Reagents

Brucella broth (BB), brain heart infusion broth (BHIB), and bacto-agar were purchased from Becton Dickinson, Inc. (Franklin Lakes, NJ). Newborn bovine serum (NBS) was purchased from Hyclone (Longan, UT). Tryptone and yeast extract were provided by Merck (Kenilworth, NJ) and glucose was provided by HiMedia (Kennett Square, PA). Amoxicillin (≥ 98%) was provided by Santa Cruz Biotechnology Inc. (Dallas, TX). Solvents, hexane 96%, ethyl acetate (EtOAc) 99.8%, absolute ethanol (EtOH), and methanol (MeOH) 99.9% were purchased from Scharlau (Barcelona, Spain). All other chemicals and reagents used in this study were of analytical grade quality and available commercially.

Broth culture of and solvent extraction of the mycelium and culture filtrate

mycelium was provided and identified by Dr. Pham Thanh Ho, Laboratory of Biotransformation, Faculty of Biology and Biotechnology, VNUHCM University of Science, Ho Chi Minh City, Vietnam. mycelium could produce fruiting bodies in the laboratory conditions. The mycelium and fruiting bodies were cultured and stored at 4 C.

Four pieces of mycelium (1 cm/each) from 8 day-old culture on agar medium were inoculated in 500 mL bottles containing 200 mL liquid medium with the following composition: 20% potato extract, 2% glucose, 0.2% yeast extract, and 0.2% tryptone. The broth cultures were incubated at 25 ± 2°C and shaken at 150 rpm for 7 days11. After that, the culture broths were filtered to obtain the mushroom mycelium and culture filtrate for solvent extraction, as shown in Figure 1.

The dry mycelium was disrupted by liquid nitrogen in 30 minutes. The mycelium was then finely grounded and extracted with 200 mL hexane for 1 day (3 times) and further extracted sequentially with 200 mL EtOAc for 1 day (3 times) and 500 mL hot water for 2 hours. The extracts were filtered, and a rotary evaporator was used to remove the solvents to obtain a hexane-soluble fraction (mHexane Fr.), EtOAc-soluble fraction (mEtOAc Fr.), and water-soluble fraction (mWater Fr.) (Figure 1).

The culture filtrate was precipitated with 4 times volume of cold absolute EtOH (1 °C) overnight and then centrifuged to obtain the crude polysaccharide fraction (precipitant). The polysaccharide fraction (PS Fr.) was dissolved in sodium phosphate buffer buffer (30 mM, pH 7) to a 200 mg/mL stock concentration and stored at — 20 °C until further use. After the precipitation, the filtrate was then fractionated sequentially three times with an equal volume of hexane for 2 hours, and then three times with an equal volume of EtOAc for 2 hours using the liquid-liquid method. The solvent-soluble fractions were condensed to dryness by a rotary evaporator at 42 °C to gain the hexane fraction (fHexane Fr.), EtOAc fraction (fEtOAc Fr.), and water fraction (fWater Fr.) (Figure 1).

strain and culture condition

The strain (ATCC 43504) was provided by the Oxford University Clinical Research Unit in Vietnam (OUCRU-VN) and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI. The bacterial strain was stored in BHIB medium supplemented with 25% glycerol and placed in a nitrogen liquid container until use.

The strain was cultured on Brucella agar supplemented with 10% newborn bovine serum, and then incubated at 37°C in 3 days under microaerophilic condition created by a 2.5 L Oxoid anaerojar and Oxoid Campygen sachet (Thermo Fisher Scientific, Waltham, MA, USA). The bacterial suspensions used for bioassay were suspended in Brucella broth supplemented with 5% NBS using a 72-h subculture of on Brucella agar. Bacterial density was determined using McFarland turbidity standards.

Antibacterial assay

Preparation of urease and inhibition of urease activity

urease crude extract was prepared as reported previously21 with slight modification. Briefly, an amount of 500 mg of cell mass from 72-h cultures on NBS-supplemented Brucella agar was spread as thinly as possible on the wall of a polypropylene tube and stored in a nitrogen liquid container for 15 minutes. The cell mass was then thawed at room temperature and added with 5 mL of 20 mM sodium phosphate buffer (pH 7.3) containing EDTA (1 mM). Finally, the mixture was centrifuged at 12,000×g, 4°C for 30 minutes, and the supernatant was filtered using a 0.22 µm Millex GV Millipore filter. The protein content was determined using Bradford protein assay. Bovine serum albumin (BSA) was used as a protein standard.

urease inhibitory activity of the fractions extracted frommycelium and culture filtrate was assayed in 96-well plates using the salicylate-hypochlorite method with minor modification22. In brief, 50 µL of 0.25 µg urease crude preparation (0.04 urease units) in EDTA-sodium phosphate buffer (pH 7.3) was added to each well containing 50 µL of the test fraction at various concentrations. The plates were preincubated at 37°C and shaken at 50 rpm for 90 minutes. An amount of 50 µL of 5 mM urea in phosphate buffer saline (PBS) was added into each well. After 30 minutes of incubation, a stop solution consisting of 35 µL of solution A (146% Na salicylate + 0.1% sodium nitroprusside) and 65 µL of solution B (1.78% NaOH + 11.57% Na citrate + 0.54% active NaOCl) were supplemented in sequence to each well. The plates were incubated for 30 minutes at 37°C for color development. The ammonia production released from the hydrolysis of urea by urease activity was quantified by measuring absorbance on the microplate reader at 625 nm using ammonium chloride as a standard. Thiourea as a standard reference was similarly prepared. Inhibition rate (I%) was calculated by the following formula: I (%) = [1 — (OD test sample — OD corresponding background)/(ODcontrol — OD blank)] × 100. Urease inhibition activity of the fractions was displayed as 50% inhibitory concentration (IC), which was defined as the concentration of the fractions required to decrease urease activity to 50% of the control value. The assay was performed in duplicate three times.

DPPH free radical scavenging assay

The antioxidant activities of the fractions extracted from mycelium and culture filtrate were evaluated via 2,2,1-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity with slight modification11. The experiment was performed in 96-well plates. The fraction solutions in MeOH at various concentrations from 0 – 50 mg/mL were placed in the wells, and 150 µL of 380 µM DPPH in MeOH was then added to each well. Blank and background wells, which contained MeOH and the fractions, respectively, without DPPH, were also prepared. The plates were placed in the dark and incubated at 37°C with rotation at 75 rpm for 30 minutes. Subsequently, the absorbance measurements were determined at 515 nm on a Microlisa Plus microplate reader (Micro Lab Instruments, India). Acid ascorbic (0 – 0.5 mg/mL) served as a positive control and was similarity prepared. The percent radical scavenging activity was calculated as follows: percent scavenging effect (%) = [1 — (OD— OD)/(OD — OD)] × 100, where OD, OD, OD and OD are absorbance values of the controls, blanks, fraction treatments, and background wells, respectively.

Data analysis

All bioassays were repeated three to five times in triplicate. The inhibition zones of the test fractions were presented in mean values (± SD). DPPH ICand urease IC(half maximal inhibitory concentration) values were calculated using GraphPad Prism 5 software program (GraphPad Software, Inc., La Jolla, CA, USA). The IC values of the treatments would be declared significantly different if their 95% confidence intervals did not overlap.

Solvent extraction of the mycelium and culture filtrate of

After 7 days of incubations, the culture broths of were filtered to obtain 57.7 g dry weight of the mycelium and 24 L of its culture filtrate. Solvent-soluble fractions obtained from the dry mycelium and culture filtrate are presented in Figure 1.

From the dry mycelium (57.7 g), the three fractions of mHexane Fr., mEtOAc Fr. and mWater Fr. were obtained, and weighed as 0.162, 0.091 and 0.509 g, respectively (Figure 1).

From 24 L of the culture filtrate, the four fractions of PS Fr. (26.4 g), fHexane Fr.(0.12 g), fEtOAc Fr. (11.04 g) and fWater Fr. (72.48 g) were obtained (Figure 1). The yields of the Hexane and EtOAc fractions were lower than those of the PS and Water fractions. This implied that there were very few non-polar components in the mycelium and culture filtrate.

Antibacterial assays

The results from the paper disc diffusion method are summarized in Table 1 and Figure 2. The results revealed that the EtOAc fractions from both the mycelium (mEtOAc Fr.) and filtrate (fEtOAc Fr.) had strong anti-activity with inhibition zones of 18 – 19 mm, while mHexane Fr. and PS Fr. showed weak activity with 12 and 9 mm inhibition zones, respectively. However, mWater Fr., fHexane Fr. and fWater Fr. showed no inhibitory activity (Table 1). All fractions displayed weaker inhibitory activity than the positive control amoxicillin (inhibition zone of 37.5 mm).

The MIC and MBC results also yielded similar results (Table 1). The fractions mEtOAC Fr. and fEtOAC Fr. showed strong anti- activity with MIC (MBC) values of 1.5 (7.5) and 1.25 (5.0) mg/mL, respectively. However, mHexane Fr. and PS Fr. displayed weak anti- activity with MIC (MBC) values of 7.5(≥ 10) mg/mL, while fHexane Fr., mWater Fr. and fWater Fr. showed no inhibitory activity (MIC ≥ 10 mg/mL). The ATCC 43504 strain was susceptible to the positive control amoxicillin with MIC (MBC) values of 0.032 (0.063) µg/mL.

Urease inhibition

Based on urease IC data presented in Table 2, mEtOAc Fr. and fEtOAc Fr. displayed strong urease inhibitory activity with IC values of 0.34 and 0.35 mg/mL, respectively, with a non-significant difference (p > 0.05). This was followed by the inhibitory activity of mWater Fr. and fWater Fr. (IC values of 1.26 and 1.40 mg/mL, respectively). Besides, both mEtOAc Fr. and fEtOAc Fr. caused a steep dose-response curve of the percentage of urease inhibitory activity and produced a complete inhibition against the urease at 2.5 mg/mL, whereas mWater Fr. and fWater Fr. showed a complete inhibition against the urease at 3 and >5 mg/mL, respectively (Figure 3). Moreover, PS Fr., mHexane Fr. and fHexane Fr. gave very weak or no inhibitory activity towards the enzyme. All the fractions exhibited lower urease inhibitory activity than the positive control thiourea (IC, 0.055 mg/mL) (Table 2).

DPPH free radical scavenging

Based on DPPH IC values presented in Table 3, fEtOAc Fr. displayed the greatest antioxidant activity in the DPPH assay (IC of 11.83 mg/mL), followed by mEtOAc Fr. (IC of 14.75 mg/mL), with a significant difference (p < 0.01). Moreover, PS Fr., mWater Fr. and fWater Fr. showed weak or very weak antioxidant activity (IC, 28.66 – 34.51 mg/mL). In addition, fEtOAc Fr. caused a steep dose-response curve of percentage of DPPH radical scavenging activity and complete inhibition at 40 mg/mL (Figure 4). Fractions mHexane Fr. and fHexane Fr. had no radical scavenging activity. Overall, all of these fractions showed significantly weaker DPPH radical scavenging activity than the positive control acid ascorbic (IC of 0.057 mg/mL) (Table 3).