There is a vast amount of nutritional studies of wild and cultivated mushrooms across the world. However, relatively little data exist in the literature on the nutrient content of Pleurotus giganteus. Herein, it was intended to compare only the highly appreciated and most cultivated culinary-medicinal mushrooms, for example the Pleurotus genus and Agaricus genus. Generally, mushrooms have high (19 – 35%) protein contents . In Brazil, it was reported that the fruiting bodies of Pleurotus ostreatus and Pleurotus sajor-caju presented protein content ranging from 13.1% to 18.4%, depending on the substrates used . The present study showed that the protein level of P. giganteus is 5.3-time lower than that of Agaricus bisporus (white button mushroom) with reference to a study from Portugal . On the other hand, the carbohydrate content in P. giganteus is 4-, 6-, 7.2-, 7.5-, 8-, 11-time higher than that of Lentinula edodes, shiitake (17.12 g/100 g), Flammulina velutipes, golden needle mushroom (10.57 g/100 g), Pleurotus ostreatus, oyster mushroom (9.30 g/100 g), Pleurotus eryngii, king oyster mushroom (8.95 g/100 g), Agaricus bisporus white button mushroom (8.25 g/100 g) (30), and Agaricus bisporus brown mushroom (5.98 g/100 g) . This suggested that carbohydrates (glucose, mannitol, trehalose, oligosaccharide groups, and reserved polysaccharide like glycogen) account for the prevailing component of P. giganteus fruiting body. Reports related to the nutritional evaluation of Pleurotus genus carried out by other researchers from different regions (Japan, India, Bangladesh, Turkey, Finland, and Italy) can be retrieved from [32–37], respectively. Nevertheless, the differences between the nutrient values may be attributed to the type of mushroom, strain of mushroom, environmental factors, and composition of growth media .
MTT assay is by far the most convenient colorimetric assay based on the metabolic activity of a viable cell [24, 38]. Basically, only viable cell has the mitochondrial dehydrogenase system that can cleave the yellow MTT tetrazolium salt and yield MTT formazan which is blue in colour. Thus, the optical density of the amount of solubilised MTT formazan is quantitatively correlated to the percentage of cell viability. The present study showed that cytotoxic effect of P. giganteus aqueous and ethanolic extracts towards PC12 cells were concentration dependant. This is consistent with the finding by Cheung et al.  whereby viability of PC12 cells was dose-dependently decreased by increasing Ganoderma lucidum extracts.
On-going studies show that the aqueous extract of P. giganteus contains bioactive secondary metabolites like sterols and triterpenes (unpublished data). These compounds are reported to have neutrophic NGF-like properties and caused neurite outgrowth activity in PC12 cells . We have shown for the first time that P. giganteus extract can stimulate neurite outgrowth by using PC12 cell line model. It was shown that 25 μg/ml of aqueous extract and 15 μg/ml of ethanolic extract induced the highest percentage of neurite outgrowth in PC12 cells at day 3. The number of neurite bearing cells was significantly higher than that of NGF. The mushroom extracts may contain bioactive compounds either mimic NGF or trigger the production of NGF, hence resulting in neurite outgrowth. Further, the potassium level in P. giganteus was 1345.7 mg/100 g and according to Kalac , potassium level in fruiting bodies is between 20- and 40-fold higher than in the substrates used for mushroom cultivation. In the study by Cohen-Cory et al. , the cell number of Purkinje cells, the major efferent neurons of the brain cerebellum increased by 40% when treated with potassium. Besides, potassium alone or potassium coupled with NGF markedly increased the cell survival, cell differentiation and neurite outgrowth. In this study, the potassium present in P. giganteus extracts may be involved in the regulation of the morphological differentiation of PC12 cells by acting as a depolarising agent.
The present study extends recent findings that some mushroom extracts can have neuritogenesis effects. Prior studies by our group have shown that 0.2% (v/v) aqueous extract of freeze dried fruiting bodies from Hericium erinaceus caused maximal stimulation of neurite outgrowth (17.3% of neurite bearing cells and 88.2% increase compared to control) in NG108-15 cell line after 24 hours of incubation . Besides, freeze drying was found to be the best approach to preserve the bioactive compounds in mushroom as compared to oven-dried method . It had been reported that PC12 cells responded well to water extract of sclerotium of Lignosus rhinocerus (Cooke) Ryvarden . It was found that synergistic effect, i.e. 42.12% of neurite bearing PC12 cells was elicited when the cells were treated with 20 μg/ml of water extract combined with 30 ng/ml of NGF. Some other medicinal mushrooms that induced neurite outgrowth included Grifola frondosa (Maitake) , Tricholoma sp , Termitomyces albuminosus[45, 46], Dictyophora indusiata, Tremella fuciformis, and Ganoderma lucidium (Lingzhi) .
The involvement of the MAPK/ERKs signaling pathway in neuronal differentiation by mushroom extracts has been reported. Neuroprotective and neuritogenesis effect of Ganoderma lucidium extracts on PC12 was stipulated to be mediated via the MAPK/ERK signalling pathway . Besides, lysophosphatidylethanolamine from Grifola frondosa induced activation of ERK1/2 of PC12 cells thus stimulated neurite outgrowth and inhibited serum withdrawal-induced apoptosis . Neurotrophins like NGF are mostly mediated by the Trk family of receptor tyrosine kinase, TrKA. However, discrepancy did occur in the case of Ganoderma lucidium extracts, whereby there was no direct involvement of TrkA . Similarly, α-Phenyl-N-tert-butylnitron was also found to induce neurite outgrowth in PC12 independent of TrkA . It is thus predicted, based on the ability of P. giganteus extract to stimulate neurite outgrowth of PC12 without NGF, that activation of TrKA receptor tyrosine kinase may not be necessary. According to Sweatt , the mitogen-activated protein kinase (MAP kinase, MAPK) cascade is a superfamily of signalling cascade and is a vital regulator of cell division and differentiation. Recently, MAPK was specified as the extracellular signal-regulated kinase (ERK) comprising ERK 1 and 2, or as ERK1/2. It has been demonstrated that ERK-cascade was necessary and sufficient enough for NGF-induced neuronal differentiation of PC12 cells. In the present study, upon inhibition by MEK-selective inhibitor U0126 and PD98059, the percentage of neurite outgrowth decreased significantly. This suggested that ERK1/2 phosphorylation was affected and this indirectly implied that activation of ERK1/2 is necessary for P. giganteus-mediated neuritogenesis. Inhibition of PI3K/Akt signalling by LY294002 also negatively affected neurite outgrowth of PC12. This finding suggested that neurite outgrowth potentiated by P. giganteus in PC12 cells is also regulated by PI3K/Akt signaling pathway. However, it was noted that PI3K/Akt inhibitor did not markedly affect the activities of ERK , therefore neurite extension of PC12 still could be observed at lower concentrations of LY294002. According to Naidu et al. , phospho-Akt and phospho-MAPK were expressed during neurodevelopment and nerve regeneration following sciatic nerve crush on rats. Collectively, these results demonstrated that P. giganteus-induced neurite extension is regulated at least by part between MEK/ERK and PI3K/Akt pathways. For the future work, confirmation by immunoblot analysis to detect the phosphorylation of TrKA, ERK, and Akt, is necessary.