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Anti-inflammatory meroterpenoids from the mangrove endophytic fungus

Talaromyces amestolkiae YX1

Article  in  Phytochemistry · November 2017

DOI: 10.1016/j.phytochem.2017.11.011

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Phytochemistry 146 (2018) 8e15

Contents lists avai

Phytochemistry

journal homepage: www.elsevier .com/locate/phytochem

Anti-inflammatory meroterpenoids from the mangrove endophyticfungus Talaromyces amestolkiae YX1

Senhua Chen a, 1, Meng Ding a, 1, Weiyang Liu c, Xishan Huang a, *, Zhaoming Liu a,Yongjun Lu b, Hongju Liu a, c, **, Zhigang She a, ***

a School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, Chinab School of Life Sciences and Biomedical Center, Sun Yat-Sen University, Guangzhou 510275, Chinac School of Pharmacy, Guangdong Medical University, Dongguan, 523808, China

a r t i c l e i n f o

Article history:Received 21 June 2017Received in revised form3 November 2017Accepted 22 November 2017Available online 1 December 2017

Keywords:Talaromyces amestolkiaeTrichocomaceaeMeroterpenoidsMangrove endophytic fungusAnti-inflammatory activity

* Corresponding author.** Corresponding author. School of Chemistry, Sun Y510275, China.*** Corresponding author.

E-mail addresses: huangxishan13@foxmail.com (Xedu.cn (H. Liu), cesshzhg@mail.sysu.edu.cn (Z. She).

1 S.C. and M.D. contributed equally.

https://doi.org/10.1016/j.phytochem.2017.11.0110031-9422/© 2017 Elsevier Ltd. All rights reserved.

a b s t r a c t

Four previously undescribed meroterpenoids, amestolkolides A�D, along with three known compoundswere isolated from the mangrove endophytic fungus Talaromyces amestolkiae YX1 cultured on wheatsolid-substrate medium culture. Their structures were elucidated by a combination of spectroscopicanalyses. The absolute configurations of amestolkolides B and C, and purpurogenolide E were determinedby single-crystal X-ray diffraction using Cu Ka radiation, and those of amestolkolides A and D wereelucidated on the basis of experimental and calculated electronic circular dichroism spectra. The absoluteconfiguration of amestolkolides A-D, and purpurogenolide E (9R) at C-9 was different from that of an-alogues (9S) in references, so that their plausible and distinct biosynthetic pathways were proposed.Amestolkolide B showed strong anti-inflammatory activity in vitro by inhibiting nitric oxide (NO) pro-duction in lipopolysaccharide activated in RAW264.7 cells with IC50 value of 1.6 ± 0.1 mM.

© 2017 Elsevier Ltd. All rights reserved.

1. Introduction

Meroterpenoids were hybrid natural products partially derivedfrom mevalonic acid pathways and widely derived from animals,plants, bacteria, and fungi (Geris and Simpson, 2009; Matsuda andAbe, 2015). The meroterpenoids with the source of fungi exhibiteddiverse structural features and a wide range of biological activities,such as asperterpenes A and B with promising inhibitory activitiesagainst BACE1 (Qi et al., 2016), austalides with strong inhibition ofendo-1,3-b-D-glucanase (Zhuravleva et al., 2014), mycophenolicacid as a strong inhibitor of inosine 50-monophosphate dehydro-genase (IMPDH) (Sintchak et al., 1996), territrem B as a potent in-hibitor of acetylcholinesterase (AChE) (Peng, 1995), berkeleyacetalC exhibited promising anti-inflammatory activity (Etoh et al., 2013).Among them, berkeleyacetals are the polyketide-terpenoid hybrid

at-Sen University, Guangzhou

. Huang), liuhj8@mail2.sysu.

meroterpenoid class, possessing a unique and congested polycyclicskeleton with 6/7/6/5/6 system. Since paraherquonin was isolatedfrom Penicillium paraherquei in 1983 (Okuyama et al., 1983), about13 analogues, berkeleyacetals A�C (Stierle et al., 2007), mini-olutelides A�B (Iida et al., 2008), 4,25-dehydrominiolutelide B,4,25-dehydro-22-deoxyminiolutelide B, isominiolutelide A (Zhanget al., 2012), and purpurogenolides A�E (Sun et al., 2016) havebeen discovered mainly several fungi in the genus Penicillium (Liet al., 2014).

The genus Talaromyces was widespread around plants, foods,soil, as well as sponges (Zhai et al., 2016). The fungus could producea wide range of secondary metabolites, such as anthraquinones(Bara et al., 2013), prenylated indole alkaloids (Chu et al., 2010),norsesquiterpene peroxides (Li et al., 2011), sesquiterpene lactones(Ngokpol et al., 2015), and meroterpenoids (Kaur et al., 2016).

Endophytic fungi have been demonstrated to be an importantsource of pharmacologically active metabolites (Debbab et al.,2013). In the last decade, our research group has focused on themangrove endophytic fungi isolated from the South China Sea todiscover novel and bioactive compounds (Chen et al., 2016a, 2016b,2017a, 2017b; Li et al., 2011; Liu et al., 2016; Tan et al., 2016; Xiaoet al., 2013). Talaromyces amestolkiae YX1 was cultured on solidwheat medium, which led to obtain four previously undescribed

mailto:huangxishan13@foxmail.commailto:liuhj8@mail2.sysu.edu.cnmailto:liuhj8@mail2.sysu.edu.cnmailto:cesshzhg@mail.sysu.edu.cnhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.phytochem.2017.11.011&domain=pdfwww.sciencedirect.com/science/journal/00319422www.elsevier.com/locate/phytochemhttps://doi.org/10.1016/j.phytochem.2017.11.011https://doi.org/10.1016/j.phytochem.2017.11.011https://doi.org/10.1016/j.phytochem.2017.11.011

Table 11H (500 MHz) and 13C (125 MHz) NMR data for compounds 1e2 in CDCl3.

no. 1 2

dC, type dH, (J in Hz) dC, type dH, (J in Hz)

1 163.4, C 162.5, C2 115.7, CH 6.02, d, (1.5) 117.8, CH 6.11, d, (1.5)3 155.2, C 152.2, C4 58.0, C 59.4, C

S. Chen et al. / Phytochemistry 146 (2018) 8e15 9

meroterpenoids, amestolkolides A�D (1e4), along with threeknown compounds 5e7 (Fig. 1). Amestolkolides A and B (1 and 2)exhibited anti-inflammatory activity in vitro by inhibiting nitricoxide (NO) production in lipopolysaccharide activated inRAW264.7 cells with IC50 values of 30 ± 1.2 and 1.6 ± 0.1 mM,respectively. The isolation, structure elucidation, plausible biosyn-thetic pathways, and bioactivities of the isolates from the fungusare described herein.

5 37.5, CH 2.56, dd, (12.2, 4.3) 32.4, CH 3.02, dd, (12.8, 4.9)6 27.6, CH2 1.70, dd, (14.7, 4.3)

1.62, dd, (14.7, 12.2)27.5, CH2 2.02, dd, (14.7, 4.9)

1.51, dd, (14.6, 12.9)7 46.6, C 43.4, C8 177.7, C 176.1, C9 63.6, CH 4.44, dq, (6.7, 2.4) 74.3, CH 4.27, q, (7.1)10 150.4, C 211.7, C11 105.2, C 51.7, CH 3.66, d, (9.9)12 47.7, C 48.1, C13 90.5, CH 4.97, d, (2.6) 204.9, C14 128.1, CH 6.14, dd, (2.6, 1.5) 130.1, CH 6.34, d, (1.5)15 133.4, C 141.8, C16 82.8, C 82.9, C17 26.2, CH3 1.47, s 26.8, CH3 1.61, s18 25.8, CH3 1.66, s 26.4, CH3 1.67, s19 19.5, CH3 1.31, s 17.6, CH3 1.17, s20 17.7, CH3 1.36, d, (6.7) 17.2, CH3 1.32, d, (7.1)21 40.9, CH 2.91, dd, (2.5, 4.5) 46.0, CH 3.15, dd, (9.9, 5.4)22 97.9, CH 6.01, d, (4.5) 98.8, CH 6.14, d, (5.4)23 24.2, CH3 1.30, s 27.0, CH3 1.34, s24 55.8, CH2 3.14, d, (5.5)

2.42, d, (5.5)55.1, CH2 3.19, d, (5.0)

2.63, d, (5.0)

2. Results and discussion

The mangrove endophytic fungus Talaromyces amestolkiae YX1were cultured on solid wheat medium with artificial seawater for28 days, respectively. The EtOAc extract of the wheat fermentationwas fractionated by repeated silica gel chromatography andSephadex LH-20 column chromatography to afford four previouslyundescribed meroterpenoids, amestolkolides A�D (1e4), togetherwith three known meroterpenoids, purpurogenolide E (5) (Sunet al., 2016), chrodrimanin B (6) (Wei et al., 2011), and chro-drimanin A (7) (Hayashi et al., 2012).

Amestolkolide A (1) was obtained as white powder. The mo-lecular formula was deduced to be C24H26O7 on the basis ofpositive-ion HRESIMS (m/z 427.1746 [M þ H]þ, calcd for C24H27O7,427.1751), implying 12 degrees of unsaturation. The IR spectrumindicated the presence of carbonyl (1778 and 1712 cm�1) andolefinic (1612 cm�1) functional groups. The 1H NMR spectrumshowed resonances for two olefinic protons [dH 6.02 (d, J ¼ 1.5 Hz,H-2) and 6.14 (dd, J¼ 2.6,1.5 Hz, H-14)], three oxygenatedmethines[dH 4.44 (dq, J ¼ 6.7, 2.4 Hz, H-9), 4.97 (d, J ¼ 2.6 Hz, H-13), and 6.01(d, J¼ 4.5 Hz, H-22)], twomethines [dH 2.56 (dd, J¼ 12.2, 4.3 Hz, H-5) and 2.91 (dd, J ¼ 4.5, 2.5 Hz, H-21)], one oxygenated methylene[dH 3.14 (d, J ¼ 5.5 Hz, H-24); 2.42 (d, J ¼ 5.5 Hz, H-24)], onemethylene [dH 1.70 (dd, J ¼ 14.7, 4.3 Hz, H-6); dH 1.62 (dd, J ¼ 14.7,12.2 Hz, H-6)], five methyls [dH 1.47 (s, H-17), 1.66 (s, H-18), 1.31 (s,H-19),1.36 (d, J¼ 6.7 Hz, H-20), and 1.30 (s, H-23)] (Table 1). The 13CNMR spectrum (Table 1) revealed the presence of 24 carbons cor-responding to two ester carbonyl groups (dC 163.4, 177.7), one ketal(dC 97.9), three double bonds (dC 105.2, 115.7, 128.1, 133.4, 150.4,155.2), five methyls, two methylenes, five methines, one oxygen-ated tertiary carbon (dC 82.8), and two quaternary carbons (Table 1).These 1D NMR data (Table 1) with the help of the molecular for-mula suggested that 1 belonged to a heptacyclic meroterpenoidcontaining a hemiketal, three double bonds.

Analysis of the 1H-1H COSY spectrum suggested the presence offour isolated proton spin systems, CH(5)�CH2(6), CH(9)�CH3(20),CH(13)�CH(14), and CH(21)�CH(22) (Fig. 2). Key HMBC correla-tions from H-17 to C-16 and C-18, H-18 to C-15, and H-2 to C-1, C-3and C-15 established a hexatomic ring lactone fragment (A ring)(Fig. 2). The HMBC correlations of H-5 with C-4, H-19with C-5, C-12and C-13, H-14 with C-3, H-13 with C-15 indicated a heptatomic

Fig. 1. Chemical str

ring fragment (B ring). The three rings system (C/D/E) was assignedby the correlations of H-6 to C-12 and C-21, H-23 to C-6, C-7, and C-8, H-21 to C-8, H-22 to C-8, C-9, and C-11, as well as H-20 to C-9 andC-10. The oxirane moiety was located at C-4, which was supportedby the HMBC correlations of H-24 with C-4 and C-3, as well as thechemical shifts of C-4 (dC 58.0) and C-24 (dC 55.8). An ether linkagebetween C-10 and C-13 was fused as a furan unit according to thechemical shifts of C-10 (dC 150.4) and C-13 (dC 90.5), as well as therequired degrees of unsaturation.

The relative configuration of 1 was determined on the basis ofNOESY data (Fig. 3). NOE interactions H-21 with H-19, H-22, and H-23, H-22 with H-20 and H-23, H-13 with H-19 indicated theircofacial orientation. Furthermore, the correlations of H-5 with H-24suggested that these protons were located on the opposite face(Fig. 3). The absolute configuration of 1 was assigned by compari-son of the experimental and theoretical ECD spectra, which wascalculated by a quantum chemical method at the [B3LYP/6-311þg(2d,p)] level. The predicted ECD spectrum of 4R, 5R, 7R, 9R,12S, 13S, 21S, and 22R was in good agreement with that of theexperimental one (Fig. 4). Thus, compound 1 was established as(1R,2aR,2a1S,4aR,5aR,5a1S,6R,11aS)-1,4a,5a1,10,10-pentamethyl-2a,2a1,4a,5,5a,5a1,10,11a-octahydro-8H-2,3,9,12-tetraoxaspiro

uctures of 1e7.

Fig. 2. Key HMBC (black arrows) and COSY (black bold lines) correlations of compounds 1e4.

Fig. 3. Key NOE (dashed arrows) correlations of compounds 1e4.

S. Chen et al. / Phytochemistry 146 (2018) 8e1510

Fig. 4. Experimental and calculated ECD spectra of compound 1 (in MeOH).

S. Chen et al. / Phytochemistry 146 (2018) 8e15 11

[benzo[5,6]cyclohepta[1,2,3-bc]cyclopenta[fg]acenaphthylene-6,20-oxiran]-6a,10a,12a-triene-4,8(1H)-dione and named ames-tolkolide A.

Amestolkolide B (2) was obtained as colorless crystals, analyzedfor the molecular formula C24H26O8 by interpretation of HRESIMSdata (m/z 443.1695 [M þ H]þ, calcd for C24H27O8, 443.1700). The 1Hand 13C NMR spectra showed a structure closely related to 1 exceptfor the presence of two additional keto carbonyl groups (dC 211.7,204.9) and absence of two olefinic carbons (dC 150.4, 105.2) in 2.This evidence suggested that compound 2 lacked a furan ring andwas most likely be derived from the furan ring-opening andoxidation of compound 1. The HMBC spectrum showed long-rangeH-C correlations fromH-20 and H-21 to C-10, H-19 and H-5 to C-13,indicating that two keto carbonyl groups were located at C-10 andC-13, respectively. The key HMBC and 1H-1H COSY correlations(Fig. 2) permitted further elucidation for the planar structure of 2.The relative configuration of 2 was determined by analysis of theNOESY data. NOESY correlations of H-21 with H-11, H-22, and H-23,H-11 with H-19, H-22 with H-20 and H-23 indicated that they werecofacial and were assigned to be a-oriented, while the b-orienta-tion of H-5 was supported by the correlations of H-5 and H-24(Fig. 3). The absolute configuration of 2 was assigned as(4R,5R,7R,9R,11R,12S,21S,22R) by a single-crystal X-ray diffractionexperiment using Cu Ka radiation (Flack parameter ¼ 0.03(12) andHooft parameter ¼ 0.02 (4)) (Fig. 5) (Flack and Bernardinelli, 2008).Hence, compound 2 was elucidated as (2R,3aR,3a1S,5aR,6-aR,7R,13aS,13bR)-2,5a,11,11,13a-pentamethyl-3a1,5a,6,6a,13a,13b-hexahydro-9H-spiro[furo[4,3,2-ij]pyrano[40,3':4,5]cyclohepta[1,2-f]isochromene-7,20-oxirane]-1,5,9,13(2H,3aH,11H)-tetraone andnamed amestolkolide B.

Amestolkolide C (3) was obtained as colorless crystals. Themolecular formula was determined as C26H30O9 on the basis ofpositive-ion HRESIMS (m/z 509.1774 [M þ Na]þ, calcd forC26H30O9Na, 509.1782). Detailed analysis of its NMR spectroscopicdata (Table 2) suggested that 3 was similar to 5 belonging to thepolyketide-terpenoid hybrid meroterpenoid class, except for thepresence of additional signals attributed to an acetyl group (dC170.4 for C-25, dH for H-26 and dC 20.9 for C-26) in 3. The key HMBCcorrelations fromH-24 and H-26 to C-25were allowed to assign theattachment of the acetyl group to C-24 in 3 (Fig. 2). The relativeconfigurations at C-4, -5, -7, -9, -11, �12, �21, and �22 were the

same as those of 5 according to the NOESY data (Fig. 3). The ab-solute configuration of 3was unambiguously determined by single-crystal X-ray diffraction analysis as 7R, 9R, 11R, 12S, 21S, and 22R(Flack parameter ¼ �0.01(14) and Hooft parameter ¼ 0.04 (8))(Fig. 5) (Flack and Bernardinelli, 2008). Therefore, compound 3wasidentified as ((2R,3aR,3a1S,5aR,6aS,13aS,13bR)-2,5a,11,11,13a-pen-tamethyl-1,5,9,13-tetraoxo-1,2,3a,3a1,5,5a,6,6a,8,9,11,13,13a,13b-tetradecahydrofuro[4,3,2-ij]pyrano[40,3':4,5]cyclohepta[1,2-f]iso-chromen-7-yl)methyl acetate and named amestolkolide C.

Amestolkolide D (4) was obtained as white powder. The mo-lecular formula was assigned as C29H36O9 according to the HRE-SIMS peak m/z 551.2252 [M þ Na]þ (calcd for C29H36O9Na,551.2251). The 1H and 13C NMR spectra (Table 2) were almostidentical to those of 3 except for the replacement of an acetyl groupin 3 by a 3-methylbutanoyl group [dC 172.6, 43.2, 25.8, 22.6, 22.6; dH2.17 (2H, d, J ¼ 7.1 Hz), 2.06 (1H, m), 0.94 (6H, d, J ¼ 6.6 Hz)].Detailed analysis of the 1H-1H COSY and HMBC spectra (Fig. 2), inparticular HMBC correlations from the H-24 and H-26 to C-25, wasused to locate the 3-methylbutanoyl group at C-24. The relativeconfiguration of 4 was identified to be identical to 3 by interpre-tation of its NOESY spectrum (Fig. 3). Compounds 3 and 4 showedquite similar ECD spectra (Fig. 6), with two strongminima of Cottoneffects (CEs) at 216 and 284 nm, indicating that they share the sameabsolute configurations as 7R, 9R, 11R, 12S, 21S, and 22R. Therefore,compound 4 was established as ((2R,3aR,3a1S,5aR,6aS,13aS,13bR)-2,5a,11,11,13a-pentamethyl-1,5,9,13-tetraoxo-1,2,3a,3a1,5,5a,6,6a,8,9,11,13,13a,13b-tetradecahydrofuro[4,3,2-ij]pyrano[40,3':4,5]cyclohepta[1,2-f]isochromen-7-yl)methyl 3-methylbutanoate and named amestolkolide D.

Amestolkolides A¡D (1e4) and purpurogenolide E (5) werefirstly isolated from the fungi in the genus Talaromyces andbelonged to 3,5-dimethylorsellinic acid (DMOA)-derived mer-oterpenoids possessing a unique and congested polycyclic skeletonwith 6/7/6/5/6 system. Their structures were similar to berkeleya-cetals (Stierle et al., 2007) and miniolutelides (Iida et al., 2008),while the absolute configuration of 1�5 (9R) at C-9 was distinctfrom that of berkeleyacetals and miniolutelides (9S), so that it wasallowed us to propose that their biosynthetic pathways was similarbut slightly different (Fig. 7). In the proposed biosyntheses of 1�5and berkeleyacetal C, Iwas utilized as the pathway intermediate aswell, but underwent C-9 epimerization to generate intermediates IIand III, respectively. Finally, the biosynthetic pathways of com-pounds 1e5 were proposed on the basis of the references (Fig. 7)(Geris and Simpson, 2009; Matsuda and Abe, 2015; Matsuda et al.,2016).

Meroterpenoids 1e7 were tested for their inhibitory activitiesagainst LPS-activated NO production in RAW264.7 cells in vitrousing the Griess assay with indomethacin as a positive control(IC50 ¼ 26.3 ± 1.2 mM). Amestolkolide B (2) displayed stronginhibitory effects on NO productionwith IC50 values of 1.6 ± 0.1 mM.Amestolkolide A (1) showed weaker activity (30 ± 1.2 mM). Com-pound 1 was approximately 10 times more potent than compound2, indicating that the presence of two keto carbonyl group at C-10and C-13 in the molecule enhanced the activity. However, com-pounds 3e7 were inactive (

Fig. 5. X-ray crystallographic analysis of compounds 2, 3 and 5.

S. Chen et al. / Phytochemistry 146 (2018) 8e1512

3. Conclusions

The chemical investigation of the mangrove endophytic fungusTalaromyces amestolkiae YX1 afforded four previously undescribedmeroterpenoids, amestolkolides A�D (1e4), along with threeknown compounds (5e7) on wheat solid-substrate medium cul-ture. The absolute configuration of 1�5 (9R) at C-9 was differentfrom that of analogues (9S) in references, it was speculated that thekey epimerization of intermediate I would make the distinctbiosynthetic pathways. Compounds 1e5 belonged to 3,5-dimethylorsellinic acid (DMOA)-derived meroterpenoids possess-ing a unique and congested polycyclic skeleton with 6/7/6/5/6system, which were firstly isolated from the fungi in the genusTalaromyces. Compounds 1 and 2 exhibited potent anti-inflammatory activity in vitro.

4. Experimental

4.1. General experimental procedures

Melting points were recorded on a Fisher-Johns hot-stageapparatus and were uncorrected. Optical rotations were measuredon a MCP 300 (Anton Paar) polarimeter at 28 �C. UV data wererecorded with MeOH as the solvent using a PERSEE TU-1900

spectrophotometer, and ECD data were obtained on a Chirascan™CD spectrometer (Applied Photophysics). IR spectra were carriedout on a Nicolet Nexus 670 spectrophotometer, in KBr discs. AllNMR experiments were performed on a Bruker Avance 500 spec-trometer (1H 500 MHz, 13C 125 MHz) at room temperature, usingCDCl3 as the solvent. All chemical shifts (d) were given in ppmwithreference to the solvent signal (dC 77.1/dH 7.26 for CDCl3), andcoupling constants (J) were given in Hz. ESIMS spectra wererecorded on a Finnigan LCQ-DECAmass spectrometer and HRESIMSspectra were measured on a Thermo Fisher Scientic Q-TOF massspectrometer. Column chromatography (CC) was performed onsilica gel (200e300 mesh, Qingdao Marine Chemical Factory) andSephadex LH-20 (Amersham Pharmacia). Thin-layer chromatog-raphy (TLC) was performed on silica gel plates (Qingdao Huang HaiChemical Group Co., G60, F-254). Single-crystal data were carriedout on an Agilent Gemini Ultra diffractometer (Cu Ka radiation).

4.2. Fungal material

The fungus used in this studywas isolated fromhealthy leaves ofthe marine mangrove Kandelia obovata, which were collected inApril 2012 from Zhanjiang Mangrove Nature Reserve (coordinates21.3167�N, 109.8102�E) in Guangdong Province, China. The funguswas obtained using the standard protocol for isolation (Chen et al.,

Table 21H (500 MHz) and 13C (125 MHz) NMR data for compounds 3e4 in CDCl3.

no. 3 4

dC, type dH, (J in Hz) dC, type dH, (J in Hz)

1 168.2, C 168.2, C2 31.4, CH2 3.07, dd, (20.9, 2.2)

3.52, dd, (20.9, 1.5)31.5, CH2 3.08, dd, (20.9, 2.2)

3.54, dd, (20.9, 1.6)3 126.7, C 126.7, C4 133.7, C 134.0, C5 143.2, C 143.0, C6 34.4, CH2 2.08, d, (16.2)

2.86, d, (16.2)34.5, CH2 2.10, d, (16.3)

2.91, d, (16.3)7 42.7, C 42.7, C8 178.7, C 178.7, C9 72.5, CH 4.24, q, (6.9) 72.5, CH 4.25, q, (6.9)10 212.4, C 212.4, C11 52.9, CH 3.52, d, (8.3) 53.0, CH 3.53, d, (8.4)12 52.7, C 52.7, C13 210.3, C 210.2, C14 44.6, CH2 3.26, d, (12.0)

3.77, d, (12.0)44.6, CH2 3.27, d, 11.9

3.76, d, 11.915 129.4, C 129.5, C16 85.4, C 85.4, C17 27.8, CH3 1.56, s 27.8, CH3 1.56, s18 26.0, CH3 1.50, s 26.2, CH3 1.51, s19 27.1, CH3 1.25, s 27.2, CH3 1.27, s20 16.4, CH3 1.36, d, (6.9) 16.5, CH3 1.37, d, (6.9)21 41.5, CH 3.21, t, (8.3) 41.5, CH 3.22, t, (8.3)22 98.4, CH 6.26, d, (8.3) 98.4, CH 6.26, d, (8.3)23 26.1, CH3 1.37, s 26.1, CH3 1.38, s24 60.9, CH2 4.67, d, (12.9)

4.77, d, (12.9)60.6, CH2 4.73, d, (13.0)

4.78, d, (13.0)25 170.4, C 172.6, C26 20.9, CH3 2.05, s 43.2, CH2 2.17, d, (7.1)27 25.8, CH 2.06, m28/29 22.6, CH3 0.94, d, (6.6)

Fig. 6. Experimental ECD spectra of compounds 3e5 (in MeOH).

S. Chen et al. / Phytochemistry 146 (2018) 8e15 13

2016a,b). Fungal identification was carried out using a molecularbiological protocol by DNA amplification and sequencing of the ITSregion. The sequence data obtained from the fungal strain havebeen deposited at Gen Bank with accession no. KP975419. A BLASTsearch result showed that the sequence was the most similar (99%)to the sequence of Talaromyces amestolkiae (compared toJX965214.1 JN899315.1). Talaromyces is a genus of fungi in thefamily Trichocomaceae. A voucher strain was deposited in the

China Center for Type Culture Collection under patent depositorynumber CCTCC M 2016218.

4.3. Fermentation, extraction and isolation

The fungus was cultured on autoclaved wheat solid-substratemedium (sixty 500 mL Erlenmeyer flasks; each containing 50 g ofwheat, 1.5 g of artificial sea salts, and 50 mL of distilled H2O) atroom temperature under static conditions and daylight for 28 days.Following incubation, the mycelia and solid wheat medium wereextracted with EtOAc. The extract was evaporated under reducedpressure to yield 101 g of residue. The residuewas then divided into20 fractions (Fr. 1eFr. 20) by column chromatography on silica gel,eluting with a gradient of petroleum ether/EtOAc from 1:0 to 0:1.Initially, it was eluted with petroleum ether, then petroleum ether/EtOAc (v/v, 9:1). Each step of the gradient increased polarity by 10percent (v/v). Finally, it was eluted with EtOAc. Fr. 6 (153 mg) wasapplied to silica gel CC, eluting with petroleum ether/EtOAc (v/v,7:3), to obtain compounds 1 (2.5 mg) and 2 (8.1 mg). Fr. 8 (119 mg)was subsequently separated by Sephadex LH-20 CC eluting withCH2Cl2/MeOH (v/v, 1:1) to give subfraction Fr. 8.6, which was pu-rified on silica gel (CH2Cl2/MeOH v/v, 98:2) to yield compounds 3(4.3 mg) and 4 (2.2 mg). Fr. 10 (148 mg) was applied to silica gel CC,eluting with CH2Cl2/MeOH (v/v, 97:3), to obtain compounds 6(2.1 mg) and 7 (5.4 mg). Fr. 14 (127 mg) was subsequently separatedby Sephadex LH-20 CC eluting with CH2Cl2/MeOH (v/v, 1:1) to yieldcompound 5 (6.3 mg).

4.3.1. Amestolkolide A (1)Amorphous powder; ½a�20D �139 (c 0.02, MeOH); UV (MeOH)

lmax (log ε): 263 (3.63) nm; ECD (MeOH) lmax (Dε): 206 (�59), 271(25) nm; IR (KBr) nmax: 3057, 2921, 2852, 1778, 1712, 1612, 1460,1394, 12924, 1166, 1120, and 979 cm�1; 1H and 13C NMR data,Table 1; ESIMS m/z 427 [M þ H]þ; HREIMS m/z 427.1746 [M þ H]þ(calcd for C24H27O7, 427.1751).

4.3.2. Amestolkolide B (2)Colorless crystals; mp 215e217 �C; ½a�20D þ133 (c 0.02, MeOH);

UV (MeOH) lmax (log ε): 272 (4.06) nm; ECD (MeOH) lmax (Dε): 226(�64), 299 (26) nm; IR (KBr) nmax: 2956, 2923, 1783, 1716, 1655,1292,1162,1122, 980 cm�1; 1H and 13C NMR data, Table 1; ESIMSm/z 443 [M þ H]þ; HRESIMS m/z 443.1695, (calcd for C24H27O8,443.1700).

4.3.3. Amestolkolide C (3)Colorless crystals; mp 233e235 �C; ½a�20D �210 (c 0.02, MeOH);

UV (MeOH) lmax (log ε): 211 (4.45), 248 (3.58) nm; ECD (MeOH)lmax (Dε): 216 (�10), 284 (�21) nm; IR (KBr) nmax: 2982, 2942, 1774,1738, 1716, 1655, 1386, 1226, 1148, 969 cm�1; 1H and 13C NMR data,Table 2; ESIMS m/z 509 [M þ Na]þ; HRESIMS m/z 509.1774 [M þNa]þ (calcd for C26H30O9Na, 509.1782).

4.3.4. Amestolkolide D (4)Amorphous powder; ½a�20D �198 (c 0.02, MeOH); UV (MeOH)

lmax (log ε): 212 (4.44), 247 (3.54) nm; ECD (MeOH) lmax (Dε): 216(�12), 284 (�24) nm; IR (KBr) nmax: 2973, 2940, 1774, 1729, 1712,1459, 1351, 1282, 1147, 970 cm�1; 1H and 13C NMR data, Table 2;ESIMSm/z 551 [Mþ Na]þ; HRESIMSm/z 551.2252 [Mþ Na]þ (calcdfor C29H36O9Na, 551.2251).

4.4. X-ray crystallographic analysis of compounds 2, 3 and 5

The single crystal X-ray diffraction data was collected on anAgilent Gemini Ultra diffractometer with Cu Ka radiation(l ¼ 1.54178 Å). The structures were solved by direct methods

Fig. 7. Plausible biosynthetic pathways of compounds 1e5 and berkeleyacetal C.

S. Chen et al. / Phytochemistry 146 (2018) 8e1514

(SHELXS-97) and refined using full-matrix least-squares differenceFourier techniques (Sheldrick, 2008). Hydrogen atoms bonded tocarbons were placed on the geometrically ideal positions by the“ride on”method. Hydrogen atoms bonded to oxygen were locatedby the difference Fourier method and were included in the calcu-lation of structure factors with isotropic temperature factors.Crystallographic data for 2, 3 and 5 have been deposited with theCambridge Crystallographic Data Center. Copies of the data can beobtained, free of charge, on application to the Director, CCDC, 12Union Road, Cambridge CB2 1EZ, UK (fax: 44-(0)1223-336033, or e-mail: deposit@ccdc.cam.ac.uk).

4.4.1. Crystal data of (2)C24H26O8, Mr ¼ 442.45, orthorhombic, a ¼ 9.64915(5) Å,

b ¼ 11.23380(5) Å, c ¼ 19.70911(10) Å, a ¼ 90.00, b ¼ 90.00,g ¼ 90.00, V ¼ 2136.401(19) Å3, space group P212121, Z ¼ 4,Dcalcd¼ 1.376mg/m3, m¼ 0.863mm�1, and F(000)¼ 936.0. Crystaldimensions: 0.42 � 0.36 � 0.25 mm3. Independent reflections:4272 (Rint ¼ 0.0192). The final R1 values were 0.0288, uR2 ¼ 0.0794(I > 2s(I)). The goodness of fit on F2 was 1.045. Flack parametervalue was 0.03(12). CCDC number: 1520914.

4.4.2. Crystal data of (3)C26H30O9, Mr ¼ 486.50, monoclinic, a ¼ 12.4083(2) Å,

b ¼ 6.66510(10) Å, c ¼ 15.0875(3) Å, a ¼ 90.00, b ¼ 100.174(2),g ¼ 90.00, V ¼ 1228.15(4) Å3, space group P21, Z ¼ 4,Dcalcd ¼ 1.316 mg/m3, m ¼ 0.739 mm�1, and F(000) ¼ 516.0. Crystal

dimensions: 0.39 � 0.34 � 0.12 mm3. Independent reflections:1987 (Rint ¼ 0.0180). The final R1 values were 0.0319, uR2 ¼ 0.0821(I > 2s(I)). The goodness of fit on F2 was 1.048. Flackparameter ¼ �0.01(14). CCDC number: 1520994.

4.4.3. Crystal data of (5)C24H28O8 (2CH3OH),Mr ¼ 508.55, monoclinic, a ¼ 12.6816(4) Å,

b ¼ 6.5333(2) Å, c ¼ 15.5587(4) Å, a ¼ 90.00, b ¼ 100.022(3),g ¼ 90.00, V ¼ 1269.41(6) Å3, space group P21, Z ¼ 2,Dcalcd ¼ 1.330 mg/m3, m ¼ 0.851 mm�1, and F(000) ¼ 544.0. Crystaldimensions: 0.41 � 0.38 � 0.34 mm3. Independent reflections:4428 (Rint ¼ 0.0337). The final R1 values were 0.0491, uR2 ¼ 0.1372(I > 2s(I)). The goodness of fit on F2 was 1.070. Flackparameter ¼ 0.1(2). CCDC number: 1520932.

4.5. Calculation of ECD spectra

Molecular Merck force field (MMFF) and DFT/TD-DFT calcula-tions were carried out with Spartan' 14 software (WavefunctionInc.) and Gaussian 09 program, respectively. Conformers weregenerated and optimized using DFT calculations at the B3LYP/6-31G(d) level. Conformers with a Boltzmann distribution over 1%were chosen for ECD calculations in MeOH at the B3LYP/6-311þg(2d,p) level. The IEF-PCM solvent model for MeOH wasused. ECD spectra were generated using the program SpecDis 3.0(University of Würzburg) and Origin Pro 8.5 (Origin Lab, Ltd.) fromdipole-length rotational strengths by applying Gaussian band

mailto:deposit@ccdc.cam.ac.uk

S. Chen et al. / Phytochemistry 146 (2018) 8e15 15

shapes with sigma ¼ 0.30 ev. All calculations were performed byTianhe-2 in the National Super Computer Center in Guangzhou.

4.6. Cell culture, measurement of NO production, and cell viability

RAW264.7 cells were purchased from Cell bank of ChineseAcademy of Sciences (Shanghai, People's Republic of China). Cellmaintenance, experimental procedures, and data determination forthe inhibition of NO production and the viability assay in vitro arethe same as literature (Etoh et al., 2013). The IC50 values weredetermined using Origin 8 Pro. software from experiments per-formed in triplicate. Indomethacin (IC50 value of 26.3 ± 1.2 mM)wasused as a positive control, and was purchased from SigmaeAldrichCo. (CAS number: 53-86-1, EINECS number: 200-186-5). HPLCprofile and 1H NMR spectrum were used to determine the puritiesof the pharmacologically tested compounds. Except the purity ofcompound 1 was 90%, the other tested compounds' purities was atleast 95%. All tested compoundswere prepared as stock solutions inDMSO and final solvent concentration was less than 0.2% in allassays.

Notes

The authors declare no competing financial interest.

Acknowledgements

We thank the National Natural Science Foundation of China(21472251, 41276146, 41404134), the Science & Technology PlanProject of Guangdong Province of China (2013B021100011), the Keyproject of Natural Science Foundation of Guangdong Province(2016A040403091), Special Financial Fund of Innovative Develop-ment of Marine Economic Demonstration Project (GD2012-D01-001) for generous support.

Appendix A. Supplementary data

Supplementary data related to this article can be found athttps://doi.org/10.1016/j.phytochem.2017.11.011.

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Anti-inflammatory meroterpenoids from the mangrove endophytic fungus Talaromyces amestolkiae YX11. Introduction2. Results and discussion3. Conclusions4. Experimental4.1. General experimental procedures4.2. Fungal material4.3. Fermentation, extraction and isolation4.3.1. Amestolkolide A (1)4.3.2. Amestolkolide B (2)4.3.3. Amestolkolide C (3)4.3.4. Amestolkolide D (4)

4.4. X-ray crystallographic analysis of compounds 2, 3 and 54.4.1. Crystal data of (2)4.4.2. Crystal data of (3)4.4.3. Crystal data of (5)

4.5. Calculation of ECD spectra4.6. Cell culture, measurement of NO production, and cell viability

NotesAcknowledgementsAppendix A. Supplementary dataReferences