α-Glucosidase inhibitory fatty acids from Morchella fluvialis mushroom

The fruiting bodies of M. fluvialis were collected from the local market at Yunnan, China, in April 2018. The mushroom was first identified by Prof. Hyun You Chang of the Korea National College of Agriculture and Fisheries. In addition, a DNA-based approach was used. In the process of DNA extraction, Morchella specimens were pulverised into a fine powder using a grinder. The extraction process was subsequently carried out using a commercially available kit (DNeasy^® Plant Mini Kit, Qiagen Hilden, Germany) following the protocol provided by the manufacturer. The quantification of DNA was performed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Voucher specimens (CBNU2018-MF) were deposited in a specimen room of the herbarium of the College of Pharmacy, Chungbuk National University.

Dried fruiting bodies of M. fluvialis (10.2 g) were subjected to two extractions using 80% MeOH, resulting in a methanolic extract weighing 1.4 g. Subsequently, the methanolic extract was diluted in H₂O and subjected to sequential partitioning with n-hexane, CH₂Cl₂, EtOAc and n-BuOH. The n-hexane fraction (0.2 g) underwent filtration using MPLC RP-Silica to characterise nine distinct fractions (HE1-HE9). HE4 was subjected to column chromatography over Sephadex LH-20 eluting with CH₂Cl₂-MeOH (1:1) to give three fractions (HE4A-HE4C). Compounds 1 (8.7 mg), 2 (2.5 mg) and 3 (3.1 mg) were obtained from HE4C by semi-preparative HPLC eluting with acetonitrile. A sample of an EtOAc fraction weighing 0.2 g was subjected to column chromatography using silica gel. The elution process involved a mixture of n-hexane and EtOAc, resulting in the separation of the sample into nine distinct fractions, labelled EA1 to EA9. The compound EA3 underwent column chromatography with Sephadex LH-20 as the stationary phase and a mixture of CH₂Cl₂ and MeOH in a 1:1 ratio as the eluent. This process resulted in the separation of EA3 into five distinct fractions, namely, EA3A-EA3E. Compounds 4 (2.8 mg), 5 (3.1 mg), 6 (5.3 mg) and 7 (3.4 mg) were obtained from EA3E by semi-preparative HPLC eluting with acetonitrile-water (90:10). Compounds 8 (0.7 mg) and 9 (0.7 mg) were obtained from EA3C by semi-preparative HPLC eluting with acetonitrile–water (80:20).

For the determination of linoleic acid (1), the total extract was prepared by extracting the fruiting bodies of M. fluvialis with 80% MeOH. n-Hexane, EtOAc and n-BuOH fractions were prepared from the total extract by partitioning successively with n-hexane, EtOAc and n-BuOH.

The HPLC analysis was conducted utilising a Waters HPLC system that was equipped with Waters 600Q pumps, a 996 photodiode array detector and Waters Empower software. The analysis employed a Phenomenex Gemini-NX 3μ C18 110A column with a dimension of 150 mm x 4.60 mm. The process of chromatographic separation was successfully achieved by employing a mixture of acetonitrile and water in a ratio of 85:15. The separation was conducted at a flow rate of 2.0 mL · min^–1. Calibration solutions were prepared using linoleic acid (1) at varying concentrations. The relationship between concentration and peak area was examined by employing the regression line derived from the least square analyses.

The evaluation of the inhibitory effects of the samples on α-glucosidase activity was conducted utilising α-glucosidase derived from Saccharomyces cerevisiae. In brief, α-glucosidase (1.0 U · mL^–1) was added to the sample, and then it was incubated at 37 °C for 15 min. The enzyme reaction was performed at 37 °C for 20 min after adding 10 μL of p-nitrophenyl α-_D-glucopyranoside solution. Absorbance at 405 nm in a 96-well microplate reader was used to calculate the quantity of p-nitrophenol cleaved by the enzyme. The calculation of the percentage of α-glucosidase inhibition for the compounds was performed as follows: [(Absorbance without sample – absorbance with sample)/absorbance without sample] x 100. Acarbose was employed as a positive control in the study.

Statistical significance was assessed using one-way analysis of variance (ANOVA), where a significance level of p < 0.05 was deemed to indicate statistical significance.

Morchella was first identified by morphological characteristics using DNA-based approaches (Figure 1). Morphologically, a yellow morel was similar to M. esculenta, but with a slender habit, a stipe longer than the pileus and a pileus that is normally conical, with the top slanted to one side. At the pileus base, there is no sinus. The pileus primary pits are spherical or elongated, with crests that stain orange when injured and are orientated somewhat lengthwise.

Figure 1.

For additional DNA-based approaches, ITS sequencing for Morchella authentication was used. Because the ITS regions have different rates of evolution, they are common markers in evolutionary studies at different taxonomic levels (Balasubramani et al., 2010). PCR amplification was performed with the primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’)forITS,whileEF1-983 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and EF1-1492R (5’-TACGGYTACCTTGTTACGACTT-3’) were used to amplify the translation elongation factor 1-α(tef1) gene.

The number of reports on this mushroom species is quite limited in the literature. Most of them especially focused on M. esculenta, but there are no record of M. fluvialis. Therefore, we revealed a new interesting finding of M. fluvialis, which suggests these species to be a good candidate for a highly nutritive source of mushrooms.

For the investigation of fruiting bodies of M. fluvialis for the beneficial effects on metabolic diseases, α-glucosidase inhibitory and antioxidant activities of its methanolic extract were measured. As shown in Figure 2, the total extract of M. fluvialis showed α-glucosidase inhibitory and antioxidant activities. To characterise the active constituents, the total extract was further fractionated into n-hexane, EtOAc and n-BuOH fractions according to the polarity. Among the fractions, the n-hexane fraction showed the most potent α-glucosidase inhibitory activity. On the other hand, the EtOAc fraction exerted mild α-glucosidase inhibitory and antioxidant activities in an assay system (Figure 2).

Figure 2.

For the characterisation of active constituents, the isolation of compounds from n-hexane and EtOAc-soluble fractions was conducted using chromatographic techniques (Supplementary Scheme S1). As a result, nine compounds (Figure 3) were isolated from n-hexane and EtOAc-soluble fractions. The structures of isolated compounds were determined as three fatty acids, linoleic acid (1), (9Z,12Z)-3-hydroxy-9,12-octadecadienoic acid (2) and (6Z,9Z)-13-hydroxy-6,9-octadecadienoic acid (3); four sterols, stellasterol (4), ergosterol peroxide (5), ergosterol (6) and brassicasterol (7); one sugar alcohol, arabitol (8); and nicotinamide (9) by spectroscopic analysis including ¹H, ¹³C NMR and MS analyses (Supplementary Figures S1–S6), which were confirmed by the comparison of literature values (Dong et al., 2000; Jinming et al., 2001; Seo et al., 2009; Kim et al., 2011). Among them, compounds 2–3 and 7 were first reported from this mushroom.

Figure 3.

The biological activity of isolated compounds was evaluated (Table 1). Among the isolated compounds, compound 1 showed the most potent α-glucosidase inhibitory, with an IC₅₀ value of 14.8 μM (4.2 μg · mL^–1). Compounds 2 and 3, fatty acid derivatives, also showed α-glucosidase inhibition by 53.1 and 32.9% at a concentration of 100 μM. However, sterol derivatives (4–6) showed weak α-glucosidase inhibitory activity. Related to antioxidant activity, all the isolated compounds exerted weak activities.

Compound 1 is the major compound of morel and showed potent α-glucosidase inhibitory activity. Therefore, the content in the total extract and each fraction were quantitated by HPLC analysis (Figure 4). The content of compound 1 was 1.2 mg · g^–1 extract in the total extract of morel mushroom and was much higher as 15.9 mg · g^–1 extract in the n-hexane fraction, which is consistent with α-glucosidase inhibitory activity. However, compound 1 was not detectable in EtOAc and n-BuOH-soluble fractions. These results showed that compound 1 is a major active compound for α-glycosidase inhibitory activity of Morchella mushroom.

Figure 4.

Interestingly, the n-hexane-soluble fraction of morel contained 1.59% of compound 1 and showed inhibitory activity of 75% at a concentration of 200 μg · mL^–1. The amount of compound 1 contained in 200 μg · mL^–1 of the n-hexane-soluble fraction was about 3.2 μg · mL^–1, which is lower than the IC₅₀, but exhibits more than 50% inhibitory effect. As mentioned earlier, morel contains several fatty acids with α-glucosidase inhibitory activity such as (9Z,12Z)-3-hydroxy-9,12-octadecadienoic acid (2) and (6Z,9Z)-13-hydroxy-6,9-octadecadienoic acid (3). From these results, it can be inferred that the α-glucosidase inhibitory activity of Morchella mushroom is synergistic with compound 1 and other compounds.

Previous studies on the strain of Morchella related to anti-diabetic activity have been mainly focused only on the organic acid profile of Morchella. Dietary fat, which is a large component of the typical diet and hence a tight feedback regulator, is required to maintain lipid homeostasis. Many studies have investigated the fatty acid content of a variety of mushrooms to better understand their nutritional roles in human diets (Lv et al., 2015). Polyunsaturated fatty acids, which are also present in mushrooms, play a crucial role in human nutrition and the prevention of diseases. Linoleic acid (1), a polyunsaturated fatty acid, has been consumed for the prevention of various diseases, such as cardiovascular and inflammatory diseases, as well as depression. A higher level of linoleic acid in diet is also suggested to contribute to insulin sensitivity (Lv et al., 2015). Consistent with previous research, linoleic acid (1) of morel mushroom showed potent inhibitory activity, with an IC₅₀ value of 14.73 μM in an assay system. The present study also yielded another fatty acid derivative, (9Z,12Z)-3-hydroxy-9,12-octadecadienoic acid (2) and (6Z,9Z)-13-hydroxy-6,9-octadecadienoic acid (3), with α-glucosidase inhibitory activity. The observation highlights the importance of fatty acids and their synergic contribution to α-glucosidase inhibition activity.