Major Antioxidant Compound of Polyporus Parvovarius Culture Filtrate

Abstract

During the screening of natural antioxidants from the culture broths of fungi of mushroom origin, the culture broth of Polyporus parvovarius was found to exhibit potent antioxidant activity. An antioxidative substance, compound 1, was isolated by solvent partitioning and column chromatography, and its chemical structure determined by nuclear magnetic resonance spectroscopic methods to be 3,4-dihydroxybenzaldehyde. Compound 1 exhibited potent free radical scavenging activity with a half-maximal inhibitory concentration of 101.4 µg/mL against the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical. These results indicate that the culture filtrate of P. parvovarius has antioxidant properties, with the major antioxidant compound confirmed as a protocatechuic aldehyde; these findings provide novel insights regarding mushroom-induced antioxidant resources.

Traditionally, mushrooms are considered functional foods that exert health benefits, in addition to having nutritional value, and are important sources of medicines. They produce diverse secondary metabolites, such as polyphenols and polysaccharides.^1,2 The components from mushrooms exhibit numerous pharmacological properties, such as immunostimulatory, anti-carcinogenic, anti-inflammatory, antimicrobial, anti-allergic, and antioxidant activities owing to their phenolic nature.^3,4 Oxidative stress induced by energy production in living organisms causes serious health concerns such as cancer, rheumatoid arthritis, leukemia, and diabetes.^5,6 Secondary metabolites generated in mushrooms play numerous vital roles, particularly in antioxidant defense mechanisms in the human body. ⁷ Many mushrooms also contain diverse molecules, such as free radical scavengers or reactive oxygen species, including polysaccharides and phenolic compounds.^8,9

The genus Polyporus P. Micheli ex Adans., family Polyporaceae, class Basidiomycetes, is widely applied as a medicinal mushroom. Polyporus species possess cytotoxic nephroprotective, hepatoprotective, immune-enhancing, anti-inflammatory, antioxidative, hair-growing, and antimicrobial activities.^10-13 Polyporus parvovarius H. Lee, N.K. Kim, and Y.W. Lim was first reported from Ulleungdo Island in Korea (Figure 1); according to phylogenetic analysis, this species is closely related to P. brevibasidiosus, but is morphologically more similar to P. varius. ¹⁴ As P. brevibasidiosus was only reported in 2017, its bioactivity has not been examined. However, the antioxidant activity of P. varius was confirmed in fruiting body extracts collected in Turkey. ¹⁵ P. parvovarius, which is predicted to exhibit high antioxidant activity, has been analyzed using different types of media and methods to confirm the presence of major antioxidant compounds.

Figure 1.

Morphology of Polyporus parvovarius collected from Ulleungdo islands in Korea.

Four types of culture media [dextrose and yeast extract (DY), malt extract broth (MEB), malt extract and yeast extract (MY), and potato dextrose broth (PDB)] were used to evaluate the correlation between the produced components from P. parvovarius and medium ingredients. The results show that the antioxidant activity depends on the media; the optimal carbon and other nutritional sources were dextrose and above 0.2% of yeast extract, respectively (Figure 2). The cultivation of P. parvovarius strain proceeded for 60 days to ensure sufficient production of secondary metabolites. The ABTS and DPPH assays were employed to evaluate the antioxidant activity of P. parvovarius culture filtrate, and ascorbic acid was used as a positive antioxidant.

Figure 2.

Scavenging activities of the samples on ABTS + radicals (A) and DPPH radicals (B). Control: ascorbic acid, DY: dextrose and yeast extract, MEB: malt extract broth, MY: malt extract and yeast extracts, PDB: potato dextrose broth.

Figure 2A shows the results of the ABTS assay for the culture filtrate of P. parvovarius cultivated in DY and MEB; almost 95% activity of the positive control, ascorbic acid, was achieved. Additionally, the DY sample showed almost 90% of the activity of ascorbic acid in the DPPH assay (Figure 2B). The results of the two antioxidant assays for P. parvovarius showed a significantly high value, suggesting that the optimal medium for antioxidant production is DY. To identify the major antioxidant compound, large-scale cultivation was performed using DY medium and antioxidant activity was evaluated by ABTS assay. The results were similar to those of a small-scale assay in the current study. Antioxidant substances were isolated by solvent partitioning and column chromatography using Diaion HP-20 resin and silica gel. The active fraction (compound 1) was concentrated and subjected to Sephadex LH-20 column chromatography. The yield of antioxidant compound 1 obtained by column chromatography was 9.9 mg. Compound 1 exhibited potent free radical scavenging activity with a half-maximal inhibitory concentration (IC₅₀) of 101.4 µg/mL in the ABTS radical scavenging assay. In addition, 1 exhibited free radical scavenging activity with an IC₅₀ of 73.0 µg/mL in the DPPH radical scavenging assay.

Compound 1, obtained as a colorless solid, was proved to be 3,4-dihydroxybenzaldehyde by electrospray ionization-mass spectrometry measurement, and from NMR spectroscopic data, which matched those previously published. ¹⁶ Compound 1 was identified as a protocatechuic aldehyde, a natural polyphenol compound isolated from plants and foods and associated with diverse biological effects, such as anti-inflammatory, anti-cancer, antibacterial, anti-aging, and antioxidant properties.^17–19 Three other Polyporus species, namely P. squamosus, P. tenuiculus, and P. sanguineus, have been reported to contain large amounts of polyphenols.^20–22

This study indicates that the culture filtrate of P. parvovarius has antioxidant properties, with the highest production of antioxidants observed when cultured in DY medium. In addition, the major antioxidant compound was identified to be a protocatechuic aldehyde, which have been reported from Polyporales species extracts such as Phellinus igniarius, Ganoderma applanatum, Hymenochaete xeratica and Polyporus umbellatus; such compounds are known to have antioxidant properties.^23–26 Therefore, P. parvovarius may be useful as an antioxidant supplement.

Experimental

Sample Preparation for Antioxidant Assay

Polyporus parvovarius NIBRFGC0000500557 was isolated from a specimen collected in the Nari Basin of Ulleungdo Island, Korea. A voucher specimen was deposited at the National Institute of Biological Resources. To confirm the differences resulting from the composition of the medium, medium containing dextrose and yeast extract (DY, BD Difco Laboratories, Detroit, MI, USA), malt extract broth (MEB, BD Difco), malt extract and yeast extract (MY, BD Difco), and potato dextrose broth (PDB, BD Difco) were prepared. Pre-cultivated mycelial discs (5 mm) were inoculated in each Erlenmeyer flask containing 700 mL of medium and incubated at 25 °C with shaking at 170 rpm for 60 days. To obtain the culture filtrate, the liquid cultures containing mycelia were filtered through Miracloth (Calbiochem, La Jolla, CA, USA). The separated culture filtrate was freeze-dried, and the dried powder extracted with 70% aqueous ethanol for analysis.

General Antioxidant Activity Assay

Antioxidant activity of culture filtrates was evaluated via 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical scavenging ²⁷ and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. ²⁸

ABTS Radical Scavenging Assay

The ability of the culture filtrate extract to scavenge ABTS radical cations (ABTS + •) was evaluated using an Antioxidant Assay Kit (CS0790, Sigma-Aldrich, St. Louis, USA), according to the manufacturer's instructions. The extracts were dissolved at a concentration of 10 mg/mL in 70% aqueous ethanol. The ethanol extract (10 µL) and 20 µL of myoglobin working solution were added to a 96-well plate. ABTS substrate working solution (150 µL) was added to each well. After vortex mixing, the mixture was incubated for 10 min at 25 °C, after which 100 µL of stop solution was added to each well. Absorbance was measured at 405 nm using a microplate reader (SpectraMax190, Molecular Devices, Sunnyvale, CA, USA). Each assay was performed in triplicate. Ascorbic acid was used as a standard.

Free radical scavenging activity was calculated after measuring the decrease in absorbance and using the following equation:

ABTS radical scavenging activity (%) = {(A_control - A_sample)/A_control} × 100 (%)

where A_control is the absorbance of the control reaction and A_sample is the absorbance in the presence of the test sample.

DPPH Radical Scavenging Assay

The extracts were dissolved in 70% aqueous ethanol to a concentration of 10 mg/mL, and 0.2 mM DPPH in ethanol was prepared. The ethanol extract (20 µL) and 100 µL of DPPH solution were added to each well. After vortex mixing, the mixture was incubated for 20 min at 25 °C, and absorbance was measured at 517 nm using a microplate reader (SpectraMax190). Ascorbic acid was used as a standard. Each assay was performed in triplicate. Free radical scavenging activity was calculated after measuring the decrease in absorbance and using the following equation:

DPPH radical scavenging activity (%) = {(A_control - A_sample)/A_control} × 100 (%)

where A_control is the absorbance of the control reaction and A_sample is the absorbance in the presence of the test sample.

Large-Scale Cultivation for major Antioxidant Compounds

P. parvovarius was grown on potato dextrose agar medium at 25 °C for 2 weeks. Agar plugs of P. parvovarius were then inoculated into 16 flasks containing 1 L of DY and cultured at 25 °C for 8 weeks with agitation at 170 rpm.

Isolation and Purification of Compound 1

The culture broth of P. parvovarius (16 L) was adsorbed onto a Diaion HP-20 resin and eluted stepwise with 30%, 70%, and 100% aqueous methanol. An active fraction (70% aqueous methanol) showing ABTS radical scavenging activity was partitioned between ethyl acetate and water. This fraction was concentrated under reduced pressure, separated by column chromatography using silica gel, and eluted with CHCl₃:MeOH (100:1→0:100, v/v). The active fraction was concentrated and subjected to Sephadex LH-20 column chromatography and then eluted with methanol to obtain antioxidant compound 1. The IC₅₀ value was calculated as the concentration of antioxidant needed to decrease the initial ABTS concentration by 50%.

Structure Determination of Compound 1

Compound 1 was characterized by electrospray ionization mass spectrometry, and UV, ¹H NMR, ¹³C NMR, heteronuclear multiple quantum coherence, and ¹H-¹H correlation spectroscopy.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X211072336 - Supplemental material for Major Antioxidant Compound of Polyporus Parvovarius Culture Filtrate

Supplemental material, sj-docx-1-npx-10.1177_1934578X211072336 for Major Antioxidant Compound of Polyporus Parvovarius Culture Filtrate by Minkyeong Kim, Chorong Ahn and Changmu Kim in Natural Product Communications