Effects of Heat Treatment on Total Phenolic Contents, Antioxidant and Anti-Inflammatory Activities of Pleurotus Sajor-Caju Extract

Abstract

Untreated and heat treated Pleurotus sajor-caju methanolic extracts were examined for total phenolic content, antioxidant and anti-inflammatory activities. Total phenolic content was determined by using the Folin-Ciocalteu method, and results were expressed as mg gallic acid equivalent per g of sample extract. Antioxidant activity was measured by using the DPPH free radical scavenging method while anti-inflammatory activity was measured by using the Griess assay. Methanolic extracts of fresh fruiting bodies were heated at 100 and 121°C for 15 and 30 min. The results showed that total phenolic contents of heat-treated samples did not increase significantly when compared to the control sample. Total phenolic contents were within the range of 0.06 ± 0.05 to 0.08 ± 0.05 mg gallic acid/equivalents g of dry weight. The samples heated at 100°C for 15 and 30 min showed the highest radical scavenging activity at 500 μg/ml with the lowest IC₅₀ values of 10.4 ± 3.11 and 11.67 ± 2.75 μg/ml, respectively. The results suggested that the increased antioxidant activity of P. sajor-caju after the heat treatment might have increased the health beneficial effects to humans.

Keywords:

• Pleurotus sajor-caju
• Heat treatment
• Total phenolic content
• Antioxidant activity
• Anti-inflammatory activity

INTRODUCTION

Mushrooms have been widely used since ancient times as a functional food as well as for therapeutic values.[1,2] Many types of mushrooms have been studied and some of the examples of therapeutic properties include anticarcinogenic, anti-inflammatory, and antimicrobial activities.^[3] Pleurotus sajor-cajuan edible mushroom is also known as oyster mushroom with good flavour and taste. The aqueous extract of Pleurotus sajor-caju contains vitamin B₁, B₂, and C.[4]

Phenolics are one of the major groups of non-essential dietary components. The ability of these compounds to act as antioxidants has been well established.[5–7] The bioactivity of phenolics may be related to their ability to chelate metals, inhibit lipoxygenase, and scavenge free radicals. Mushrooms contain various polyphenolic compounds with excellent antioxidant activity due to their ability to scavenge free radicals by single electron transfer.[8] A significant correlation between the total phenol of commercial fruit juices and their total antioxidant capacities was reported by Herken and Guzel.[9] Heat treated shiitake mushroom increased the total content of free polyphenolic and flavonoid compounds.[10] Jeong et al.[11] reported that heat treatment may liberate low molecular weight of phenolic compounds that increase the antioxidant activity of citrus peels.

Several types of inflammatory tissue injury are mediated by reactive oxygen metabolites. The most likely sources of these oxidizing agents are the phagocytic leukocytes that invade the tissue. The nitric oxide (NO) is a free radical and function as a mediator of inflammation or tissue destruction in inflammatory and arthritic disorders, and the excessive production of NO may lead to chronic inflammation. The reaction between NO and reactive oxygen species may lead to formation of reactive nitrogen species, such as peroxynitrite. In this study, the effects of heat treatment on the total phenolic contents (TPC), antioxidant and anti-inflammatory activities of Pleurotus sajor-caju mushroom extract was investigated.

MATERIALS AND METHODS

Heat Treatment of Mushroom

Fresh Pleurotu sajor-caju fruiting bodies were purchased from the mushroom farm at the University Agricultural Park, Universiti Putra Malaysia. Approximately 500 g of raw Pleurotus sajor-caju were sliced into small pieces and divided into five groups of 100 g for each group. Four groups were treated at 100 and 121°C for 15 and 30 min and one group was unheated as control.

Extraction

A sample of 20 g from each group of mushroom was extracted by shaking with 200 ml of 95% methanol at 150 rpm overnight using a shaker incubator. The mixture was filtered through Whatman No. 4 filter paper and the filtrate obtained was concentrated in a rotary evaporator at 40°C to dryness and stored at 4°C for further use.

Total Phenolic Contents (TPC)

TPC in the mushroom extracts were determined by using the Folin-Ciocalteu method.[12] A volume of 0.1 ml of sample (1 mg/ml) was mixed with 0.5 ml of Folin-Ciocalteu reagent. After 3 min, 0.4 ml of 7.5% (w/v) sodium bicarbonate was added and incubated at room temperature in the dark for 90 min. The absorbance was measured at 725 nm and the results were expressed in mg of gallic acid (GA) equivalents per g of dry weight sample. A calibration curve was constructed with different concentrations of GA at 0.01–0.1 mg/ml.

DPPH Radical Scavenging Activity

The scavenging activity of diphenylpicryl-hydrazyl (DPPH) radical was measured according to the method of Mau et al.[13] A sample extract of 1 mg/ml in methanol was prepared. The solutions were two-fold diluted at seven concentrations (7.81 to 500 μg/ml in methanol) in a 96-well microtiter plate. A volume of 5 μl of DPPH reagent (2.5 mg/ml in methanol) was added into each well. The plate was incubated at room temperature in the dark for 30 min and the absorbance was measured at 517 nm. Vitamin C and quercetin were used as positive control in this assay. The percentage of scavenging activity (%) was calculated by comparing the absorbance value of the control.

Anti-Inflammatory Assay

The murine monocytic macrophages cell line (RAW 264.7) purchased from ATCC (Manassas, VA, USA) was cultured and maintained in plastic culture flask in Dulbecco's Modified Eagle Medium (DMEM) with phenol red supplemented with 10% foetal calf serum (FCS), 4.5 g/L glucose, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 μg/ml streptomycin and 50 U/ml penicillin. Cells were grown in 37°C humidified incubator supplemented with 5% CO₂ and subcultured twice a week.

The RAW 264.7 cell culture at 95% confluency was scraped out and centrifuged at 110 × g at 4°C for 10 min. The supernatant was discarded and the pellet was resuspended with fresh DMEM without phenol supplemented with 10% FCS. Cell concentrations were then adjusted to 1 × 10⁶ cells/ml using a haemacytometer by adding 1:1 ratio of cells with trypan blue (0.031 M in phosphate buffer saline [PBS], pH 7.2). Then, 50 μl of cell suspension was dispensed into each well of a 96-well microtiter plate except for blank and incubated in an incubator at 37°C, 5% CO₂ for 3 h to attach the cell. After 3 h, the unattached cells were discarded and the attached cells were induced with 200 U/ml of recombinant mouse IFN-γ (BD Pharmingen, San Diego, CA, USA) plus 10 μg/ml of E. coli lipopolyssaccharide (LPS) (055:B5) in the presence or absence of sample extract (100 mg/ml in dimethyl sulfoxide [DMSO]). The plate was incubated for 17–20 h at 37°C, 5% CO₂ in a humidified incubator. After incubation, cell culture supernatants were collected for nitrite measurement to assess the anti-inflammatory response by the Griess assay and the cells remaining in the well were tested for cell viability by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent.

Nitrite Determination

To measure NO production, 50 μl of treated cell culture supernatant was assayed for stable nitrite (NO₂ ⁻) content using Griess assay according to Syahida et al.[14] with some modifications. Initially, 50 μl of cell culture supernatant from each well (including controls) were transferred into a corresponding well of a clean, non-sterile 96-well microplate. An equal volume of 50 μl of Griess reagent (1% sulfanilamide/0.1% N-(1-naphtyl)ethylene diamine dihydrochloride in 2.5% H₃PO₄) was mixed with cell culture supernatants and colour development was measured at 550 nm using a microplate reader (SpectraMax, Plus 384, Molecular Devices, Inc., Sunnyrale, CA, USA). The amount of nitrite (NO₂ ⁻) in the samples was calculated from a sodium nitrite (NaNO₂) standard curve (3.1–200 mM) prepared in deionized water. Percentage inhibition (%) was calculated by comparing with the absorbance value of IFN-γ/LPS-induced as the control.

Cell Viability

After transferring the supernatant for Griess assay, the excess supernatant was discarded. Then, 100 μl of DMEM with phenol red containing 5% FCS was added into each well followed by 20 μl of MTT at a concentration of 5 mg/ml diluted in sterile PBS, pH 7.2. The cells were incubated at 37°C, 5% CO₂ for 4 h. After incubation, the medium was discarded and the formazan salt formed at the bottom of the well was dissolved with 100 μl of DMSO. The absorbance was measured at 570 nm. Percentage of cell viability was calculated by comparing with the absorbance value of IFN-γ/LPS-induced as the control.

Statistical Analysis

All the experiments were repeated three times and the results were expressed as mean ± standard error of mean (S.E.M.). The IC₅₀ values were calculated using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Differences between groups were determined by one-way analysis of variance (ANOVA) followed by Dunnett test.

RESULTS AND DISCUSSION

Effect of Heat Treatment on TPC

Table 1 shows the TPC of P. sajor-caju extract at different heat treatments. The TPC of an unheated sample (control) expressed as mg GA equivalents per g of dry weight (DW) sample extracts was 0.063 ± 0.05 mg. Pleurotus sajor-caju extracts treated at 100°C for 15 and 30 min showed 0.080 ± 0.05 and 0.074 ± 0.03 mg GA equivalent per g of DW sample, respectively, while samples treated at 121°C for 15 min and 30 min showed 0.064 ± 0.05 mg and 0.072 ± 0.02 mg GA equivalents per g of DW sample, respectively. There was no significant difference (P > 0.05) between heated and unheated samples. Thus, the present results suggested that the level phenolic compounds were not affected by heat. Phenolics were too low for any effects compared to other mushroom or plant extracts. On the other hand, other reports have shown that the bound phenolic compounds might be more easily released from the mushroom cell wall after heat treatment.[10]

Table 1 The IC₅₀ value of DPPH radical scavenging activity, total phenolic contents, nitrite concentration, and percentage of cell viability at different heat treatments of Pleurotus sajor-caju methanolic extracts

Samples		DPPH radical scavenging activity IC50 value (μg/ml)	Total phenolic contents (mg GA equivalents/g of DW sample)	Nitrite concentration (μM)	Cell viability (%)
Pleurotus sajor-caju extracts	100°C/15 min	10.4 ± 3.11^b	0.080 ± 0.05^a	46.7 ± 16.7^b	117.0 ± 5.9^a
	100°C/30 min	11.6 ± 2.57^b	0.074 ± 0.03^a	44.8 ± 10.5^b	110.3 ± 7.7^a
	121°C/15 min	12.2 ± 3.08^b	0.064 ± 0.05^b	48.6 ± 13.8^b	114.2 ± 18.5^a
	121°C/30 min	13.7 ± 3.08^b	0.072 ± 0.02^a	39.2 ± 8.5^b	111.4 ± 10.5^a
	Unheated	12.5 ± 1.26^b	0.063 ± 0.05^b	43.4 ± 9.6^b	113.2 ± 5.3^a
Positive controls	Vitamin C	3.68 ± 0.44^a	—	—	—
	Quercetin	4.84 ± 0.67^a	—	—	—
	L-NAME	—	—	17.8 ± 2.4^a	112.9 ± 12.3^a
All values are mean ± S.E.M. at three different experiments. The means with different letters are significantly different (P < 0.001).

Effect of Heat Treatment on Antioxidant Activities

Antioxidant activity of mushroom extracts was evaluated by using DPPH assay. Table 1 shows the 50% inhibitory concentration (IC₅₀ value) of samples as compared to positive controls. It was observed that all samples tested with different heat treatments showed scavenging activity with IC₅₀ value of 10.4 ± 3.11 and 11.7 ± 2.57 μg/ml for 100°C/15 and 30 min, 12.2 ± 3.08 and 13.7 ± 3.05 for 121°C/15 and 30 min. IC₅₀ value for control was 12.5 ± 1.26.

Table 1 shows the DPPH radical scavenging activity percentage of samples and positive controls at 500 μg/ml. Among the methanolic extracts from four different heat treatments, the heat-treated sample at 100°C for 15 min showed the highest antioxidant activity (85.6 ± 3.1%) followed by 121°C for 15 min (83.2 ± 3.1%), 121°C for 30 min (82.7 ± 3.1%), and 100°C for 30 min (77.1 ± 2.6%). The DPPH radical scavenging activity of the positive controls ascorbic acid and quercetin were significantly different (P < 0.001) relative to those of the unheated sample (60.3 ± 1.2%).

The results showed an increase in the percentage of scavenging activity in the heated sample compared to the control suggesting that the heating process may release the antioxidants compounds in the mushroom. Similarly, Choi et al.[10] reported that heated Shiitake mushroom may have increased health beneficial effects associated with the increase of antioxidant activity. It was also reported that thermal processing enhances the nutritional value of tomatoes by increasing the total antioxidant activity and the content of bioaccessible lycopene in tomatoes, while at the same time it reduced the ascorbic acid, vitamin C, and folate content.[15]

Effect of Heat Treatment on Nitric Oxide (NO) Production

Anti-inflammatory activity of samples was evaluated by the ability of the mushroom extracts to inhibit NO production in RAW 264.7 cells. Table 1 shows the effect of P. sajor-caju extract on NO production in stimulated RAW 264.7 cells tested at the highest concentration of 200 μg/ml. Induced cells, treated with IFN-γ and LPS produced the highest amount of NO at 59.4 ± 0.9 μg/ml, while uninduced cells produced the least concentration of NO at 200 μg/ml. The results showed that there was no significant difference among all treatments. Thus, the present study demonstrated that there was no anti-inflammatory activity in all heated mushroom extracts. On the other hand, N^w-nitro-L-arginine methyl ester (L-NAME), a NO synthase (NOS) inhibitor was used as a positive control. L-NAME is an L-arginine analog, which can compete with L-arginine (substrate) to bind with NOS.[3] Several L-arginine analogs like N^w-methyl-L-arginine (L-NMMA)[16] and N-iminoethyl-L-ornithine (L-NIO) can also inhibit NO generation in activated murine macrophage. Results showed that L-NAME produced a significantly (P < 0.001) low amount of NO at 200 μg/ml.

Cell Viability (MTT Assay)

A cytotoxicity test was performed by using MTT assay in order to determine the toxicity of samples. Table 1 shows the percentage of cell viability on RAW 264.7 cells of the methanolic extracts of P. sajor-caju in unheated and heated sample (100 and 121°C for 15 and 30 min). The results demonstrated that all mushroom extracts were nontoxic to the RAW 264.7 cells.

CONCLUSION

The present study demonstrated that heat treatment did not change the phenolic content of P. sajor-caju mushroom. However, there was a significant increase in the antioxidant activities of heated mushroom, hence, other bioactive compounds may contribute to the antioxidant properties. There was no anti-inflammatory activity detected in unheated and heated P. sajor-caju extracts. However, MTT assay results showed that all unheated and heated mushroom extracts were nontoxic to RAW 264.7 cells.

ACKNOWLEDGMENTS

This work was supported by a grant from the Ministry of Higher Education Malaysia under the Fundamental Research Grant Scheme (FRGS) (05-10-07-366FR).