A new fatty acid ester from an edible mushroom Rhizopogon luteolus

Abstract Phytochemical investigation of the Rhizopogon luteolus Fr. led to the isolation of one new fatty acid ester, 3-hydroxy-2,4-dimethylheptacosyl acetate (1) together with two known compounds tetracosanoic acid (2) and ergosterol (3). 1D and 2D NMR, and MS techniques were used for structural elucidation. Phenolic and fatty acid compositions were identified using HPLC–DAD and GC–MSD, respectively. Fumaric acid was the major phenolic acid, whereas linoleic, stearic and oleic acids were the most abundant fatty acids. Antioxidant and anticholinesterase activities of the extracts and compounds (1–3) were tested spectrophotometrically. Among the extracts, hexane extract showed the highest activity in all tests, particularly in β-carotene-linoleic acid assay (IC50: 16.65 ± 1.12 μg/mL). Furthermore, compound 3 exhibited higher antioxidant and anticholinesterase activities. The study indicates that R. luteolus can be used in food, cosmetic and pharmaceutical industries.


Introduction
Mushrooms are used in food and pharmaceutical industries due to their unique taste, flavour and biological activities. They are the sources of carbohydrates, proteins, amino acids, vitamins, minerals and fats. Till date, various compounds having significant biological and nutraceutical properties have been isolated from mushroom species (Duru & Tel-Çayan 2015;Öztürk et al. 2015).
Fatty acids are important components of mushrooms. They exhibit cytotoxic (Kim et al. 2012), antimicrobial (Ivanova et al. 2013), anticholinesterase (Özturk et al. 2014) and antitumour (Gao et al. 2012) activities. Polyunsaturated fatty acids especially the omega-3, and -6 series, are necessary for human health due to their positive effects on health and chronic diseases. They reduce cardiovascular risks and regulate cholesterol levels and immune system (Benatti et al. 2004).
Steroids are lipophilic significant biological players. Several steroids possessing cytotoxic (Hien et al. 2013), antimicrobial (Niedermeyer et al. 2005), anticomplement (Seo et al. 2009) and antitubercular activities (Akihisa et al. 2005) were isolated from various mushrooms. Ergosterol, an unsaturated hydrocarbon of vitamin D is a well-known steroid that plays an important role in the eukaryotic cell membrane of fungi and yeast (Ivanova et al. 2013). Additionally, it was reported as a anticomplement (Seo et al. 2009), antitubercular (Akihisa et al. 2005), anti-inflammatory and anticancer (Ma et al. 2013).
Rhizopogon species are ectomycorrhizal and are assumed to play an important role in the ecology of coniferous forests. Rhizopogon luteolus is an edible mushroom species belonging to Rhizopogonaceae family. Literature survey revealed that no phytochemical studies on R. luteolus have been carried out. Herein, we report the first isolation study and structure elucidation of 3-hydroxy-2,4-dimethylheptacosyl acetate (1), tetracosanoic acid (2) and ergosterol (3) together with possible antioxidant and anticholinesterase activities.

Identification of compounds
Compound 1 was isolated as a colourless gum with a molecular formula of C 31 H 62 o 3 by positive mode FAB ⊕ MS with m/z 483.3. IR spectrum showed characteristic vibration peak at 3096 cm −1 for hydroxyl group, 2951 and 1689 cm −1 for carbonyl vibration. The 1 H/ 13 C NMR spectrum of (1) displayed typical signals for methine protons resonances as multiplets at δ 4.04/65.5 (C-3) and δ 2.34/33.3 (C-4). The hydroxyl methylene protons appeared at δ 3.63 (1H, d, J = 6.0 Hz, H-1a) and 3.55 (1H, d, J = 6.0 Hz, H-1b). The acetoxyl moiety methyl protons in acetyl group were observed at δ 2.08, which were also confirmed through 13 C NMR by observing signals at δ 173.3 for ester carbonyl and δ 20.1 for methyl group (Muhammad et al. 2012). In HMBC experiment, methyl protons at δ 2.08 showed correlation with the ester carbonyl (δ 173.3). In addition, hydroxy methylene protons at δ 2.33 and 2.34 also showed 3 J connectivity with ester carbonyl that was observed at δ 173.3 (Kim et al. 2012). Protons of two methyl groups (2-Me, 4-Me) were appeared at δ 1.13 showed cross-peaks ( 2 J & 3 J) with C-2 (δ 34.4), C-4 (δ 33.3) and with C-3 (δ 70.6) confirming the position of hydroxyl and methyl groups. The FAB ⊕ MS spectrum showed [M] + peak at m/z 483.3 as a base peak, while other fragments were found at m/z 425, which was due to the losses of methyl and acetyl moiety from the molecular ion, establishing the presence of acetyl moiety. Thus, 1 was characterised as 3-hydroxy-2,4-dimethylheptacosyl acetate.
Compound 3 was obtained as white needles aCompound 3 was obtained as white needles and identified using GC-MS, ESI-MS and NMR (1D, 2D NMR). ESI-MS showed m/z 396 molecular ion peak that established a molecular formula of C 28 H 44 o. The 1 H NMR spectrum of 3 exhibited signals for one oxygenated methine proton at δ 3.50 (1H, s, H-3); six methyl at δ 0.54 (3H, s, H-18 .5 (C-23). The structure of 3 was concluded as ergosterol. It was also affirmed by comparing its spectral data with the literature (Kanagasabapathy et al. 2011;Ivanova et al. 2013).
Compound (2) was identified as tetracosanoic acid by comparing its chemical and spectral data with the literature (Zhang et al. 2004). The structures of compounds were given in Figure 1. The important HMBC interactions of 1 were also given in Figure S1.

Phenolic profile
Phenolics and flavonoids show a wide range of biological activities including antioxidant, antibacterial, antimutagenic, cytotoxic and anticarcinogenic (Heim et al. 2002;Modak et al. 2005). As shown in Table S1, fumaric acid (102 ± 0.002 μg/g), gallic acid (1.50 ± 0.002 μg/g) Figure 1. structures of the isolated compounds from R. luteolus. and protocatechuic acid (1.15 ± 0.001 μg/g) were major compounds found in R. luteolus. Fumaric acid is an antioxidant, antimicrobial, have acidifying properties (Ribeiro et al. 2008) and also widely used in food and other industries. It is also reported as the main organic acid in some edible mushrooms (Valentão et al. 2005).
The total phenolic contents of the methanol, hexane, ethyl acetate and water extracts were found 31.0 ± 0.01, 54.4 ± 0.01, 31.9 ± 0.001 and 26.4 ± 0.01 μg PEs/mg, respectively, while the total flavonoid contents were found to be 8.9 ± 0.001, 50.9 ± 0.002, 12.6 ± 0.01 and 4.5 ± 0.001 μg QEs/mg, respectively (Figure 2). The total phenolic and flavonoid contents of extracts were found in the following order: hexane > ethyl acetate > methanol > water. The hexane extract was the richest, both in phenolic and flavonoid content, while the water extract was the poorest.

Fatty acid composition
A total of eight fatty acids, ranging from C 15:0 to C 24:0 were detected in R. luteolus (Table S2). Major fatty acids were linoleic acid (45.8%), stearic acid (23.7%) and oleic acid (16.2%). It is known that linoleic acid is the precursor of 1-octen-3-ol (the alcohol of mushroom). It is the main aromatic compound that contributes the mushroom flavour (Heleno et al. 2009). The percentages of other fatty acids such as C 15:0 , C 16:1 , C 16:0 , C 20:4 and C 24:0 were found to be less than 7%. The total unsaturation was calculated as 68.2%, and the amount of unsaturated fatty acids are directly proportional to the nutritional value of mushroom. Linoleic and oleic acid ratio (L/o) which could provide an important criterion from a chemotaxonomic viewpoint was calculated as 2.80.

Conclusion
We describe here the phytochemical investigation of R. luteolus. The anticholinesterase activities of the extracts and compounds (1-3) were also investigated for the first time. Hexane extract showed the highest antioxidant and anticholinesterase activities. Compound 3 exhibited good anticholinesterase activity against BChE. R. luteolus is a good source for food because it contains a major portion of linoleic acid (45.8%) and other bioactive compounds such as vitamin D and fumaric acid.

Supplementary material
Experimental details relating to this paper are available online, alongside Tables S1-S4 and Figures S1-S7.