Antimicrobial, antioxidant, anti-inflammatory, and cytotoxic activities of Cordyceps militaris spent substrate

Danyu Zhang; Qingjiu Tang; Xianzhe He; Yipeng Wang; Guangyong Zhu; Ling Yu

doi:10.1371/journal.pone.0291363

Abstract

Cordyceps militaris is a medicinal mushroom and has been extensively used as a traditional medicine in East Asia. After the chrysalis seeds are matured and harvested, the spent substrate of C. militaris still contains active ingredients but is usually discarded as waste. This study aimed to determine the antioxidant and anti-inflammatory activities of C. militaris spent substrate extract and its inhibitory activity on the Malassezia commensal yeasts that can cause dandruff and seborrheic dermatitis. Active substances in the spent substrate of C. militaris were extracted using a hot water extraction method and were used for the determination of antioxidant activity by measuring their ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radicals, hydrogen peroxide, and superoxide anions. The ability to inhibit Malassezia was analyzed using the broth microdilution method, and the reparative effect on oxidative damage in HaCaT cells was measured using in vitro cell analysis. Respiratory burst evaluation was used to determine the anti-inflammatory capacity of extracts. Analysis of the Malassezia-inhibiting activity of the extracts showed that the minimum inhibitory concentration was 6.25 mg/mL. The half maximal inhibitory concentration (IC50) values of DPPH, O₂^-, H₂O₂ and OH^- were 3.845 mg/mL, 2.673 mg/mL, 0.037 mg/mL and 0.046 mg/mL, respectively. In the concentration range of 2 to 50%, the extract was non-toxic to cells and was able to protect HaCaT cells from H₂O₂ damage. When the volume fraction of the extract was 20.96%, its anti-inflammatory ability reached 50%. These results demonstrated that the extract may be a safe and efficacious source for pharmaceutical or cosmetic applications, with Malassezia-inhibiting, antioxidant and anti-inflammatory activities.

Citation: Zhang D, Tang Q, He X, Wang Y, Zhu G, Yu L (2023) Antimicrobial, antioxidant, anti-inflammatory, and cytotoxic activities of Cordyceps militaris spent substrate. PLoS ONE 18(9): e0291363. https://doi.org/10.1371/journal.pone.0291363

Editor: Brahma Nand Singh, National Botanical Research Institute CSIR, INDIA

Received: May 20, 2022; Accepted: August 29, 2023; Published: September 8, 2023

Copyright: © 2023 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and Supporting Information files.

Funding: The authors received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

1. Introduction

Malassezia spp. are commensal yeasts that can cause cutaneous ailments such as dandruff and seborrheic dermatitis. Malassezia secretes lipases and hydrolases that act on human sebum and cause the release of diglycerides and unsaturated and saturated fatty acids on the scalp. Some of these unsaturated fatty acids penetrate the stratum corneum, inducing inflammation and enhancing abnormal keratinization [1]. Studies showed that certain natural materials from mushrooms [2], neem, licorice, pomegranate, and ghirtkumari [3] have antimicrobial activities and can be used as raw materials in skincare products.

The generation of free radicals or reactive oxygen species from the incomplete reduction of molecular oxygen during aerobic respiration is closely related to cellular damage. An excess of reactive oxygen species leads to severe oxidative stress, which leads to nucleic acid damage, polyunsaturated fatty acid oxidation in lipids, and amino acid oxidation in proteins [4]. The antioxidant properties of many phytochemicals (e.g., phenolic compounds) allow them to reduce reactive oxygen species production [5]. Mycelium extracts of some mushrooms reduce oxidative stress by increasing the activity of antioxidant enzymes [6]. Normal physiological processes require a balance between cellular processes that produce reactive oxygen species and antioxidant defense systems that remove them [7]. Due to their safety and widespread distribution, plant-based antioxidants have gained considerable attention in recent years. Studies have demonstrated that they are highly effective scavengers of a broad spectrum of oxidants and inhibitors of lipid peroxidation. In addition to antioxidants, antimicrobial agents have been reported in a variety of medicinal plants.

The cultivation substrate left after harvesting edible mushroom seed entities is edible mushroom bran, which consists of mycelium, straw, other crude fibers and fermentation metabolites. Edible mushroom bran contains high concentrations of carbohydrates, proteins, amino acids, vitamins, and trace elements. One kilogram of fresh mushrooms generates approximately 5 kg of spent mushroom substrate [8]. Cordyceps militaris, an entomopathogenic fungus belonging to the class Ascomycetes, is a valuable medicinal fungus and a modern rare herbal medicine in China [9]. Many bioactive molecules of nutraceutical interest, such as cordycepin(3’-deoxyadenosine), ergosterol, trehalose, mannitol, and several polysaccharides, nucleosides, and amino acids have been detected in or isolated from C. militaris, and these are thought to have potential antiaging, whitening, antitumor, anti-inflammatory, immunomodulatory, and blood glucose and cholesterol-lowering activities [10]. Of these, cordycepin is the main active constituent that is most widely studied for its medicinal value and nutraceutical potential. Cordycepin exhibits a wide range of beneficial health effects, such as antimicrobial [11], antioxidant, anti-inflammatory, immunomodulatory [12], and antitumor activities [13]. Almost all biologically active components are extracted from the fermented solution or fruit bodies, while the spent cultivation substrate used to cultivate the C. militaris fruit body is rarely used after harvest. Due to the advantage of the spent substrate in many applications, compared with the C. militaris fruit body, there is great potential for its scientific study and commercial value [14]. While the fruiting bodies and mycelium of C. militaris have been investigated, there is limited information on the biochemical properties of the spent substrate of C. militaris (bacteriostatic, antioxidant, anti-inflammatory, and cytotoxic activities).

In this study, we aimed to explore the bacteriostatic, antioxidant and anti-inflammatory effects of the spent substrate extract of C. militaris and to clarify whether cordycepin is the main Malassezia inhibitory component. Our results may provide new ideas for further investigation and development of C. militaris waste substrate and lay a solid foundation for its application in cosmetics and as therapeutic agent.

2. Materials and methods

2.1. Materials

Spent substrate was obtained from the Shanghai Academy of Agricultural Sciences (Shanghai, China). The dried spent substrate of C. militaris was ground into a fine powder using an 80-mesh and a high-speed grinder (DSY-9002, Yongkang Jiu shunying Trading Co., Ltd., China) and stored in a desiccator.

2.2. Reagents and cell lines

Methanol, cordycepin, 97% luminol, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and phorbol 12-myristate 13- acetate (PMA) were purchased from Sigma (USA). Phenol, concentrated sulfuric acid, ethanol, trifluoroacetic acid, cuprous chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate were purchased from Sinopharm Chemical Reagent Co. Ltd (CN). NKA-II macroporous adsorption resin was purchased from Shanghai Yuanye Biotechnology Co., Ltd (CN). D101 macroporous adsorption resin was purchased from Cangzhou Baoen Adsorbent Material Technology Co. Ltd (CN). 30% hydrogen peroxide was from Shanghai Taopu Chemical Company, and o-phenanthroline was from Shanghai Jingdian Chemical Technology Co. Ltd (CN). AlamarBlue reagent was purchased from Thermo Scientific (USA). All the reagents were of analytical grade.

Mouse macrophage RAW 264.7 and immortalized human keratinocytes (HaCaT cells) were provided by the Institute of Edible Mushrooms, Shanghai Academy of Agricultural Sciences. Malassezia furfur ATCC 14521 was purchased from the China Industrial Microbial Strain Collection Management Center. Dulbecco’s modified Eagle’s medium (DMEM), high glucose DMEM, heat-inactivated fetal bovine serum, glutamine, penicillin, and streptomycin were purchased from Gibco (USA).

2.3. Preparation of extracts

The spent substrate of C. militaris was incubated with distilled water (1:20, w/v) at 100°C for 2 h, and the extract was collected by filtration. The material was vacuum filtered, and the precipitate was subjected to the same process. After two extractions, the aqueous filtrates were combined. The obtained extract was stored at −20°C until use. The extractions were performed in triplicates.

Different components of the extract were separated through adsorption on macroporous resin. Briefly, 80 g each of D101 and NKA-II macroporous adsorption resin were mixed with 200 mL of C. militaris spent extract and incubated for 8 h at 26°C on a shaker with protection from light. The resin and the adsorption supernatant fraction were then collected by filtration, and the macroporous adsorption resin was eluted with 95% ethanol to obtain the eluted fraction. Adsorption supernatants and ethanol-eluted fractions from each resin were lyophilized.

To test the antimicrobial effects of C. militaris spent extract, NKA-II macroporous adsorption resin was used to gradually elute the extract using 30%, 50%, 70%, and 90% ethanol, and the prepared components were lyophilized.

2.4. Characterization of the extract

Total sugar content was determined with the phenol-sulfuric acid method using glucose as the standard [15].

The content of cordycepin in the spent substrate extract of C. militaris was determined by a Waters high-performance liquid chromatography (HPLC) system (Waters 2695) [16] using the Venusil MP C18 column (250 mm × 4.60 mm, 5 μm) at room temperature. Chromatographic parameters were as follows: mobile phase, methanol-water (20:80); flow rate, 1 mL/min; UV detection wavelength, 254 nm; and injection volume, 10 μL. The prepared cordycepin solution was weighed and passed through a microporous filter membrane (0.22 μm), and then the sample was tested. The peak area Y was linearly regressed with the mass concentration X of cordycepin (mg/mL).

2.5. Antimicrobial evaluation

The antimicrobial evaluation was performed according to the American Clinical and Laboratory Standards Institute (CLSI) judgment criteria [17]. Briefly, 100 μL of Malassezia suspension (3 × 10⁸ CFU/ mL), 100 μL of the sample, and 10 μL of olive oil were added to each well of a 96-well plate. For the negative control group, 100 μL of sterile water was added instead of the sample. Ketoconazole (0.16 ug/mL) was used as a positive control for the in vitro inhibition test, and testing was performed in triplicate. The samples were incubated at 32°C for 48 h. Because Malassezia can only grow when floating on the surface of an oil layer, the naked eye observation method was used. The concentration of the C. militaris solid fermentation extract in the sample with complete fungal inhibition was used for the minimum bactericidal concentration (MBC) value, and the concentration of the extract in the sample with 50% fungal growth inhibition was taken as the minimum inhibitory concentration (MIC) value.

2.6. Antioxidant evaluation

The in vitro antioxidant potential of the spent substrate extract of C. militaris was evaluated by assessing its free radical quenching ability using DPPH, hydroxyl radical, hydrogen peroxide, and superoxide anion scavenging assays.

2.6.1. DPPH radical scavenging assay.

The DPPH radical scavenging activities of the spent substrate extract of C. militaris were measured as reported by Blois [18, 19], with some modifications. A volume of 0.2 mL of sample solution (sample group) or 80% ethanol (blank group) was mixed with 2.8 mL of a 0.1 mmol/L DPPH solution and incubated for 30 min in the dark at 25°C. The optical density (OD) value at 517 nm was measured using a microplate reader (Synergy HT, Bio-TEK, USA). Three replicate wells were used for each group and the average value was calculated. The formula for the DPPH clearance rate was as follows: (1) where A1 is the absorbance value of the sample solution at 517 nm, and A0 is the absorbance value of the blank group at 517 nm.

2.6.2. Superoxide anion scavenging assay.

The superoxide anion scavenging activity of the spent substrate extract of C. militaris was assayed using an o-phenothrin-luminol system [20, 21]. A carbonate buffer solution (pH 10) at a concentration of 50 mmol/L and a luminol solution of 1 mmol/L were mixed well in a 2:1 ratio (working solution). In a 96-well plate, 150 μL of the working solution was then mixed with 10 μL of different concentrations of the spent substrate extract of C. militaris, and with 10 μL of a 0.625 mmol/L catechol solution. Chemiluminescence was evaluated with a luminometer (Clarity-2PC, Bio-TEK, USA). The reaction time was set for 1 s, and the data were recorded continuously for 30 s. The highest value A1 was obtained, and the peak control value was recorded as A0 with deionized water as the blank control. Three replicate wells were used in each group and the average value was calculated. The formula for the superoxide anion clearance rate was: (2)

2.6.3. Hydroxyl radical scavenging assay.

The hydroxyl radical scavenging activity was analyzed using a chemiluminescence method [22, 23]. A carbonate buffer solution (pH 8.5) with a concentration of 50 mmol/L and a luminol solution of 1 mmol/L was mixed at a ratio of 17:1 (working solution) away from light. For the assay, 10 μL of different concentrations of the spent substrate extract of C. militaris, 10 μL of a 6% H₂O₂ solution, 10 μL of a 1 mmol/L CuCl solution, 10 μL of a 1 mmol/L o-phenanthroline solution, and 150 μL of the working solution were added to a 96-well plate. The reaction time was set for 2 s. Chemiluminescence was measured and recorded continuously for 15 s to obtain the highest value, A1, with deionized water as blank control. The peak control value was measured and recorded as A0. Three replicate wells were used in each group, and the average value was calculated. The formula for hydroxyl radical clearance was: (3)

2.6.4. Hydrogen peroxide scavenging assay.

The H₂O₂-scavenging activities were analyzed using a chemiluminescence method [24]. A carbonate buffer solution (pH 9.5) with a concentration of 50 mmol/L and a luminol solution of 1 mmol/L were mixed at a ratio of 17:1 away from light (working solution). For the measurement, 10 μL of different concentrations of the spent substrate extract of C. militaris, 10 μL of a 6% H₂O₂ solution, and 150 μL of the luminol-carbonate system luminescence working solution were added to the a 96-well plate, and the chemiluminescence was measured. The reaction time was set to 30 s, and the experimental data were measured every 0.3 s to obtain the highest value, A1, with deionized water as a blank control. The peak control value was recorded as A0.

Three replicate wells were used for in each group and the average value was obtained. The formula for hydrogen peroxide anion clearance was: (4)

2.7. In vitro cellular analysis of the spent substrate extract of C. militaris

In vitro cell analysis of the spent substrate extract of C. militaris was performed to determine its protective and reparative effects on H₂O₂-damaged cultured human keratinocyte cells (HaCaT cells).

2.7.1. Effects of the spent substrate extract of C. militaris on the activity of H₂O₂-damaged HaCaT cells.

HaCaT cells were cultured in 5% CO₂ at 37°C in regular DMEM supplemented with 10% heat-inactivated fetal bovine serum, glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 μg/mL). Cells were grown to 80% confluence, trypsinized, and centrifuged at 1000×g for 3 min (centrifuge 5415D, Eppendorf). The pellets were diluted to 1 × 10⁵/mL with a growth medium and transferred into 96-well cell culture plates at a concentration of 1 × 10⁴ cells/well, and 100 μL of culture medium with different concentrations of C. militaris spent extract were added. PBS was used as control. Cells were cultured in a CO₂ incubator for 24 h. After 24 h, cell viability was assessed using AlamarBlue reagent. The cells were placed in the incubator for 6 h, and the resulting fluorescence was read on a plate reader (Synergy HT, Bio-TEK, USA) to detect cell viability and calculate cell proliferation rate. The calculation formula was (5)

2.7.2. Establishment of an H₂O₂ oxidative stress model.

Cells were cultured in a 96-well cell culture plate. The culture medium was replaced with 100 μL of medium containing six concentration gradients of H₂O₂ (range of 0.1–0.6 mmol/L) and cells were incubated for 24 h in a CO₂ incubator. PBS was used as a control. Cells were stained with AlamarBlue reagent and incubated for 6 h in the CO₂ incubator, at which time cell activity and proliferation rate were calculated. An H₂O₂ concentration leading to cell damage of 50% was selected as an indicator.

2.7.3. Protective effect of the spent substrate extract of C. militaris on H₂O₂-damaged HaCaT cells.

The spent substrate extract of C. militaris was diluted with sterile PBS to 50%, 15%, 8%, 4%, and 2% v/v. HaCaT cells were cultured overnight in a 96-well cell culture plate and incubated with the medium containing different volume fractions of the extract for 24 h. Cells were then incubated with a fresh culture medium containing the appropriate concentration of H₂O₂ for 24 h. PBS was used as control.

2.8. Determination of the anti-inflammatory capacity

RAW 264.7 cells were cultured in a 5% CO₂ incubator at 37°C in high glucose DMEM supplemented with 10% heat-inactivated fetal bovine serum, glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 mg/mL). A respiratory burst assay [23] was used to test the anti-inflammatory capacity of chrysalis solid fermentation extracts by incubating 180 μL of 5 × 10⁵ RAW 264.7 cells/mL, 50 μL of the luminol working solution, 2.5 μL (2 × 10⁻² mg/mL) of PMA, and 10 μL of chrysalis solid fermentation extracts. The reaction was performed for 40 min, and data were measured every 1 min to obtain the peak A1. PBS was substituted for the sample to obtain the peak A0. Three replicate wells were used for each group and the average value was obtained. The formula for hydrogen peroxide anion clearance was: (6)

2.9. Statistical analysis

Excel 2007 software was used to process the data, and statistical analysis was performed using SPSS 22 software. Triplicate studies were performed, and the results were expressed as the mean ± standard deviation. P < 0.05 was considered statistically significant.

3. Results and discussion

3.1. Active matter content of the extract

The total sugar content of C. militaris spent extract was determined using the phenol-sulfuric acid method. The total sugar regression equation was as follows:: Y = 6.3413X + 0.0401; R² = 0.9998. (p = 0.010 < 0.05) (Fig 1).

Download:

Fig 1. Standard curve for total sugar content determined using the phenol-sulfuric acid method.

https://doi.org/10.1371/journal.pone.0291363.g001

The cordycepin content was determined using HPLC and showed a good correlation with the peak area in the range of approximately 0.04–0.2 mg/mL. The regression equation was as follows: Y = 3 × 10⁷X + 86,514; R² = 0.9999 (p = 0.035 < 0.05) (Fig 2).

Download:

Fig 2. Cordycepin mass concentration and HPLC peak area standard curve.

https://doi.org/10.1371/journal.pone.0291363.g002

The extraction rate of cordycepin was 0.846 ± 0.0058%, and the extraction rate of the total sugar was 5.829 ± 0.087%.

3.2. Analysis of the antimicrobial ability and main active ingredients of the extract

3.2.1. Inhibitory effect of different fractions on Malassezia.

The ability of the C. militaris spent substrate extract to inhibit Malassezia was determined using the broth microdilution method. The results showed that the raw extract had an MIC value of 6.25 mg/mL and an MBC value of 12.5 mg/mL at the concentration of the raw drug.

As summarized in Table 1, the adsorption separation of the inhibitory components was greater when NKA-II macroporous adsorption resin was used, and the inhibition ability of the NKA-II 95% ethanol-eluted fraction was the highest. The inhibition ability of the NKA-II adsorption supernatant was relatively poor, indicating that the main inhibitory components in the extract of the spent substrate of C. militaris were effectively adsorbed and enriched by this resin. The adsorption effect of the D101 macroporous resin was relatively weak, and the adsorption effect of the supernatant was greater than that of the 95% ethanol eluted-fraction, suggesting that the adsorption effect of the D101 macroporous resin on the effective antimicrobial components was not as high as that of the NKA-II macroporous resin. NKA-II, therefore, was used for all the subsequent experiments. As shown in Table 2, 30% ethanol was used to elute most of the effective inhibitory components adsorbed by NKA-II resin. The MIC of this fraction was 0.75 mg/mL, the MBC was 0.75 mg/mL, and the inhibition of Malassezia was slightly less effective than that of the 95% ethanol elution fraction. The abilities of 50%, 70%, and 90% ethanol elution fractions to inhibit Malassezia were relatively similar, and therefore, it was presumed that the difference in the content of the effective inhibitory components was not significant.

Download:

Table 1. MIC and MBC results of samples separated using different macroporous adsorption resins against Malassezia.

https://doi.org/10.1371/journal.pone.0291363.t001

Download:

Table 2. MIC and MBC results of different concentrations of ethanol stepwise elution samples against Malassezia.

https://doi.org/10.1371/journal.pone.0291363.t002

3.2.2. Determination of nucleoside components in the 95% elution fraction of NKA-II.

The adsorption characteristics of NKA-II resin suggested that the inhibition component of C. militaris extract was a small molecule. Previous studies showed that the nucleosides in C. militaris have a broad-spectrum inhibitory effect on a variety of bacteria and fungi [11]. Therefore, the 95% eluted fraction of NKA-II with the highest inhibitory effect was then analyzed using 13 nucleoside standards with a standard method to determine the content of nucleosides in the fraction. The results are shown in Fig 3.

Download:

Fig 3. Comparison of nucleoside components in the 95% elution fraction of NKA-II with using 13 nucleoside standards.

(A) HPLC chromatogram of the 13 nucleoside standards (1: cytosine, 2: uracil, 3: cytidine, 4: hypoxanthine, 5: uridine, 6: thymine, 7: adenine, 8: inosine, 9: guanosine, 10: thymidine, 11: adenosine, 12: cordycepin, and 13: N-hydroxyethyl adenosine). (B) HPLC chromatogram of the 95% eluted fraction of NKA-II.

https://doi.org/10.1371/journal.pone.0291363.g003

The comparison with the mixed standards showed that the fraction contained the highest content of cordycepin, small amounts of guanosine, uridine, adenosine, N-hydroxyethyl adenosine, and adenine. Yuan et al. [25] used gradient reversed-phase HPLC for the determination of adenosine, cordycepin, cytidine, guanosine, thymidine, uridine, inosine, adenine, cytosine, thymine, uracil, and hypoxanthine in Cordyceps militaris. It was found that the main component of the nucleosides was cordycepin and that they also contained a small amount of uridine, adenosine and guanosine. Therefore, it was inferred that cordycepin was one of the main antimicrobial components in this fraction. To verify the correlation between cordycepin and the inhibition of Malassezia, the HPLC determination of the cordycepin content was performed for each sample after separation through by adsorption on NKA-II resin. The linear regression equation was similar to the results of the cordycepin standard: Y = 3 × 10⁷X+80,123; R² = 0.997 (p = 0.035) (Fig 4).

Download:

Fig 4. Cordycepin mass concentration and HPLC peak area standard curve.

https://doi.org/10.1371/journal.pone.0291363.g004

3.2.3. Separation of cordycepin content of each fraction through adsorption on NKA-II resin.

The cordycepin content of different fractions is shown in Table 3 and Fig 5. The 95% ethanol-eluted fraction with the highest inhibitory effect also had the highest cordycepin content (89.9%), followed by the 30% ethanol-eluted fraction with the second highest inhibitory effect and relatively lower cordycepin content (70.33%). Fractions eluted with 50%, 70%, and 90% ethanol had similarly low inhibitory effects, and similarly low cordycepin content (Table 3).

Download:

Fig 5. HPLC cordycepin profile of the components adsorbed and separated using NKA-II.

https://doi.org/10.1371/journal.pone.0291363.g005

Download:

Table 3. Separation of the cordycepin content of each fraction through adsorption on NKA-II resin.

https://doi.org/10.1371/journal.pone.0291363.t003

Because the inhibitory properties of the extracts positively correlated with the content of cordycepin, the inhibitory effect of the cordycepin standard on Malassezia was measured and showed an MBC value of 0.125 mg/mL (Table 2), suggesting a significant inhibitory effect. In addition, it was found that the adsorption supernatant fraction with the lowest content of cordycepin was more effective than the stepwise elution fraction, indicating that the spent substrate extract of C. militaris had multiple active components working together to inhibit the proliferation of Malassezia, and cordycepin was one of the main inhibitory components.

Numerous studies demonstrated that many edible mushroom extracts are nutritionally safe and easily degradable sources of antimicrobial agents against human pathogens [26]. For example, the Pleurotus florida mycelial extract showed very effective activity against gram-positive, and gram-negative bacteria and yeast [27]. The methanolic extract of C. militaris showed strong antibacterial and antifungal properties [28]. In literature, cordycepin appeared as a key component exhibiting antibacterial activity, especially against Escherichia coli, Staphylococcus aureus [29], and Bacillus subtilis [30]. The highly pure cordycepin obtained using prep-HPLC demonstrated inhibitory activity against NAD+-dependent DNA ligase (LigA) from various bacteria in vitro. The cordycepin antibiotic could be potentially useful as a broad-spectrum antibiotic targeting LigA in a variety of bacterial species [11]. Some studies related anti-Malassezia activity from the extract of natural plants. Han et al. [31] found that chestnut shell and oil-soluble Glycyrrhiza extracts exhibited particularly high anti-Malassezia activity with MIC values ≤ 0.5 mg/mL, indicating that their extracts could be used as active ingredients in anti-seborrheic dermatitis and anti-dandruff shampoos. Onlom et al. [32] studied the antimicrobial activity of Asparagus root extracts against Malassezia furfur. A Saponin extract had an MIC of 0.20 mg/mL, while an ethanol extract had an MIC of 25 mg/mL, and both extracts did not interact negatively with the antifungal drugs ketoconazole and zinc azole. Malassezia spp. are commensal yeasts that can cause cutaneous ailments such as dandruff and seborrheic dermatitis. Thus, the spent substrate extract of C. militaris could be a safe and effective herbal treatment for various cutaneous fungal infections, including dandruff.

3.3. Antioxidant evaluation

It was determined that the spent substrate extract of C. militaris effectively scavenged a variety of free radicals (Figs 6–9). A direct correlation between the concentration and DPPH, superoxide anion, hydroxyl radical, and hydrogen peroxide scavenging abilities was observed for the spent substrate extract of C. militaris (p = = 0.0002, 0.003, 0.0004, and 0.0006, respectively; p < 0.05). The IC50 is a half-scavenging concentration that indicates a concentration of a sample that results in a free radical scavenging rate of 50%. IC50 values of the spent substrate extract of C. militaris for DPPH, superoxide anion radicals, hydroxyl radicals, and hydrogen peroxide were 15.38%, 10.7%, 0.18% and 0.15% (w/v), respectively; converted into mg/mL, they were 3.85, 2.68, 0.05, and 0.04 mg/mL, respectively. The extract showed good scavenging abilities for all four types of free radicals, especially for the hydroxyl radical and hydrogen peroxide.

Download:

Fig 6. Scavenging effect of the spent substrate extract of C. militaris on DPPH.

Results are expressed as the mean ± SD (n = 3).

https://doi.org/10.1371/journal.pone.0291363.g006

Download:

Fig 7. Scavenging effect of the spent substrate extract of C. militaris on superoxide anions.

Results are expressed as the mean ± SD (n = 3).

https://doi.org/10.1371/journal.pone.0291363.g007

Download:

Fig 8. Scavenging effect of the spent substrate extract of C. militaris on hydroxyl radical.

Results are expressed as the mean ± SD (n = 3).

https://doi.org/10.1371/journal.pone.0291363.g008

Download:

Fig 9. Scavenging effect of the spent substrate extract of C. militaris on hydrogen peroxide.

Results are expressed as the mean ± SD (n = 3).

https://doi.org/10.1371/journal.pone.0291363.g009

Studies have shown that free radicals damage biomolecules, causing the progression of aging, cancer, inflammation, diabetes, metabolic disorders, atherosclerosis, and cardiovascular diseases [30]. Numerous studies have demonstrated the antioxidant potential of C. militaris. The ethanol extract of C. militaris has shown antioxidant properties against DPPH, superoxide radicals, hydroxyl radicals, and low-density lipoproteins in vitro [33]. Zhang et al. [34] showed that polysaccharide-iron (II) extracted from Cordyceps militaris had significant radical scavenging activity on DPPH, hydroxyl, and superoxide species. The clearance rate of the polysaccharide-iron (II) of DPPH reached 74.02% at 2.5 mg/mL, but it had no significant effect on the clearance rate of hydroxyl and superoxide species. Compared with the polysaccharide-iron (II), our extract showed a similar scavenging ability for DPPH but stronger scavenging abilities for hydroxyl free radicals and hydrogen peroxide. Chen et al. [35] demonstrated that the polysaccharides extracted from Cordyceps militaris (W-CBP50) were capable of scavenging radicals such as hydroxyl, superoxide, and DPPH. The IC50 values were 1.485, 0.144 and 0.042 mg/mL. Thanh et al. [36] optimized the extraction conditions of cordycepin from C. militaris using ultrasonic-assisted enzyme extraction (UAEE) and obtained extracts with IC50 values of 0.260 ± 0.004 mg/mL for DPPH and 2.63 ± 0.22 mg/mL for H₂O₂. Our extract showed a greater ability to scavenge hydroxyl free radicals and hydrogen peroxide than W-CBP50 and UAEE, with IC50 values of 0.05 mg/mL and 0.04 mg/mL, respectively.

3.4. Effect of the spent substrate extract of C. militaris on HaCaT cells

3.4.1. Effects of the spent substrate extract of C. militaris on the activity of H₂O₂-damaged HaCaT cells.

The procedure for the evaluation of cytotoxicity initially determines the potential of already cultured cells to multiply in the presence of a test compound. As shown in Table 4, spent substrate extracts of C. militaris in the 50–60% concentration range were associated with a certain level of toxicity to HaCaT cells. Therefore, a non-toxic concentration range of approximately 2–50% was selected to determine the efficiency of the spent substrate extract in preventing H₂O₂ damage to HaCaT cells. There was a direct correlation between the extract concentration and the rate of cell proliferation (p = 0.033 < 0.05).

Download:

Table 4. Cytotoxicity of the spent substrate extract of C. militaris.

https://doi.org/10.1371/journal.pone.0291363.t004

3.4.2. Establishment of an H₂O₂ oxidative stress model.

The effect of different concentrations of H₂O₂ on the cell proliferation rate is shown in Table 5 and Fig 10. A cell proliferation rate of 50% was next selected as a damage model, and 0.2 mmol/L H₂O₂ was used to induce peroxidative damage to the cells. There was a direct correlation between H₂O₂ concentration and the rate of cell proliferation (p = 0.031 < 0.05).

Download:

Fig 10. The damaging effect of H₂O₂ on cells.

Results are expressed as the mean ± SD (n = 3).

https://doi.org/10.1371/journal.pone.0291363.g010

Download:

Table 5. The damaging effect of H₂O₂ on cells.

https://doi.org/10.1371/journal.pone.0291363.t005

3.4.3. Protective effect of the spent substrate extract of C. militaris on H₂O₂-damaged HaCaT cells. In the range of sample concentrations that were found safe in testing, the protective ability in terms of growth and proliferation of H₂O₂-damaged cells increased with increasing sample concentrations. As shown in Table 6 and Fig 11, a 50% volume fraction of extract was associated with a cell proliferation rate of 89.79 ± 0.45%.

Download:

Fig 11. Protective effect of the spent substrate extract of C. militaris on H₂O₂ injured cells.

Results are expressed as the mean ± SD (n = 3). *: Compared with the control group, the p-value of the extract of different concentrations were less than 0.05. (*p < 0.05; **p < 0.01).

https://doi.org/10.1371/journal.pone.0291363.g011

Download:

Table 6. Protective effect of the spent substrate extract of C. militaris on H₂O₂ injured cells.

https://doi.org/10.1371/journal.pone.0291363.t006

H₂O₂ is extensively used as an indicator of oxidative stress-induced cell injury in several in vitro models. It was previously demonstrated that a polysaccharide derived from C. militaris protected the human hepatic cell line HL-7702 from hydrogen peroxide-stimulated apoptosis. The tested polysaccharide inhibited cell apoptosis induced by H₂O₂, which may have correlated with scavenging free radicals [37]. He et al. [38] investigated the anti-inflammatory properties of an ethanol extract of C. militaris based on its suppression of H₂O₂-related cell injury caused by Reactive Oxygen Species (ROS) production and by downregulating mitogen-activated protein kinases in C6 glial cells.

3.5. Anti-inflammatory capacity assay

In the presence of PMA, RAW 264.7 macrophages produce large quantities of reactive oxygen species and inflammatory factors, and the resulting respiratory burst response can be measured using the luminol-dependent chemiluminescence assay. The anti-inflammatory activity of the solid fermentation extracts is shown in Table 7 and Fig 12. The blank group without PMA stimulant showed that different concentrations of the spent substrate extract of C. militaris had no effect on the autoluminescence of RAW 264.7 macrophages. In the presence of PMA, a gradual increase in the spent substrate extract concentration (10% to 90% by volume) correlated with a similar increase in the inhibition of a respiratory burst. SPSS calculations showed that the volume fraction of 40% of the extract was associated with an 85.6% respiratory burst inhibition, and a volume fraction of 20.96% resulted in 50% inhibition. A direct correlation between the concentration and the rate of respiratory burst inhibition was observed for the spent substrate extract of C. militaris (p = 0.005 < 0.05).

Download:

Fig 12. Anti-inflammatory activity of the spent substrate extract of C. militaris.

https://doi.org/10.1371/journal.pone.0291363.g012

Download:

Table 7. Anti-inflammatory activity of the spent substrate extract of C. militaris.

https://doi.org/10.1371/journal.pone.0291363.t007

Inflammation is a key function in biological processes and is initiated by various stimuli and noxious factors such as irradiation by ultraviolet light, irritants, infections, and cell injury. Currently, many researchers are interested in natural sources such as mushrooms, which contain medicinally important biofunctional components that may be able to reduce the severity of inflammatory ailments by regulating oxidative stress within physiological ranges and regulating pro-inflammatory cytokines [39]. There is both in vitro and in vivo evidence that C. militaris and its active constituents prevent inflammation in several experimental models. Cordycerebroside A, soyacerebroside I, and glucocerebroside have been isolated from C. militaris as cerebrosides (glycosphingolipids). These compounds inhibited the accumulation of pro-inflammatory iNOS protein and reduced the expression of COX-2 protein in Lipopolysaccharide-stimulated RAW 264.7 macrophages [40]. Recent studies showed that cordycepin, one of the prominent components of C. militaris, inhibited inflammation-associated expression of the genes encoding COX-2 and iNOS [41]. Lei et al. [42] demonstrated that cordycepin suppressed LPS-induced malondialdehyde (MDA) content and inflammatory cytokine (IL-1β and TNF-α) production. Maya et al. [43] used membrane stabilization of human red blood cells (HRBCs) and were able to show and to quantify anti-inflammatory activity in plant extracts. Their experiment revealed that at a dose of 500 μg/mL, the anti-inflammatory activity of a hot water extract of Wattakaka volubilis leaves was 54.27%, as indicated by its ability to protect HRBC membranes from hyposaline-induced lysis. C. militaris and its bioactive components are being investigated for biomedical applications due to their minimal side effects, high nutritional content, and suppression of inflammation.

4. Conclusions

The present study highlighted that the spent substrate leftovers from the cultivation of C. militaris can potentially be used as a new material source for the preparation of crude extracts for use in pharmaceutical or cosmetic applications. The results obtained from this study revealed that the spent substrate extract of C. militaris has a certain inhibitory effect on Malassezia and that cordycepin was the main antimicrobial active component in the extract.The results of this study also indicated that the extract has strong antioxidant and certain anti-inflammatory activities, can reduce oxidative damage and inhibit the subsequent inflammatory response to a certain extent, and is non-toxic to cells in the range of 2–50%. These results suggested that the extract could be potentially used to develop safe and effective functional medicines and cosmetics for cutaneous fungal infections. Our data strengthens a need for further research into the spent substrate of C. militaris for its therapeutic and repair uses.

Supporting information

S1 Raw data. Cordycepin content.

https://doi.org/10.1371/journal.pone.0291363.s001

(XLSX)

S2 Raw data. DPPH, O2−, -OH, and H₂O₂.

https://doi.org/10.1371/journal.pone.0291363.s002

(XLSX)

S3 Raw data. Cytotoxic activities.

https://doi.org/10.1371/journal.pone.0291363.s003

(XLSX)

S4 Raw data. Anti-inflammatory activities.

https://doi.org/10.1371/journal.pone.0291363.s004

(XLSX)

Acknowledgments

We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

References

1. Dawson TL. Malassezia globosa and restricta: Breakthrough Understanding of the Etiology and Treatment of Dandruff and Seborrheic Dermatitis through Whole-Genome Analysis—ScienceDirect. J Invest Derm Symp P. 2007;12(2):15–9. https://doi.org/10.1038/sj.jidsymp.5650049
- View Article
- Google Scholar
2. Taofiq O, Heleno SA, Calhelha RC, Alves MJ, Barros L, Barreiro MF, et al. Development of Mushroom-Based Cosmeceutical Formulations with Anti-Inflammatory, Anti-Tyrosinase, Antioxidant, and Antibacterial Properties. Molecules. 2016;21(10). pmid:27754433
- View Article
- PubMed/NCBI
- Google Scholar
3. Mishra RC, Kumari R, Yadav JP. Comparative antidandruff efficacy of plant extracts prepared from conventional and supercritical fluid extraction method and chemical profiling using GCMS. J Dermatol Treat. 2022;33(2):989–95. pmid:32691649
- View Article
- PubMed/NCBI
- Google Scholar
4. Zhou J, Yang Q, Zhu X, Lin T, Xu J. Antioxidant activities of Clerodendrum cyrtophyllum Turcz leaf extracts and their major components. PloS one.15(6):e0234435. https://doi.org/10.1371/journal.pone.0234435.
- View Article
- Google Scholar
5. Tian Y, Liimatainen J, Alanne AL, Lindstedt A, Liu PZ, Sinkkonen J, et al. Phenolic compounds extracted by acidic aqueous ethanol from berries and leaves of different berry plants. Food Chem. 2017;220:266–81. pmid:27855899
- View Article
- PubMed/NCBI
- Google Scholar
6. Balaji P, Madhanraj R, Rameshkumar K, Veeramanikandan V, Eyini M, Arun A, et al. Evaluation of antidiabetic activity of Pleurotus pulmonarius against streptozotocin-nicotinamide induced diabetic wistar albino rats. Saudi J Biol Sci. 2020;27(3):913–24. pmid:32127771
- View Article
- PubMed/NCBI
- Google Scholar
7. Vikram P, Chiruvella KK, Ripain IH, Arifullah M. A recent review on phytochemical constituents and medicinal properties of kesum (Polygonum minus Huds.). Asian Pac J Trop Biomed. 2014;4(6):430–5. pmid:25182942
- View Article
- PubMed/NCBI
- Google Scholar
8. Williams BC, McMullan JT, McCahey S. An initial assessment of spent mushroom compost as a potential energy feedstock. Bioresource Technol. 2001;79(3):227–30. pmid:11499576
- View Article
- PubMed/NCBI
- Google Scholar
9. Zhang JX, Wen CT, Duan YQ, Zhang HH, Ma HL. Advance in Cordyceps militaris (Linn) Link polysaccharides: Isolation, structure, and bioactivities: A review. Int J Biol Macromol. 2019;132:906–14. pmid:30954592
- View Article
- PubMed/NCBI
- Google Scholar
10. Yu SH, Chen SYT, Li WS, Dubey NK, Chen WH, Chuu JJ, et al. Hypoglycemic Activity through a Novel Combination of Fruiting Body and Mycelia of Cordyceps militaris in High-Fat Diet-Induced Type 2 Diabetes Mellitus Mice. J Diabetes Res. 2015;2015. https://doi.org/Artn 723190.10.1155/2015/723190.
- View Article
- Google Scholar
11. Zhou XF, Cai GQ, He Y, Tong GT. Separation of cordycepin from Cordyceps militaris fermentation supernatant using preparative HPLC and evaluation of its antibacterial activity as an NAD(+)-dependent DNA ligase inhibitor. Exp Ther Med. 2016;12(3):1812–6. pmid:27588098
- View Article
- PubMed/NCBI
- Google Scholar
12. Tuli HS, Sharma AK, Sandhu SS, Kashyap D. Cordycepin: A bioactive metabolite with therapeutic potential. Life Sci. 2013;93(23):863–9. pmid:24121015
- View Article
- PubMed/NCBI
- Google Scholar
13. Wada T, Sumardika IW, Saito S, Ruma IMW, Kondo E, Shibukawa M, et al. Identification of a novel component leading to anti-tumor activity besides the major ingredient cordycepin in Cordyceps militaris extract. J Chromatogr B. 2017;1061:209–19. pmid:28750234
- View Article
- PubMed/NCBI
- Google Scholar
14. Shi H, Zhang M, Bhandari B, Wang YC, Yi SF. Effects of superfine grinding on the properties and qualities of Cordyceps militaris and its spent substrate. J Food Process Pres. 2019;43(11). https://doi.org/10.1111/jfpp.14169.
- View Article
- Google Scholar
15. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric Method for Determination of Sugars and Related Substances. Analytical Chemistry. 1956;28(3):350–6. https://doi.org/10.1021/AC60111A017.
- View Article
- Google Scholar
16. Fothergill AW. Antifungal Susceptibility Testing: Clinical Laboratory and Standards Institute (CLSI) Methods: Interactions of Yeasts, Moulds, and Antifungal Agents; 2012. https://doi.org/10.1007/978-1-59745-134-5_2
- View Article
- Google Scholar
17. Leong LP, Shui G. An investigation of antioxidant capacity of fruits in Singapore markets. Food Chem. 2002;76(1):69–75. https://doi.org/ 10.1016/S0308-8146(01)00251-5.
- View Article
- Google Scholar
18. Blois MS. Antioxidant Determinations by the Use of a Stable Free Radical. Nature. 1958;181(4617):1199–200. https://doi.org/10.1038/1811199a0.
- View Article
- Google Scholar
19. Ardestani A, Yazdanparast R. Antioxidant and free radical scavenging potential of Achillea santolina extracts. Food Chem. 2007;104(1):21–9. https://doi.org/10.1016/j.foodchem.2006.10.066.
- View Article
- Google Scholar
20. Du XJ, Wang XD, Chen Y, Tian SY, Lu SF. Antioxidant Activity and Oxidative Injury Rehabilitation of Chemically Modified Polysaccharide (TAPA1) from Tremella aurantialba. Macromol Res. 2018;26(6):479–83. https://doi.org/10.1007/s13233-018-6078-0.
- View Article
- Google Scholar
21. Du XJ, Mu HM, Zhou S, Zhang Y, Zhu XL. Chemical analysis and antioxidant activity of polysaccharides extracted from Inonotus obliquus sclerotia. Int J Biol Macromol. 2013;62:691–6. pmid:24145301
- View Article
- PubMed/NCBI
- Google Scholar
22. Fu HF, Xie B, Gang F, Ma SJ, Zhu XR, Pan SY. Effect of esterification with fatty acid of β-cryptoxanthin on its thermal stability and antioxidant activity by chemiluminescence method. Food Chemistry. 2010;122(3):602–9. https://doi.org/10.1016/j.foodchem.2010.03.019.
- View Article
- Google Scholar
23. Qi HM, Zhang QB, Zhao TT, Chen R, Zhang H, Niu XZ, et al. Antioxidant activity of different sulfate content derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta) in vitro. International Journal of Biological Macromolecules. 2005;37(4):195–9. pmid:16310843
- View Article
- PubMed/NCBI
- Google Scholar
24. Guo SS, Deng QC, Xiao JS, Xie BJ, Sun ZD. Evaluation of antioxidant activity and preventing DNA damage effect of pomegranate extracts by chemiluminescence method. J Agr Food Chem. 2007;55(8):3134–40. pmid:17381116
- View Article
- PubMed/NCBI
- Google Scholar
25. Yuan JP, Zhao SY, Wang JH, Kuang HC, Liu X. Distribution of nucleosides and nucleobases in edible fungi. J Agric Food Chem. 2008;56(3):809–15. pmid:18183952
- View Article
- PubMed/NCBI
- Google Scholar
26. Mostafa AA, Al-Askar AA, Almaary KS, Dawoud TM, Sholkamy EN, Bakri MM. Antimicrobial activity of some plant extracts against bacterial strains causing food poisoning diseases. Saudi J Biol Sci. 2018;25(2):361–6. pmid:29472791
- View Article
- PubMed/NCBI
- Google Scholar
27. Illuri R, Eyini M, Kumar M, Babu RS, Prema P, Nguyen V, et al. Bio-prospective potential of Pleurotus djamor and Pleurotus florida mycelial extracts towards Gram positive and Gram negative microbial pathogens causing infectious disease. J Infect Public Heal. 2022;15(2):297–306. pmid:34690095
- View Article
- PubMed/NCBI
- Google Scholar
28. Reis FS, Barros L, Calhelha RC, Ciric A, Van Griensven LJLD, Sokovic M, et al. The methanolic extract of Cordyceps militaris (L.) Link fruiting body shows antioxidant, antibacterial, antifungal and antihuman tumor cell lines properties. Food Chem Toxicol. 2013;62:91–8. pmid:23994083
- View Article
- PubMed/NCBI
- Google Scholar
29. Joshi M, Sagar A, Kanwar SS, Singh S. Anticancer, antibacterial and antioxidant activities of Cordyceps militaris. Indian J Exp Biol. 2019;57(1):15–20.
- View Article
- Google Scholar
30. Quy TN, Xuan TD. Xanthine Oxidase Inhibitory Potential, Antioxidant and Antibacterial Activities of Cordyceps militaris (L.) Link Fruiting Body. Medicines (Basel). 2019;6(1). pmid:30699961
- View Article
- PubMed/NCBI
- Google Scholar
31. Han SH, Hur MS, Kim MJ, Jung WH, Park M, Kim JH, et al. In Vitro Anti-Malassezia Activity of Castanea crenata Shell and Oil-Soluble Glycyrrhiza Extracts. Ann Dermatol. 2017;29(3):321–6. pmid:28566909
- View Article
- PubMed/NCBI
- Google Scholar
32. Onlom C, Khanthawong S, Waranuch N, Ingkaninan K. In vitro anti-Malassezia activity and potential use in anti-dandruff formulation of Asparagus racemosus. Int J Cosmetic Sci. 2014;36(1):74–8. pmid:24117781
- View Article
- PubMed/NCBI
- Google Scholar
33. Wu HC, Chen ST, Chang JC, Hsu TY, Cheng CC, Chang HS, et al. Radical Scavenging and Antiproliferative Effects of Cordycepin-Rich Ethanol Extract from Brown Rice-Cultivated Cordyceps militaris (Ascomycetes Mycelium on Breast Cancer Cell Lines. International Journal of Medicinal Mushrooms. 2019;21(7):657–69. pmid:31679300
- View Article
- PubMed/NCBI
- Google Scholar
34. Zhang X, Zhang X, Gu S, Pan L, Sun H, Gong E, et al. Structure analysis and antioxidant activity of polysaccharide-iron (III) from Cordyceps militaris mycelia. Int J Biol Macromol. 2021;178:170–9. pmid:33639188
- View Article
- PubMed/NCBI
- Google Scholar
35. Chen X, Wu G, Huang Z. Structural analysis and antioxidant activities of polysaccharides from cultured Cordyceps militaris. Int J Biol Macromol. 2013;58:18–22. pmid:23537797
- View Article
- PubMed/NCBI
- Google Scholar
36. Thanh VH, My PLT, Dat TD, Viet ND, Phuc NCM, Thao NH, et al. Integrating of Aqueous Enzymolysis with Ethanolic Ultrasonication to Ameliorate Cordycepin Content from Vietnamese Cordyceps militaris and Biological Analysis of Extracts. Chemistryselect. 2021;6(1):16–31. https://doi.org/10.1002/slct.202003995.
- View Article
- Google Scholar
37. Liu Y, E Q , Zuo J, Tao Y, Liu W. Protective effect of Cordyceps polysaccharide on hydrogen peroxide-induced mitochondrial dysfunction in HL-7702 cells. Mol Med Rep. 2013;7(3):747–54. https://doi.org/10.3892/mmr.2012.1248.
- View Article
- Google Scholar
38. He MT, Lee AY, Park CH, Cho EJ. Protective effect of Cordyceps militaris against hydrogen peroxide-induced oxidative stress in vitro. Nutr Res Pract. 2019;13(4):279–85. pmid:31388403
- View Article
- PubMed/NCBI
- Google Scholar
39. Phull AR, Ahmed M, Park HJ. Cordyceps militaris as a Bio Functional Food Source: Pharmacological Potential, Anti-Inflammatory Actions and Related Molecular Mechanisms. Microorganisms. 2022;10(2). pmid:35208860
- View Article
- PubMed/NCBI
- Google Scholar
40. Chiu CP, Liu SC, Tang CH, Chan Y, El-Shazly M, Lee CL, et al. Anti-inflammatory Cerebrosides from Cultivated Cordyceps militaris. J Agric Food Chem. 2016;64(7):1540–8. pmid:26853111
- View Article
- PubMed/NCBI
- Google Scholar
41. Cheng YH, Hsieh YC, Yu YH. Effect of Cordyceps militaris Hot Water Extract on Immunomodulation-associated Gene Expression in Broilers, Gallus gallus. J Poult Sci. 2019;56(2):128–39. pmid:32055207
- View Article
- PubMed/NCBI
- Google Scholar
42. Lei J, Wei Y, Song P, Li Y, Zhang T, Feng Q, et al. Cordycepin inhibits LPS-induced acute lung injury by inhibiting inflammation and oxidative stress. Eur J Pharmacol. 2018;818:110–4. pmid:29054740
- View Article
- PubMed/NCBI
- Google Scholar
43. Maya MR, Rameshkumar K, Veeramanikandan V, Boobalan T, Kumar M, Eyini M, et al. Evaluation of antioxidant, anti-inflammatory, and anti-hyperglycemic effects of Wattakaka volubilis Linn. f. Process Biochem. 2022;112:183–91. https://doi.org/10.1016/j.procbio.2021.12.001.
- View Article
- Google Scholar

[ref1] 1. Dawson TL. Malassezia globosa and restricta: Breakthrough Understanding of the Etiology and Treatment of Dandruff and Seborrheic Dermatitis through Whole-Genome Analysis—ScienceDirect. J Invest Derm Symp P. 2007;12(2):15–9. https://doi.org/10.1038/sj.jidsymp.5650049
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Taofiq O, Heleno SA, Calhelha RC, Alves MJ, Barros L, Barreiro MF, et al. Development of Mushroom-Based Cosmeceutical Formulations with Anti-Inflammatory, Anti-Tyrosinase, Antioxidant, and Antibacterial Properties. Molecules. 2016;21(10). pmid:27754433
View Article
PubMed/NCBI
Google Scholar

[5] View Article

[6] PubMed/NCBI

[7] Google Scholar

[ref3] 3. Mishra RC, Kumari R, Yadav JP. Comparative antidandruff efficacy of plant extracts prepared from conventional and supercritical fluid extraction method and chemical profiling using GCMS. J Dermatol Treat. 2022;33(2):989–95. pmid:32691649
View Article
PubMed/NCBI
Google Scholar

[9] View Article

[10] PubMed/NCBI

[11] Google Scholar

[ref4] 4. Zhou J, Yang Q, Zhu X, Lin T, Xu J. Antioxidant activities of Clerodendrum cyrtophyllum Turcz leaf extracts and their major components. PloS one.15(6):e0234435. https://doi.org/10.1371/journal.pone.0234435.
View Article
Google Scholar

[13] View Article

[14] Google Scholar

[ref5] 5. Tian Y, Liimatainen J, Alanne AL, Lindstedt A, Liu PZ, Sinkkonen J, et al. Phenolic compounds extracted by acidic aqueous ethanol from berries and leaves of different berry plants. Food Chem. 2017;220:266–81. pmid:27855899
View Article
PubMed/NCBI
Google Scholar

[16] View Article

[17] PubMed/NCBI

[18] Google Scholar

[ref6] 6. Balaji P, Madhanraj R, Rameshkumar K, Veeramanikandan V, Eyini M, Arun A, et al. Evaluation of antidiabetic activity of Pleurotus pulmonarius against streptozotocin-nicotinamide induced diabetic wistar albino rats. Saudi J Biol Sci. 2020;27(3):913–24. pmid:32127771
View Article
PubMed/NCBI
Google Scholar

[20] View Article

[21] PubMed/NCBI

[22] Google Scholar

[ref7] 7. Vikram P, Chiruvella KK, Ripain IH, Arifullah M. A recent review on phytochemical constituents and medicinal properties of kesum (Polygonum minus Huds.). Asian Pac J Trop Biomed. 2014;4(6):430–5. pmid:25182942
View Article
PubMed/NCBI
Google Scholar

[24] View Article

[25] PubMed/NCBI

[26] Google Scholar

[ref8] 8. Williams BC, McMullan JT, McCahey S. An initial assessment of spent mushroom compost as a potential energy feedstock. Bioresource Technol. 2001;79(3):227–30. pmid:11499576
View Article
PubMed/NCBI
Google Scholar

[28] View Article

[29] PubMed/NCBI

[30] Google Scholar

[ref9] 9. Zhang JX, Wen CT, Duan YQ, Zhang HH, Ma HL. Advance in Cordyceps militaris (Linn) Link polysaccharides: Isolation, structure, and bioactivities: A review. Int J Biol Macromol. 2019;132:906–14. pmid:30954592
View Article
PubMed/NCBI
Google Scholar

[32] View Article

[33] PubMed/NCBI

[34] Google Scholar

[ref10] 10. Yu SH, Chen SYT, Li WS, Dubey NK, Chen WH, Chuu JJ, et al. Hypoglycemic Activity through a Novel Combination of Fruiting Body and Mycelia of Cordyceps militaris in High-Fat Diet-Induced Type 2 Diabetes Mellitus Mice. J Diabetes Res. 2015;2015. https://doi.org/Artn 723190.10.1155/2015/723190.
View Article
Google Scholar

[36] View Article

[37] Google Scholar

[ref11] 11. Zhou XF, Cai GQ, He Y, Tong GT. Separation of cordycepin from Cordyceps militaris fermentation supernatant using preparative HPLC and evaluation of its antibacterial activity as an NAD(+)-dependent DNA ligase inhibitor. Exp Ther Med. 2016;12(3):1812–6. pmid:27588098
View Article
PubMed/NCBI
Google Scholar

[39] View Article

[40] PubMed/NCBI

[41] Google Scholar

[ref12] 12. Tuli HS, Sharma AK, Sandhu SS, Kashyap D. Cordycepin: A bioactive metabolite with therapeutic potential. Life Sci. 2013;93(23):863–9. pmid:24121015
View Article
PubMed/NCBI
Google Scholar

[43] View Article

[44] PubMed/NCBI

[45] Google Scholar

[ref13] 13. Wada T, Sumardika IW, Saito S, Ruma IMW, Kondo E, Shibukawa M, et al. Identification of a novel component leading to anti-tumor activity besides the major ingredient cordycepin in Cordyceps militaris extract. J Chromatogr B. 2017;1061:209–19. pmid:28750234
View Article
PubMed/NCBI
Google Scholar

[47] View Article

[48] PubMed/NCBI

[49] Google Scholar

[ref14] 14. Shi H, Zhang M, Bhandari B, Wang YC, Yi SF. Effects of superfine grinding on the properties and qualities of Cordyceps militaris and its spent substrate. J Food Process Pres. 2019;43(11). https://doi.org/10.1111/jfpp.14169.
View Article
Google Scholar

[51] View Article

[52] Google Scholar

[ref15] 15. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric Method for Determination of Sugars and Related Substances. Analytical Chemistry. 1956;28(3):350–6. https://doi.org/10.1021/AC60111A017.
View Article
Google Scholar

[54] View Article

[55] Google Scholar

[ref16] 16. Fothergill AW. Antifungal Susceptibility Testing: Clinical Laboratory and Standards Institute (CLSI) Methods: Interactions of Yeasts, Moulds, and Antifungal Agents; 2012. https://doi.org/10.1007/978-1-59745-134-5_2
View Article
Google Scholar

[57] View Article

[58] Google Scholar

[ref17] 17. Leong LP, Shui G. An investigation of antioxidant capacity of fruits in Singapore markets. Food Chem. 2002;76(1):69–75. https://doi.org/ 10.1016/S0308-8146(01)00251-5.
View Article
Google Scholar

[60] View Article

[61] Google Scholar

[ref18] 18. Blois MS. Antioxidant Determinations by the Use of a Stable Free Radical. Nature. 1958;181(4617):1199–200. https://doi.org/10.1038/1811199a0.
View Article
Google Scholar

[63] View Article

[64] Google Scholar

[ref19] 19. Ardestani A, Yazdanparast R. Antioxidant and free radical scavenging potential of Achillea santolina extracts. Food Chem. 2007;104(1):21–9. https://doi.org/10.1016/j.foodchem.2006.10.066.
View Article
Google Scholar

[66] View Article

[67] Google Scholar

[ref20] 20. Du XJ, Wang XD, Chen Y, Tian SY, Lu SF. Antioxidant Activity and Oxidative Injury Rehabilitation of Chemically Modified Polysaccharide (TAPA1) from Tremella aurantialba. Macromol Res. 2018;26(6):479–83. https://doi.org/10.1007/s13233-018-6078-0.
View Article
Google Scholar

[69] View Article

[70] Google Scholar

[ref21] 21. Du XJ, Mu HM, Zhou S, Zhang Y, Zhu XL. Chemical analysis and antioxidant activity of polysaccharides extracted from Inonotus obliquus sclerotia. Int J Biol Macromol. 2013;62:691–6. pmid:24145301
View Article
PubMed/NCBI
Google Scholar

[72] View Article

[73] PubMed/NCBI

[74] Google Scholar

[ref22] 22. Fu HF, Xie B, Gang F, Ma SJ, Zhu XR, Pan SY. Effect of esterification with fatty acid of β-cryptoxanthin on its thermal stability and antioxidant activity by chemiluminescence method. Food Chemistry. 2010;122(3):602–9. https://doi.org/10.1016/j.foodchem.2010.03.019.
View Article
Google Scholar

[76] View Article

[77] Google Scholar

[ref23] 23. Qi HM, Zhang QB, Zhao TT, Chen R, Zhang H, Niu XZ, et al. Antioxidant activity of different sulfate content derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta) in vitro. International Journal of Biological Macromolecules. 2005;37(4):195–9. pmid:16310843
View Article
PubMed/NCBI
Google Scholar

[79] View Article

[80] PubMed/NCBI

[81] Google Scholar

[ref24] 24. Guo SS, Deng QC, Xiao JS, Xie BJ, Sun ZD. Evaluation of antioxidant activity and preventing DNA damage effect of pomegranate extracts by chemiluminescence method. J Agr Food Chem. 2007;55(8):3134–40. pmid:17381116
View Article
PubMed/NCBI
Google Scholar

[83] View Article

[84] PubMed/NCBI

[85] Google Scholar

[ref25] 25. Yuan JP, Zhao SY, Wang JH, Kuang HC, Liu X. Distribution of nucleosides and nucleobases in edible fungi. J Agric Food Chem. 2008;56(3):809–15. pmid:18183952
View Article
PubMed/NCBI
Google Scholar

[87] View Article

[88] PubMed/NCBI

[89] Google Scholar

[ref26] 26. Mostafa AA, Al-Askar AA, Almaary KS, Dawoud TM, Sholkamy EN, Bakri MM. Antimicrobial activity of some plant extracts against bacterial strains causing food poisoning diseases. Saudi J Biol Sci. 2018;25(2):361–6. pmid:29472791
View Article
PubMed/NCBI
Google Scholar

[91] View Article

[92] PubMed/NCBI

[93] Google Scholar

[ref27] 27. Illuri R, Eyini M, Kumar M, Babu RS, Prema P, Nguyen V, et al. Bio-prospective potential of Pleurotus djamor and Pleurotus florida mycelial extracts towards Gram positive and Gram negative microbial pathogens causing infectious disease. J Infect Public Heal. 2022;15(2):297–306. pmid:34690095
View Article
PubMed/NCBI
Google Scholar

[95] View Article

[96] PubMed/NCBI

[97] Google Scholar

[ref28] 28. Reis FS, Barros L, Calhelha RC, Ciric A, Van Griensven LJLD, Sokovic M, et al. The methanolic extract of Cordyceps militaris (L.) Link fruiting body shows antioxidant, antibacterial, antifungal and antihuman tumor cell lines properties. Food Chem Toxicol. 2013;62:91–8. pmid:23994083
View Article
PubMed/NCBI
Google Scholar

[99] View Article

[100] PubMed/NCBI

[101] Google Scholar

[ref29] 29. Joshi M, Sagar A, Kanwar SS, Singh S. Anticancer, antibacterial and antioxidant activities of Cordyceps militaris. Indian J Exp Biol. 2019;57(1):15–20.
View Article
Google Scholar

[103] View Article

[104] Google Scholar

[ref30] 30. Quy TN, Xuan TD. Xanthine Oxidase Inhibitory Potential, Antioxidant and Antibacterial Activities of Cordyceps militaris (L.) Link Fruiting Body. Medicines (Basel). 2019;6(1). pmid:30699961
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref31] 31. Han SH, Hur MS, Kim MJ, Jung WH, Park M, Kim JH, et al. In Vitro Anti-Malassezia Activity of Castanea crenata Shell and Oil-Soluble Glycyrrhiza Extracts. Ann Dermatol. 2017;29(3):321–6. pmid:28566909
View Article
PubMed/NCBI
Google Scholar

[110] View Article

[111] PubMed/NCBI

[112] Google Scholar

[ref32] 32. Onlom C, Khanthawong S, Waranuch N, Ingkaninan K. In vitro anti-Malassezia activity and potential use in anti-dandruff formulation of Asparagus racemosus. Int J Cosmetic Sci. 2014;36(1):74–8. pmid:24117781
View Article
PubMed/NCBI
Google Scholar

[114] View Article

[115] PubMed/NCBI

[116] Google Scholar

[ref33] 33. Wu HC, Chen ST, Chang JC, Hsu TY, Cheng CC, Chang HS, et al. Radical Scavenging and Antiproliferative Effects of Cordycepin-Rich Ethanol Extract from Brown Rice-Cultivated Cordyceps militaris (Ascomycetes Mycelium on Breast Cancer Cell Lines. International Journal of Medicinal Mushrooms. 2019;21(7):657–69. pmid:31679300
View Article
PubMed/NCBI
Google Scholar

[118] View Article

[119] PubMed/NCBI

[120] Google Scholar

[ref34] 34. Zhang X, Zhang X, Gu S, Pan L, Sun H, Gong E, et al. Structure analysis and antioxidant activity of polysaccharide-iron (III) from Cordyceps militaris mycelia. Int J Biol Macromol. 2021;178:170–9. pmid:33639188
View Article
PubMed/NCBI
Google Scholar

[122] View Article

[123] PubMed/NCBI

[124] Google Scholar

[ref35] 35. Chen X, Wu G, Huang Z. Structural analysis and antioxidant activities of polysaccharides from cultured Cordyceps militaris. Int J Biol Macromol. 2013;58:18–22. pmid:23537797
View Article
PubMed/NCBI
Google Scholar

[126] View Article

[127] PubMed/NCBI

[128] Google Scholar

[ref36] 36. Thanh VH, My PLT, Dat TD, Viet ND, Phuc NCM, Thao NH, et al. Integrating of Aqueous Enzymolysis with Ethanolic Ultrasonication to Ameliorate Cordycepin Content from Vietnamese Cordyceps militaris and Biological Analysis of Extracts. Chemistryselect. 2021;6(1):16–31. https://doi.org/10.1002/slct.202003995.
View Article
Google Scholar

[130] View Article

[131] Google Scholar

[ref37] 37. Liu Y, E Q , Zuo J, Tao Y, Liu W. Protective effect of Cordyceps polysaccharide on hydrogen peroxide-induced mitochondrial dysfunction in HL-7702 cells. Mol Med Rep. 2013;7(3):747–54. https://doi.org/10.3892/mmr.2012.1248.
View Article
Google Scholar

[133] View Article

[134] Google Scholar

[ref38] 38. He MT, Lee AY, Park CH, Cho EJ. Protective effect of Cordyceps militaris against hydrogen peroxide-induced oxidative stress in vitro. Nutr Res Pract. 2019;13(4):279–85. pmid:31388403
View Article
PubMed/NCBI
Google Scholar

[136] View Article

[137] PubMed/NCBI

[138] Google Scholar

[ref39] 39. Phull AR, Ahmed M, Park HJ. Cordyceps militaris as a Bio Functional Food Source: Pharmacological Potential, Anti-Inflammatory Actions and Related Molecular Mechanisms. Microorganisms. 2022;10(2). pmid:35208860
View Article
PubMed/NCBI
Google Scholar

[140] View Article

[141] PubMed/NCBI

[142] Google Scholar

[ref40] 40. Chiu CP, Liu SC, Tang CH, Chan Y, El-Shazly M, Lee CL, et al. Anti-inflammatory Cerebrosides from Cultivated Cordyceps militaris. J Agric Food Chem. 2016;64(7):1540–8. pmid:26853111
View Article
PubMed/NCBI
Google Scholar

[144] View Article

[145] PubMed/NCBI

[146] Google Scholar

[ref41] 41. Cheng YH, Hsieh YC, Yu YH. Effect of Cordyceps militaris Hot Water Extract on Immunomodulation-associated Gene Expression in Broilers, Gallus gallus. J Poult Sci. 2019;56(2):128–39. pmid:32055207
View Article
PubMed/NCBI
Google Scholar

[148] View Article

[149] PubMed/NCBI

[150] Google Scholar

[ref42] 42. Lei J, Wei Y, Song P, Li Y, Zhang T, Feng Q, et al. Cordycepin inhibits LPS-induced acute lung injury by inhibiting inflammation and oxidative stress. Eur J Pharmacol. 2018;818:110–4. pmid:29054740
View Article
PubMed/NCBI
Google Scholar

[152] View Article

[153] PubMed/NCBI

[154] Google Scholar

[ref43] 43. Maya MR, Rameshkumar K, Veeramanikandan V, Boobalan T, Kumar M, Eyini M, et al. Evaluation of antioxidant, anti-inflammatory, and anti-hyperglycemic effects of Wattakaka volubilis Linn. f. Process Biochem. 2022;112:183–91. https://doi.org/10.1016/j.procbio.2021.12.001.
View Article
Google Scholar

[156] View Article

[157] Google Scholar

Figures

Abstract

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Reagents and cell lines

2.3. Preparation of extracts

2.4. Characterization of the extract

2.5. Antimicrobial evaluation

2.6. Antioxidant evaluation

2.6.1. DPPH radical scavenging assay.

2.6.2. Superoxide anion scavenging assay.

2.6.3. Hydroxyl radical scavenging assay.

2.6.4. Hydrogen peroxide scavenging assay.

2.7. In vitro cellular analysis of the spent substrate extract of C. militaris

2.7.1. Effects of the spent substrate extract of C. militaris on the activity of H2O2-damaged HaCaT cells.

2.7.2. Establishment of an H2O2 oxidative stress model.

2.7.3. Protective effect of the spent substrate extract of C. militaris on H2O2-damaged HaCaT cells.

2.8. Determination of the anti-inflammatory capacity

2.9. Statistical analysis

3. Results and discussion

3.1. Active matter content of the extract

3.2. Analysis of the antimicrobial ability and main active ingredients of the extract

3.2.1. Inhibitory effect of different fractions on Malassezia.

3.2.2. Determination of nucleoside components in the 95% elution fraction of NKA-II.

3.2.3. Separation of cordycepin content of each fraction through adsorption on NKA-II resin.

3.3. Antioxidant evaluation

3.4. Effect of the spent substrate extract of C. militaris on HaCaT cells

3.4.1. Effects of the spent substrate extract of C. militaris on the activity of H2O2-damaged HaCaT cells.

3.4.2. Establishment of an H2O2 oxidative stress model.

3.5. Anti-inflammatory capacity assay

4. Conclusions

Supporting information

S1 Raw data. Cordycepin content.

S2 Raw data. DPPH, O2−, -OH, and H2O2.

S3 Raw data. Cytotoxic activities.

S4 Raw data. Anti-inflammatory activities.

Acknowledgments

References

2.7.1. Effects of the spent substrate extract of C. militaris on the activity of H₂O₂-damaged HaCaT cells.

2.7.2. Establishment of an H₂O₂ oxidative stress model.

2.7.3. Protective effect of the spent substrate extract of C. militaris on H₂O₂-damaged HaCaT cells.

3.4.1. Effects of the spent substrate extract of C. militaris on the activity of H₂O₂-damaged HaCaT cells.

3.4.2. Establishment of an H₂O₂ oxidative stress model.

S2 Raw data. DPPH, O2−, -OH, and H₂O₂.