A New Ergosterol Analog, a New Bis-Anthraquinone and Anti-Obesity Activity of Anthraquinones from the Marine Sponge-Associated Fungus Talaromyces stipitatus KUFA 0207

A new ergosterol analog, talarosterone (1) and a new bis-anthraquinone derivative (3) were isolated, together with ten known compounds including palmitic acid, ergosta-4,6,8(14),22-tetraen-3-one, ergosterol-5,8-endoperoxide, cyathisterone (2), emodin (4a), questinol (4b), citreorosein (4c), fallacinol (4d), rheoemodin (4e) and secalonic acid A (5), from the ethyl acetate extract of the culture of the marine sponge-associated fungus Talaromyces stipitatus KUFA 0207. The structures of the new compounds were established based on extensive 1D and 2D spectral analysis, and in the case of talarosterone (1), the absolute configurations of its stereogenic carbons were determined by X-ray crystallographic analysis. The structure and stereochemistry of cyathisterone (2) was also confirmed by X-ray analysis. The anthraquinones 4a–e and secalonic acid A (5) were tested for their anti-obesity activity using the zebrafish Nile red assay. Only citreorosein (4c) and questinol (4b) exhibited significant anti-obesity activity, while emodin (4a) and secalonic acid A (5) caused toxicity (death) for all exposed zebrafish larvae after 24 h.


Introduction
The genus Talaromyces (Trichocomaceae), a sexual state of Penicillium, is important because of its ubiquity.They have been isolated from soil, plants, sponges and foods [1].The fungi of this genus produce a wide variety of secondary metabolites including alkaloids, peptides, lactones, polyketides, and miscellaneous structural type of compounds [2].Recently, we have reported the isolation of wortmin, meso-1,4-bis(4-methoxybenzyl)-2,3-butanediol and a new isocoumarin derivative tratenopyrone from the ethyl acetate extract of the culture of Talaromyces tratensis KUFA 0091, isolated from the marine sponge Mycale sp., collected from the Gulf of Thailand [3].During our ongoing research on secondary metabolites from marine-derived fungi with potential anti-obesity activity, we have investigated the secondary metabolites of a Thai collection of T. stipitatus KUFA 0207, isolated from the marine sponge Stylissa flabelliformis, collected from the coral reef at Samaesarn Island in the Gulf of Thailand.The ethyl acetate extract of the culture of this fungus furnished, in addition to two new compounds including a new analog of ergosterol: talarosterone (1) and a new bis-anthraquinone derivative (3), palmitic acid and the previously reported ergosta-4,6,8 (14),22-tetraen-3-one [4], ergosterol 5,8-endoperoxide [5], cyathisterone (2) [6], emodin (4a) [7], questinol (4b) [8], citreorosein (4c) [7], fallacinol (4d) [9], rheoemodin (4e) [10], and secalonic acid A (5) [11,12] (Figure 1).Obesity is increasing at an alarming rate and only a few medications are currently on the market [13].Recent researches demonstrated that many natural products, including secondary metabolites Obesity is increasing at an alarming rate and only a few medications are currently on the market [13].Recent researches demonstrated that many natural products, including secondary metabolites from plants, cyanobacteria, fungi and phytoplankton, possess anti-obesity activities [14,15].Although many phenolic compound-containing plant extracts and several of their phenolic constituents such as flavonoids have been demonstrated to have potential as anti-obesity agents with different modes of action [15], only a few anthraquinones have been investigated for their anti-obesity potential.Huang et al. [16] have patented (US 8247001 B2) the anti-obesity product and the method of preparation of the alcohol extract of Cassia obtusifolia seeds, which contains the anthraquinones aurantio-obtusin, obtusifolin and their glycosides, for use in the treatment of obesity and related metabolic and liver diseases.Tzeng et al. [17], in their investigation of the anti-obesity and antihyperlipidaemic effects of emodin, have found that this anthraquinone caused dose-related reductions in the hepatic triglyceride and cholesterol contents and lowered hepatic lipid droplets accumulation in high-fat diet-fed rats.
In order to investigate the potential of anthraquinone-producing marine fungi as a source of anti-obesity compounds, anthraquinones 4a-e and secalonic acid A (5) were evaluated for their anti-obesity activity using the zebrafish Nile red assay.Zebrafish is gaining popularity as a screening system for bioactive compounds with the advantages of a small whole animal model, and higher physiological relevance over cellular in vitro models [18].The zebrafish larvae Nile red assay is suitable to detect anti-obesogenic activities since zebrafish larvae were demonstrated to respond to known lipid regulator drugs similarly as humans [19].

Results and Discussion
The molecular formula C 28 H 40 O 3 of compound 1, a white crystal (mp, 145-148 • C), was established based on the (+)-HRESIMS m/z 425.3056 [M + H] + (calculated 425.3056), indicating nine degrees of unsaturation.The IR spectrum showed absorption bands for conjugated ketone carbonyl (1689, 1670 cm −1 ) and olefin (1593 cm −1 ) groups.The 1 H and 13 C NMR spectra (Table 1, Supplementary Information, Figures S1 and S2) of 1 showed similar characteristics of ergosterol derivatives.The 13 C NMR spectrum of 1 showed the presence of twenty-eight carbon signals which can be classified, by DEPTs and HSQC spectra (Supplementary Information, Figure S4), as two conjugated ketone carbonyl (δ C 199.5 and 195.9), one quaternary sp 2 (δ C 155.0), three methine sp 2 (δ C 135.0, 132.5, 129.2), one oxyquaternary sp 3 (δ C 66.2), two quaternary sp 3 (δ C 44.3, 36.8),seven methine sp 3 (δ C 56.1, 56.0, 51.8, 43.9, 42.8, 40.0, 33.0), six methylene sp 3 (δ C 39.0, 37. 1, 33.8, 27.7, 20.7, 18.7), two tertiary methyl (δ C 21.8, 12.4) and four secondary methyl (δ C 21.0, 20.0, 19.6, 17.6) groups.The 1 H NMR spectrum (Table 1, Supplementary Information, Figure S1) exhibited a doublet of an olefinic proton at δ H 6.66 (J = 0.8 Hz), a singlet of one proton at δ H 3.20, two singlets of the tertiary methyls at δ H 1.50 and 0.89, in addition to the proton signals of the (3E)-5,6-dimethylhept-3-en-2-yl side chain [δ H 0.82, d, J = 6.8 Hz (H 3 -27), δ H 0.84, d, J = 6.8 Hz (H 3 -28), δ H 1.04, d, J = 6.8 Hz (H 3 -21), δ H 5.15, dd, J = 15.3, 7.3 Hz (H-22) and δ H 5.25, dd, J = 15.3, 8.0 Hz (H-23)].As both H 3 -21 and the methyl singlet at δ H 0.89 showed HMBC cross peaks to C-17 (δ C 56.0), the methyl singlet at δ H 0.89 was assigned to CH 3 -18.Therefore, another methyl singlet (δ H 1.50, δ C 21.8) was assigned to CH 3 -19.Since the HMBC spectrum (Table 1 and Figure 2, Supplementary Information, Figure S5) also exhibited cross peaks from both H 3 -19 and the doublet of the olefinic proton at δ H 6.66 (J = 0.8 Hz, H-4) to the quaternary sp 2 carbon at δ C 155.0, the double bond was placed between C-4 (δ C 129.2) and C-5 (δ C 155.5).Moreover, H 3 -19 also showed HMBC cross peaks to the carbon signals at δ C 43.9, 37.1 and 36.8,therefore, they were assigned to C-9, C-1 and C-10, respectively.On the other hand, the carbons at δ C 44.3 and 51.8 were assigned to C-13 and C-14 since they both showed HMBC cross peaks to H 3 -18.Moreover, since the HMBC spectrum (Table 1 and Figure 2, Supplementary Information, Figure S5) also exhibited cross peaks from the singlet at δ H 3.20 to C-5, C-14, the carbons at δ C 66.2 and δ C 195.9, the carbon at δ C 66.2 was assigned to C-8 and one of the conjugated carbonyl (δ C 195.9) was placed on C-6.This was supported by the HMBC cross peak from H-4 to C-6.Therefore, another conjugated carbonyl group (δ C 199.5) must be on C-3.The fact that there was one more oxygen atom to be accounted for, along with the presence of the oxyquaternary carbon (δ C 66.2), the epoxide function was placed between C-7 and C-8.Therefore, the structure of 1 was elucidated as 7,8-epoxyergosta-4,22-dien-3,6-dione.In order to determine the stereochemistry of 1, the ROESY spectrum was obtained.As the ROESY spectrum (Table 1 and Figure 3, Supplementary Information, Figure S7) showed cross peaks   In order to determine the stereochemistry of 1, the ROESY spectrum was obtained.As the ROESY spectrum (Table 1 and Figure 3, Supplementary Information, Figure S7) showed cross peaks   In order to determine the stereochemistry of 1, the ROESY spectrum was obtained.As the ROESY spectrum (Table 1 and Figure 3, Supplementary Information, Figure S7) showed cross peaks In order to determine the stereochemistry of 1, the ROESY spectrum was obtained.As the ROESY spectrum (Table 1 and Figure 3, Supplementary Information, Figure S7) showed cross peaks from H-7 to H-9, H-14 and H-15, but not to H 3 -19, it was concluded that the epoxide ring was on the same face as CH 3 -19, i.e., the relative configuration of C-7 and C-8 is 7R and 8R respectively.from H-7 to H-9, H-14 and H-15, but not to H3-19, it was concluded that the epoxide ring was on the same face as CH3-19, i.e., the relative configuration of C-7 and C-8 is 7R and 8R respectively.Since compound 1 was obtained in a suitable crystal, X-ray analysis was carried out, and the ORTEP view shown in Figure 4 revealed that the absolute configuration of C-7, C-8, C-9, C-10, C-13, C-14, C-17, C-20 and C-24 is 7R, 8R, 9R, 10R, 13R, 14R,17R, 20S, 24R.A literature search indicated that 1 has never been previously reported.Therefore, it is a new compound and was named talarosterone.The 1 H, 13 C NMR, IR and HRMS data of 2 (Supplementary information, Table S1, Figures S8-S12) are compatible with those reported for cyathiserone [6].The structure and the stereochemistry of 2 were confirmed by X-ray analysis and the ORTEP view is shown in Figure 5.   Since compound 1 was obtained in a suitable crystal, X-ray analysis was carried out, and the ORTEP view shown in Figure 4 revealed that the absolute configuration of C-7, C-8, C-9, C-10, C-13, C-14, C-17, C-20 and C-24 is 7R, 8R, 9R, 10R, 13R, 14R,17R, 20S, 24R.A literature search indicated that 1 has never been previously reported.Therefore, it is a new compound and was named talarosterone.The 1 H, 13 C NMR, IR and HRMS data of 2 (Supplementary information, Table S1, Figures S8-S12) are compatible with those reported for cyathiserone [6].The structure and the stereochemistry of 2 were confirmed by X-ray analysis and the ORTEP view is shown in Figure 5.  Since compound 1 was obtained in a suitable crystal, X-ray analysis was carried out, and the ORTEP view shown in Figure 4 revealed that the absolute configuration of C-7, C-8, C-9, C-10, C-13, C-14, C-17, C-20 and C-24 is 7R, 8R, 9R, 10R, 13R, 14R,17R, 20S, 24R.A literature search indicated that 1 has never been previously reported.Therefore, it is a new compound and was named talarosterone.Since compound 1 was obtained in a suitable crystal, X-ray analysis was carried out, and the ORTEP view shown in Figure 4   The 1 H, 13 C NMR, IR and HRMS data of 2 (Supplementary information, Table S1, Figures S8-S12) are compatible with those reported for cyathiserone [6].The structure and the stereochemistry of 2 were confirmed by X-ray analysis and the ORTEP view is shown in Figure 5.The 1 H, 13 C NMR, IR and HRMS data of 2 (Supplementary information, Table S1, Figures S8-S12) are compatible with those reported for cyathiserone [6].The structure and the stereochemistry of 2 were confirmed by X-ray analysis and the ORTEP view is shown in Figure 5.  Since compound 1 was obtained in a suitable crystal, X-ray analysis was carried out, and the ORTEP view shown in Figure 4   The 1 H, 13 C NMR, IR and HRMS data of 2 (Supplementary information, Table S1, Figures S8-S12) are compatible with those reported for cyathiserone [6].The structure and the stereochemistry of 2 were confirmed by X-ray analysis and the ORTEP view is shown in Figure 5.  Compound 3 was isolated as a reddish orange solid (mp, 258-260 • C).The IR spectrum showed absorption bands for hydroxyl (3463 cm −1 ), conjugated ketone carbonyl (1622 cm −1 ) and aromatic (1550 cm −1 ) groups.The 13 C NMR spectrum (Table 2, Supplementary Information, Figure S14) exhibited fifteen carbon signals which, in conjunction with DEPTS and HSQC spectra (Supplementary Information, Figure S16), can be categorized as two conjugated ketone carbonyls (δ C 189. 6 S13) showed one singlet at δ H 6.73 and two double doublets at δ H 7.15 (J = 1.0, 0.5 Hz) and 7.28 (J = 1.0, 0.5 Hz) of the aromatic protons and one singlet of the tertiary methyl (δ H 2.33), in addition to two singlets of the hydrogen-bonded hydroxyl groups at δ H 12.04 and δ H 12.79.* can be interchanged.
The structure of 3 can be viewed as a dimer of helminthosporin, an anthraquinone previously isolated from the subterranean stems of Aloe saponaria Haw.[21], and also from the roots of Berchemia floribunda [22].
The structures of the other known compounds, i.e., palmitic acid, ergosta-4,6,8( 14),22-tetraen-3one, ergosterol-5,8-endoperoxide, emodin (4a), questinol (4b), citreorosein (4c), fallacinol (4d), rheoemodin (4e) and secalonic acid A (5) were elucidated by analysis of their 1 H, 13 C NMR and HRMS spectra, and a rotation when the compounds have stereogenic carbons as well as by comparison of these spectroscopic data with those reported in the literature.In order to determine the stereochemistry of 1, the ROESY spectrum was obtained.As the ROESY spectrum (Table 1 and Figure 3, Supplementary Information, Figure S7) showed cross peaks   In order to determine the stereochemistry of 1, the ROESY spectrum was obtained.As the ROESY spectrum (Table 1 and Figure 3, Supplementary Information, Figure S7) showed cross peaks Literature search revealed that Tan et al. [20] have reported isolation of the structurally similar bis-anthraquinone, named 2240A, from an unidentified endophytic fungus (strain no 2240), isolated from the estuarine mangrove from the South China Sea Coast.The structure of 2240A is also a dimer of 7-methyl-1,4,5-trihydroxy-9,10-anthraquinone; however, the two anthraquinone monomers are linked between C-3 and C-3 instead of C-2 and C-2 as in 3. Surprisingly, the 1 H and 13 C NMR chemical shift values reported for 2240A by Tan et al. [20] were very similar to those of 3.Although the structures of 3 and 2240A cannot be distinguished by HMBC correlations, careful analysis of the 1 H and 13 C NMR data of compound 2240A revealed that Tan et al. [20]  The structure of 3 can be viewed as a dimer of helminthosporin, an anthraquinone previously isolated from the subterranean stems of Aloe saponaria Haw.[21], and also from the roots of Berchemia floribunda [22].

General Experimental Procedures
Melting points were determined on a Bock monoscope and are uncorrected.Optical rotations were measured on an ADP410 Polarimeter (Bellingham + Stanley Ltd., Tunbridge Wells, Kent, UK).Infrared spectra were recorded in a KBr microplate in a FTIR spectrometer Nicolet iS10 from Thermo Scientific (Waltham, MA, USA) with Smart OMNI-Transmission accessory (Software 188 OMNIC. 1 H and 13 C NMR spectra were recorded at ambient temperature on a Bruker AMC instrument (Bruker Biosciences Corporation, Billerica, MA, USA) operating at 300.13 and 75.4 Hz or at 500.13 and 125.4 MHz, respectively.High resolution mass spectra were measured with a Waters Xevo QToF mass spectrometer (Waters Corporations, Milford, MA, USA) coupled to a Waters Aquity UPLC system.A Merck (Darmstadt, Germany) silica gel GF 254 was used for preparative TLC, and a Merck Si gel 60 (0.2-0.5 mm) was used for column chromatography.

Fungal Material
The strain KUFA 0207 was isolated from the marine sponge Stylissa flabelliformis, which was collected by scuba diving at a depth of 10-15 m, from the coral reef at Samaesarn Island (12 • 34 36.64N 100 • 56 59.69 E) in the Gulf of Thailand, Chonburi Province, in April 2014.The sponge was washed with 1% sodium hypochlorite solution for 1 min, followed by sterilized seawater three times, and then dried on sterile filter paper under a laminar flow hood, cut into small pieces (5 × 5 mm), and placed on malt extract agar (MEA) plates containing 70% seawater and 300 mg/L of streptomycin sulfate.The plates were incubated at 28 • C for seven days, after which the hyphal tips were transferred onto a slant MEA and maintained as pure culture for further identification.The fungus was identified as Talaromyces stipitatus C.R. Benj., based on morphological characteristics, and was also confirmed by analysis sequence of the internal transcribed spacer (ITS) gene according to the procedure previously described by us [23].Its gene sequences were deposited in GenBank with accession number KU500028.The pure cultures were deposited as KUFA 0207 at Kasetsart University Fungal Collection, Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand.The fungus was cultured for one week at 28 • C in 10 Petri dishes (i.d.90 mm) containing 25 mL of MEA.In order to obtain the mycelial suspension, the mycelial plugs were transferred to two 500 mL Erlenmeyer flasks containing 200 mL of potato dextrose broth, and then incubated on a rotary shaker at 120 rpm at 28 • C for one week.Fifty 1000 mL Erlenmeyer flasks, each containing 300 g of cooked rice, were autoclaved at 121 • C for 15 min, and then inoculated with 20 mL of mycelial suspension of T. stipitatus and incubated at 28 • C for 30 days, after which the moldy rice was macerated in ethyl acetate (25 L total) for seven days, and then filtered.The ethyl acetate solution was concentrated under reduced pressure to yield 65.6 g of crude ethyl acetate extract.

X-ray Crystal Structure of Compounds 1 and 20
Diffraction data were collected at 293 K with a Gemini PX Ultra equipped with CuK α radiation (Oxford Diffraction, Abingdon, Oxfordshire, UK) (λ = 1.54184Å).The structures were solved by direct methods using SHELXS-97 and refined with SHELXL-97 [24].Carbon and oxygen atoms were refined anisotropically.Hydrogen atoms were either placed at their idealized positions using appropriate HFIX instructions in SHELXL, and included in subsequent refinement cycles, or were directly found from difference Fourier maps and were refined freely with isotropic displacement parameters.Full details of the data collection and refinement and tables of atomic coordinates, bond lengths and angles, and torsion angles have been deposited with the Cambridge Crystallographic Data Centre (CCDC).

Anti-Obesity Assay
Compounds 4a-e and 5 were dissolved in DMSO at a concentration of 5 mM and stored at −20 • C until analyses.Anti-obesity activity of the compounds was analyzed with the zebrafish Nile red assay as described in Jones et al. [18] with some modifications.Zebrafish adults and larvae were maintained
Mar. Drugs 2017, 15, 139 5 of 12 from H-7 to H-9, H-14 and H-15, but not to H3-19, it was concluded that the epoxide ring was on the same face as CH3-19, i.e., the relative configuration of C-7 and C-8 is 7R and 8R respectively.
Mar. Drugs 2017, 15, 139 5 of 12 from H-7 to H-9, H-14 and H-15, but not to H3-19, it was concluded that the epoxide ring was on the same face as CH3-19, i.e., the relative configuration of C-7 and C-8 is 7R and 8R respectively.
assigned the chemical shift values to some of the carbons differently from what we assigned for 3. Tan et al. [20] assigned the carbon signal at δC 108.7 to C-9a/9a′ (i.e., C-14/14′ for the numbering used by Tan et al.) instead of C-4a/4′a (i.e., C-13/13′ for the numbering used by Tan et al.) and the carbon signal at δC 131.8 to C-4a/4′a (i.e., C-13/C-13′ for the numbering used by Tan et al.) instead of C-9a/9a′ (i.e., C-13/C-13′ for the numbering used by Tan et al.
assigned the chemical shift values to some of the carbons differently from what we assigned for 3. Tan et al. [20] assigned the carbon signal at δ C 108.7 to C-9a/9a (i.e., C-14/14 for the numbering used by Tan et al.) instead of C-4a/4 a (i.e., C-13/13 for the numbering used by Tan et al.) and the carbon signal at δ C 131.8 to C-4a/4 a (i.e., C-13/C-13 for the numbering used by Tan et al.) instead of C-9a/9a (i.e., C-13/C-13 for the numbering used by Tan et al.).This assignment seems to be incorrect since the chemical shift values of C-4a and C-10a of a series of anthraquinones with the hydroxyl groups on C-4 and C-5 isolated from this extract, i.e., emodin (4a), citreorosein (4c), fallacinol (4d) and rheoemodin (4e), are ca.108 and 115 ppm, which is far from 133 ppm proposed by Tan et al. [19].Consequently, we are convinced that the structure proposed for compound 2240A is not correct.Interestingly, Tan et al. [19] reported 2240A as dextrorotatory, displaying [α] 20 D +62.50 (c 0.08, dioxin).On the contrary, 3 is levorotatory having [α] 20 D −40 (c 0.05, dioxin) and −100 (c 0.05, MeOH), respectively.Since 3 can be considered as a bridged biphenyl, it can have a phenomenon of atropisomerism due to a restricted rotation of the phenyl rings around the C-2/C-2 bond.Therefore, 3 and compound 2240A, previously reported by Tan et al. [19] are different and could probably be atropisomers.

Figure 7 .
Figure 7. Anti-obesity activity of compounds 4a-e and 5 in the zebrafish larvae Nile red assay.The solvent control had 0.1% DMSO and the positive control received 50 μM resveratrol (REV).Values are presented as mean fluorescence intensity (MFI) relative to the DMSO group, and are derived from 10 to 12 individual larvae per treatment group.Statistical differences to the solvent control are indicated with asterisks, *** = p < 0.001.

Figure 8 .
Figure 8. Representative images of the zebrafish Nile red assay.The upper images show the overlay of the fluorescence and phase contrast; the lower images show the mean fluorescence intensity (MFI) given as black and white picture.DMSO, solvent control 0.1%.

Figure 7 .
Figure 7. Anti-obesity activity of compounds 4a-e and 5 in the zebrafish larvae Nile red assay.The solvent control had 0.1% DMSO and the positive control received 50 µM resveratrol (REV).Values are presented as mean fluorescence intensity (MFI) relative to the DMSO group, and are derived from 10 to 12 individual larvae per treatment group.Statistical differences to the solvent control are indicated with asterisks, *** = p < 0.001.

Figure 7 .
Figure 7. Anti-obesity activity of compounds 4a-e and 5 in the zebrafish larvae Nile red assay.The solvent control had 0.1% DMSO and the positive control received 50 μM resveratrol (REV).Values are presented as mean fluorescence intensity (MFI) relative to the DMSO group, and are derived from 10 to 12 individual larvae per treatment group.Statistical differences to the solvent control are indicated with asterisks, *** = p < 0.001.

Figure 8 .
Figure 8. Representative images of the zebrafish Nile red assay.The upper images show the overlay of the fluorescence and phase contrast; the lower images show the mean fluorescence intensity (MFI) given as black and white picture.DMSO, solvent control 0.1%.

Figure 8 .
Figure 8. Representative images of the zebrafish Nile red assay.The upper images show the overlay of the fluorescence and phase contrast; the lower images show the mean fluorescence intensity (MFI) given as black and white picture.DMSO, solvent control 0.1%.