No Evidence Was Found for the Presence of Terreolides, Terreumols or Saponaceolides H-S in the Fruiting Bodies of Tricholoma terreum (Basidiomycota, Agaricales)

Two different collections of the gilled wild fungus Tricholoma terreum, collected in Italy, were subjected to phytochemical analysis. The fungal material was confidently identified by analysis of the ITS genomic sequences. Using both HR-LC-MS and NMR techniques, no evidence was found for the presence in the fruiting bodies of terreolides, terreumols or saponaceolides H-S, in striking contrast with the isolation of these terpenoids by Chinese authors from a mushroom collected in France and identified as T. terreum. The main cytotoxic terpenoid identified and isolated from the extracts of the specimens investigated in this work was the C30 derivative saponaceolide B, which had been previously isolated from T. saponaceum and other T. terreum collections. Although saponaceolide B is a rather labile molecule, easily degradable by heat or in acidic conditions, our study indicated that none of the extraction protocols used produced saponaceolide H-S or terreolide/terreumol derivatives, thus excluding the possibility that the latter compounds could be extraction artifacts. Considered together, these findings point to the need for the unambiguous identification of mushroom species belonging to the complex genus Tricholoma, characterized by high variability in the composition of metabolites. Moreover, based on our data, T. terreum must be considered an edible mushroom.


Introduction
Tricholoma terreum (Schaeff.: Fr.) P. Kummer (1871) is a gilled mushroom (phylum Basidiomycota, order Agaricales, family Tricholomataceae) that forms mycorrhiza with conifer trees, mainly with pines (Pinus sp.pl.).It was first described in Europe by Schaeffer in 1762 with the name Agaricus terreus and its presence was subsequently reported in other continents of the Northern Hemisphere [1]; it has also been encountered under introduced pine trees in Australia and New Zealand.The mushroom's morphological characteristics do not vary significantly with the different places in which it grows.
T. saponaceum (Fr.)P. Kummer (1871), together with saponaceolide A (2), and related saponaceolides [4,5].Saponaceolide B is strongly cytotoxic and different tests on human tumor cancer cell lines have pointed to its potential development as an antitumor drug [6].
Liu et al. published a series of papers [7][8][9] on the structures of secondary metabolites isolated from an EtOAc subextract of dried fruiting bodies of a mushroom harvested in southern France, which was identified as T. terreum, apparently based only on morphological characteristics.In the first paper of the series, Liu et al. determined the structures of four prenylated phenols named terreumols A-D [7], which bear an unusual ten-membered monoterpenoid carbocyclic ring fused to a hydroquinone aromatic ring.One or two epoxide rings are attached to the macrocycle as in terreumol A (3) (Figure 1), which exhibited an antiproliferative activity comparable to that of cisplatin in a cytotoxicity assay against human cancer cells [7].
Subsequently, the same research group determined the structures of sixteen C30 terpenoids isolated from the same mushroom [8].These compounds included saponaceolide B (1), nine new saponaceolide derivatives, H-P, and six novel related C30 terpenoids, named terreolides A-F, among which terreolide A (4) was the most abundant [8].It has been suggested that the 5/6/7 trioxaspiroketal system of terreolides A-C, e.g., compounds 4 and 5 (Figure 1), may have resulted from a ketal reaction between C3′ and C6′ of corresponding saponaceolides, while a subsequent pinacol-like rearrangement of C4′ to C2′ may have afforded the trioxaspiroketal moiety of terreolides D-F, e.g., compound 6 (Figure 1) [8].Finally, in the third paper of the series, three more undescribed saponaceolides, named saponaceolides Q, R and S, were isolated [9].The Chinese authors found that the crude CHCl3/MeOH (1:1) extract of the fruiting bodies as well as two abundant isolated Saponaceolide B is strongly cytotoxic and different tests on human tumor cancer cell lines have pointed to its potential development as an antitumor drug [6].
Liu et al. published a series of papers [7][8][9] on the structures of secondary metabolites isolated from an EtOAc subextract of dried fruiting bodies of a mushroom harvested in southern France, which was identified as T. terreum, apparently based only on morphological characteristics.In the first paper of the series, Liu et al. determined the structures of four prenylated phenols named terreumols A-D [7], which bear an unusual ten-membered monoterpenoid carbocyclic ring fused to a hydroquinone aromatic ring.One or two epoxide rings are attached to the macrocycle as in terreumol A (3) (Figure 1), which exhibited an antiproliferative activity comparable to that of cisplatin in a cytotoxicity assay against human cancer cells [7].
Subsequently, the same research group determined the structures of sixteen C 30 terpenoids isolated from the same mushroom [8].These compounds included saponaceolide B (1), nine new saponaceolide derivatives, H-P, and six novel related C 30 terpenoids, named terreolides A-F, among which terreolide A (4) was the most abundant [8].It has been suggested that the 5/6/7 trioxaspiroketal system of terreolides A-C, e.g., compounds 4 and 5 (Figure 1), may have resulted from a ketal reaction between C3 ′ and C6 ′ of corresponding saponaceolides, while a subsequent pinacol-like rearrangement of C4 ′ to C2 ′ may have afforded the trioxaspiroketal moiety of terreolides D-F, e.g., compound 6 (Figure 1) [8].Finally, in the third paper of the series, three more undescribed saponaceolides, named saponaceolides Q, R and S, were isolated [9].The Chinese authors found that the crude CHCl 3 /MeOH (1:1) extract of the fruiting bodies as well as two abundant isolated compounds, saponaceolide B (1) and saponaceolide M (7) (Figure 1), exhibited acute toxicity to mice, following oral administration, with LD 50 values of 1.51 g kg −1 , and 88.3 and 63.7 mg kg −1 , respectively [8].Moreover, compounds 1 and 7 increased serum creatine kinase levels in treated mice [8].Based on these findings, the authors concluded that T. terreum should be considered toxic, causing mushroom poisoning that ultimately leads to rhabdomyolysis [8].Indeed, this type of fungal poisoning is associated with the ingestion in consecutive meals of different mushrooms, among which T. equestre (L.) P. Kummer is one of the best known species [10].
The presumed toxicity of T. terreum has had a strong resonance among mycologists in Europe, where the mushroom has a long history as being edible.In fact, it is still widely consumed and has the vernacular name "moretta" [11], mainly in the alpine regions of Italy and France, where it is also sold at local markets.Moreover, there are no documented cases of poisoning due to the ingestion of T. terreum in Europe or elsewhere, and the medical literature has never reported that consumption of this mushroom induces any symptom related to rhabdomyolysis [12].For these reasons, the presumed toxicity of T. terreum has been debated by different scholars [10,12,13].
Currently, a scientifically sound phytochemical investigation cannot be conducted without the aid of molecular data, mainly when working with Basidiomycota fruiting bodies collected in the wild.In particular, the T. terreum species complex has been revised with the designation of sequenced neotypes, to clarify the role of some strongly related taxa, such as Tricholoma gausapatum (Fr.)Quél., T. myomyces (Pers.)Lge, and T. triste (Scop.)Quél.[14].
For this reason, we decided to reinvestigate the content of secondary metabolites of T. terreum, using specimens firmly identified by DNA analysis and not only by morphological characteristics.Moreover, to explore the possible differences in the metabolite contents of specimens collected in different places, we examined two different batches of fruiting bodies.One was collected in a pine wood in the Bolzano province (northern Italy), which was designated as T. terreum_Bz, while the other was collected in a pine wood in the Grosseto province (central Italy), designated as T. terreum_Gr.

DNA Analysis
Dried specimens from the two T. terreum collections were sent to Molecular Solutions LLC, Portland, OR, USA (Matthew Gordon), for DNA extraction, amplification of ITS regions, and sequencing.A 695 bp sequence and a 700 bp sequence were obtained from T. terreum_Bz and T. terreum_Gr, respectively.They were submitted to the BLASTn algorithm [15] at GenBank, and both were retrieved as Tricholoma terreum with 100% identity relative to other sequences of the same species present in the database.The two sequences were deposited at GenBank under accession numbers PP101944 and PP101945, respectively.

Phytochemical Analysis
At the onset of our investigation, we verified whether the extraction procedure employed by Liu et al. [7][8][9] could alter the pattern of fungal metabolites occurring in fruiting bodies.In fact, the Chinese authors extracted air-dried fruiting bodies by means of a CHCl 3 /MeOH (1:1) mixture, although this procedure has been proven to be inappropriate for the extraction of wild mushrooms because both MeOH and HCl, usually present in CHCl 3 , often promote the formation of artifacts [16].In addition, drying of the mushroom may also lead to important chemical changes in the metabolite contents.
To mimic the procedure used by Liu et al. [7][8][9], the basidiomes of T. terreum_Gr were air-dried and extracted with CHCl 3 /MeOH (1:1).Subsequently, the crude extract was then partitioned between H 2 O and EtOAc, and the organic sub-extract was analyzed by TLC (Figure 2, lane 1).For comparison, a second batch of T. terreum_Gr basidiomes was extracted according to a procedure developed by us to avoid the formation of artifacts in mushroom extraction [16].Thus, fresh fruiting bodies of T. terreum_Gr were initially frozen at −20 °C and then extracted with EtOAc (Figure 2, lane 2), followed by 2-propanol or MeOH (Figure 2, lanes 3 and 4) to also extract high polar metabolites.For T. terreum_Bz fruiting bodies, in one experiment they were frozen and extracted with EtOAc (extract A, Figure 2, lane 7), while in another experiment they were air-dried and then extracted with EtOAc (Figure 2, lane 8).
The various crude extracts were compared on a TLC silica gel plate with a crude EtOAc extract of T. saponaceum (Figure 2, lane 9) and authentic samples of saponaceolides A (2) and B (1) (Figure 2, lanes 5 and 6).The spot patterns of the four T. terreum extracts in lanes 1, 2, 7, and 8 clearly indicated that the metabolite contents were almost identical, suggesting that they were not affected by the various extraction methods and the mushroom provenance.Moreover, the sulfovanillin reagent revealed the presence of saponaceolide B (1) in all four extracts of T. terreum collected in Italy as an intense purplishreddish spot with Rf = 0.5, while saponaceolide A (2), Rf = 0.21, was clearly absent.The alcoholic extracts (Figure 2, lanes 3 and 4) did not contain other polar compounds in addition to those present in the EtOAc extracts, while the crude EtOAc extract of T. saponaceum (Figure 2, lane 9) showed, as expected, the presence of both saponaceolides A (2) and B (1).Further TLC analyses of the above-mentioned extracts, using various eluents more polar than toluene-EtOAc, 3:1, confirmed the results.
Crude extract A of T. terreum_Bz was then analyzed by HPLC-ESI + -HR QTOF MS to explore the presence of saponaceolides, terreolides, and terreumols.The total ion current (TIC) chromatogram of the extract is shown in Figure 3a.For comparison, a second batch of T. terreum_Gr basidiomes was extracted according to a procedure developed by us to avoid the formation of artifacts in mushroom extraction [16].Thus, fresh fruiting bodies of T. terreum_Gr were initially frozen at −20 • C and then extracted with EtOAc (Figure 2, lane 2), followed by 2-propanol or MeOH (Figure 2, lanes 3 and 4) to also extract high polar metabolites.For T. terreum_Bz fruiting bodies, in one experiment they were frozen and extracted with EtOAc (extract A, Figure 2, lane 7), while in another experiment they were air-dried and then extracted with EtOAc (Figure 2, lane 8).
The various crude extracts were compared on a TLC silica gel plate with a crude EtOAc extract of T. saponaceum (Figure 2, lane 9) and authentic samples of saponaceolides A (2) and B (1) (Figure 2, lanes 5 and 6).The spot patterns of the four T. terreum extracts in lanes 1, 2, 7, and 8 clearly indicated that the metabolite contents were almost identical, suggesting that they were not affected by the various extraction methods and the mushroom provenance.Moreover, the sulfovanillin reagent revealed the presence of saponaceolide B (1) in all four extracts of T. terreum collected in Italy as an intense purplish-reddish spot with R f = 0.5, while saponaceolide A (2), R f = 0.21, was clearly absent.The alcoholic extracts (Figure 2, lanes 3 and 4) did not contain other polar compounds in addition to those present in the EtOAc extracts, while the crude EtOAc extract of T. saponaceum (Figure 2, lane 9) showed, as expected, the presence of both saponaceolides A (2) and B (1).Further TLC analyses of the above-mentioned extracts, using various eluents more polar than toluene-EtOAc, 3:1, confirmed the results.
Crude extract A of T. terreum_Bz was then analyzed by HPLC-ESI + -HR QTOF MS to explore the presence of saponaceolides, terreolides, and terreumols.The total ion current (TIC) chromatogram of the extract is shown in Figure 3a.In conclusion, these preliminary experiments indicated the absence of terreolides, terreumols, and saponaceolides, except saponaceolide B (1) in the extracts of T. terreum fruiting bodies.The striking discrepancy between our findings and those reported in a previous study prompted us to isolate the compounds expressed in T. terreum fruiting bodies.To this aim, fresh T. terreum_Bz mushrooms (1 kg) were extracted with EtOAc and the crude organic extract A (0.54% w/w of fresh mushrooms) was separated by chromatography on a silica gel column.Elution with hexane/EtOAc gave, in the first eluted fractions, a mixture of triglycerides (TG fraction) followed by free fatty acids (FA fraction) and diglycerides (DG fraction).These compounds were the main components of extract A, accounting for about 65% (w/w).
To determine the overall composition of fatty acids in the latter fractions, a sample of TG + DG + FA fractions was treated with NaOH in MeOH/H2O at reflux.After acidification, the resulting mixture of free fatty acids was methylated with diazomethane, and the methyl esters, whose relative abundances are reported in Table 1, were determined by GC-MS analysis (Figures S8 and S9 in the Supplementary Materials).Oleic and linoleic acid were the main fatty acids constituents, accounting for approximately 84% of the total fatty acid content.In conclusion, these preliminary experiments indicated the absence of terreolides, terreumols, and saponaceolides, except saponaceolide B (1) in the extracts of T. terreum fruiting bodies.The striking discrepancy between our findings and those reported in a previous study prompted us to isolate the compounds expressed in T. terreum fruiting bodies.To this aim, fresh T. terreum_Bz mushrooms (1 kg) were extracted with EtOAc and the crude organic extract A (0.54% w/w of fresh mushrooms) was separated by chromatography on a silica gel column.Elution with hexane/EtOAc gave, in the first eluted fractions, a mixture of triglycerides (TG fraction) followed by free fatty acids (FA fraction) and diglycerides (DG fraction).These compounds were the main components of extract A, accounting for about 65% (w/w).
To determine the overall composition of fatty acids in the latter fractions, a sample of TG + DG + FA fractions was treated with NaOH in MeOH/H 2 O at reflux.After acidification, the resulting mixture of free fatty acids was methylated with diazomethane, and the methyl esters, whose relative abundances are reported in Table 1, were determined by GC-MS analysis (Figures S8 and S9 in the Supplementary Materials).Oleic and linoleic acid were the main fatty acids constituents, accounting for approximately 84% of the total fatty acid content.
Our findings are consistent with a previous study conducted on the nutritional value of T. terreum and T. portentosum collected in northwest Spain [17].It is worth noting that we have also discovered the presence of (11Z)-octadecenoic (asclepic, cis-vaccenic) acid in the significant amount of 4.3% (Table 1, entry 6).Although the presence of this uncommon fatty acid in different species of edible mushrooms has already been reported [18], its occurrence in T. terreum has not been described before. 1 Values are expressed as percentages of total fatty acids and were determined by GC-MS analysis of the mixture of the corresponding methyl esters.
After increasing the polarity of the eluent, the chromatographic separation of extract A yielded a fraction of C 28 sterols, made almost exclusively by ergosterol (3.7% of extract A), whose chemical structure was verified by NMR and GC-MS spectra (Figures S10-S13 in the Supplementary Materials).
Further chromatographic elution of extract A gave several minor fractions containing unidentified compounds and tentatively detected coriolic acid (8).They were followed by crude saponaceolide B (1) which was finally purified by crystallization.The 1 H and 13 C NMR spectra (Figures S14-S16 in the Supplementary Materials) of terpenoid 1 were superimposable with those reported in the literature [2,3], and the MS-ESI + spectrum (Figure S17 in the Supplementary Materials) confirmed the expected molecular weight.In addition, the sample showed the same melting point and specific rotation as saponaceolide B isolated from T. saponaceum [4], proving that T. terreum and T. saponaceum produced the same enantiomer.The yield of crystallized 1 was 9.3 mg/g of extract A (0.93%, w/w), which corresponded to 47 mg/kg of T. terreum_Bz fresh fruiting bodies.This value was consistent with the yield of 0.7% (w/w) reported for isolated saponaceolide B by Geraci et al. [2].
The HPLC-UV chromatogram of extract A from T. terreum_Bz is shown in Figure 4.The analysis confirmed the presence of saponaceolide B (1), at RT = 7.91 min, by coelution with an authentic sample.This peak was preceded by a few peaks attributable to longchain acids and C 28 sterols.The compound eluted as an intense peak at RT = 5.10 min was isolated from extract A by preparative TLC and identified as the bioactive (9Z,11E)-13hydroxy-9,11-octadecadienoic acid (coriolic acid) [19] (structure 8 in Figure 4) by NMR and ESI-MS spectra (Figures S21-S23 in the Supplementary Materials).

Saponaceolide B Chemical Stability
Saponaceolide B (1) is highly cytotoxic [6,8]; however, it is also a rather chemically labile compound, and its structure cannot survive the traditional cooking of mushrooms in a frying pan at T > 100 • C. Therefore, any study on the thermal stability of this terpenoid may give further insights about the edibility of T. terreum.In this context, saponaceolides B (1) and A (2) were converted to tricholopardins D (9) and C (10), respectively, upon heating a solution in toluene at 120 • C for 12 h (Figure 5) [20].This result indicated the thermal instability of saponaceolides A and B.
The terpenoids 9 and 10 have already been isolated from Chinese specimens of Tricholoma pardinum; moreover, compound 10 exhibited significant cytotoxicity against MCF-7 cells with an IC 50 value of 4.7 µM, by inducing apoptosis [20].

Saponaceolide B Chemical Stability
Saponaceolide B (1) is highly cytotoxic [6,8]; however, it is also a rather chemically labile compound, and its structure cannot survive the traditional cooking of mushrooms in a frying pan at T  100 °C.Therefore, any study on the thermal stability of this terpenoid may give further insights about the edibility of T. terreum.In this context, saponaceolides B (1) and A (2) were converted to tricholopardins D (9) and C (10), respectively, upon heating a solution in toluene at 120 °C for 12 h (Figure 5) [20].This result indicated the thermal instability of saponaceolides A and B.
The terpenoids 9 and 10 have already been isolated from Chinese specimens of Tricholoma pardinum; moreover, compound 10 exhibited significant cytotoxicity against MCF-7 cells with an IC50 value of 4.7 μM, by inducing apoptosis [20].Although the saponaceolide and terreolide skeletons were suggested to derive from independent biosynthetic pathways [8], it also seemed possible that the trioxaspiroketal moiety of the terreolides could derive from that of the saponaceolides through rearrangement/oxidative reactions.However, based on the experiments previously discussed, we excluded the hypothesis that such transformations occurred in T. terreum basidiomes, either enzymatically or by heating.To obtain more information about the chemical stability of saponaceolide B (1), it was dissolved in non-stabilized CHCl 3 and left in an open-air vial at room temperature for 1 week with the volume of the solution kept constant.Subsequent analysis of the reaction mixture by TLC, ESI-MS and NMR techniques indicated the disappearance of saponaceolide B and the formation of several products.However, neither the MS analysis nor the viewing of the 1 H NMR signals of the mixture indicated the possible presence of terreolides, notably of terreolides B (5) and D (6) which, in principle, could have been formed by rearrangements of compound 1.In addition, careful TLC and HPLC analysis of extract A of T.terreum_Bz fruiting bodies (see above) did not reveal the presence of the saponaceolide B derivatives formed in non-stabilized CHCl 3 , thus confirming that our extraction procedure did not produce artifacts.Although the saponaceolide and terreolide skeletons were suggested to derive from independent biosynthetic pathways [8], it also seemed possible that the trioxaspiroketal moiety of the terreolides could derive from that of the saponaceolides through rearrangement/oxidative reactions.However, based on the experiments previously discussed, we excluded the hypothesis that such transformations occurred in T. terreum basidiomes, either enzymatically or by heating.To obtain more information about the chemical stability of saponaceolide B (1), it was dissolved in non-stabilized CHCl3 and left in an open-air vial at room temperature for 1 week with the volume of the solution kept constant.Subsequent analysis of the reaction mixture by TLC, ESI-MS and NMR techniques indicated the disappearance of saponaceolide B and the formation of several products.However, neither the MS analysis nor the viewing of the 1 H NMR signals of the mixture indicated the possible presence of terreolides, notably of terreolides B (5) and D (6) which, in principle, could have been formed by rearrangements of compound 1.In addition, careful TLC and HPLC analysis of extract A of T.terreum_Bz fruiting bodies (see above) did not reveal the presence of the saponaceolide B derivatives formed in non-stabilized CHCl3, thus confirming that our extraction procedure did not produce artifacts.

General
All solvents and reagents were of commercial quality and were purchased from Merck-Sigma-Aldrich (Milan, Italy).Reference samples of saponaceolide A (2) and B (1) were obtained by the extraction of the mushroom T. saponaceum, according to the procedure described by Vidari et al. [4,5].

General
All solvents and reagents were of commercial quality and were purchased from Merck-Sigma-Aldrich (Milan, Italy).Reference samples of saponaceolide A (2) and B (1) were obtained by the extraction of the mushroom T. saponaceum, according to the procedure described by Vidari et al. [4,5].
HPLC-ESI-QTOF MS spectra were recorded on a SCIEX X500B QTOF system (SCIEX, Framingham, MA 01701, USA) equipped with the Twin Sprayer ESI probe and coupled to an ExionLC TM system.The chromatographic column was a Phenomenex Luna C18(2), 150 × 2.0 mm, 3 µm column (Castel Maggiore (Bo), Italy).The eluent flow was kept constant at 0.3 mL/min and the injection volume was 20 µL.The eluent gradient is reported in Table 2.

Extraction of Tricholoma terreum Fruiting Bodies and Isolation of Saponaceolide B (1)
Frozen fresh fruiting bodies of T. terreum_Bz (1.05 kg) were soaked in EtOAc (400 mL) and minced under the solvent.The resulting mixture was stirred for 1 h, keeping the temperature below 4 • C with the help of an external ice bath.The organic phase was removed, and further EtOAc (400 mL) was added to the mush, which was homogenized with an electric mixer.The resulting mixture was centrifuged (4 • C, 8000 rpm) and the resulting two phases were separated.The aqueous phase was extracted with EtOAc (100 mL) and the combined organic layers (extract A) were washed with brine, dried with Na 2 SO 4 and concentrated under reduced pressure to produce a brown oil (5.4 g, residue A).A small sample (50 mg) of this oil was subjected to ESI-MS, HPLC-ESI-QTOF-MS, and HPLC-UV analyses.The remaining part of the residue (5.35 g) was chromatographed over a silica gel column at atmospheric pressure.Elution with a gradient of n-hexane-EtOAc-MeOH yielded, after evaporation of the fractions, 84 residues (A1-A84), whose overall weight (5.25 g) indicated the almost complete recovery of the starting sample.The residues A1-A13 (1.82 g) contained triglycerides, whereas the following residues (A14-A33) consisted of a mixture of fatty acids and diglycerides.The residues A1-A33 were combined and a sample (200 mg) of the resulting oily mixture was treated with hydroquinone (10 mg, 0.09 mmol) and a solution of NaOH (0.8 g, 20 mmol) in MeOH/H 2 O (2:1, 20 mL).The mixture was heated at reflux under nitrogen for 1 h.Subsequently, the reaction mixture was cooled to room temperature, acidified with diluted aqueous HCl and extracted with

Figure 1 .
Figure 1.Selected terpenoids isolated from fruiting bodies of Tricholoma terreum and T. saponaceum according to published studies.

Figure 1 .
Figure 1.Selected terpenoids isolated from fruiting bodies of Tricholoma terreum and T. saponaceum according to published studies.

Figure 3 .
Figure 3. (a) HPLC-ESI + -HR QTOF MS TIC chromatogram of the crude T. terreum_Bz extract; (b) SIM extraction of the ion at m/z 525.31866, corresponding to the pseudomolecular ion [M + Na] + of saponaceolide B (1); (c) cluster of the isotopic C30H46NaO6 + ions.A series of single ion monitoring (SIM) experiments were performed in search of the pseudomolecular ions [M + Na] + of terreolides A-F, terreumols A-D, and saponaceolides A-S.Details are reported in the Supplementary Materials (Figures S1-S7).Negative responses were obtained for all these metabolites except for saponaceolide B (1).The SIM search of the ion at m/z 525.31866, corresponding to the pseudomolecular ion [M + Na] + (C30H46NaO6 + ) of saponaceolide B, allowed the extraction of a broad peak (P) from the chromatogram, at RT = 15.0-15.25 min (Figure 3b).The measured exact mass, 525.3193 a.m.u, of the monoisotopic ion 12 C30 1 H46 23 Na 16 O6 + in the cluster of the isotopic pseudomolecular ions [M + Na] + attributable to compound 1 (Figure 3c), was in agreement with the calculated accurate mass of 525.3187 a.m.u.(m = 1.14 ppm).In conclusion, these preliminary experiments indicated the absence of terreolides, terreumols, and saponaceolides, except saponaceolide B (1) in the extracts of T. terreum fruiting bodies.The striking discrepancy between our findings and those reported in a previous study prompted us to isolate the compounds expressed in T. terreum fruiting bodies.To this aim, fresh T. terreum_Bz mushrooms (1 kg) were extracted with EtOAc and the crude organic extract A (0.54% w/w of fresh mushrooms) was separated by chromatography on a silica gel column.Elution with hexane/EtOAc gave, in the first eluted fractions, a mixture of triglycerides (TG fraction) followed by free fatty acids (FA fraction) and diglycerides (DG fraction).These compounds were the main components of extract A, accounting for about 65% (w/w).To determine the overall composition of fatty acids in the latter fractions, a sample of TG + DG + FA fractions was treated with NaOH in MeOH/H2O at reflux.After acidification, the resulting mixture of free fatty acids was methylated with diazomethane, and the methyl esters, whose relative abundances are reported in Table1, were determined by GC-MS analysis (Figures S8 and S9 in the Supplementary Materials).Oleic and linoleic acid were the main fatty acids constituents, accounting for approximately 84% of the total fatty acid content.

Figure 3 .
Figure 3. (a) HPLC-ESI + -HR QTOF MS TIC chromatogram of the crude T. terreum_Bz extract; (b) SIM extraction of the ion at m/z 525.31866, corresponding to the pseudomolecular ion [M + Na] + of saponaceolide B (1); (c) cluster of the isotopic C 30 H 46 NaO 6 + ions.A series of single ion monitoring (SIM) experiments were performed in search of the pseudomolecular ions [M + Na] + of terreolides A-F, terreumols A-D, and saponaceolides A-S.Details are reported in the Supplementary Materials (Figures S1-S7).Negative responses were obtained for all these metabolites except for saponaceolide B (1).The SIM search of the ion at m/z 525.31866, corresponding to the pseudomolecular ion [M + Na] + (C 30 H 46 NaO 6 + ) of saponaceolide B, allowed the extraction of a broad peak (P) from the chromatogram, at RT = 15.0-15.25 min (Figure 3b).The measured exact mass, 525.3193 a.m.u, of the monoisotopic ion 12 C 30 1 H 46 23 Na 16 O 6 + in the cluster of the isotopic pseudomolecular ions [M + Na] + attributable to compound 1 (Figure 3c), was in agreement with the calculated accurate mass of 525.3187 a.m.u.(∆m = 1.14 ppm).In conclusion, these preliminary experiments indicated the absence of terreolides, terreumols, and saponaceolides, except saponaceolide B (1) in the extracts of T. terreum fruiting bodies.The striking discrepancy between our findings and those reported in a previous study prompted us to isolate the compounds expressed in T. terreum fruiting bodies.To this aim, fresh T. terreum_Bz mushrooms (1 kg) were extracted with EtOAc and the crude organic extract A (0.54% w/w of fresh mushrooms) was separated by chromatography on a silica gel column.Elution with hexane/EtOAc gave, in the first eluted fractions, a mixture of triglycerides (TG fraction) followed by free fatty acids (FA fraction) and diglycerides (DG fraction).These compounds were the main components of extract A, accounting for about 65% (w/w).To determine the overall composition of fatty acids in the latter fractions, a sample of TG + DG + FA fractions was treated with NaOH in MeOH/H 2 O at reflux.After acidification, the resulting mixture of free fatty acids was methylated with diazomethane, and the methyl esters, whose relative abundances are reported in Table1, were determined by GC-MS analysis (Figures S8 and S9 in the Supplementary Materials).Oleic and linoleic acid were the main fatty acids constituents, accounting for approximately 84% of the total fatty acid content.Our findings are consistent with a previous study conducted on the nutritional value of T. terreum and T. portentosum collected in northwest Spain[17].It is worth noting that we have also discovered the presence of (11Z)-octadecenoic (asclepic, cis-vaccenic) acid in the significant amount of 4.3% (Table1, entry 6).Although the presence of this uncommon fatty

Figure 5 .
Figure 5. Thermal conversion of saponaceolides A and B to tricholopardins C and D.

Figure 5 .
Figure 5. Thermal conversion of saponaceolides A and B to tricholopardins C and D.

3. 2 .
Fungal Material T. terreum_Bz: The fruiting bodies were collected in a pine wood at Collalbo (11.458E; 46.558 N) in the province of Bolzano, Italy on 3 August 2021 and stored at −20 • C until extraction.A lyophilized voucher specimen has been deposited at the CNR-SCITEC laboratories (via L. Mancinelli 7, Milan, Italy) with the accession number TT2022/1.

Table 1 .
Fatty acid composition of the combined fractions of glycerides and free fatty acids extracted from T. terreum_Bz.