Introduction
Because of the significant increase in life expectancy over sixty-five years of age in Korea, dementia diseases have also increased. Some forms of dementia are caused by a lack of neurotransmitters. Acetylcholine is one of the neurotransmitters in the peripheral nervous system and central nerve system, and it is converted into choline and acetate by acetylcholinesterase (EC.3.1.1.7, AChE) [1, 2]. Therefore, AChE is a key enzyme in the pathophysiology of dementia.
Several AChE inhibitors as anti-dementia agents have been extracted and characterized from various plants or microorganisms including Umbilicaria esculenta [3], green tea [4, 5], Securinega suffruticosa [6], Onosma hispida [7], Juglans regia [2], the Chinese herb Huperzia serrate [8], etc. However, AChE inhibitors such as Galantamine, Riv- astigmine, Donepezil, Tacrine and Memantine have been only approved by the FDA as drug therapy for dementia [9]. They also have some side effects including nausea and anorexia. Therefore, research on development of new anti-dementia agents with high efficacy and no side effects is necessary.
Meanwhile, bioactive compounds from mushrooms have been reported for their health-stimulating effects [10]. One of the edible mushroom Pholiota adiposa is classified under the genus Pholiota of the family Strophariaceae. This mushroom is cultivated in Asia including Korea, Europe, and North America. The pharmaceutical effects of P. adiposa have been reported its antihypertension [11], chole- sterol-lowering [12], antibiotic, and antitumor activities [11]. This study describes the screening of a potent AChE inhibitor found in P. adiposa and the optimization of the extraction conditions to develop a new anti-dementia agent from edible mushrooms for application in the medicinal food industry.
Materials and Methods
Mushrooms and chemicals
Nine kinds of commercial edible mushrooms were pur- chased at local oriental medicinal markets and agriculture markets which were cultivated in Korea between 2014~ 2015. Acetylcholinesterase (AChE from Electrophorus electricus), acetylcholine chloride and 5,5’-dithiobis (2-nitro- benzonic acid) were purchased from the Sigma Chemical Co. (St, Louis, MO, USA). A VERSAmax microplate rea- der (Molecular Devices, Sunnyvale, CA, USA) was used to assay the AChE inhibitory activity.
Water and ethanol extraction
Air-dried (45 o C for 48 hr) fruiting bodies were finely pulverized. The sample powders were added to water and 70% ethanol each at a 1:30 w/v ratio and then kept in a shaker for 24 hr at 30 oC. Each extract was filtered with Whatman 0.45 µm membrane filter (NO 7404-004; Whatman, Piscataway, NJ, USA) and lyophilized.
Acetylcholinesterase inhibitory activity assay
The AChE inhibitory activity was measured spectropho- tometrically as follows [2, 13, 14]. A mixture of 110 µL of assay buffer (0.1 M sodium phosphate, pH 7.3), 30 µL of AChE (0.8 unit/AChE), 30 µL of substrate (2 mM acetyl- thiocholine chloride), 20 µL of 5,5’-Dithiobis (2-nitroben- zonic acid, 2 mM DTNB) and 10 µL of sample (1 mg/mL) dissolved in the assay buffer (1 mg/mL) in a 96 well plate was kept at 37 oC for 6 min. The reaction product 5- thio-2-nitrobenzate produced was measured at 415 nm. The inhibition rate was obtained with the following equa- tion:
Inhibition (%) = [1 − {(S−S0) / (C−C0)}] × 100,
where C was the radiation of a control (enzyme, assay buffer, DTNB, and substrate) after an activation for 6 min; C0 was the radiation of the control at time zero; S was the radiation of the tested samples (enzyme, assay buffer, DTNB, and substrate) after an activation of 6 min, and S0 was the radiation of the tested samples at time zero.
To check the quenching effect of the samples, the sam- ple was added to the reaction mixture C (control), and any reduction in radiation by the sample was investigated.
Results and Discussion
Screening of potent acetylcholinesterase inhibitor-con- taining mushrooms
To select a potent anti-dementia AChE inhibitor-contai-ning mushroom, water and 70% ethanol extracts from nine kinds of edible mushrooms were prepared, and their yields and AChE inhibitory activities were determined. As shown in Table 1, the water extract from Lentinula edodes fruiting body had the highest yield of 48.7%, and the water and 70% ethanol extracts of Agaricus bisporus and the water extract of Pleurotus ostreatus also had yields over 40%.
Table 1. Yield and acetylcholinesterase inhibitory activity of water and 70% ethanol extracts from various market mushrooms
|
| Extraction condition: 1:30, 30oC, 24 hr. n.d, not detected. |
However, the AChE inhibitory activity was the highest at 30.6% for the 70% ethanol extract of P. adiposa, and its water extract also had an AChE inhibitory activity of 23.8%. Finally, P. a di p o s a was selected as a good AChE inhibitor-containing edible mushroom. This inhibitory activity was lower than those from plants and fruits such as walnut (72.6%) and job’s tears (55.1%) [2, 15, 16].
Optimal conditions for the extraction of the acetyl- cholinesterase inhibitor
The effects of the extraction temperature on the AChE inhibitory activity and yields from Pholiota adiposa fruit- ing body were determined (Table 2). The 70% ethanol extract had about twice higher yield than that of the water extract, and their yields were slightly increased as the ex traction temperature was increased to 70oC.
The AChE inhibitory activity of the 70% ethanol ext- ract from the extraction at 70oC had the highest activity at 35.0%. The water extract from the extraction at 70 oC also had an inhibitory activity of 30.9%. However, water extract from extraction of 100oC for 6 hr showed inhi- bitory activity less than 10% (data not shown).
The effect of the extraction time on the AChE inhibit- ory activity and yield was investigated. As seen in Fig. 1 and Fig. 2, the yields of the water and 70% ethanol extracts increased when the extraction time was increased to 3 and 6 hr, respectively. The AChE inhibitory activities also increased when the extraction time was increased. The maximum inhibitory activity was 33.7% for the water ext- ract at 3 hr and 35.0% for the 70% ethanol extract at 12 hr.
) and 70% ethanol extracts (
) from
Pholiota adiposa.
Lee et al. [2] reported AChE inhibitor of walnut (Jugl- ans regia L.) was maximally obtained from extraction at 40oC for 12 hr by 80% methanol but Seo et al. [13] rep- erted AChE inhibitor of job’s tears (Coix Lachrymajobi L.) was maximally extracted at 40oC for 6 hr with 60% methanol.
Meanwhile, the 95% ethanol extract had lower yield and AChE inhibitory activity than that of the 70% ethanol extract (data not shown).
