| The rich diversity of different mushroom species offers a potential source of bioactive molecules that have been utilized for curative and medicinal purposes since antiquity. Mushrooms produce many bioactive molecules such as proteins, phenolics, and various metabolites that represent potential novel sources of natural antioxidants, antiproliferants and immunomodulants. Oyster mushroom (Pleurotus spp.) is one of the major cultivated mushrooms. It has many species including Pleurotus eryngii (DC. ex Fr.) Quel, an important mushroom in terms of culinary and medicinal properties. However, there is a lack of information pertaining to the activity of P. eryngii proteins in existing literature and ethnobotanical records. The current study therefore sought to extract, purify and partially characterize protein from P. eryngii (DC. ex Fr.) Quel. The antioxidant, antitumor and immunomodulatory activities of the purified protein were also investigated. Moreover, the lethality of the protein to normal cells was also determined.Protein was isolated from the fruiting bodies of P. eryngii (DC. ex Fr.) Quel by acetic acid extraction and ammonium sulfate precipitation. To purify the isolated protein a three-step purification procedure was employed using diethylaminoethyl cellulose-52 (DEAE-52), carboxymethyl cellulose-52 (CM-52) and fast protein liquid chromatography (FPLC) using Superdex 75. The purified protein was then characterized using high performance gel permeation chromatography (HPGPC), hydrogen nuclear magnetic resonance (’H-NMR) and Fourier Transform infrared (FT-IR). On HPGPC PEP yielded a single and symmetrically sharp peak indicative of homogeneity. The developed calibration curve correlated the molecular weight with the HPGPC retention time of the standards, and was subsequently used for molecular weight determination. The HPGPC retention time for PEP was 7.49 min hence PEP had a molecular weight of about 63 kDa. The FT-IR spectra of PEP displayed the characteristic IR absorption bands of proteins at 1654 cm-1 (amide I). This band is mainly associated with C=O stretching vibration. The second protein characteristic band was observed at 1577 cm"1 (amide Ⅱ), which is mainly as a result of N-H bending. A broadly stretched intense peak at 3300 cm"1 and other peaks were also observed at 1154 and 1038 cm-1. The peak frequency of the amide I band has been shown to correspond to the secondary structure of protein where a-helices give rise to a main absorption band close to 1655 cm-1. Thus, peak 1654 cm-1 connoted a secondary (a-helix) structure of PEP. NMR spectrum showed the broad multiply signals at 3.9-4.5 ppm in the aliphatic region which corresponded to CH-O, which was mainly attributed to Ha of glycine. Moreover, the intense and sharp peak at 4.7 ppm corresponded to N-H and was mainly attributed to arginine, while the smaller peaks appearing at 4.9-5.7 ppm corresponded to O-H and were mainly attributed to serine. Based on HPGPC, FT-IR and 1H-NMR, PEP was a homogenous protein, with a molecular weight of 63 kDa, secondary a-helical structure and was mainly composed of arginine, glycine and serine.To evaluate the antioxidant activity of PEP, various assays were performed which included; total antioxidant activity, reducing power, chelating of ferrous and cupric ions, DPPH, hydroxyl and superoxide radical scavenging, β-Carotene/linoleate bleaching, and TBARS. At the highest concentration (60 μg/ml), PEP showed significant (P< 0.05) total antioxidant, reducing, cupric and ferric chelating, DPPH and superoxide radical scavenging activities of 28.34± 0.01,0.153 ± 0.004,18.89 ± 0.00,19.30 ± 0.01,27.28 ± 0.03 and 20.93 ± 0.01% respectively compared to untreated controls. Compared to the standard antioxidants, PEP had a significantly (P< 0.05) higher hydroxide ion scavenging activity (48.06 ± 0.00%) and p-carotene bleaching inhibition (55.80 ± 0.00%). In addition, PEP significantly (P< 0.05) inhibited the formation of lipid peroxidation product (MDA-TBA) compared to the untreated control. At the highest concentration (60 μg/ml) it had a lipid peroxidation inhibition activity of 43.41 ± 0.01% which was higher than the activities of standards. With regard to the findings of the present study, it is noteworthy that Pleurotus eryngii (DC. ex Fr.) Quel fruiting bodies powder protein (PEP) could serve as a mushroom component rich in antioxidant activity, which could be developed into a possible food supplement or a pharmaceutical agent.The in vitro anti-cancer activity of PEP was evaluated using the non-small cell lung cancer (NSCLC) A549, human stomach adenocarcinoma BGC-823, human liver hepatocellular carcinoma HepG2 and human gastric carcinoma HGC-27 cell lines. Cytotoxicity assays which included, MTT, alamar blue (AB), trypan blue (TB) dye exclusion, neutral red (NR) uptake, LDH release, Annexin V-FITC/propidium iodide and cellular morphology analysis were used to examine the antiproliferative activity of the cancer cells using conventional cancer drugs, paclitaxel (PTX), doxorubicin (Dox) and mitomycin C (MMC) as positive control. PEP significantly (P< 0.05) inhibited the proliferation of all cancer cells dose-dependently with an IC50 of 229.0 ± 1.2,41.2 ± 1.1 and 36.5 ± 0.8 μg/ml for A549, BGC-823 and HepG2 respectively. HGC-27 cancer cells were the least sensitive to PEP. Furthermore, presence of different organelle targets in the cancer cells was demonstrated through the assays. Our results showed that in tumor cells, lysosomes readily interacted with the PEP, hence the higher percent toxicity in NR results with up to 23.12±1.15% cell (HepG2) survival rate at 320 ug/ml. Consequently, Pleurotus eryngii (DC. ex Fr.) Quel fruiting bodies powder protein is an active antitumor agent.Macrophages, RAW 264.7 cells were used to determine the immunostimulatory activity of PEP. Cytotoxicity and immunostimulatory assays (nitric oxide (NO) and hydrogen peroxide release, lysosomal enzyme activity and pinocytosis assays) were used to determine the immunomodulatory activity of PEP. PEP significantly (P< 0.05) stimulated the proliferation, lysosomal enzyme and pinocytosis activities, nitric oxide and hydrogen peroxide release in macrophage, RAW 264.7 cells. At 160 μg/ml, PEP significantly (P<0.05) stimulated the macrophage proliferation by 12.9±1.5,17.35±2.76 and 20.83±1.84% based on MTT, AB and TB assays, respectively. This confirmed that dose-dependently, PEP could activate the macrophage-mediated immune responses. Moreover, the effect of PEP on normal cells was examined using Chang’s liver cell. Cytotoxicity assay results revealed that PEP had no significant (P<0.05) toxicity in the cells compared to the control. Therefore, PEP could be used as an active immunomodulatory agent, through its stimulation of the macrophage-mediated immune response and lack of toxicity to normal cells. |
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