Anatoluin A and B isolated from medicinal Tricholoma anatolicum are new cytotoxic ergostanoids against the most common cancers

Abstract Tricholoma anatolicum is an edible mushroom from the matsutake group growing under Cedar trees. Bioactivity-guided fractionation of Tricholoma anatolicum afforded two new (1 and 2), three known ergosterols (3–6), and four known (6–9) compounds. Structures were identified as anatoluin A (1), anatoluin B (2), 5α,6α-epoxy-ergosta-7,22-dien,3β-ol (3), ergosterol-endoperoxide (4), ergosterol,3β-ol (5), 3,5-dihydroxyfuran-2(5H)-one (6), mannitol (7), turanose (8), fumaric acid (9) using spectroscopic techniques. The cytotoxic activity of extract and isolated compounds was performed using MTT assay against MCF7, HT29, H1299, and HeLa cancerous cell lines while toxicity against PDF and L929 fibroblast healthy cell lines. The lipid peroxidation inhibitory and ABTS•+ scavenging activities were used to determine antioxidant activity. The polar extracts exhibited significant cytotoxic activity. The more perfect is that the extracts and isolated compounds (1–5) were inactive against PDF and L929 healthy cell lines. Compounds 1–3 and 4 exhibited noticeable cytotoxic activity, while 1–5 moderately inhibited lipid peroxidation. Graphical abstract


Introduction
Mushrooms have been mainly considered a food source due to their nutritional properties and unique aromas since ancient times (Venditti et al. 2017).Many communities have used mushrooms to treat many diseases, particularly cancers (Debnath and Sen 2022).Doctors have recently recommended mushrooms to enhance the immune system, especially for cancer patients.Many studies have been done on the isolation and evaluation of biological activities of the compounds from the medicinal mushrooms (Liu 2014;Duru and Tel-C ¸ayan 2015;€ Ozt€ urk et al. 2015).Tricholoma matsutake is called matsutake, which is a mushroom consumed in East Asia, USA and Japan, due to its various bioactivities.It serves cytotoxic, antitumor, and immunomodulatory activities due to containing a-glucan-protein complexes, steroids and triterpenes (Hoshi et al. 2005;Vamanu 2018;Pandya et al. 2019;Ogbole et al. 2022).According to IARC and WHO reports scientists have accelerated cancer research since cancer incidence increases daily.
Tricholoma anatolicum, a new member of the Matsutake group (Intini et al. 2003), is grown naturally under the Cedar (Cedrus libani) and Taurus fir (Abies cilicica) forests (Do gan and Akata 2011).More than 100 tons are exported to Japan (in the first place) (Do gan and Akata 2011) and other countries each year.Up to date, its metal content and nutritional values have been reported (Dogan et al. 2012).Moreover, Its cytostatic and hepatoprotective effects (Ozkaya et al. 2015) and cytotoxic activity against hepatocellular carcinoma (HEPG-2) cell lines (Arpacı et al. 2016), moderate anti-herpes simplex virus 1 (HSV-1) activity (Do gan et al. 2018) were reported.Furthermore, its extracts also possessed high in vitro modulation activity in breast cancer cell lines (MCF-7) (Do gan et al. 2020).
Since medicinal mushrooms are sources of cancer-preventing compounds, and regarding the various bioactivities, and there has been no report on the phytochemical composition, the fruiting body of Tricholoma anatolicum was studied phytochemically to isolate its cytotoxic and lipid peroxidation inhibitory compounds.

Result and discussion
Both antioxidant and cytotoxic activities of all extracts of Tricholoma anatolicum were investigated.Lipid peroxidation inhibitory and ABTS cations radical scavenging activities were selected as different antioxidant activity assays.In the former assay, the antioxidants are able to transfer H to the media to stop radicalic degradation besides scavenging singlet oxygen accelerating the radicalic degradation are measured.In the latter test antioxidants having the ability to give electrons to the media are mesured.Among the extracts acetone extract (IC 50 :68.1 ± 1.2 mg/mL) and methanol extract (IC 50 :82.5 ± 1.8 mg/mL) inhibited lipid peroxidation in a good manner.In ABTS assay, the methanol extract exhibited the best radical scavenging activity (IC 50 : 98.6 ± 1.9 mg/ mL).As for cytotoxic activity against MCF-7, HT-29, H1299, HeLa cancerous cell lines, and PDF and L929 fibroblast cell lines acetone extract exhibited the highest activity against MCF-7 (IC 50 : 22.9 ± 0.2 mg/mL), HT-29 (IC 50 : 31.6 ± 0.2 mg/mL), and HeLa (IC 50 : 11.8 ± 0.1 mg/mL) cancerous cell lines while the methanol extract against H1299 (IC 50 : 19.3 ± 0.1 mg/mL) cancerous cell lines.The methanol extract also possessed activity against MCF-7 and HT-29 cancer cell lines (IC 50 : 37.5 ± 0.8 and 41.2 ± 0.9 mg/mL, respectively) (Table S1).Therefore, acetone and methanol extracts exhibiting functional activities were fractionated over silica gel by following activity-guided fractionation.The antioxidant active fractions were selected for further isolation studies.
Considering the W 1/2 of the multiplet of H-3 proton, 3-OH was in b-configuration.As also correlated via COSY spectrum, the proton at d H 3.59 (1H, d, J ¼ 5.4 Hz, H-6) ppm and olefinic proton at d H 5.30 (1H, d, J ¼ 5.3 Hz, H-7) suggest that hydroxyl at C-6 position was in a configuration after elucidated together with compound 2. Besides, the peaks at d H 5.11 (1H, dd, J ¼ 15.3; 8.2 Hz, H-22), and d H 5.14 (1H, dd, J ¼ 15.3; 7.4 Hz, H-23) ppm corresponds the vinylic hydrogen peaks on the chain (Figure S4).The coupling constant (J ¼ 15.3 Hz) between H-22 and H-23 indicated that the D 22double bond geometry was deduced to be E.The NMR values of the compound's side chain are consistent with the literature (Yaoita et al. 1998(Yaoita et al. , 2001)).
Compound 2 was obtained as a amorphous white powder.It was colorless under UV (254 nm) while a greenish color appeared after cerium(IV)sulphate spraying.HRMS analyses gave a molecular peak at m/z 428.3280 (calculated for [C 28 H 44 O 3 ] þ : 428.3285; theoretical: 428.3290 as given in Figure S11), which suggested molecular formula C 28 H 44 O 3 having 7 unsaturation equivalents.The peak at m/z 410.3168 (calc.: 410.3185) also explains water loss in HRMS (Figure S11).The low-resolution EI-MS mass fragment m/z 428.1 [M] þ confirmed the molecular ion peak (Figure S12). 1 H-NMR (CD 3 OD 500 MHz) suggested that 2 was ergostan steroid.Compound 2 showed nearly identical chemical shift values with slight differences when compared with 1.The 1 H-NMR of 2 showed a similar pattern to that of 1, except for the chemical shifts of signals by virtue of hydroxy-bearing methine proton at C-6 and olefinic proton at C-7.Moreover, the 13 C-NMR spectrum of 2 was quite similar to that of 1.Only the chemical shifts of C-6 and C-7 have slight differences in 2. These data indicated that compound 2 was an epimer of 1 at C-6.The oxygen substituted peaks observed at d H 3.90 (1H, m, H-3) and d H 3.87 (1H, brs, H-6) ppm in 1 H-NMR (CD 3 OD 500 MHz) (Figure S13).One of these hydrogens resonated at d H 3.90 belongs to a-hydrogen of C-3 position (Yaoita et al. 2001).
The hydrogen resonated at d H 3.87 belongs to the C-6 position.In compound 1, H-6 resonated at d H 3.59 (d, J ¼ 5.4 Hz).In compound 2, however, H-6 proton deshielded 0.28 ppm and resonated d H 3.87 (br.s) ppm.On the other hand, the H-7 vinylic proton of compound 1 resonated at d H 5.30 (d, J ¼ 5.3 Hz) ppm was shielded 0.26 ppm and resonated at d H 5.05 (1H, m, H-7) in compound 2. H-6 (d H 3.87) and H-7 (d H 5.04) were the vicinal protons since a correlation was observed at COSY spectrum (Figure S19).This correlation also verified that a hydroxyl was linked to C-6 position.Comparing to compound 1, the chemical shift of C-6 was shielded, and C-7 was deshielded in compound 2. Considering the H-7 vinilyc protons, in the case of the b configuration -OH at the C-6 position as in 2, the H-7 is affected by epoxy oxygen, while in compound 2, H-7 affacted by two oxygens which lead H-7 deshielding in compound 1 comparing to 2 (Figure 2).Therefore, it can be concluded that the -OH substitution is b configuration in compound 2. The information suggested the structure of 2 as 5a,9a-epoxy-(22E)-ergosta-7,22-dien-3b,6b-diol (anatoluin B) as a new ergostanoid from nature.
All antioxidant activity tests were performed in four concentrations, and the results were presented as IC 50 values where the lower IC 50 value indicates the higher activity.Compounds 1 -5 exhibited very close activity to acetone and methanol extracts.Anatoluin A (1) was the most active compound in lipid peroxidation inhibitory activity, followed by 2, 3, 5, and 4, respectively.In ABTS assay, all isolated compounds exhibited IC 50 more than 100 mg/mL (Table S1).

General experimental procedures
The compounds were isolated and purified using JAI-Next recycling preparative HPLC-UV-RI (Japan Analytical Instruments, Japan) coupled with ODS C 18 (250 Â 20 mm ID; S-5 lM) and GS-320 (GPC, Exl:40.000Da; %70 polyvinyl alcohol; %30 C 18 ; 500 Â 20 mm ID) columns.The antioxidant activity tests were carried out 96-well microplate reader (SpectraMax 340PC 384 , Molecular Devices, CA).Cytotoxic activity was performed at Kırıkkale University using ELISA Microplate Reader (BioTek, USA).Thermo Fisher-Nicolet iS10 instrument equipped with ATR was used to record IR spectrum.NMR spectra were obtained at C ¸ankırı Karatekin University (500 and 600 MHz Agilent NMR Spectrometer).EI-MS spectrum of the compounds were run on JEOL-MS600 (Japan), and the HREI-MS of new compounds were recorded on Jeol HX110 mass spectrometer (Japan).

Collection and extraction of mushroom material
Tricholoma anatolicum H.H.Dogan&Intini were collected from Mu gla (Fethiye-Koruk€ oy), Turkey under cedar trees (Cedrus libani A.Rich.) on November 7, 2015, and identified by Dr. Ilgaz Akata.Herbarium materials were prepared from the identified samples and deposited into Ankara University Herbarium (ANK), with herbarium number AkataOzturk-15.
Successively, the dried and grinded mushroom sample (1.0 kg) was extracted at room temperature with petroleum ether, acetone, and methanol (24hx5 times with each solvent).The solvents were evaporated under vacuum using a rotary evaporator at 40 C. The yields of crude extracts were 1.35%, 1.56%, and 8.62%, respectively.

Cytotoxic activity assay
Cytotoxic activity was performed against MCF-7 (breast cancer), HT-29 (colorectal adenocarcinoma), H1299 (lung carcinoma), HeLa (cervical cancer) cell lines while toxicity against PDF (dermal fibroblast) and L929 (mouse fibroblast) cell lines using MTT test (Boke Sarikahya et al., 2022).For each cell series, the number of cells to be placed on the platelets was optimized.After incubation for 24 h, the cells were treated with 10 lL of extracts at different concentrations for 48 h.After addition of 10 lL of MTT reagent, and then incubation for 4 h, purple precipitate occurred.After one day under humidity and CO 2 atmosphere the absorbances were measured using a microplate reader at 570 and 690 nm.The control group's cell viability (absorbance) was accepted as %100 and % cell viability was calculated.
Figure 1.The structures of isolated compounds from Tricholoma anatolicum.

Figure 2 .
Figure 2. Space views of Anatoluin A and Anatoluin B.