Evaluation of phenolic profile , antioxidant and anticholinesterase effects of Fuscoporia torulosa

In this study, we investigated antioxidant and anticholinesterase activities of the hexane, chloroform, acetone, methanol and water extracts of F. torulosa mushroom with total phenolic contents. Also, HPLC-DAD was used to identify phenolic profile of F. torulosa. The acetone and methanol extracts of F. torulosa with the highest total phenolic contents showed the highest antioxidant activity in all assays except metal chelating assay. Furthermore, antioxidant activities of the acetone and methanol extract were found to be higher than αtocopherol and BHA used as standards in DPPH•, ABTS• and CUPRAC assays. When F. torulosa hexane extract (41.34±1.50 %) showed moderate AChE inhibitory activity, the acetone (40.78±0.30 %) and methanol (45.39±0.65 %) extracts of F. torulosa indicated moderate BChE inhibitory activity. Major phenolic compounds were identified as trans-2-hydroxy cinnamic acid (10.05 μg/g), gallic acid (5.01 μg/g) and p-coumaric acid (3.04 μg/g). These results suggest that F. torulosa mushroom could be used as a valuable natural antioxidant source for pharmaceutical industry. ARTICLE HISTORY Received: December 12, 2018 Revised: February 25, 2019 Accepted: February 28, 2019


INTRODUCTION
Free radicals are produced from oxygen during aerobic respiration and excessive amount of formation and accumulation causes oxidative stress [1].The formation of oxidative stress in living organisms, in particular damages the biomolecules such as DNA, proteins and lipids, resulting in many diseases such as hypertension, ischemia, neurodegenerative diseases and rheumatoid arthritis [2].Antioxidants prevent or reduce the harmful effects of oxidative stress.The use of synthetic antioxidants is an old practice and the safety of these substances is questioned by consumers.At present, interest in alternative natural compounds with high antioxidant effect is increasing [3].
Alzheimer's disease (AD) eliminates neurons in the cortex and limbic structure in the brain, causing learning and memory loss and behavioral disorders in humans.AD is characterized by a reduction of acetylcholine (ACh) due to damage to cholinergic neurons in some specific parts of the brain, such as the hippocampus and cortex (cholinergic hypothesis) [4].One effective approach in the treatment of AD is the inhibition of acetylcholinesterase (AChE), which is responsible for the hydrolysis of ACh [5,6].During aging, a gradual decrease in antioxidant defense mechanism and increased oxidative stress cause neuronal injury and death, which is another neurotoxic pathway causing AD [7].Previous studies have demonstrated that antioxidant therapy is successful in improving cognitive function and behavioral deficits in patients with mild to moderate AD [8].
It is well known that mushrooms have therapeutic properties since ancient times.Up to know, many bioactive compounds such as lectins, polysaccharides, terpenoids, alkaloids, sterols and phenolics having anticancer, antioxidant, antitumor, antiinflammatory, antifungal, antibacterial, antiviral, anti-immunomodulatory activities have been isolated from mushrooms.When literature studies are examined, it is seen that mushrooms are used especially due to anticancer activity [9][10][11][12][13][14][15].Mushrooms show beneficial effects on cancer, either directly as antioxidants or preventing genetic factors that cause cancer [16].
Recently, studies on the discovery of bioactive compounds from mushrooms have become more important because of their functional and therapeutic properties.Therefore, in this report, we focused to evaluate antioxidant and anticholinesterase activities of the hexane, chloroform, acetone, methanol and water extracts of F. torulosa mushroom with total phenolic contents.Also, phenolic profile of F. torulosa were determined by HPLC-DAD.

Mushroom Material
F. torulosa (Pers.)T. Wagner & M. Fisch.was collected from Muğla, Turkey, in November-December, 2014 and January, 2015.The voucher specimen has been deposited at the herbarium of Natural Products Laboratory of Muğla Sıtkı Koçman University with Fungarium No AT-2436.

Instruments and Chemicals
Bioactivity measurements were carried out on a 96-well microplate reader, SpectraMax 340PC384 (Molecular Devices, Silicon Valley, CA).The measurements and calculations of the activity results were evaluated by using Softmax PRO v5.2 software (Molecular Devices, Silicon Valley, CA).

Extraction
The aerial parts of F. torulosa (2.8 kg) were extracted separately with different solvents according to their increasing polarity: hexane, chloroform, acetone, methanol at room temperature for 24 h and four times.Solvents were evaporated on a rotary evaporator at 40°C.The hexane (10.5 g), chloroform (29.8 g), acetone (38.4 g) and methanol (55.6 g) extracts were obtained.The remaining mushroom part was allowed to stand for one day with water at 80°C.The water extract of (17.6 g) were obtained by lyophilisation using a freeze-drier.All extracts were stored at +4°C until analysis.

Determination of Total Phenolic Content
The phenolic content of extracts was stated as microgram of pyrocatechol equivalents (PEs) [17].The phenolic contents were calculated according to the following equation that was obtained from standard pyrocatechol graph: Absorbance=0.0176[pyrocatechol(µg)] -0.355 (r 2 , 0.9992)

Determination of Antioxidant Activity
Total antioxidant activity by β-carotene-linoleic acid test, DPPH free radical scavenging assay, ABTS cation radical scavenging assay, cupric reducing antioxidant capacity (CUPRAC) assay and metal chelating activity on Fe 2+ assays were carried out according to our earlier publication [19].BHA, α-tocopherol and EDTA were used as antioxidant standards for comparison of the activities.The antioxidant activity results are expressed as 50 % inhibition concentration (IC50) and inhibition percentage (%) at 200 µg/mL and A0.50 which corresponds to the concentration producing 0.500 absorbance for CUPRAC assay.

Determination of Anticholinesterase Activity
Acetylcholinesterase and butyrylcholinesterase inhibitory activity were determined the spectrophotometric method developed by Ellman et al. [20].AChE from electric eel and BChE from horse serum were used, acetylthiocholine iodide and butyrylthiocholine chloride were employed as substrates of the reaction.DTNB was used for the measurement of the cholinesterase activity.Briefly, 150 μL of 100 mM sodium phosphate buffer (pH 8.0), 10 μL of the sample solution dissolved in ethanol at different concentrations and 20 μL AChE or BChE solution in buffer were mixed and incubated for 15 min at 25 °C, and 10 μL of 0.5 mM DTNB was added.The reaction was then initiated by the addition of 0.71 mM, 10 μL of acetylthiocholine iodide or 0.2 mM, 10 μL of butyrylthiocholine chloride.The hydrolysis of these substrates was monitored spectrophotometrically by the formation of yellow 5-thio-2nitrobenzoate anion as the result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine iodide or butyrylthiocholine chloride, respectively, at a wavelength of 412 nm utilizing a 96-well microplate reader.Galantamine was used as reference compounds.The results were given as inhibition percentage (%) of the enzyme at 100 μg/mL concentration of the extracts.

Statistical Analysis
All data on antioxidant and anticholinesterase tests were the average of three parallel sample measurements.Data were recorded as mean ± S.E.M. Significant differences between means were determined by student's test, p values <0.05 were regarded as significant.

Total Phenolic Content
The calibration curve of pyrocatechol (0.0176[pyrocatechol (μg)] -0.355; r 2 , 0.9992) was used to determine the total phenolic content.Table 1 presents the total phenolic contents of the hexane, chloroform, acetone, methanol and water extracts of F. torulosa.
The methanol extract (131.35±0.29 µg PEs/mg) of F. torulosa has the highest level of the phenolic compounds among the other extracts.The total phenolic contents of the extracts were decreased in the order of methanol> acetone> water> hexane> chloroform.Total phenolic content of Inonotus obliquus (Fuscoporia obliqua) ethanol extract was found as 55.94±1.08 mg GAE/g extract by Zhang et al. [21].The content of total phenols of 80 % ethanol, 80 % methanol and 95 % ethanol extracts of Inonotus obliquus (Fuscoporia obliqua) expressed as μg of gallic acid equivalents extracted from 100 mg extracts were found as 1388.505,1404.907 and 662.184 respectively [22].In the report of Seephonkai et al. [23], total phenolic contents of 50 % EtOH, 80 % EtOH, EtOH, EtOAc extracts of F. torulosa were studied and found as 43.80±0.78,54.86±0.21,62.51±0.65,16.56±0.29mg GA/100 mg of extract, respectively.The results obtained are consistent with the literature.Total phenolic contents of hexane, chloroform, acetone, methanol and water extracts of F. torulosa were studied for the first time in this research.

Antioxidant Activity
β-carotene-linoleic acid, DPPH free radical scavenging, ABTS cation radical scavenging, cupric-reducing antioxidant capacity (CUPRAC) and metal chelating activity assays were used to determine antioxidant activities of the hexane, chloroform, acetone, methanol and water extracts of F. torulosa.All of the extracts showed antioxidant activities in a dose-dependent manner.Table 3 shows the IC50 values and inhibition percentage (%) at 200 µg/mL concentration of the extracts and standard compounds (BHA, α-tocopherol, and EDTA).

Anticholinesterase Activity
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of the hexane, chloroform, acetone, methanol and water extracts of F. torulosa were screened by using Ellman method.All of the extracts showed anticholinesterase activities in a dosedependent manner.Inhibition percentage (%) at 100 µg/mL concentration of the extracts and standard compound (galantamine) are given in Table 4.
Among the extracts, the hexane extract (41.34±1.50%) of F. torulosa was found as the most active against AChE enzyme.The chloroform, acetone and methanol extracts indicated moderate activity against BChE enzyme with inhibition value of 35.18±0.55,40.78±0.30and 45.39±0.65 %, respectively, when the hexane and water extracts were found to be inactive.According to our knowledge, there is no study about anticholinesterase activities of Fuscoporia species in the literature.

CONCLUSION
In this research, antioxidant and anticholinesterase activities of various extracts of F. torulosa were determined with the total phenolic contents.Also, phenolic profile of the mushroom analyzed by HPLC-DAD.The acetone and methanol extracts with the highest content of total phenolic contents displayed higher antioxidant activity than standards in DPPH • , ABTS • and CUPRAC assays.Totally, thirteen phenolic compounds were identified by using HPLC-DAD and trans-2-hydroxy cinnamic acid, gallic acid, p-coumaric acid were found as major phenolic compounds.In conclusion, this study reveals that extracts obtained from F. torulosa mushroom could be used as promising antioxidant and anticholinesterase agents.However, it is necessary to carry out isolation studies to discover the compounds responsible for these bioactivities.

Table 1 .
Total phenolic contents of the extracts of F. torulosa a

Table 4 .
Cholinesterase inhibitory activities of the extracts of F. torulosa a a Inhibition % of 100 µg/mL concentration of the extracts and compounds.Values represent the means ± S.E.M. of three parallel measurements (p < 0.05).b NA: not active