Acetylcholinesterase inhibition effects of marine fungi

Abstract

Context: To this day, there are no reports that marine compounds isolated from microorganisms of the Lianyungang area of China have been used for the treatment of Alzheimer’s disease.

Objective: The present study was to isolate fungi from the sea sediment of the Lianyungang area and screen for acetylcholineseterase inhibition activities of ethyl acetate extracts.

Materials and methods: Fungi were isolated from the sea sediment and fermented. After centrifugation, the supernate was extracted with ethyl acetate. The ethyl acetate extract was then fractionated into five fractions. Acetylcholinesterase inhibition activities of the ethyl acetate extracts and five sub-fractions were tested at a concentration of 500 μg/mL with the Ellman’s method.

Results: Forty-three marine fungi were isolated; 15 extracts inhibited acetylcholinestrease >50% and 3 extracts inhibited the acetylcholinesterase >80% at the concentration of 500 μg/mL. The 3 extracts (L1705, S1101, SH0701) inhibited AChE dose-dependently with IC₅₀ values of 11.3 ± 1.2, 72.1 ± 2.3, and 7.8 ± 2.8 μg/mL, respectively. After the extract of SH0701 was fractionated into five fractions, the ethyl acetate fraction possessed the highest acetylcholinesterase inhibitory activity with an inhibition rate of 71.55% at the concentration of 10 μg/mL. The fungus SH0701 was identified as Aspergillus ochraceus SH0701 according to morphology and molecular identification.

Discussion and conclusion: The present results indicates that some ethyl acetate extracts of marine fungi isolated from Lianyungang area of China could inhibit AChE potently. Therefore, some novel AChE inhibitors might exist in those extracts.

• Alzheimer’s disease
• Lianyungang area of China
• sea sediment

Introduction

Marine microorganisms are a potential source of novel natural products due to their extreme living conditions and unique physiological metabolism. Nowadays, global research focused on the therapeutic potential of the marine natural products remained very active (Lee et al., 2011). Among them, marine fungi have received more attention. Recently, a number of novel structures with various potent biological activities have been found continuously from marine fungi (Zhuang et al., 2012). More and more marine compounds for the therapeutic treatment of neurological disorders such as Alzheimer’s disease (AD) have been isolated from the marine organisms (Alonso et al., 2005). Some novel AChE inhibitors were isolated from marine microbes (Qiao et al., 2011; Sangnoi et al., 2008).

Based on the cholinergic hypothesis, acetylcholinesterase (AChE, EC 3.1.1.7) inhibitors were developed for the treatment of AD (Lahiri et al., 2004). Four AChE inhibitors (tacrine, donepezil, rivastigmine and galantamine) were approved for the treatment of AD by US Food and Drug Administration (Cummings, 2000). Though AChE inhibitors have become the mainstays for treating AD (Cummings, 2004), the non-selectivity of these drugs, poor bioavailability, adverse cholinergic side effects in the periphery, narrow therapeutic ranges and hepatotoxicity were some of the severe limitations for their therapeutic success (Bores et al., 1996; Forette et al., 1999). Additional studies on the AChE inhibitors derived from the natural products or from design or synthesis are necessary (Rodrigues et al., 2005; Zheng et al., 2009).

To find novel AChE inhibitors from the marin microbes, we isolated fungi from sea sediment of the Lianyungang area of China and screened AChE inhibition activities of the ethyl acetate extracts from the fermented broth of the isolated fungi with Ellman’s method. The isolation, identification and AChE inhibitory activities of marine fungi are reported in this article.

Materials and methods

Chemicals

5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB) and acetylthiocholine iodide (ATCI) were purchased from Sigma-Aldrich (St. Louis, MO). All other reagents used in the study were of analytical grade.

Sample collection

The sea sediment was collected and identified by J.-T.X. (Huaihai Institute of Technology, Lianyungang, P.R. China) from the Lianyungang area in April 2011. The voucher specimens were registered and preserved in the herbarium of Huaihai Institute of Technology at 4 °C. After the collection, the sea sediment was processed for the isolation of fungi immediately.

Media preparation

Sea water potato dextrose agar (PDA) medium (replaced the water with sea water of the PDA medium) was used for isolation and purification of marine fungi. Antibiotics, penicillin (100 μg/mL) and streptomycin sulfate (100 μg/mL), were added to the media to avoid bacterial contamination.

Isolation of marine fungi

The sediment grab was immediately sampled using a sterile syringe, plated in triplicate on sea water PDA media with antibiotics and incubated at 27 °C. After 1 week, the emerging hyphae were cut and transferred into new seawater PDA Petri dish for purification. The isolate fungi were stored by covering a culture on PDA slants with sterile liquid paraffin at 27 °C.

Extraction of metabolites from marine fungi

The fungi were cultured in a 500 mL fermentor containing 300 mL of seawater potato dextrose broth (PDB). The inoculation amount was 5% (220 rpm, 28 °C, 7 d). After the fermentation, the culture was centrifuged to remove the mycelia. After centrifugation, the supernate was extracted with ethyl acetate three times at room temperature. The ethyl acetate was removed under reduced pressure using a rotary evaporator (45 °C) to yield the ethyl acetate extract.

Polarity gradient fractionation of the ethyl acetate extract

The ethyl acetate extract from fungal strain SH0701 was suspended in 1 L of water and then extracted three times with equal volumes of petroleum ether, chloroform, ethyl acetate and n-butanol separately at room temperature. The solvent layer was removed and evaporated to dryness. Different polarity fractions were collected.

AChE inhibitory assay

AChE activities were measured through Ellman’s colorimetric method with a slight modification (Ellman et al., 1961). AChE was prepared from rabbit cortex homogenate (Pereira et al., 2004). All procedures involving the use of laboratory animals were in accordance with National Institutes of Health guidelines and were approved by the Committee for the Ethical Use of Animals of Huaihai Institute of Technology. The rabbits were sacrificed under anesthesia by decapitation and the brain was quickly removed and placed on an inverted Petri dish on ice. The forebrain was dissected, weighed and homogenized in four volumes of a sodium phosphate buffer (100 mM, pH 7.4). The total homogenate was centrifuged at 1000 g (4 °C) for 10 min. Then the supernatant was collected and diluted with the sodium phosphate buffer (100 mM, pH 7.4) to get a solution with a protein concentration of 10 mg/mL and used as AChE. Protein was determined by the method of Bradford (1976) using bovine serum albumin as standard.

In assays, 10 μL of rabbit cortex homogenate was incubated with 10 μL of tested sample and 140 μL of 0.1 M phosphate-buffered saline (pH 8.0) for 10 min in 96-well microplates before addition of 20 μL of 3.33 mM DTNB solution and 20 μL of 5.30 mM ATCI solution. After the addition of DTNB and ATCI, the 96-well microplates were read at 412 nm with a microplate reader (SPECTRAFLUOR, TECAN, Sunrise, Austria) for 15 min. One triplicate sample without inhibitors was always present to yield 100% of AChE activity. One triplicate sample with 20 μM tacrine was always present to yield 100% AChE inhibition (to obviate the ChE-independent substrate hydrolysis). The reaction rates were compared and the percent inhibition due to the presence of tested compounds was calculated. Tacrine was applied as a positive drug. All the samples were assayed in triplicate. To determine the IC₅₀ of the ethyl acetate extracts from fermentation broth of the marine fungi, the AChE inhibition activities of the extracts were tested at the final concentrations of 0.01, 0.1, 1, 10, 100 and 1000 μg/mL.

Identification of marine fungal strain SH0701

The fungal strain SH0701 was incubated in PDA medium at 27 °C in darkness for 6–8 d. Mycelia were scraped from the plate and grounded to a powder under liquid nitrogen. Fungal DNA was extracted using the UNIQ-10 kit (Sangon Corp., Shanghai, China) following the manufacturer’s instructions. β-Tubulin gene and the internal transcribed spacer (ITS) of ribosomal DNA were amplified and sequenced. Similar taxon retrieved by basic local alignment search tool (BLAST) in GenBank/NCBI was used as a reference for further morphological examination and identification. β-Tubulin gene was amplified using the primers Bt2a and Bt2b (Glass & Donaldson, 1995) with the following conditions: denaturing at 94 °C for 5 min, followed by 35 cycles at 94 °C for 40 s, 54 °C for 40 s and 72 °C for 1 min, then a final extension at 72 °C for 10 min after cycling. ITS gene was amplified using the primers ITS1 and ITS4 (White et al., 1990) with the following conditions: denaturing at 98 °C for 5 min, followed by 35 cycles at 95 °C for 35 s, 55 °C for 35 s and 72 °C for 40 s, then a final extension at 72 °C for 8 min.

Results and discussion

Forty-three marine fungi were isolated from the sea sediment of the Lianyungang area of China. The ethyl acetate extracts from the fermentation broth of these fungi were tested for their AChE inhibitory activities with Ellman’s method. The results are summarized in Table 1. Among them, 15 extracts possessed remarkable AChE inhibitory activities at a concentration of 500 μg/mL with inhibitory rates of >50%. Three extracts inhibited AChE >80% at a concentration of 500 μg/mL. The three extracts (L1705, S1101 and SH0701) inhibited AChE dose-dependently with the IC₅₀ values of 11.3 ± 1.2, 72.1 ± 2.3 and 7.8 ± 2.8 μg/mL, respectively (Table 1). Under the same condition, the positive control tacrine could inhibit the AChE with an IC₅₀ of 0.27 μM. The extract of SH0701 was studied further. After polarity gradient fractionation, the anti-AChE activities of different polarity fractions were tested at a concentration of 10 μg/mL. The results showed that different polarity fractions possessed different AChE inhibition activity. Among them, the ethyl acetate fraction possessed the highest AChE inhibition activity with an inhibition rate of 71.55% at a concentration of 10 μg/mL (Figure 1). This suggested that the AChE inhibitors could be isolated from the ethyl acetate fraction.

Figure 1. AChE inhibition by different polarity fractions of the ethyl acetate extract from fungal strain SH0701. Values are means ± SD, n = 3.

Table 1. AChE inhibitory activities of extracts of marine fungi.

	Inhibition
Strains	Inhibition (%)^a	IC50 (μg/mL)
G0301	17.87 ± 3.09	–
G0302	35.73 ± 5.36	–
HS0106	37.51 ± 8.18	–
HS0108	25.01 ± 5.36	–
L0101	25.01 ± 5.36	–
L0102	38.56 ± 21.14	–
L0301	25.01 ± 9.28	–
L1701	54.28 ± 6.55	–
L1703	44.28 ± 1.45	–
L1705	94.82 ± 0.31	11.3 ± 1.2
L1707	50.01 ± 8.18	–
L2001	71.42 ± 8.92	–
L2004	44.28 ± 7.42	–
L2005	17.13 ± 5.69	–
L2006	46.44 ± 5.36	–
L2007	25.70 ± 2.47	–
L2008	61.42 ± 7.42	–
S0501	35.71 ± 8.57	–
S0503	55.71 ± 5.69	–
S0504	7.16 ± 1.15	–
S0505	28.56 ± 8.92	–
S0506	28.58 ± 8.18	–
S0914	42.87 ± 3.09	–
S0917	19.66 ± 0.21	–
S1001	17.87 ± 3.09	–
S1002	38.56 ± 6.55	–
S1003	39.30 ± 8.18	–
S1004	16.09 ± 3.09	–
S1005	20.96 ± 0.83	–
S1006	52.85 ± 4.29	–
S1007	16.09 ± 6.18	–
S1008	12.84 ± 4.95	–
S1009	75.00 ± 3.09	–
S1010	7.16 ± 8.18	–
S1011	64.29 ± 6.18	–
S1012	19.66 ± 5.36	–
S1013	53.58 ± 8.18	–
S1014	28.58 ± 6.36	–
S1101	95.71 ± 0.00	72.1 ± 2.3
S1105	62.85 ± 6.23	–
S1109	55.71 ± 4.95	–
SH0701	96.29 ± 0.99	7.8 ± 2.8
SH0709	66.08 ± 3.09	–
Tacrine	–	0.27 ± 0.3 (nmol/L)

^aThe inhibitory rate of the extracts at the concentration of 500 μg/mL. Values are mean ± SD, n = 3.

Colonies of marine fungal strain SH0701 were round and light ochraceous-buff to buff from the abundant conidial heads on sea water PDA medium (Figure 2A). The microscopical observation showed that the mycelia were branched, septate and transparent with rhizoid development (Figure 2B). The conidiophore stripes were brownish with smooth walls. The vesicles spherical were thin-walled, hyaline with metulae covering the entire vesicle (Figure 2C). The conidia spherical were subspherical, smooth-walled to finely roughened (Figure 2D). Based on sequencing of β-tubulin gene and ITS region of rDNA, the fungus was identified as Aspergillus ochraceus SH0701. The β-tubulin gene and ITS region of rDNA sequences of strain SH0701 determined in this study has been deposited in the GenBank database under the accession numbers JX244861 and JX244862, respectively. The results demonstrated that composite analysis based on sequencing of β-tubulin gene and ITS region of rDNA and microscopic examination was helpful to discriminate this marine fungus. This strain has been preserved in the China Center for Typical Culture Collection (CCTCC) with the collection number of CCTCC No. M 2012003.

Figure 2. Colony morphology of the marine fungal strain SH0701.

Alzheimer’s disease is a highly complex neurodegenerative disorder of the aged that has multiple factors that contribute to its etiology in terms of initiation and progression (Rosales-Corral et al., 2011). The AChE inhibitiors are the main drugs for the treatment of AD. However, most natural AChE inhibitors were isolated from terrestrial plants (Williams et al., 2011). Nowadays, the study of marine natural products has becoming one of the hottest research topics (Blunt et al., 2012). More and more marine natural products have been developed for the treatment of neurodegenerative diseases (Alonso et al., 2005). In this study, marine fungus SH0701 was isolated from the sea sediment of the Lianyungang area of China. The fungus was identified as A. ochraceus. It was reported that some potent AChE inhibitors had been isolated from Aspergillus sp. For example, terreulactone A, terreulactone B, terreulactone C, terreulactone D, isoterreulactone A, territrem A, territrem B, territrem C, territrem A′, territrem B′ and 16α-hydroxy-5-N-acetylardeemin, were isolated from A. terreus (Ge et al., 2010; Hostettmann et al., 2006; Houghton et al., 2006). AChE inhibitors, (8E,12Z)-10,11-dihydroxyoctadeca-8,12-dienoic acid, 3β,4α-dihydroxy-26-methoxyergosta-7,24(28)-dien-6-one and aflatoxin B1, were isolated from A. flavus (Houghton et al., 2006; Qiao et al., 2011). This study is the first to report the AChE inhibition activity of A. ochraceus. The ethyl acetate extract of A. ochraceus SH0701 could inhibit the AChE potently. Therefore, some novel AChE inhibitors might exist in the ethyl acetate extract of the fungal strain SH0701. An intensive study on A. ochraceus SH0701 for the treatment of AD will be performed in future.

Declaration of interest

This work was financially supported by the Open-End Funds of Jiangsu Key Laboratory of Marine Biotechnology, Huaihai Institute of Technology (2010HS04), Science and Technology Project of Lianyungang (CG1202), Collegiate Natural Science Fund of Jiangsu Province (12KJB350001) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.