Termitomyces mushroom extracts and its biological activities

Termitomyces mushrooms are affluent in bioactive components contributing to their therapeutic properties. This study aimed to investigate the antioxidant, antibacterial, antifungal activities and the toxicity of Termitomyces extracts. Termitomyces mushrooms were collected from the South of Vietnam, separated into different parts, air-dried, ground into powder, and extracted with methanol and ethanol at a divergent temperature of evaporation. Termitomyces mycelial biomass extract was discovered to be efficient in scavenging the free radicals through DPPH assay. The extract exhibited potent efficacy against Gram-negative ( Escherichia coli , Pseudomonas aeruginosa , and Salmonella typhimurium ) and Gram-positive ( Bacillus cereus , Staphylococcus aureus ) bacteria along with Candida albicans fungus applying the disc-diffusion method. Termitomyces extract underwent in vitro and in vivo experiments revealed no toxicity. The Termitomyces mycelial biomass extract is a potential source in developing novel antioxidant, antibacterial and antifungal agents.


Introduction
Mushrooms play a significant role in curing various degenerative diseases and food processing by their chemical composition, nutritional value, and therapeutic properties. Mushrooms were determined to be affluent in unsaturated fatty acids, fat-soluble vitamins, ergosterol, vitamin D, along with bioactive molecules consisting of β-glucans, triterpenoids, antioxidants (Rathore et al., 2017).
According to the research results of Ulziijargal & Mau (2011), the difference in the content of the essential components involving fruiting bodies and mycelia among fungal species of genera named Agaricus, Auricularia, Cordyceps, Flammulina, Ganoderma, Lentinus, Pleurotus, and so on showed close resemblance. Therefore, the study on the production process of termite mycelium implemented a liquid culture method guiding a premise for future studies on culturing other species.

Termitomyces mushroom extracts
100 g of dry biomass powder and fruiting body of 5 samples were extracted by methanol as a solvent with a sample ratio 5:1 at 40 °C, shaken for 24 h at 150 rpm, and filtered. The residual material underwent a double extraction with 300 mL of methanol afterward. The total extracts were evaporated at 50 °C to release the solvent, as reported by Giri et al. (2012).

Evaluation of DPPH radical scavenging activity assay
The mixture consisted of 0.5 mL of DPPH (2, 2-diphenyl-1-picrylhydrazyl) solution 2 mL of the extract with different concentrations. After incubation for 30 min, the absorbance of the solution was measured in a spectrophotometer at 517 nm and compared with the methanol control sample.

Evaluation of antibacterial activity
The antibacterial capacity was assessed using the agar well diffusion method (Palaksha et al., 2010). Microbial strains, namely Escherichia coli ATCC 8739, Bacillus cereus ATCC 11778, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 14028, Candida albicans ATCC 10231 were activated in meat peptone broth for 24 h. The bacterial density was determined by optical density at 660 nm, adjusted in the range of 106-107 CFU/mL, and inoculated in solid mediumfor validation afterward.
A volume of 40 µL of the extract was diluted into different concentration ranges and added to the wells on the agar plate covered with bacteria. Chloramphenicol (10 mg/mL) was utilized as a positive control. The antibacterial potency was the distinction between the diameter of the inhibition zone and that of the well after 24 h. The increase the difference, the greater the antibacterial capacity.
The MIC (Minimum Inhibitory Concentration) on a 96-well plate method was employed to quantify the antibacterial effect of the extracts (Clinical & Laboratory Standards Institute, 2010). The solution involving 100 µL of liquid-form bacteria and 100 µL of different concentration ranges extract was incubated at 37 °C for 16-24h. Then, 20 µL of 0.01% resazurin reagent was made up serially to each well. The conversion of the resazurin solution from blue to pink indicated bacterial presence. MIC was defined as the lowest concentration of extracts inhibiting bacterial growth (without discoloring resazurin).

In-vitro MTT assay
The cytotoxic activity of the extract was investigated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method as described by Mosmann (1983). LO-2 hepatocytes were seeded in 96 well microtiter plates at a density of 1 × 10 5 cells/mL at 37 ºC and 5% CO 2 . Cells were transferred to medium without bovine serum and treated with Termitomyces strains biomass extracts in various gradient concentrations. After 24 h incubation, LO-2 hepatocytes were rinsed, and MTT solution was applied simultaneously (0.5 mg/mL, final concentration) for 4 h. 100 µL dimethyl sulfoxide (DMSO) was placed in each well after eliminating the supernatant and resuspended until all formazan crystals were dissolved. The absorbance value was measured on a microplate reader using a filter at a wavelength of 540 nm. The percentage of cellular viability was quantified as a proportion of the absorbance between the test sample and negative controls.

In-vivo acute and semi-chromic toxicity
Evaluation of acute toxicity of the reagent in white mice orally was executed by the description of Litchfield & Wilcoxon (1949). White mice were plotted and numbered in order of offspring and drank water freely during the fasting time of 12 h. Then, rats had applied reagents with increasing doses, from the lowest dose of 0 mg/kg body weight to the highest dose of 10000 mg/kg body weight. The analogous volume of both distilled water and different dose levels of biomass extracts were inserted directly into the stomach of each rat in test plots by a blunt-tipped needle. Each rat drank 3 hours/time x 3 times/24 h. Mice should be monitored concerning general condition, dead mice, weight, and hematological index in 72 h, then all dead mice were dissected to evaluate the gross damage. White mice were further observed for up to 14 days.
According to Vietnam Health Ministry's regulation, the semichronic toxicity was evaluated on white mice. In total, 32 mice were used for this experiment, where 4 mice were stabilized for 24 h and taken blood to analyze physiological indicators. The remaining 28 rats were divided into 2 plots located in 4 cages. The same volume of distilled water and extracts were inserted directly into the stomach of each rat by a curved needle once a day in the mornings from 8-10 h and drank continuously for 30 days. Test rats drank 1000 mg/kg body weight of extracts while control mice consumed distilled water. Parameters including body weight, blood physiological, and biochemical blood index were verified before utilizing the extract within 30 days from the first dose.

Antibacterial potential
Studies on the antibacterial ability of plant extracts prioritized methanol and ethanol solvents by their capability to dissolve natural compounds (El-Mahmood & Doughari, 2008); hence the extracts will be highly effective in both Gram-negative and Gram-positive bacteria (Turker et al., 2009).
The antibacterial activity of Termitomyces mushroom biomass and fruiting body extract against six strains of pathogenic bacteria, including Escherichia coli ATCC 8739 (EC), Bacillus cereus ATCC 11778 (BC), Staphylococcus aureus ATCC 6538 (SA), Pseudomonas aeruginosa ATCC 27853 (PA), Salmonella typhimurium ATCC 14028 (ST), Candida albicans ATCC 10231(CA) was evaluated in Table 1. The biomass extract expressed potent efficacy against all strains of pathogenic bacteria with the inhibition zone diameters ranging from 12-14.5 mm, in which the ability against EC was weaker with the respective inhibition zone 8 mm. On the other hand, remaining extracts from the fruiting body were determined inactive against SA and PA and showed moderate resistance to other strains. In particular, extract M2 prevented the growth of EC, BC, ST, CA with the inhibition zone ranging from 6.5-10 mm. Extract M3 exposed a similar activity to extract M2 to EC, BC, and CA but strongly against ST with the inhibition zone diameter of 12 mm. Extract M4 exhibited weak activity inhibiting BC and CA but dynamically against ST (inhibition zones 12 mm). Extract M5 revealed no activity or weak efficacy resist the bacteria enumerated.
The tested bacterial strains were susceptible to 5 extracts from Termitomyces mushroom with low minimum inhibitory concentration (MIC) values ranging from 10.21 mg/mL to 46.2 mg/mL (Table 2). This result was in accordance with other studies on the antibacterial activity of mushrooms (Jiamworanunkul & Chomcheon, 2019). In particular, common puffball mushroom (Lycoperdon perlatum) extracted with ethanol methanol had the potential against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus cereus with MICs ranging from 15.63 mg/mL to 125 mg/mL (Akpi et al., 2017); wood ear mushroom (Auricularia auricula-judae) resisted to Staphylococcus aureus, and Escherichia coli with MICs of 12.5 mg/mL and 6.25 mg/mL, respectively (Cai et al., 2015); white oyster mushroom (Pleurotus Florida) had the MIC value of 25 mg/mL for Escherichia coli, 50 mg/mL for Streptococcus sp., and 75 mg/mL for Proteus murabilus (Thillaimaharani et al., 2013); Caterpillar fungus (Cordyceps Sinensis) inhibited the growth of Bacillus subtilis and Streptococcus epidermidis with MICs were 938 μg/mL and 469 μg/mL, respectively (Ren et al., 2014).

Toxicity
Before applying for large-scale production, termite mushroom biomass extract was tested for cytotoxic activity to ensure efficacy and safety. In the in-vitro model, the cytotoxicity of the mushroom was evaluated by the MTT method on LO-2 human hepatocytes. Cell survival was recorded above 90% at the concentration range from 100,200,300,500,1000,1500,2000 to the highest concentration of 2500 μg/mL, proving that all concentrations of termite mushroom extract had no toxicity on the hepatoma cell line LO-2. According to Youn et al. (2008) research, the extract of Chaga mushroom (Inonotus obliquus) at the concentration of 1000 μg/mL had no effect on the hepatocytes, and cell survival rate attained over 80% in 48 h. However, the termite mushroom biomass extract should be further inspected in multiple mouse models at different concentration ranges to have entire toxicity data.
The acute toxicity results monitored after 72 h indicated that at the maximum oral dose of 10,000 mg/kg body weight, 100% of mice were normal without extraordinary expressions or dead. The next 14 days showed no abnormalities. Thus, termite mushroom biomass extract did not cause oral toxicity in white mice with the consumed dose of D max = 10,000 mg/kg body weight. There was no statistical difference in the weight of mice before and after 72 h of oral administration and physiological  blood indices between the experimental and control groups (Tables 3-4).
The semi-permanent toxicity observed after 30 days of applying extract at the concentration of 1000 mg/kg body weight revealed that the weight of mice in the treated group (29.787 ± 0.446) was inferior to that in the control group (30.563 ± 0.792). The difference between the 2 groups was not statistically significant but remarkably dissimilar to the initial mice weight (20.690 ± 0.264) ( Table 5). The result proved that termite mushroom biomass extract did not affect mice's weight during 30 days.
A large amount of blood was required to check the biochemical indices in mice; hence, the blood of mice in the group was combined before centrifugation. The biochemical blood indices between the non-consumed extract and the consumed extract groups differed, but the difference and fluctuation were not too much. The fluctuation levels among biochemical blood indices of mice shown in Table 6 expressed the normal liver and kidney function. The liver and kidney specimens of the tested mice were not damaged compared to the control group after dissecting and observing under the microscope at the magnification of 400x.
Blood physiological indices between the control and test samples after 30 days utilizing pooled T-test and T-Satterthwaite exposed no statistical difference. On the other hand, MCV and RDW-SD indices in the experimental group were elevated in comparison with the control group (p < 0.05) ( Table 7). However,   (Heatley & Harris, 2009).
Termitomyces extracts were evaluated to possess valuable nutrition as well as pharmacological properties contributing to health benefits. Nakalembe et al. (2015) discovered high levels of thiamin, folic acid, vitamin C, and niacin within 3 species of edible Termitomyces mushrooms in Uganda namely Termitomyces microcarpus (Berk and Broom) R. Heim, Termitomyces tyleranus (Otieno), Termitomyces clypeatus (Heim). According to Zhao et al. (2017), depolymerized-exopolysaccharides and exopolysaccharides derived from Termitomyces albuminosus exposed scavenging Table 5. Weight and lethal rate of mice after treated with mushroom extracts in semi-chronic toxicity experiment.   free DPPH radicals activities at a concentration of 400 μg/ mL. Besides, methanolic extract of Termitomyces albuminosus mycelia showed high antioxidant activity (Mau et al., 2004). However, varietal differences leaded to divergent biological activities, in particular, Termitomyces heimii and Termitomyces mummiformis displayed good antioxidant activity while Termitomyces microcarpus was contrary (Puttaraju et al., 2006). Aqueous extract of Termitomyces striatus were determined to inhibit several strains of bacteria and fungi due to the presence of phytochemicals namely alkaloids, flavonoids, sterols and steroids, saponins, phenols, carbohydrates, and proteins inside (Sitati et al., 2021). Both endopolysaccharide and exopolysaccharide derived from Termitomyces heimii RFES 230662 (THR2) were verified as β-glucan, which played a key role in the antibacterial activity of the mushroom extract (Ahmad et al., 2021). The research of Rathore et al. (2017) verified that Termitomyces eurhizus including significant homopolysaccharides supported anti-aging effects. Serine protease AkP isolated from Termitomyces clypeatus selectively prevented the growth of Hep-G2 cells by cleaving cell surface HSPG with the concomitant induction of apoptosis orchestrated by activation of caspase-3 (Majumder et al., 2016).

Conclusions
This study concluded that the methanol extract of Termitomyces mycelial biomass expressed antioxidant potential and potent efficacy against Gram-negative (Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium) and Gram-positive (Bacillus cereus, Staphylococcus aureus) bacteria. Besides, the extract showed an antifungal effect that inhibits Candida Albicans without causing toxicity. Therefore, the extract of Termitomyces mycelial biomass can be utilized as an antioxidant, antibacterial, and antifungal agent.