table 3, showed evident dose- and time-dependent impacts of the O. canarii mycelial extract on zebrafish embryos. Complete mortality (100%) was observed within 24 h at 10,000 and 100,000 µg/mL. Hatchability was also entirely inhibited (0%) at these concentrations, as well as at 1,000 µg/mL, paralleling the outcome in the ethanol control. Meanwhile, the water control consistently yielded 0% mortality and 100% hatchability, thereby validating the reliability of the assay. At a concentration of 1,000 µg/mL, the extract killed 50% of the zebrafish embryos within 24 h, which increased to 100% mortality within 48 h. In comparison, the lowest concentration (0.1 µg/mL) exhibited no mortality and a hatchability of 91.67%.

As indicated in Table 4, the O. canarii extract also induced dose-dependent teratogenic effects in zebrafish embryos. Under high (0.34 µg/mL) and medium (0.17 µg/mL) doses, the mycelial extract caused delayed growth in the zebrafish embryos. In contrast, no abnormalities were observed in zebrafish embryos that were exposed to low dosage (0.08 µg/mL), similar to the outcome observed in embryo water, which confirms assay reliability.

Artemia salina cytotoxicity

A brine shrimp (Artemia salina) lethality assay was carried out to evaluate the cytotoxic potential of the O. canarii extract. Results revealed that the extract induced cytotoxic effects on brine shrimp larvae in a dose-dependent manner (Table 5). Exposure to the highest concentrations (1,000–100,000 µg/mL) of O. canarii extract showed 100% mortality, similar to ethanol-water. At intermediate concentrations (10 and 100 µg/mL), varying effects were observed, ranging from 33.33% to 46.67% mortality. By contrast, the lowest concentrations (0.1 and 1 µg/mL) demonstrated 15.00% and 20% mortality, respectively. No mortality was recorded in the saline solution, validating the assay's reliability.

In the submerged fermentation method of mushroom cultivation, the optimization of factors such as media composition, pH, temperature, and agitation is critical to achieving higher biomass yields and enhanced bioactive compound synthesis. The present study identified coconut water as the most suitable liquid culture medium for promoting mycelial growth of O. canarii among the treatments tested. Coconut water likely supports favorable growth conditions due to the presence of phytohormones—such as auxin, cytokinin, and gibberellins—that enhance nutrient absorption and stimulate cell division, processes that are essential for plant development and also for fungal mycelial proliferation, given the close resemblance of fungal histidine kinases to plant hormone receptors^[23]. Moreover, coconut water possesses a balanced composition of minerals and vitamins, along with a relatively high sugar content, which collectively supports fungal growth and development^[24]. Several studies have also reported that coconut water supports the growth of other mushroom species. For example, it was reported that submerged cultures in coconut water yielded optimal biomass production in 14 Lentinus isolates^[25]. Comparable outcomes were observed in a different study, with coconut water gulaman producing the greatest mycelial growth diameter in 10 mushroom species/strains^[26].

The pH of the culture medium is another critical factor influencing mycelial proliferation, as it is a potent regulator of fungal physiology, influencing enzyme activity, nutrient uptake, and biomass yield^[27]. In this study, O. canarii exhibited optimal growth at pH values between 6.0 and 7.0, aligning with reports that most fungi favor weakly acidic to neutral conditions. Similar findings were observed that three O. canarii isolates have the best mycelial growth performance at pH 6^[17]. Furthermore, it was also demonstrated that both Cyathus striatus and Xylaria hongkongensis grow best at pH 7.0, although their biomass yields were found statistically comparable to those observed at pH 6^[28].

Temperature is another critical determinant of growth. Among the three temperature conditions tested, O. canarii exhibited optimal development at 30 °C, confirming its pantropical nature and supporting previous reports on the thermophilic tendencies of many basidiomycetes^[29]. In contrast, mycelial growth was completely suppressed at 10 °C, demonstrating the adverse effects of low temperatures on protein dynamics, metabolic activity, and enzymatic functions critical for nutrient cycling and fungal growth^[30].

The present study shows that shake-flask culture at 150 rpm yielded the highest biomass, confirming agitation as a crucial factor for O. canarii growth. These results align with the study that reported four O. canarii isolates that achieved maximum biomass yield under the same agitation condition^[17]. Similarly, a study reported that Pleurotus djamor achieved its peak biomass yield and exopolysaccharide production at 150 rpm agitation^[31]. Given this context, agitation in submerged fermentation is an important growth factor as it improves oxygen transfer, nutrient distribution, and homogeneity of the culture medium, leading to higher mycelial biomass and metabolite production^[13]. However, it should also be noted that agitation beyond the optimal level can impose shear stress that compromises the mycelial structure and leads to reduced biomass production^[32]. For instance, certain Lentinus isolates attained maximum biomass yields under static-flask culture conditions, with yields declining as agitation rates increased^[25].

Under optimized cultivation conditions, the mycelia of O. canarii were successfully mass-produced and extracted, providing samples for a series of biological assays. Among these, the antidiabetic potential of the extract was assessed using an α-glucosidase inhibition assay. This assay suppresses α-glucosidase, the enzyme responsible for hydrolyzing complex carbohydrates into glucose, to slow down carbohydrate digestion and delay intestinal glucose absorption, thereby reducing sharp postprandial spikes in blood sugar^[33]. This mechanism is particularly relevant for the management of diabetes mellitus (type 2 diabetes), a global health challenge whose prevalence continues to rise at an alarming rate. Given the growing burden of diabetes mellitus, the search for novel anti-diabetic agents has become a pressing medical priority. Several studies highlight mushrooms as promising natural reservoirs of bioactive compounds, many of which demonstrate α-glucosidase inhibitory activity. At the tested concentration, the O. canarii extract demonstrated a minimal activity of 1.42% inhibition, which is not significant based on the 50% inhibition threshold required for therapeutic consideration. Similar findings were reported in a study by Nacpil et al., wherein none of the mycelial extracts of four O. canarii isolates exhibited significant inhibitory activity^[17]. The relatively minimal activity of the O. canarii mycelial extract observed in this study may reflect methodological constraints, particularly the extraction approach and the concentration range tested. Having said that, further work employing alternative extraction solvents and refined concentration gradients is warranted to more accurately assess the inhibitory potential of this species

The acetylcholinesterase (AChE) inhibition assay is a widely used biochemical test to evaluate compounds that block AChE activity, an enzyme responsible for breaking down the neurotransmitter acetylcholine. This assay is crucial in drug discovery for Alzheimer's disease and other neurodegenerative disorders, as it enhances cholinergic neurotransmission, which refers to the fundamental mechanism of neurons using acetylcholine to release and send signals across synapses for the modulation of cognitive processes^[34,35]. In the present study, the O. canarii extract yielded a negative inhibition value of −4.35 at a concentration of 10 µg/mL, indicating a lack of detectable AChE inhibitory activity under these conditions. The negative value observed may reflect interference from certain components present in the extract that influenced the assay system^[36]; however, since no chemical analysis of the extract was performed in this preliminary screening, the specific compounds responsible for such effects cannot be determined. Nevertheless, many other mushroom species have been documented to exhibit acetylcholinesterase inhibitory effects. A study involving 51 different extracts from five mushroom species indicated that inhibitory strength varies significantly based on the extraction medium used, with the butanolic extract of Agaricus bisporus exhibiting the highest anti-cholinesterase activity^[37]. Furthermore, the specific chemical constituents of these fungi are central to their inhibitory success. While polysaccharide-peptide complexes from Flammulina velutipes and Hypsizygus marmoreus show direct activity^[38], a broader range of compounds, such as alkaloids and terpenoids, are increasingly recognized for their combined neuroprotective and enzyme-inhibitory roles^[39].

The enzymes cyclooxygenase-1 and 2 are responsible for the synthesis of prostaglandins, which play essential roles in inflammation, pain regulation, and the fever response^[40]. In this study, the O. canarii extract demonstrated moderate activity against COX-1, whereas its activity toward COX-2 was comparatively low. This trend aligns with the observations in another study, which noted greater COX-1 inhibition in O. canarii isolates, though the difference relative to COX-2 was not statistically significant^[17]. Interestingly, one isolate from Nueva Ecija exhibited strong activity, exceeding 50% inhibition for both COX-1 and COX-2. This suggests variability in the bioactive compound profiles among different sources of O. canarii, possibly reflecting ecological or genetic influences on metabolite production. Meanwhile, the relatively greater COX-1 selectivity observed in most isolates may be attributed to the presence of specific fatty acids or terpenoids that preferentially interact with this isoenzyme, a mechanism also reported in Grifola frondosa^[41]. However, from a clinical perspective, the transition from COX-1 to selective COX-2 inhibition represents a more rational approach to suppress pain and inflammation without the adverse gastrointestinal effects typical of non-selective NSAIDs^[42]. Nonetheless, these results point toward the presence of selective enzyme inhibitors within the extract of O. canarii, warranting further investigation.

Free radicals, which are inevitably generated within biological systems and acquired from external sources, are strongly linked to degenerative conditions such as mutagenesis, carcinogenesis, cardiovascular disorders, and ageing^[43]. As a result, extensive research has been directed toward the discovery and characterization of antioxidants, which are compounds capable of neutralizing free radicals and mitigating oxidative stress. Mushrooms, in particular, have been widely recognized as valuable reservoirs of antioxidant metabolites. In the present study, the antioxidant potential of O. canarii extract was assessed using the DPPH radical scavenging assay. The extract exhibited strong radical scavenging activity against DPPH, though its effect was not significantly greater than that of acarbose, which served as the reference drug. Previous studies have demonstrated that the antioxidant activity of mushrooms is often associated with their phenolic content. For instance, a 78% positive correlation between total phenolic levels and antioxidant capacity was reported in a study by Kim et al.^[44], suggesting that species with higher phenolic concentrations generally display stronger antioxidant properties. In this context, the relatively strong DPPH scavenging activity of O. canarii may be attributed to its inherently high phenolic content; however, it is recommended that future studies conduct detailed mycochemical analysis to confirm the presence of phenols along with other bioactive compounds. Beyond phenolics, previous studies have highlighted that the significant antioxidant capacity of basidiomycetous fungi is also largely driven by their mycelial polysaccharides and secondary metabolites, including terpenoids and flavonoids^[45−47].

The bioactivities of mushrooms extend beyond inhibitory effects, with several species also documented to possess embryotoxic and teratogenic properties. This study used zebrafish (Danio rerio) as an embryonic and larval model to perform in vitro experiments and developmental toxicity studies, wherein results revealed O. canarii extract's dose- and time-dependent effects. Exposure to the highest concentrations tested (100,000 and 10,000 µg/mL) resulted in complete mortality within 24 h, whereas at 1,000 µg/mL, 100% mortality was achieved only after 48 h. Accordingly, hatchability was entirely suppressed (0%) across all three concentrations mentioned. The 0.1 µg/mL concentration caused no mortality, yet hatchability was limited to 91.67% as compared to the 100% hatchability recorded in embryo water, which served as the positive control. Regarding the teratogenicity of O. canarii extract, no abnormalities were observed in zebrafish embryos across all concentrations tested, only delayed growth, which was observed at medium and high doses. These results align with the findings of a study by Dulay et al., which reported that aqueous extracts of L. strigosus induced time- and dose-dependent embryotoxic and teratogenic effects in zebrafish, manifested as increased mortality and delayed development^[48]. In parallel, another study by Dumale & Gamoso observed that water extracts of Pleurotus citrinopileatus similarly caused growth retardation and lethality in zebrafish embryos at elevated concentrations^[49]. The decline in hatching success could be attributed to impaired embryonic development, plausibly resulting from inhibition of critical developmental enzymes, suppression of growth factors, or disruption of physiological pathways necessary for normal embryogenesis^[50]. Overall, the results establish that O. canarii extract is highly embryotoxic at elevated concentrations, moderately disruptive at intermediate levels, minimally impactful at the lowest dose, and non-teratogenic, with growth retardation being the only developmental effect observed. Future studies should, however, incorporate standardized morphological scoring systems to systematically document teratogenic endpoints (e.g., pericardial edema, yolk sac edema, spinal curvature, delayed development), thereby providing more detailed and reproducible characterization of developmental effects.

Artemia salina (brine shrimp) has long been recognized as a valuable model organism for toxicity screening. The brine shrimp lethality assay relies on the sensitivity of brine shrimp nauplii (larvae) to bioactive compounds, measuring their mortality to screen for general toxicity and potential anticancer or pharmacological activity of substances like plant extracts or nanomaterials^[51]. In this study, O. canarii extract produced dose-dependent cytotoxic responses in brine shrimp nauplii, ranging from complete mortality at the highest concentrations, substantial lethality at intermediate levels, and moderate activity even at the lowest concentrations tested. The calculated LC₅₀ value of 17.87 µg/mL highlights its cytotoxic potency. Notably, several other mushroom species have likewise been reported to exhibit cytotoxicity in brine shrimp assays. Schizophyllum commune, for instance, was reported to induce cytotoxic effects on brine shrimp with an LC₅₀ value of 55.64 μg/mL^[52]. Likewise, the n-hexane, ethyl acetate, and methanolic extracts of Ganoderma applanatum displayed potent toxicity even at low concentration, comparable to the standard potassium dichromate^[53]. The cytotoxic potency observed in mushrooms is largely attributed to their polysaccharides, particularly glucans, which constitute major structural components of fungal cell walls. These polysaccharides have been documented to inhibit oncogenesis not through directly targeting cancer cells, but by stimulating host immune mechanisms that elicit antitumor responses against diverse allogeneic and syngeneic tumors, while also suppressing tumor metastasis^[54]. Steroidal compounds, including ergosterol derivatives, have likewise been reported to exert cytotoxic effects. Ergosterol peroxide, for example, that was isolated from the marine basidiomycete Fulvifomes sp., has induced apoptosis in the MCF-7 cell line by promoting reactive oxygen species (ROS) generation^[55]. Collectively, these results suggest that mushrooms such as O. canarii may serve as potential sources of bioactive compounds, although the activities observed in this study were generally modest and warrant further investigation to clarify their therapeutic relevance.

The study demonstrated that submerged fermentation using coconut water at pH 7, 30 °C, and 150 rpm significantly enhanced the mycelial biomass production of Oudemansiella canarii. The ethanolic extract exhibited moderate antioxidant and partial anti-inflammatory activities, but showed minimal antidiabetic and neuroprotective effects. Toxicological evaluations in zebrafish embryos and brine shrimp nauplii demonstrated pronounced dose-dependent embryotoxicity, developmental delay, and cytotoxicity. Overall, while O. canarii mycelial extracts hold promise for functional food and therapeutic applications, careful consideration of their toxicological profile is essential before practical use. Therefore, further mechanistic studies, fractionation of active compounds, and comprehensive safety assessments are recommended.