Fatty Acids from Ganoderma Lucidum Spores: Extraction, Identification

applied sciences

Article Fatty Acids from Ganoderma lucidum Spores: Extraction, Identiﬁcation and Quantiﬁcation

Maria Michela Salvatore 1 , Alessandro Elvetico 1, Monica Gallo 2 , Francesco Salvatore 1, Marina DellaGreca 1 , Daniele Naviglio 1,* and Anna Andolﬁ 1,*

1 Department of Chemical Sciences, University of Naples ‘Federico II’, 80126 Naples, Italy; [email protected] (M.M.S.); [email protected] (A.E.); [email protected] (F.S.); [email protected] (M.D.) 2 Department of Molecular Medicine and Medical Biotechnology, University of Naples ‘Federico II’, 80131 Naples, Italy; [email protected] * Correspondence: [email protected] (D.N.); andolﬁ@unina.it (A.A.); Tel.: +39-081-2539179 (A.A.)

Received: 19 May 2020; Accepted: 27 May 2020; Published: 5 June 2020

Featured Application: This study sought to contribute to the improvement of the extraction, identification and quantification of the fatty acids component in Ganoderma lucidum spores in order to find simple and non-expensive tools for laboratory studies aiming at future applications, such as the easy comparison of lipids produced by different strains at diverse growth conditions.

Abstract: Ganoderma lucidum is a rich and promising source of bioactive compounds capable of positively influencing the human health. For this reason, G. lucidum is the target of vigorous investigational activities, focusing on the extraction of substances with potential biomedical applications, as well as on the improvement of analytical methodologies for their screening. In the present study, three different extraction processes (i.e., extraction with n-hexane, in Soxhlet apparatus with n-hexane and reflux with chloroform) were employed to evaluate the lipid content of G. lucidum spores. The experimental data from the produced extracts were compared with ones from the commercial liquid extract obtained with supercritical CO2 extraction of G. lucidum spores. To achieve this goal, the gas chromatography-mass spectrometry (GC-MS) methodology was optimized for an accurate and reliable identification and quantification of fatty acids in triglycerides of G. lucidum spores. Significant amounts of oleic and palmitic acids were found in the examined extracts. In particular, our findings show that economical and convenient extraction procedures, which are simple, low cost and no need for expensive tools, allow to produce extracts with a lipid composition similar to the one obtained with hard strategies of extraction.

Keywords: Ganoderma; bioactive compounds; GC-MS; triglycerides; mushroom; fatty acids

1. Introduction Ganoderma lucidum is a saprophytic mushroom belonging to the Basidiomycetes phylum, which has been extensively used in oriental medical culture for the general promotion of health and longevity. In fact, it is considered the “immortality mushroom” in China, Japan, Korea and other Asian countries [1,2]. It has received an increasing attention in mycological research for the biomedical applications of the fruiting bodies, cultured mycelia and spores [3]. In fact, it has been used in the treatment of diverse human diseases including allergy, insomnia, inﬂammation, fungal, bacterial and viral infections, hypertension and cancer [4–6]. The numerous pharmacological eﬀects of this mushroom may be related to its ability to produce various bioactive substances, such as proteins, polysaccharides,

Appl. Sci. 2020, 10, 3907; doi:10.3390/app10113907 www.mdpi.com/journal/applsci Appl. Sci. 2020, 10, 3907 2 of 12 triterpenes, nucleosides, triglycerides and mineral salts [7–11]. Although the bioactivity of the spores may be higher than that of fruiting body [12], spores were recognized and utilized only in the last century [13]. For this reason, the spores of G. lucidum have been rarely studied, but over the last decade this important source of bioactive compounds is receiving an increasing attention from the scientific community [14]. The bioavailability of active compounds in the spores depends on the status of sporoderm. In fact, the spores are protected by a double chitin wall and this polysaccharidic barrier prevents a correct digestion by the human and, consequently, the absorption of the macro- and micronutrients present in the spores. Hence, a crucial step is the break of the sporoderm because there are fewer pharmacological effects observed when the sporoderm is not broken [15]. With the development of spore collection, sporoderm-broken technology and modern analytical technology, many constituents were identified in G. lucidum spores. Moreover, the quality and content of bioactive compounds vary from strain-to-strain, and may be affected by culture conditions and processing procedures [4]. Enriching the existing literature with new data on compounds produced by different isolates and on the strategy to extract them, especially from spores, may be valuable for the use of G. lucidum in the biomedical field. Although a few investigators have already studied the fatty acids in the triglycerides from G. lucidum [16], in general referred to the fruiting body [17], there are few information on the extraction processes. In the present study, three different processes were employed in order to perform a reliable and accurate identification and quantification of the lipid content of the spores of G. lucidum. Nuclear magnetic resonance (NMR) spectroscopy investigation was conducted on oily extracts of G. lucidum obtained in Soxhlet apparatus with n-hexane, to reflux with chloroform and with n-hexane at room temperature. The data of the extraction processes were compared with the data obtained from the commercially available liquid extract from supercritical CO2 extraction of G. lucidum spores. Triglycerides were submitted to a common transesterification reaction in order to obtain fatty acid methyl esters (FAMEs) and finally identified and quantified by gas chromatography-mass spectrometry (GC-MS). Particularly relevant in the present work is the optimization of the GC-MS methodology to provide the best peak resolution and minimum run time for FAMEs identification and quantification.

2. Materials and Methods

2.1. General Experimental Procedures

NMR spectra were recorded at 400 MHz in CDCl3 on a Bruker spectrometer (AscendTM400) (Rheinstetten, Germany). All reagents and solvents were analytical grade and purchased from Carlo Erba (Milan, Italy), Sigma-Aldrich (Saint Louis, MO, USA) and Fluka (Buchs, Switzerland). The GC-MS analysis for fatty acids methyl esters (FAMEs) was performed with an Agilent 6850 GC equipped with an HP-5MS capillary column (5% phenyl methyl polysiloxane stationary phase) and the Agilent 5973 Inert MS detector (Milan, Italy) (used in the scan mode). The ﬂow rate of helium carrier 1 gas was kept at 1 mL min− . Solutions of standard compounds were prepared in n-hexane at a concentration range of about 1 25–150 mg L− and analyzed by GC-MS. Methyl ester of nonanoic acid (Sigma-Aldrich, Saint Louis, MO, USA) was used as internal standard (IS), a stock solution of which in n-hexane at concentration of 1 g L 1 was prepared and stored at 20 C. − − ◦ 2.2. Spore Source The spores of G. lucidum were supplied by ItalianWay (Verbano, Italy) and cultivated using the traditional method within the DaXingAnLing natural park region, in China. The manufacturing company guaranteed 95% spore opening. Liquid extract of spores of G. lucidum obtained using supercritical CO2 extraction was supplied by ItalianWay. Appl. Sci. 2020, 10, 3907 3 of 12

2.3. Extraction of the Lipid Component

2.3.1. Extraction with n-Hexane 20 g of spores of G. lucidum were suspended in 50 mL of n-hexane, at room temperature, under magnetic stirring and in the dark for 30 min. The suspension was ﬁltered and the exhausted was recovered to repeat the extraction process in the same condition. The organic phases were combined and the solvent evaporated under reduced pressure.

2.3.2. Extraction in Soxhlet Apparatus with n-Hexane 100 g of spores of G. lucidum were placed in a thimble of ﬁlter paper and extracted with 450 mL of n-hexane for 18 h for a total of 720 cycles. At the end, the organic phase was ﬁltered and evaporated under reduced pressure. The experiment was carried out in duplicate.

2.3.3. Reflux Extraction with Chloroform 1 g of spores of G. lucidum were suspended in 30 mL of chloroform. The mixture was heated to reflux for 2 h, set to cool and filtered under reduced pressure. The exhausted was recovered and extracted in the same condition. The organic phases were then combined, and the solvent evaporated under reduced pressure. The experiment was carried out in duplicate.

2.4. Sample Preparation NMR data suggest the presence, as main components, of triglycerides in the commercial sample of G. lucidum and in the extracts obtained as reported in previous sections. The triglycerides were submitted to a transesteriﬁcation reaction with methanol in the presence of a basic catalyst (i.e., potassium hydroxide), as described by [18]. In this way, the fatty acids chains are released from glycerol skeleton and recombine with the alcohol to yield fatty acids methyl esters (FAMEs).

2.5. Qualitative and Quantitative Analysis of the Lipid Components FAMEs were identified via GC-MS by Kovats non-isothermal retention indices (KI) and comparing their mass spectra with those present in databases by employing the National Institute of Standards and Technology (NIST) Mass Spectral Search Program v.2.0 g which explores the NIST 14 Mass Spectral library (2014) [19]. Kovats indices of FAMEs were calculated by the Kovats equation, using the standard n-alkane mixture in the range C7-C40 (Sigma-Aldrich) [20]. The FAME quantification was carried out with a calibration curve five points in the range of about 1 25–150 mg L− using methyl ester of nonanoic acid as internal standard at the constant concentration of 1 122.4 mg L− [20]. The concentrations of individual FAME in each sample were converted to percentage contributions of the total FAMEs [21].

2.6. Optimization of GC-MS Method The optimization of instrumental parameters was performed using standard mixture of 37 FAMEs, purchased by Sigma-Aldrich, and added with internal standard (methyl ester of nonanoic acid). In order to avoid compounds co-elution which may provide identification and quantification problems, the separation of FAMEs in the chromatogram was optimized. The optimal injector temperature was 250 ◦C and during the run a temperature ramp raised the column temperature from 50 ◦C to 240 ◦C: the 1 initial column temperature was 50 ◦C and held for 2 min, ramped to 180 ◦C at the rate of 10 ◦C min− 1 and held 5 min and then finally increased to 240 ◦C at the rate of 5 ◦C min− , held 25 min. The ionization was carried out with the electron impact (EI) ion source at 70 eV and at 200 ◦C. The quadrupole mass filter was kept at 250 ◦C and, in the scan mode, was programmed from 45 to 550 m/z with a frequency of 3.9 Hz. The detectability of compounds in the matrix is significantly improved with deconvolution. Appl. Sci.Appl. 2020 Sci., 102020, x FOR, 10, 3907 PEER REVIEW 4 of 12 4 of 13

3. ResultsThis function is supported by the NIST deconvolution program automated mass spectral deconvolution The& identification extraction system processes (AMDIS) of [Ganoderma22,23]. lucidum spores gave oily residues, as reported in experimental3. Results section. The extracts weights obtained by extraction with n-hexane, in Soxhlet apparatus withThe extractionn-hexane processes and reflux of extractionGanoderma lucidumwith chloroformspores gave are, oily respectively, residues, as reported5.3 ± 0.31 in g (yield = 26.43%),experimental 32.7 ± 0.24 section. g (yield The extracts = 32.65%) weights and obtained 0.27 by± 0.02 extraction g (yield with n=-hexane, 26.83%). in Soxhlet The oily apparatus extracts were submittedwith ton-hexane spectroscopic and reflux investigations extraction with chloroformvia NMR. are,The respectively, 1H NMR 5.3spectra0.31 showed g (yield = typical26.43%), signals of ± triglycerides32.7 0.24containing g (yield = chains32.65%) of and saturated 0.27 0.02 and g (yield unsaturated= 26.83%). Thefatty oily acids extracts [24]. were Moreover, submitted even to in the ± ± 1 commerciallyspectroscopic available investigations liquid extract via NMR. of TheG. lucidumH NMR.spectra In fact, showed their typicalproton signals spectra of triglyceridesshowed the signals containing chains of saturated and unsaturated fatty acids [24]. Moreover, even in the commercially related availableto fatty acid liquid residues extract of atG. δ: lucidum 5.46–.5.33 In fact, (m, their -CH proton=CH spectra-); 2.78 showed (brt, J = the 6.6, signals Hz, =CH related-C toH fatty2-CH=); 2.37– 2 2 2 2 2.29 (m,acid -O residues-CO-CH at);δ :2.16 5.46–5.33–1.70 (m,(m, -C CHH=C-CH=CHH-); 2.78- (brt,); 1.67J =–6.6,1.62 Hz, (m,= CH-C-O-COH2--CHCH=-);C 2.37–2.29H -); 1.39 (m,–1.21 (m, - (CH2)n--O-CO-C); 0.93–0.88H2); 2.16–1.70(m, CH3 (m,CH C2H-)2 and-CH= ofCH-); residue 1.67–1.62 of glycerols (m, -O-CO-CH triesterified2-CH2-); 1.39–1.21 at δ: 5.28 (m, (tt, -(CH J 2=)n 4.0,-); 6.0 Hz, ROCH20.93–0.88-CHOR’- (m,CH C2OR”);H3CH2 4.30-) and (dd, of residueJ = 4.0, of12.0 glycerols Hz, ROC triesterifiedH2A-CHOR at δ’:-C 5.28H2A (tt,OR”);J = 4.154.0, 6.0(dd, Hz, J = 6.0, 12.0 ROCH -CHOR’-CH OR”); 4.30 (dd, J = 4.0, 12.0 Hz, ROCH2A-CHOR’-CH OR”); 4.15 (dd, J = 6.0, Hz, ROCH2B-2CHOR’-CH22BOR”) (Figure 1). 2A 12.0 Hz, ROCH2B-CHOR’-CH2BOR”) (Figure1).

1 Figure 1. H NMR spectra recorded at 400 MHz in CDCl3 of extracts obtained from Ganoderma lucidum spores: (A) commercial liquid extract; (B) by n-hexane; (C) by n-hexane in Soxhlet apparatus; (D) by Figure 1. 1H NMR spectra recorded at 400 MHz in CDCl3 of extracts obtained from Ganoderma lucidum reﬂux in CHCl spores: (A) commercial3. liquid extract; (B) by n-hexane; (C) by n-hexane in Soxhlet apparatus; (D) by

reflux inTriglycerides CHCl3. in the oily extracts of G. lucidum were subjected to a common transesterification reaction, called alcoholysis. In transesterification, triglycerides react with methanol in the presence of a Triglyceridesbasic catalyst in to formthe oily a mixture extracts of glycerol of G. andlucidum fatty acidwere methyl subjected esters (FAMEs)to a common [18]. FAMEs transesterification were reaction,identified called alcoholysis. and quantified In via transesterification, GC-MS in all samples. triglycerides react with methanol in the presence of For accurate identification and quantification of FAMEs, the GC-MS method was optimized for the a basic bestcatalyst peak resolutionto form a and mixture minimum of glycerol run time usingand afa 37-componentstty acid methyl FAMEs esters standard (FAMEs) mixture. [18]. As FAMEs can were identifiedbeseen and from quantified Figure2, thevia optimizedGC-MS in method all samples. (reported in a previous section) provides separation on For accurate identification and quantification of FAMEs, the GC-MS method was optimized for the best peak resolution and minimum run time using a 37-components FAMEs standard mixture. As can be seen from Figure 2, the optimized method (reported in a previous section) provides separation on the base line of all components of the standard FAMEs mixture. In Table 1 are exposed retention data both in the form of retention times (tr) and Kovats retention indices (KI) of the twelve FAMEs obtained from G. lucidum spores. Appl. Sci. 2020, 10, 3907 5 of 12 the base line of all components of the standard FAMEs mixture. In Table1 are exposed retention data both in the form of retention times (tr) and Kovats retention indices (KI) of the twelve FAMEs obtained from G. lucidum spores. Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 13

5 x 106 C12:0

4.5 x 106 C10:0

4 x 106

C14:0

- C16:0

3.5 x 106

C18:3n

C20:0

C18:0

C13:0 9

6 - C11:0

3 x 10 5

C8:0

C18:1n

C14:1n

Abundance 6

6 - C22:0

2.5 x 10 C20:3n

C20:1n

C20:4n

C21:0

C18:3n

C17:1n C15:1n

6 C16:1n

C15:0

6 C20:4n

2 x 10 C20:2n

C22:1n

C17:0

C18:2n

C20:5n 6

1.5 x 106 -

C24:0

C23:0

C22:2n -

6 C6:0

1 x 10 C24:1n

5 x105

10 15 20 25 30 35 40 Time (min) Figure 2. TotalTotal ion chromatogram (TIC) of a standard mixture of fatty acid methyl esters (FAMEs) obtained with the optimized method.

Table1.For quantificationPerformance dataof detected of the optimized FAMEs, gas the chromatography-mass internal standard spectrometry calibration (GC-MS) method separation (ISTD) was and quantiﬁcation method. employed, as is usual in quantitative GC-MS work.

To this end, the standard FAMEs mixtureIntercept was diluted with n-hexane1 to2 prepareLOD five calibrationLOQ Name * tr (min) KI Slope (b sb) L mg− R (a sa) ± (mg L 1) (mg L 1) solutions containing accurately known concentrations± of each target analyte. Furthermore,− − each C14:0 Myristic acid 17.42 1720 0.000 0.024 0.01306 0.00032 0.998 6.0 18.3 calibration solution was made 122.4 mg L-1 of ±methyl ester of nonanoic± acid internal standard (IS). C15:0 – 19.58 1821 0.016 0.011 0.00707 0.00015 0.998 5.1 15.6 For each target analyte, a calibration −plot wa± s constructed± by reporting (at each calibration level) C16:1n-7 Palmitoleic acid 22.00 1916 0.0095 0.0093 0.00665 0.00012 0.999 4.7 14.3 the area of its chromatographic peak, normalized− ± with the area± of the IS peak, as a function of the C16:0 Palmitic acid 22.51 1936 0.005 0.024 0.01408 0.00026 0.998 5.6 17.1 known concentration of the analyte (mg −L-1). The± signal of a reagent± blank containing only the IS in C17:0 Margaric acid 24.62 2024 0.0033 0.0094 0.00481 0.00012 0.998 6.4 19.4 n-hexane was also measured as control during− ± calibration. ± C18:2n-6 Linoleic acid 25.18 2094 0.0148 0.0093 0.00686 0.00011 0.999 4.5 13.6 − ± ± C18:1n-9Visual inspection Oleic acid of each 26.81 calibration 2124 plot0.018 readily0.045 showed 0.02674 a 0.00057linear dependence 0.998 of 5.6 the normalized 16.8 − ± ± signalC18:0 from the Stearic concentration. acid 27.14 In fact, 2140 calibration 0.000 0.025 data could 0.0149 be0.00033 excellently 0.998 interpolated 5.6 with 16.9 a least ± ± -1 squareC20:1n-9 regression Gadoleic line acid (y = 30.24a + bx) in 2299 which0.021 a represents0.019 0.00793the intercept0.00025 and 0.996b the slope 7.9 (L mg 24.0) of the − ± ± leastC20:0 square Arachidic line through acid 30.71 the calibration 2326 0.002 points.0.026 This 0.00838 is easily0.00046 deduced 0.988 from the 10.2 determination 31.0 ± ± coefficientC22:0 (R Behenic2) reported acid in 34.47Table 1 2523which is 0.006 very0.035 close to 0.01417 one. 0.00045 0.996 8.2 24.7 ± ± C24:0In Table Lignoceric 1 are acidalso reported 40.17 2724intercepts 0.030 and0.030 slopes 0.02064deduced0.00050 from the 0.997 twelve 4.8calibration 14.5 plots ± ± together with the corresponding* fatty acids standard were analyzed deviations via GC-MS (respectively,as methyl esters. 푠푎 and 푠푏 ) which were calculated from the calibration data according to standard statistical procedures [25]. ForThe quantiﬁcationstandard deviation of detected of the intercept, FAMEs, 푠 the푎, is internal then used standard to calculate calibration the method method detection (ISTD) limit was employed,(LOD) according as is usual to the in formula: quantitative LOD GC-MS= 3.3 × work.푠푎⁄푏; and the quantification limit (LOQ) according to the formula:To this end, LOQ the= 10 standard× 푠푎⁄푏 FAMEs[25]. mixture was diluted with n-hexane to prepare ﬁve calibration solutions containing accurately known concentrations of each target analyte. Furthermore, Table 1. Performance data of the optimized1 gas chromatography-mass spectrometry (GC-MS) each calibration solution was made 122.4 mg L− of methyl ester of nonanoic acid internal standard (IS). separation and quantification method.

Slope~~ Intercept ~ LOQ~~ tr LOD~~~ Name * KI ~~~ (풃 ± R2 ~ (min) (mg L-1) -1 (풂 ± 풔풂) 풔풃) ~~~ (mg L ) 퐋 퐦퐠−ퟏ Appl. Sci. 2020, 10, x FOR PEER REVIEW 6 of 13

0.000 ± 0.01306 ± C14:0 Myristic acid 17.42 1720 0.998 6.0 18.3 0.024 0.00032 -0.016 ± 0.00707 ± C15:0 – 19.58 1821 0.998 5.1 15.6 0.011 0.00015 Palmitoleic -0.0095 ± 0.00665 ± C16:1n-7 22.00 1916 0.999 4.7 14.3 acid 0.0093 0.00012 -0.005 ± 0.01408 ± C16:0 Palmitic acid 22.51 1936 0.998 5.6 17.1 0.024 0.00026 -0.0033 ± 0.00481 ± Appl.C17:0 Sci. 2020 , 10,Margaric 3907 acid 24.62 2024 0.998 6.4 19.46 of 12 0.0094 0.00012 -0.0148 ± 0.00686 ± C18:2n-6 Linoleic acid 25.18 2094 0.999 4.5 13.6 For each target analyte, a calibration plot was0.0093 constructed 0.00011 by reporting (at each calibration level) the area of its chromatographic peak, normalized-0.018 with ± the area0.02674 of the ± IS peak, as a function of the C18:1n-9 Oleic acid 26.81 21241 0.998 5.6 16.8 known concentration of the analyte (mg L− ). The0.045 signal of a reagent0.00057 blank containing only the IS in n-hexane was also measured as control during calibration.0.000 ± 0.0149 ± C18:0 Stearic acid 27.14 2140 0.998 5.6 16.9 Visual inspection of each calibration plot readily0.025 showed 0.00033 a linear dependence of the normalized signal from the concentration. In fact, calibration- data0.021 could ± 0.00793 be excellently ± interpolated with a least C20:1n-9 Gadoleic acid 30.24 2299 0.996 7.9 24.01 square regression line (y = a + bx) in which a represents0.019 the0.00025 intercept and b the slope (L mg− ) of the least square lineArachidic through the calibration points.0.002 This ± is easily0.00838 deduced ± from the determination C20:0 2 30.71 2326 0.988 10.2 31.0 coefficient (R ) reportedacid in Table1 which is very close0.026 to one. 0.00046 In Table1 are also reported intercepts and slopes0.006 ± deduced 0.01417 from ± the twelve calibration plots C22:0 Behenic acid 34.47 2523 0.996 8.2 24.7 together with the corresponding standard deviations0.035 (respectively, 0.00045sa and sb) which were calculated from the calibrationLignoceric data according to standard statistical0.030 ± procedures0.02064 ± [ 25]. C24:0 40.17 2724 0.997 4.8 14.5 The standard deviationacid of the intercept, sa, is0.030 then used to0.00050 calculate the method detection limit (LOD) according to the formula: LOD = 3.3 sa/b; and the quantification limit (LOQ) according to * fatty acids were analyzed× via GC-MS as methyl esters. the formula: LOQ = 10 sa/b [25]. × InIn FigureFigure3 3are are compared compared total total ion ion chromatograms chromatograms (TICs) (TICs) of the of three the three extracts extracts of G. lucidum of G. lucidumspores sporesobtained obtained in this in study this withstudy di withfferent dif processesferent processes (Figure (Figure3A–C) and3A, theB, C) commercial and the commercial liquid extract liquid of G. lucidum spores obtained using supercritical CO extraction (Figure3D). extract of G. lucidum spores obtained using supercritical2 CO2 extraction (Figure 3D).

A) B)

1.4 x 107 1.2 x 107 1.2 x 107 1 x 107 1 x 107 8 x 106

8 x 106

Abundance Abundance 6 x 106 6 x 106 4 x 106 4 x 106

2 x 106 2 x 106

0 0 5 10 15 20 25 30 35 40 45 50 55 5 10 15 20 25 30 35 40 45 50 55 Time (min) Time (min) C) D)

1.4 x 107 1.2 x 107 1.2 x 107 1 x 107 1 x 107 8 x 106 8 x 106

Abundance 6 x 106 Abundance 6 x 106

6 4 x 10 4 x 106

2 x 106 2 x 106

0 0 5 10 15 20 25 30 35 40 45 50 55 5 10 15 20 25 30 35 40 45 50 Time (min) Time (min) Figure 3. TotalTotal ion ion chromatograms chromatograms (TICs) (TICs) profiles profiles of of FAMEs obtained after transesterificationtransesterification of triglyceridestriglycerides contained contained in in samples samples of of Ganoderma lucidum extracted with didifferentfferent processes: ( (AA)) extraction wi withth n--hexane;hexane; ( (B)) extraction extraction in in Soxhlet Soxhlet apparatus apparatus with with nn--hexane;hexane; ( (CC)) reflux reflux extraction extraction with with chloroformchloroform;; ( D) commercial oil .

An example of annotated TIC relative to fatty acids contained in samples of G. lucidum spores is shown in Figure4. Appl. Sci. 2020, 10, x FOR PEER REVIEW 7 of 13

Appl.An Sci. 2020example, 10, 3907 of annotated TIC relative to fatty acids contained in samples of G. lucidum spores7 of is 12 shown in Figure 4.

4 x 106 C16:0

C18:1n-9 C18:0

2 x 106 C16:1n-7 IS C20:0 C18:2n-6 C15:0 C14:0 C17:0 C20:1n-9 C22:0 C24:0 0 10 15 20 25 30 35 40

1.2 x 107

1 x 107

8 x 106 Abundance 6 x 106

4 x 106

2 x 106

0 5 10 15 20 25 30 35 40 45 50 55 Time (min) FigureFigure 4. 4. RepresentativeRepresentative annotated annotated t totalotal ion ion chromatogram chromatogram (TIC) (TIC) of of FAMEs FAMEs in in samples samples of of GanodermaGanoderma lucidumlucidum spores.spores.

TableTable2 2 shows shows complete complete lipid lipid profiles profiles corresponding corresponding to to the the fatty fatty acid acid chains chains of triglyceridesof triglycerides in the in theform form of percentage of percentage concentrations concentrations and and the the corresponding corresponding standard standard deviations deviations (SD). (SD). Reported SDs were evaluated by first calculating the standard deviation of each target analyte Table 2. Fatty acids composition (percentage of FAMEs) contained in samples of Ganoderma lucidum concentration by using the standard deviation 푠푦/푥 of the signal about the regression line. 푠푦/푥 is a statisticobtained which with is commonly different extraction called “residual processes. deviation about the regression” and which can be readily obtained from calibration dataExtraction under with the assumptionExtraction in of homoscedasticReflux Extraction data with [26].Commercial The standard Liquid Name 1 n-Hexane (% SD 2) Soxhlet (% SD) Chloroform (% SD) Extract (% SD) deviations on analyte concentrations± are finally converted± to the reported± standard deviations± on C14:0 Myristic acid 0.14 0.03 0.24 0.06 0.14 0.03 0.09 0.04 percentage by a standard uncertainty± propagation formula.± ± ± C15:0 – 0.55 0.03 1.01 0.05 0.61 0.03 0.51 0.05 All analyzed samples contain± a significant amount± of oleic (C18:1n±-9) and palmitic (C16:0)± acids C16:1n-7 Palmitoleic acid 1.4 0.15 5.83 0.07 4.3 0.4 2.3 0.3 in the range from 60.1% to 68.4%± and from 16.3% to± 21.3%, respectively.± In the examined± samples, C16:0 Palmitic acid 18.2 0.15 21.3 0.24 17.7 0.21 16.3 0.2 stearic (C18:0), palmitoleic (C16:1n± -7) and linoleic± (C18:2n-6) acids ± are present in much± lower C17:0 Margaric acid 0.55 0.02 0.80 0.03 0.39 0.02 0.33 0.03 amounts (< 10%). In addition, a small± amount of lignoceric± (C24:0), myristic± (C14:0) and± arachidic C18:2n-6 Linoleic acid 3.19 0.03 3.00 0.03 2.68 0.03 9.5 0.10 (C20:0) acids were detected in all± the samples (< 1%).± Interestingly,± our findings show± that the C18:1n-9 Oleic acid 68.4 0.97 60.1 0.84 66.9 0.90 65.4 0.91 commercially available liquid extract± and the extracts± of spores of G. lucidum± produced in ±this study C18:0 Stearic acid 5.77 0.03 5.94 0.04 5.52 0.04 4.38 0.03 employing different processes, have± similar lipid contents± as revealed by± NMR and GC-MS± data. C20:1n-9 Gadoleic acid 0.52 0.02 0.61 0.03 0.54 0.02 0.45 0.03 ± ± ± ± C20:0Table 2. ArachidicFatty acids acid composition 0.45 (percentage0.02 of FAMEs) 0.56 0.05 contained in 0.50 samples0.03 of Ganoderma 0.42 lucidum0.04 ± ± ± ± C22:0obtained Behenicwith different acid extraction 0.48 0.02processes. 0.41 0.04 0.47 0.02 0.28 0.03 ± ± ± ± C24:0 Lignoceric acid 0.30 0.02 0.11 0.03 0.32 0.01 0.09 0.03 ± Extraction± Reflux± Commercial± 1 fatty acids analyzedExtraction via GC-MS with as methyl esters; 2 SD= standard deviation. in Extraction with Liquid Name 1 n-hexane~~~ Soxhlet~~ Chloroform~~ Extract~~~ Reported SDs were evaluated(% by first± SD calculating 2) the standard deviation of each target analyte ~ ~ (% ± SD) concentration by using the standard deviation sy/x of the signal about the regression line. sy/x is Appl. Sci. 2020, 10, 3907 8 of 12 a statistic which is commonly called “residual deviation about the regression” and which can be readily obtained from calibration data under the assumption of homoscedastic data [26]. The standard deviations on analyte concentrations are finally converted to the reported standard deviations on percentage by a standard uncertainty propagation formula. All analyzed samples contain a significant amount of oleic (C18:1n-9) and palmitic (C16:0) acids in the range from 60.1% to 68.4% and from 16.3% to 21.3%, respectively. In the examined samples, stearic (C18:0), palmitoleic (C16:1n-7) and linoleic (C18:2n-6) acids are present in much lower amounts (<10%). In addition, a small amount of lignoceric (C24:0), myristic (C14:0) and arachidic (C20:0) acids were detected in all the samples (<1%). Interestingly, our findings show that the commercially available liquid extract and the extracts of spores of G. lucidum produced in this study employing different processes, have similar lipid contents as revealed by NMR and GC-MS data. Comparing the processes adopted to extract the spores (Figure5), some interesting results can be observed. The extract of spores of G. lucidum obtained through the Soxhlet apparatus in n-hexane has the highest amount of palmitic acid (C16:0, 21.3%), but also the lowest amount of oleic acid (C18:1n-9, 60.1%). Unlike the examined extracts, the commercial oil is particularly rich in linoleic acid (C18:2n-6,

9.5%),Appl. Sci. which 2020, 10 is, x the FOR only PEER polyunsaturated REVIEW fatty acid detected. 9 of 13 68.4

68 66.9 65.4 Extraction with n-hexane 66 Extraction in Soxhlet with n-hexane 64

Reflux extraction with chloroform 60.1 62 Commercial liquid extract 60

22 21.3

19 18.2

18 17.7

17 16.3

Abundance % Abundance 12

10 9.5

5.94 5.83

6 5.77 5.52

5 4.3 4.38

3.19 3.00

3 2.7 2.3

1.4 1.01

1 0.8

0.61

0.52

0.55

0.50

0.55 0.51 0.54

0.56

0.61

0.45

0.47

0.48 0.41

0.39

0.45 0.32

0.42

0.28

0.33 0.30

0.14 0.24

0.14

0.09

0.11 0.09

C14:0 C15:0 C16:1n-7 C16:0 C17:0 C18:2n-6 C18:1n-9 C18:0 C20:1n-9 C20:0 C22:0 C24:0

FigureFigure 5.5. Graphical illustration of fatty acids profiles proﬁles of lipids from spores of G. lucidum.

TheThe contents contents of of the the saturated saturated fatty acids (SFAs), monounsaturated fatty fatty acids acids (MUFAs) (MUFAs) and and polyunsaturatedpolyunsaturated fattyfatty acidsacids (PUFAs)(PUFAs) werewere calculatedcalculated forfor eacheach extractextract (Table(Table3 3).).

Table 3. Groups of fatty acids from the composition of samples of Ganoderma lucidum obtained with different extraction processes.

% Total FA % SFA ± SD % MUFA ± % PUFA ±

1 2 SD SD Extraction with n-hexane 26.43 26.4 ± 0.16 70.3 ± 0.98 3.19 ± 0.03 Extraction in Soxhlet 32.65 30.4 ± 0.27 66.5 ± 0.84 3.00 ± 0.03 Reflux extraction with 26.83 25.7 ± 0.22 71.7 ± 0.99 2.68 ± 0.03 chloroform Commercial liquid extract – 22.0 ± 0.22 68 ± 1.00 9.5 ± 0.1 1 values reported calculated from the extraction yield; 2 SD = standard deviation.

MUFAs are the most represented fatty acids in all examined samples, which account for 60%– 70% of the total fatty acids. Meanwhile, SFAs are present in approximately 20%–30% of the total fatty acids. As anticipated, the only PUFA present in the samples is linoleic acid contained in the greater amount (9.5%) in the commercial oil of G. lucidum (Figure 6). Appl. Sci. 2020, 10, 3907 9 of 12

Table 3. Groups of fatty acids from the composition of samples of Ganoderma lucidum obtained with diﬀerent extraction processes.

% Total FA 1 % SFA SD 2 % MUFA SD % PUFA SD ± ± ± Extraction with n-hexane 26.43 26.4 0.16 70.3 0.98 3.19 0.03 ± ± ± Extraction in Soxhlet 32.65 30.4 0.27 66.5 0.84 3.00 0.03 ± ± ± Reﬂux extraction with chloroform 26.83 25.7 0.22 71.7 0.99 2.68 0.03 ± ± ± Commercial liquid extract – 22.0 0.22 68 1.00 9.5 0.1 ± ± ± 1 values reported calculated from the extraction yield; 2 SD = standard deviation.

MUFAs are the most represented fatty acids in all examined samples, which account for 60%–70% of the total fatty acids. Meanwhile, SFAs are present in approximately 20%–30% of the total fatty acids. As anticipated, the only PUFA present in the samples is linoleic acid contained in the greater amount (9.5%)Appl. Sci. in 20 the20, 10 commercial, x FOR PEER oilREVIEW of G. lucidum (Figure6). 10 of 13

SFA MUFA PUFA

71.7

70.3

66.5

30.4

Abundance % Abundance

25.4

26.83

22.0

9.5

3.19

3.00 2.68

Extraction with n--hexane Extraction in Soxhlet Reflux extraction with Commercial liquid extract with n-hexane chloroform

FigureFigure 6.6. Graphical illustration of groups of fatty acids from the composition composition of of spores spores of of G.G. lucidum. lucidum.

4.4. Discussion InIn orientaloriental countries,countries, therethere is a long and historical tradition of of using using G.G. lucidum lucidum asas a a folk folk remedy remedy forfor health health promotion. promotion. I Itsts pharmacological pharmacological activ activitiesities [3] [3] are are recognized recognized by by the the American American Herbal Herbal PharmacopoeiaPharmacopoeia andand TherapeuticTherapeutic Compendium Compendium [ 27[27].]. In In fact, fact, this this mushroom mushroom produces produces a a large large variety variety of bioactiveof bioactive compounds, compounds, representing representing a “factory” a “factory” of compounds of compounds to be to used be in used biomedical in biomedical field [ 11 field,28]. Currently,[11,28]. Currently, most therapeutic most therapeutic formulations formulations mainly mainly involve involve the use the of use the ofG. the lucidum G. lucidumfruiting fruit bodying andbody mycelium, and mycelium, but in but recent in recent years, years, the spores the spores are becoming are becoming increasingly increasing interesting,ly interesting, thanks thanks to their to beneficialtheir beneficial effects effects and innovative and innovative extraction extraction techniques techniques [4,14]. [4,14].The main The components main components of G. lucidum of G. sporeslucidum are spores triterpenes, are triterpenes, polysaccharides polysaccharides and triglycerides and triglycerides [14]. [14]. InIn thisthis study,study, analysesanalyses ofof lipidlipid compositioncomposition of G. lucidum spores were performed comparing comparing didifferentfferent extractionextraction processes. processes. To To achieve achieve this this goal, goal, we we optimized optimized the the analytical analytical methodology methodology based based on theon identification the identification and quantification and quantification of FAMEs of FAMEs by GC-MS. by GC The-MS. optimization The optimization of the method of the required method somerequired adjustment some adjustment of the temperature of the temperature ramp of GC-MS ramp of to GC obtain-MS optimalto obtain conditions, optimal conditions, best peak resolutionbest peak andresolution the minimum and the runminimum time. run time. Interestingly, our findings show that the procedures used (i.e., extraction with n-hexane, extraction in Soxhlet with n-hexane, reflux extraction with chloroform) are economical and convenient, in terms of equipment costs, management of the instrumentation and request for qualified personnel for their use. Furthermore, they are easy to implement and allow to produce extracts with a lipid composition similar to the one detected with hard strategies. In this respect, a commercially available liquid extract of G. lucidum obtained with supercritical CO2 extraction was also analyzed. Furthermore, Liu and co-workers [16] reported the extraction and detection of fatty acids in triglycerides from spores of G. lucidum obtained with an extraction process conducted ultrasonically with CH2Cl2 for 2 h. Our findings are in accordance with the constituents previously detected in spores of G. lucidum [16], but the oleic acid amount is generally higher in the extracts produced in this work, in particular when the spores were extracted with reflux with chloroform (66.9%) or extracted with n-hexane (68.4%). The beneficial effects of spores of G. lucidum may be related to the synergic action of the fatty acids in triglycerides and in particular to the high abundance of oleic acid, which properties have been extensively reviewed [29]. In fact, oils rich in oleic acid is supposed to present diverse physiological functions confirmed by several studies on its help in cancer, autoimmune and Appl. Sci. 2020, 10, 3907 10 of 12

Interestingly, our findings show that the procedures used (i.e., extraction with n-hexane, extraction in Soxhlet with n-hexane, reflux extraction with chloroform) are economical and convenient, in terms of equipment costs, management of the instrumentation and request for qualified personnel for their use. Furthermore, they are easy to implement and allow to produce extracts with a lipid composition similar to the one detected with hard strategies. In this respect, a commercially available liquid extract of G. lucidum obtained with supercritical CO2 extraction was also analyzed. Furthermore, Liu and co-workers [16] reported the extraction and detection of fatty acids in triglycerides from spores of G. lucidum obtained with an extraction process conducted ultrasonically with CH2Cl2 for 2 h. Our findings are in accordance with the constituents previously detected in spores of G. lucidum [16], but the oleic acid amount is generally higher in the extracts produced in this work, in particular when the spores were extracted with reflux with chloroform (66.9%) or extracted with n-hexane (68.4%). The beneficial effects of spores of G. lucidum may be related to the synergic action of the fatty acids in triglycerides and in particular to the high abundance of oleic acid, which properties have been extensively reviewed [29]. In fact, oils rich in oleic acid is supposed to present diverse physiological functions confirmed by several studies on its help in cancer, autoimmune and inflammatory diseases [30,31]. Controversial studies have reported potential biologic effects of the second most abundant fatty acid (i.e., palmitic acid) in the extract of spores of G. lucidum [32]. In this respect, the percentage of palmitic acid is approximately 17%–18% in the extract obtained with n-hexane, reflux with chloroform and commercial oil, but is much higher (21.3%) when the spores where extracted in Soxhlet. With reference to the extract yield, the extraction with Soxhlet seems to be the most efficient, among the extraction processes studied, with a yield of 32.65%, but it gives the lower amount of oleic acid (60.1%) and the higher amount of palmitic acid (21.3%).

5. Conclusions In this work, a GC-MS method for the identification and quantification of FAMEs in triglycerides of G. lucidum spores was successfully optimized, and a comparative study of the most used techniques for the lipid extraction from spores was performed. As revealed by NMR and GC-MS data, conventional extraction procedures (i.e., extraction with n-hexane, in Soxhlet apparatus with n-hexane and reflux with chloroform) gave similar lipid compositions as hard extraction strategies, such as supercritical CO2 extraction. Our findings demonstrated that the commonly used extraction techniques represent an efficient and feasible tool to obtain extracts from G. lucidum spores. This result is particularly relevant in view of future applications of the qualitative and quantitative study of lipids in G. lucidum spores. In fact, the opportunity to study the lipid composition of spores with simple, low cost and no expensive tools is useful to carry out laboratory studies on different strains, to compare different growth conditions or to investigate effect of abiotic factors on biosynthetic pathway of the lipid component.

Author Contributions: Conceptualization, D.N. and A.A.; methodology, M.M.S., F.S., M.D., D.N. and A.A.; investigation, M.M.S., A.E. and A.A.; data curation, M.M.S., F.S. and A.A.; writing—original draft preparation, M.M.S., M.G.; writing—review and editing, M.M.S., F.S., M.G., M.D., D.N. and A.A.; supervision D.N. and A.A.; project administration, D.N. and A.A.; funding acquisition, D.N. and A.A. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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