Two New Secondary Metabolites from the Endophytic Fungus ... - MDPI
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Two New Secondary Metabolites from the Endophytic Fungus Endomelanconiopsis endophytica Zhang-Hua Sun † , Hao-Hua Li † , Fa-Liang Liang † , Yu-Chan Chen, Hong-Xin Liu, Sai-Ni Li, Guo-Hui Tan and Wei-Min Zhang * State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangdong Institute of Microbiology, Guangzhou 510070, China; [email protected] (Z.-H.S.); [email protected] (H.-H.L.); [email protected] (F.-L.L.); [email protected] (Y.-C.C.); [email protected] (H.-X.L.); [email protected] (S.-N.L.); [email protected] (G.-H.T.) * Correspondence: [email protected]; Tel.: +86-20-8768-8309 † These authors contributed equally to this work. Academic Editor: Derek J. McPhee Received: 24 May 2016; Accepted: 15 July 2016; Published: 20 July 2016
Abstract: Two new secondary metabolites, endomeketals A–B (1–2), a new natural product (3), and a known compound (4) were isolated from the ethyl acetate extract of the endophytic fungus Endomelanconiopsis endophytica A326 derived from Ficus hirta. Their structures were determined on the basis of extensive spectroscopic analysis. All compounds were evaluated for their cytotoxic activities against SF-268, MCF-7, NCI-H460, and HepG-2 tumor cell lines. However, no compound showed cytotoxic activity against these human tumor cell lines. Keywords: endophytic fungus; Endomelanconiopsis endophytica; ketals; Ficus hirta
1. Introduction Endophytes are microorganisms that reside in the tissues of living plants asymptomatically, and the mutualistic interaction that occurs between the two may have benefits to each other. These organisms represent a huge and largely untapped resource of natural products with chemical structures that have been optimized by evolution for biological and ecological relevance [1]. Over the past 20 years, a surprisingly high number of metabolites have been described from endophytes [2–8]. Several secondary metabolites, including alkaloids [9], sesquiterpenoids [10], and azaphilones [11], exhibit a variety of biological activities. Ficus hirta Vahl. (Moraceae), endemic to Guangdong province of China, is known as “Wu Zhi Mao Tao” in traditional Chinese medicine for the treatment of cough, asthma, and inflammatory diseases [12]. As part of an ongoing program aimed at exploring bioactive secondary metabolites from endophytic fungi [13–15], we undertook a detailed chemical analysis on the ethyl acetate (EtOAc) extract of Endomelanconiopsis endophytica A326 derived from the medicinal plant Ficus hirta, which led to the isolation of two new secondary metabolites (1–2) and two known compounds (3–4). All compounds were evaluated for their cytotoxic activities against SF-268, MCF-7, NCI-H460, and HepG-2 tumor cell lines. Herein, details of the isolation and structural elucidation of these compounds are described. 2. Results and Discussion The liquid culture of the fungus E. endophytica was centrifuged to separate broth and mycelia. The broth was exhaustively extracted with ethyl acetate (EtOAc), and the concentrated EtOAc extract was further purified by various chromatographic methods to yield four compounds (1–4) (Figure 1). Two new compounds, endomeketals A–B (1–2), were identified by spectroscopic analyses Molecules 2016, 21, 943; doi:10.3390/molecules21070943
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and physicochemical properties, while a new natural product a known compound were identified physicochemical properties, while a new natural product andand a known compound were identified as as 2,3-dimethylcyclopent-2-en-1-one (3) [16,17] and 2-hydroxymethyl-3-methylcyclopent-2-enone 2,3-dimethylcyclopent-2-en-1-one (3) [16,17] and 2-hydroxymethyl-3-methylcyclopent-2-enone (4) (4) by comparison of their spectroscopic data with those in the literature. [18][18] by comparison of their spectroscopic data with those in the literature.
Figure 1. The chemical structures of compounds 1–4.
2.1. 2.1. Identification Identification of of New New Compounds Compounds 13C nuclear magnetic Compound was assigned assigned aamolecular molecularformula formulaof ofCC14H H20O3 on the basis of 13 Compound 11 was 14 20 O3 on the basis of C nuclear magnetic resonance spectroscopy (HRESIMS) ionion at resonance (NMR) (NMR) data dataand andhigh highresolution resolutionelectrospray electrosprayionization ionizationmass mass spectroscopy (HRESIMS) + (calcd 1H-NMR + 1 m/z 259.1308 [M + Na] 259.1310). The spectrum (Table 1) showed one methyl doublet at m/z 259.1308 [M + Na] (calcd 259.1310). The H-NMR spectrum (Table 1) showed one methyl [δ H 1.04 (d, J = 6.7 Hz, H3-7′)], one vinylic H 2.31 (H3-7)], four protons bonded to carbons doublet [δH 1.04 (d, J = 6.7 Hz, H3 -71 )], one methyl vinylic [δ methyl [δH 2.31 (H3 -7)], four protons bonded to bearing heteroatoms (δ H 5.36 (s, H-6), 4.25 (dd, J = 4.2, 4.1 Hz, H-1′), 4.07 (dd,1 J = 11.9, 3.1 Hz, H-6′a), and carbons bearing heteroatoms (δH 5.36 (s, H-6), 4.25 (dd, J = 4.2, 4.1 Hz, H-1 ), 4.07 (dd, J = 11.9, 3.1 Hz, 13C-NMR 1 b), of 4.03 (d, and J = 11.9 series aliphatic multiplets (Figure S1). The H-61 a), 4.03Hz, (d, H-6′b), J = 11.9and Hz,aH-6 and a seriesmethylene of aliphatic methylene multiplets (Figure S1). 13 spectrum (Figure S2), in combination with heteronuclear single quantum coherence (HSQC, Figure S3) The C-NMR spectrum (Figure S2), in combination with heteronuclear single quantum coherence experiments, showed 14 carbon resonances attributable to one carbonyl (δ C 206.8, C-1), two sp2 quaternary (HSQC, Figure S3) experiments, showed 14 carbon resonances attributable to one carbonyl (δC 206.8, 3 methines [δC 94.4 (C-6), 81.0 (C-1′), 2 quaternary carbons (δCsp177.8 (C-3) andcarbons 137.4 (C-2), 47.2 (C-2′), and(C-6), 32.5 C-1), two (δC four 177.8sp(C-3) and 137.4 (C-2), four sp3 methines [δC 94.4 1 ), 47.2 1 (C-3′)], methylenes [δ32.5 C 66.3 (C-6′), 34.4 (C-5), 32.4[δ (C-4), 31.81 (C-4′), and 31.4 (C-5′)], and two 81.0 (C-1five (C-21 ), and (C-31 )], five methylenes C 66.3 (C-6 ), 34.4 (C-5), 32.4 (C-4), 31.8 (C-4 ), 1 1 methyls [δ C 19.0 (C-7′) and 18.3 (C-7)]. As two of the five degrees of unsaturation were accounted for and 31.4 (C-5 )], and two methyls [δC 19.0 (C-7 ) and 18.3 (C-7)]. As two of the five degrees of by a double bond a carbonyl group, the remaining degrees of unsaturation that 1 unsaturation wereand accounted for by a double bond and athree carbonyl group, the remainingrequired three degrees was tricyclic. of unsaturation required that 1 was tricyclic. 13C (125 MHz)-NMR data of 1−2 in CDCl3 (J in Hz, δ in ppm). Table1.1.11H H(500 (500MHz) MHz)and and13 Table C (125 MHz)-NMR data of 1´2 in CDCl3 (J in Hz, δ in ppm).
Position
Position
1 21 2 3 3
44 55 66 7 7 11 1′21 2′31 3′41 4′51 5′61 71
6′ 7′
1 δH
1
δH
2.52 2.52(1H, (1H,m) m) 2.41 2.41(1H, (1H,m) m) 2.36(1H, (1H,m) m) 2.36 5.36(1H, (1H,s)s) 5.36 2.31 (3H, s) 2.31 4.25(3H, (1H,s)m) 4.25 1.18(1H, (1H,m) ddd, 10.7, 4.5, 3.0) 2.42(1H, (1H,ddd, m) 10.7, 4.5, 3.0) 1.18 2.12(1H, (1H,m) m) 2.42 1.25 (1H, m) 2.12 (1H, m) 1.70 (1H, ddd, 14.3, 9.1, 4.9) 1.25 1.87(1H, (1H,m) m) 1.70 14.3, 9.1, 4.9) 4.04(1H, (1H,ddd, d, 11.8) 4.07 (1H, dd, 11.8, 3.0) 1.87 (1H, m) 1.04 (3H, d, 6.7) 4.04 (1H, d, 11.8) 4.07 (1H, dd, 11.8, 3.0) 1.04 (3H, d, 6.7)
δC 206.8 206.8 137.4 137.4 177.8 δC
δH
2
2
δH
177.8
δC 207.1 207.1 136.0 136.0 177.5 δC
177.5
32.4 2.55 2.55 (2H, ddd, 32.5 32.4 (2H, ddd, 5.6, 5.6, 2.3, 2.3, 1.1) 1.1) 32.5 34.4 34.4 94.4 94.4 18.3 18.3 81.0 81.0 47.2 32.5 47.2
2.37 (1H, m) m) 2.37 (1H, 5.80 (1H, s) s) 5.80 (1H, 2.25 (3H, s) 2.25 (3H, s) 1.27 (3H, d, 6.1) 1.27 (3H, 6.1) 3.82 (1H, dd,d, 7.8, 6.1) 3.70 (1H, dd,dd, 7.8,7.8, 6.0) 6.1) 3.82 (1H,
34.5 34.5 95.9 95.9 17.8 17.8 17.3 17.3 78.6 80.4 78.6
32.5 1.36 3.70 (1H, dd, 7.8, 6.0) 31.8 (3H, d, 6.0)
80.4 16.5
31.8 31.4 66.3 31.4 19.0
66.3 19.0
1.36 (3H, d, 6.0)
16.5
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(HSQC, HMBC the Detailed and afforded Detailed Detailed 2D-NMR 2D-NMR 2D-NMR studies studies studies (HSQC, (HSQC, 1111HH-111H-COSY, H-COSY, and and HMBC HMBC experiments) experiments) experiments) afforded afforded the the gross gross gross 1H-1H-COSY, and HMBC experiments) afforded the gross Detailed 2D-NMR studies (HSQC, 1Hstructures of two correlation H-4 and structures structuresof oftwo twosub-units sub-units sub-units(A (A (Aand and andB) B) B)as as asdepicted depicted depictedin in inFigure Figure Figure2.2. 2.111HH-1111H-COSY H-COSYcorrelation correlationbetween betweenH-4 H-4and and structures of two sub-units (A and B) as depicted in Figure 2. 1H-1H-COSY correlation between H-4 and H-5 33-7 C-1, H-5 from H-5 H-5and and andHMBC HMBC HMBCcorrelations correlations correlationsfrom from fromHH H -7to to toC-2/C-3/C-4, C-2/C-3/C-4, C-2/C-3/C-4, from H-5 to toC-1, C-1,and and andfrom from fromH-6 H-6 H-6toto toC-1/C-2/C-3, C-1/C-2/C-3, C-1/C-2/C-3, 33-7 H-5 and HMBC correlations from H3-7 to C-2/C-3/C-4, from H-5 to C-1, and from H-6 to C-1/C-2/C-3, revealed unit A was which revealedunit unitA Awas washighly highly highlysimilar similar similarto to to2-hydroxymethyl-3-methylcyclopent-2-enone 2-hydroxymethyl-3-methylcyclopent-2-enone 2-hydroxymethyl-3-methylcyclopent-2-enone(4) (4) (4)(Figure (Figure (Figure2), 2), 2),which which revealed revealed unit A was highly similar to 2-hydroxymethyl-3-methylcyclopent-2-enone (4) (Figure 2), which was by of their data. As wasfurther further furtherconfirmed confirmed confirmed by bycomparison comparison comparison of of their their1D-NMR 1D-NMR 1D-NMR data. data. As Asfor for forunit unit unitB, B, B,the the thestructural structural structuralfragments fragments fragments was was further confirmed by comparison of their 1D-NMR data. As for unit1 B, the1 structural fragments 1 ÑC-71 ) were 1 H1 H-COSY 1 /C-1 1, 111H111H-COSY (C-1′→C-7′) (C-1′→C-7′) were established by the HH-COSY correlations correlations of of C-1′/C-2′/C-3′/C-4′/C-5′/C-1′, C-1′/C-2′/C-3′/C-4′/C-5′/C-1′, and and C-2′/ C-2′/ (C-1 wereestablished establishedby bythe the correlations of C-1 /C-2 /C-31 /C-41 /C-5 (C-1′→C-7′) were established by the 1H-1H-COSY correlations of C-1′/C-2′/C-3′/C-4′/C-5′/C-1′, and C-2′/ 1 1 1 1 C-6′ C-6′ and and C-3′/C-7′. C-3′/C-7′. The connectivities of oftwo twosub-units sub-units were weremainly mainly mainlyachieved achieved achieved by by HMBC HMBC interactions interactions from from and C-2 /C-6 andThe C-3connectivities /C-7 . The connectivities ofwere two sub-units were by mainly achieved by from HMBC C-6′ and C-3′/C-7′. The connectivities of two sub-units HMBC interactions 1 1 H-6 to C-1′ and C-6′. The relative configuration of 1 was determined by a NOESY experiment. NOESY H-6 relative of11was was determinedby a NOESY experiment. NOESY interactions from H-6The to C-1 and configuration C-6 . The relative configuration ofby 1a was determined by NOESY a NOESY H-6totoC-1′ C-1′and andC-6′. C-6′. The relative configuration of determined NOESY experiment. 1 /H-2 1 /H-31 that 1 /H-6 1 indicated correlations correlations of of H-6/H-1′/H-2′/H-3′ H-6/H-1′/H-2′/H-3′ and and Me-7′/H-6′ Me-7′/H-6′ indicated indicated that H-6, H-6, H-1′, H-1′, H-2′, H-2′, and and H-3′ H-3′ were were cofacial cofacial experiment. NOESY correlations of H-6/H-1 and Me-7 that H-6, H-11 , correlations of H-6/H-1′/H-2′/H-3′ and Me-7′/H-6′ indicated that H-6, H-1′, H-2′, and H-3′ were cofacial 1 1 and and designated as α-oriented (Figure S6). S6).Thus, Thus,compound compound compound was was determined determined as as depicted depicted and and given given the the H-2 ,designated and H-3 as were cofacial(Figure and designated as α-oriented (Figure S6).asThus, compound 1the was and designated asα-oriented α-oriented (Figure S6). Thus, 111was determined depicted and given trivial trivial name name endomeketal endomeketal A. A. determined as depicted and trivial name endomeketal A. given the trivial name endomeketal A.
1H-COSY ( Figure 2. Key1111 1HFigure 2. Figure2. 2. Key Key HH-1111H-COSY H-COSY ((
), HMBC ( ),), HMBC HMBC ((
), and NOE ( ),), and and NOE NOE ((
) correlations of compounds 1–2. correlations of compounds 1–2. )))correlations correlationsof ofcompounds compounds1–2. 1–2.
HRESIMS analysis of 2 revealed an adduct ion [M + Na]+ ++consistent with a molecular formula HRESIMS HRESIMS analysis analysis analysis of of of222revealed revealed revealedan an anadduct adduct adduction ion ion[M [M [M+++Na] Na] Na]++ consistent consistent consistent with with withaaamolecular molecular molecularformula formula formula HRESIMS (C11H16O3) requiring four double bond equivalents (Figure S14). The 1D-NMR data of 2 were similar 11 16 (C (C 11 11H H 16 16O O 333)) requiring requiring four four double double bond bond equivalents equivalents (Figure (Figure S14). S14). The The 1D-NMR 1D-NMR data data of of 2 2 were were similar (Cto ) requiring four equivalents (Figure S14). The 1D-NMR of 2 wereofsimilar similar 11 H 16 O3of those 1 except for thedouble absencebond of signals for the saturated pentacyclic unit anddata the presence the to tosignals those thoseof of 11except except for for the absence absence of of signals for for the the saturated saturated pentacyclic pentacyclic unit unit and and the theS9). presence presence of ofthe the to those of except forthe the ofsignals signals for the saturated pentacyclic unit presence bearing heteroatoms (Table 1, Figures S8and and the The gross of for1two methyls andabsence two sp3 methines 333methines methines bearing bearing heteroatoms heteroatoms (Table (Table 1, 1, Figures Figures S8 S8 and and S9). S9). The The gross gross signals signals for for two two methyls methyls and and two two sp sp 3 thestructure signals of for2two andbytwo sp methines bearingdata heteroatoms (Table 1,COSY Figures S8 and S9). wasmethyls established analyses of its 2D-NMR (Figures S10–S13). correlations structure structure of was established by byC-1′ analyses analyses of ofso its its 2D-NMR 2D-NMR data (Figures (Figures COSY COSY correlations correlations revealed a 212Hwas spinestablished system from to C-4′, that theof sub-structure B data in S10–S13). 1S10–S13). was substituted in 2 by a The grossof structure of 2 was established by analyses itsdata 2D-NMR (Figures S10–S13). COSY 111H revealed revealed a a H spin spin system system from from C-1′ C-1′ to to C-4′, C-4′, so so that that the the sub-structure sub-structure B B in in 1 1 was was substituted substituted in in 2 2 by 1 1 1 butane-2,3-diyl residue. assigned unit A in 2 wereBnearly identical toby aa correlations revealed a H The spincarbon systemchemical from C-1shifts to C-4 , so thattothe sub-structure in 1 was substituted butane-2,3-diyl butane-2,3-diyl residue. residue. The The carbon carbon chemical chemical shifts shifts assigned assigned to to unit unit A A in in 2 2 were were nearly nearly identical identical to carbon chemical assigned to shifts this fragment The A HMBC interactions from to in the 2 bycorresponding a butane-2,3-diyl residue. The shifts carbon chemical assignedinto1.unit in 2 were nearly identical the corresponding carbon chemical shifts assigned to this fragment in 1. The HMBC interactions from the corresponding carbon chemical shifts to this fragment 1.in The HMBC interactions from H-6 to C-1, C-2, and C-3, and from H-2′ to assigned C-6assigned connected Bfragment to unitinA. As of the four degrees to the corresponding carbon chemical shifts tounit this 1.three The HMBC interactions H-6 H-6 to to C-1, C-1, C-2, C-2, and and C-3, C-3, and and from from H-2′ H-2′ to to C-6 C-6 connected connected unit unit B B to to unit unit A. A. As As three three of of the the four four degrees degrees 1 of unsaturation were accounted for by the unsaturated pentacyclic unit, the remaining double bond from H-6 to C-1, C-2, and C-3, and from H-2 to C-6 connected unit B to unit A. As three of the four of ofequivalent unsaturation unsaturation were were accounted accounted for by byunit the the unsaturated unsaturated pentacyclic pentacyclic unit, unit, the the remaining remaining double double bond bond required that unit Bfor and inthe 2 unsaturated were connected via a 1,3-dioxolane. The relative degrees of unsaturation were accounted for A by pentacyclic unit, the remaining double configuration ofrequired 2 was a unit NOESY NOESY correlations of H-6/H-3′ and equivalent equivalent required required that thatdetermined unit unitunit BB and and unit unit A A in in in 22experiment. were connected via via aaa1,3-dioxolane. 1,3-dioxolane. The The relative relative relative bond equivalent that B by and A 2were wereconnected connected via 1,3-dioxolane. The H-3′/H3-1′ indicated that H3-1′, H-3′, and H-6 were cofacial and designated as α-oriented, while configuration configuration of of 2 2 was was determined determined by by a a NOESY NOESY experiment. experiment. NOESY NOESY correlations correlations of of H-6/H-3′ H-6/H-3′ and and configuration of 2 was determined by a NOESY experiment. NOESY correlations of H-6/H-31 and correlation of H-2′/H 3 -4′ assigned H-2′ and H 3 -4′ as β. Thus, compound 2 was assigned as depicted 3 3 H-3′/H H-3′/H 3 3 -1′ -1′ indicated indicated that that H H 3 3 -1′, -1′, H-3′, H-3′, and and H-6 H-6 were were cofacial cofacial and and designated designated as as α-oriented, α-oriented, while while H-31 /H3 -11 indicated that H3 -11 , H-31 , and H-6 were cofacial and designated as α-oriented, while and named B. correlation correlation of ofendomeketal H-2′/H H-2′/H -4′1assigned assigned H-2′ H-2′1 and and H H333-4′ -4′1as as β. β. Thus, Thus, compound compound 22 was was assigned assigned as as depicted depicted 1 /H333-4′ correlation of H-2 3 -4 assigned H-2 and H3 -4 as β. Thus, compound 2 was assigned as depicted By comparison of their spectroscopic data (Figures S15–S18) with those reported [16–18], and and named named endomeketal endomeketal B. B. and named endomeketal B. theBy structures of compounds 3 and 4 were assigned as 2,3-dimethylcyclopent-2-en-1-one (3)[16–18], and comparison of their spectroscopic data (Figures S15–S18) with those reported By comparison of their their spectroscopic spectroscopic data (Figures (Figures S15–S18) with with those those reported reported [16–18], By comparison of data S15–S18) [16–18], 2-hydroxymethyl-3-methylcyclopent-2-enone (4), respectively. Although compound 3 was previously the the structures structures structures of of compounds compounds compounds 333 and and and 444were were were assigned assigned as as 2,3-dimethylcyclopent-2-en-1-one 2,3-dimethylcyclopent-2-en-1-one (3) (3) and and the reported as a of synthetic compound, this is the assigned first reportasof2,3-dimethylcyclopent-2-en-1-one its isolation from a natural source. (3) and 2-hydroxymethyl-3-methylcyclopent-2-enone 2-hydroxymethyl-3-methylcyclopent-2-enone (4), (4), respectively. respectively. Although Although compound compound 3 3 was was previously previously 2-hydroxymethyl-3-methylcyclopent-2-enone (4), respectively. Although compound 3 was previously reported reportedas as asaaasynthetic synthetic syntheticcompound, compound, compound,this this thisis isisthe the thefirst first firstreport report reportof of ofits its itsisolation isolation isolationfrom from fromaaanatural natural naturalsource. source. source. reported 2.2. Cytotoxicity Assay The in vitro cytotoxicities of compounds 1–4 were evaluated against four tumor cell lines, 2.2. Assay 2.2. Cytotoxicity Cytotoxicity Assay 2.2. Assay including SF-268 (human glioblastoma cell line, ATCC HTB-14), MCF-7 (human breast adenocarcinoma The in against four tumor cell lines, The in vitro vitro cytotoxicities cytotoxicities of of compounds compounds compounds 1–4 1–4 1–4 were were were evaluated evaluated against four tumor tumor cell celland lines, vitro cytotoxicities of evaluated against four lines, cellThe line,in ATCC HTB-22), NCI-H460 (human non-small cell lung cancer cell line, ATCC HTB-177), including including SF-268 SF-268 (human (human glioblastoma glioblastoma cell cell line, line, ATCC ATCC HTB-14), HTB-14), MCF-7 MCF-7 (human (human breast breast adenocarcinoma adenocarcinoma HepG-2 (human hepatocellular carcinoma cell line, ATCC HB-8065). However, no compound showed cell cell line, line,ATCC ATCC HTB-22), HTB-22), NCI-H460 NCI-H460 (human (human non-small non-small cell cell lung lung cancer cancer cell cellcell line, line,line, ATCC ATCC HTB-177), HTB-177), and and cell line, HTB-22), NCI-H460 (human non-small cell lung cancer ATCC HTB-177), obvious cytotoxic activity against these human tumor cell lines at the concentration of 100 µM. HepG-2 HepG-2 (human (human hepatocellular hepatocellular carcinoma carcinoma cell cellline, line, ATCC HB-8065). HB-8065). However, However, no nocompound compound showed showed and HepG-2 (human hepatocellular carcinoma cellATCC line, ATCC HB-8065). However, no compound obvious obviousobvious cytotoxic cytotoxic activity activityagainst againstthese these human human tumor cell lines linescell at atthe the concentration concentration of of100 100µM. µM. showed cytotoxic activity against thesetumor humancell tumor lines at the concentration of 100 µM.
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3. Materials and Methods 3.1. General Experimental Procedures Optical rotation was measured on an Anton Paar MCP-500 spectropolarimeter (Anton Paar, Vienna, Austria). The IR spectrum was recorded on an IRAffinity-1 spectrophotometer in cm´1 (Shimadzu Corporation, Kyoto, Japan). UV spectra were measured on a SHIMADZU UV-2600 UV-VIS spectrophotometer (Shimadzu Corporation). 1D- and 2D-NMR spectra were recorded on a Bruker Avance-600 spectrometer with TMS as internal standard (Bruker BioSpin International, Geneva, Switzerland), δ in ppm, J in Hz. HRESIMS was measured on a Thermo MAT95XP high-resolution mass spectrometer. A Shimadzu LC-20 AT (Shimadzu Corporation) equipped with an SPD-M20A PDA detector (Shimadzu Corporation) was used for HPLC, a YMC-pack ODS-A column (250 m ˆ 10 mm, 5 µm, 12 nm) was used for semi-preparative HPLC separation. Silica gel (200–300 mesh) was used for column chromatography, and precoated silica gel GF254 plates (Qingdao Marine Chemical Inc., Qingdao, China) were used for TLC spotting. All solvents were analytical grade (Guangzhou Chemical Reagents Company, Ltd., Guangzhou, China). 3.2. Fungal Material The endophytic fungal strain A326 was isolated from the twigs of Ficus hirta, which was collected at Luofu Mountain Nature Reserve, Huizhou, Guangdong province of China, in October 2010. The isolated strain was identified as Endomelanconiopsis endophytica based on a morphological study, and sequence analysis of rDNA ITS (internal transcribed spacer), showing 100% similarity to the strain of E. endophytica (Accession No. EU687005). The strain is preserved at the Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology. 3.3. Extraction and Isolation E. endophytica A326 was grown on potato-dextrose agar (PDA) medium at 28 ˝ C for five days and then inoculated into five flasks (500 mL) containing potato-dextrose (PD) medium (250 mL). After five days of incubation at 28 ˝ C on a rotary shaker at 120 r/min, a portion of the liquid culture was aseptically transferred into each of a total of 200 flasks (500 mL) containing potato-dextrose (PD) medium (250 mL). Following seven days of cultivation at 28 ˝ C and 120 r/min on a rotary shaker, the culture (a total of 50 L) was filtered to give the filtrate and mycelia. The broth was exhaustively extracted with ethyl acetate (EtOAc) for four times, and then the EtOAc layers were combined and evaporated under reduced pressure at a temperature not exceeding 40 ˝ C to yield a dark brown gum (11.7 g). The crude extract was subjected to silica gel column chromatography with a CH2 Cl2 /MeOH gradient (9:1Ñ1:9) to afford six fractions (Fr. I–VI). Fr. II (1.1 g) was subjected to column chromatography on silica gel using n-hexane as the first eluent, and then EtOAc of increasing polarity, to give nine sub-fractions (Fr. IIa ´Fr. IIi ). Fr.IId was further separated by RP-HPLC equipped with a semi-preparative column (CH3 CN, 3 mL/min) to afford 2 (5 mg, tR 7.6 min) and 3 (19 mg, tR 9.1 min). Fr. IIe was chromatographed by RP-HPLC equipped with a semi-preparative column (MeOH/H2 O, 70:30, 3 mL/min) to afford 4 (36 mg, tR 7.2 min) and 1 (24 mg, tR 13.0 min). 3.4. Spectroscopic Data Endomeketal A (1): white, amorphous powder (MeOH); rαs25 D +33.8 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 229 (3.83) nm; IR (KBr) νmax = 1693, 1435, 1409, 1259, 1092, 1018 cm´1 ; HRESIMS m/z 259.1308 [M + Na]+ (calcd for C14 H20 O3 Na, 259.1310, Figure S7); 1 H- and 13 C-NMR data, see Table 1. Endomeketal B (2): white, amorphous powder (MeOH); rαs25 D +23.0 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 222 (3.56) nm; IR (KBr) νmax = 1691, 1381, 1257, 1087, 1022 cm´1 ; HRESIMS m/z 219.1002 [M + Na]+ (calcd for C11 H16 O3 Na, 219.0997, Figure S14); 1 H- and 13 C-NMR data, see Table 1.
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3.5. Cytotoxicity Assay The cell growth inhibitory activities of compounds 1–4 against human tumor cell lines SF-268, MCF-7, NCI-H460, and HepG-2, were tested using the previously-published method [19]. 4. Conclusions Fungal endophytes live inside plant tissues for all, or part, of their life without causing apparent disease symptoms. Endophytic fungi have been found within host tissues at high density and diversity in all tropical and temperate ecosystems where endophytes have been sought [20]. Endomelanconiopsis is a new anamorph genus in the Botryosphaeriaceae, and usually lives inside plant tissues [21]. Previous investigation onto E. endophytica A326 in our research group led to the isolation of a series of xyloketals, which were the first chemical constituents of the genus Endomelanconiopsis [22]. As the fermentation products of the fungus E. endophytica displayed considerable structural diversity, continued investigation of this fungus has resulted in the isolation of two further new secondary metabolites, which were named as endomeketals A and B. The structures were determined by spectroscopic analysis. All of the isolates were evaluated for in vitro cytotoxicity against SF-268, MCF-7, NCI-H460, and HepG-2 cell lines. This study not only enriches the chemical diversity of the genus Endomelanconiopsis, but also implies that this endophytic fungus may be a potential source for producing a series of ketals. Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/ 21/7/943/s1. Acknowledgments: This work was supported financially by the National Basic Research Program of China (973 Program, 2014CB460613), the Natural Science Foundation of Guangdong Province (2015A030313710), and the Guangdong Provincial Project for Science and Technology (2014A030304050, 2015A030302060). Author Contributions: Z.-H.S. and H.-H.L. elucidated structures and wrote the paper. F.-L.L. fractionated the extract, isolated the compounds. Y.-C.C. performed the bioassays. H.-X.L., S.-N.L. and G.-H.T. performed the experiments and analyzed the data. W.-M.Z. designed and coordinated the study and reviewed the manuscript. Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–4 are available from the authors. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
Two New Secondary Metabolites from the Endophytic Fungus Endomelanconiopsis endophytica Zhang-Hua Sun † , Hao-Hua Li † , Fa-Liang Liang † , Yu-Chan Chen, Hong-Xin Liu, Sai-Ni Li, Guo-Hui Tan and Wei-Min Zhang * State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangdong Institute of Microbiology, Guangzhou 510070, China; [email protected] (Z.-H.S.); [email protected] (H.-H.L.); [email protected] (F.-L.L.); [email protected] (Y.-C.C.); [email protected] (H.-X.L.); [email protected] (S.-N.L.); [email protected] (G.-H.T.) * Correspondence: [email protected]; Tel.: +86-20-8768-8309 † These authors contributed equally to this work. Academic Editor: Derek J. McPhee Received: 24 May 2016; Accepted: 15 July 2016; Published: 20 July 2016
Abstract: Two new secondary metabolites, endomeketals A–B (1–2), a new natural product (3), and a known compound (4) were isolated from the ethyl acetate extract of the endophytic fungus Endomelanconiopsis endophytica A326 derived from Ficus hirta. Their structures were determined on the basis of extensive spectroscopic analysis. All compounds were evaluated for their cytotoxic activities against SF-268, MCF-7, NCI-H460, and HepG-2 tumor cell lines. However, no compound showed cytotoxic activity against these human tumor cell lines. Keywords: endophytic fungus; Endomelanconiopsis endophytica; ketals; Ficus hirta
1. Introduction Endophytes are microorganisms that reside in the tissues of living plants asymptomatically, and the mutualistic interaction that occurs between the two may have benefits to each other. These organisms represent a huge and largely untapped resource of natural products with chemical structures that have been optimized by evolution for biological and ecological relevance [1]. Over the past 20 years, a surprisingly high number of metabolites have been described from endophytes [2–8]. Several secondary metabolites, including alkaloids [9], sesquiterpenoids [10], and azaphilones [11], exhibit a variety of biological activities. Ficus hirta Vahl. (Moraceae), endemic to Guangdong province of China, is known as “Wu Zhi Mao Tao” in traditional Chinese medicine for the treatment of cough, asthma, and inflammatory diseases [12]. As part of an ongoing program aimed at exploring bioactive secondary metabolites from endophytic fungi [13–15], we undertook a detailed chemical analysis on the ethyl acetate (EtOAc) extract of Endomelanconiopsis endophytica A326 derived from the medicinal plant Ficus hirta, which led to the isolation of two new secondary metabolites (1–2) and two known compounds (3–4). All compounds were evaluated for their cytotoxic activities against SF-268, MCF-7, NCI-H460, and HepG-2 tumor cell lines. Herein, details of the isolation and structural elucidation of these compounds are described. 2. Results and Discussion The liquid culture of the fungus E. endophytica was centrifuged to separate broth and mycelia. The broth was exhaustively extracted with ethyl acetate (EtOAc), and the concentrated EtOAc extract was further purified by various chromatographic methods to yield four compounds (1–4) (Figure 1). Two new compounds, endomeketals A–B (1–2), were identified by spectroscopic analyses Molecules 2016, 21, 943; doi:10.3390/molecules21070943
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and physicochemical properties, while a new natural product a known compound were identified physicochemical properties, while a new natural product andand a known compound were identified as as 2,3-dimethylcyclopent-2-en-1-one (3) [16,17] and 2-hydroxymethyl-3-methylcyclopent-2-enone 2,3-dimethylcyclopent-2-en-1-one (3) [16,17] and 2-hydroxymethyl-3-methylcyclopent-2-enone (4) (4) by comparison of their spectroscopic data with those in the literature. [18][18] by comparison of their spectroscopic data with those in the literature.
Figure 1. The chemical structures of compounds 1–4.
2.1. 2.1. Identification Identification of of New New Compounds Compounds 13C nuclear magnetic Compound was assigned assigned aamolecular molecularformula formulaof ofCC14H H20O3 on the basis of 13 Compound 11 was 14 20 O3 on the basis of C nuclear magnetic resonance spectroscopy (HRESIMS) ionion at resonance (NMR) (NMR) data dataand andhigh highresolution resolutionelectrospray electrosprayionization ionizationmass mass spectroscopy (HRESIMS) + (calcd 1H-NMR + 1 m/z 259.1308 [M + Na] 259.1310). The spectrum (Table 1) showed one methyl doublet at m/z 259.1308 [M + Na] (calcd 259.1310). The H-NMR spectrum (Table 1) showed one methyl [δ H 1.04 (d, J = 6.7 Hz, H3-7′)], one vinylic H 2.31 (H3-7)], four protons bonded to carbons doublet [δH 1.04 (d, J = 6.7 Hz, H3 -71 )], one methyl vinylic [δ methyl [δH 2.31 (H3 -7)], four protons bonded to bearing heteroatoms (δ H 5.36 (s, H-6), 4.25 (dd, J = 4.2, 4.1 Hz, H-1′), 4.07 (dd,1 J = 11.9, 3.1 Hz, H-6′a), and carbons bearing heteroatoms (δH 5.36 (s, H-6), 4.25 (dd, J = 4.2, 4.1 Hz, H-1 ), 4.07 (dd, J = 11.9, 3.1 Hz, 13C-NMR 1 b), of 4.03 (d, and J = 11.9 series aliphatic multiplets (Figure S1). The H-61 a), 4.03Hz, (d, H-6′b), J = 11.9and Hz,aH-6 and a seriesmethylene of aliphatic methylene multiplets (Figure S1). 13 spectrum (Figure S2), in combination with heteronuclear single quantum coherence (HSQC, Figure S3) The C-NMR spectrum (Figure S2), in combination with heteronuclear single quantum coherence experiments, showed 14 carbon resonances attributable to one carbonyl (δ C 206.8, C-1), two sp2 quaternary (HSQC, Figure S3) experiments, showed 14 carbon resonances attributable to one carbonyl (δC 206.8, 3 methines [δC 94.4 (C-6), 81.0 (C-1′), 2 quaternary carbons (δCsp177.8 (C-3) andcarbons 137.4 (C-2), 47.2 (C-2′), and(C-6), 32.5 C-1), two (δC four 177.8sp(C-3) and 137.4 (C-2), four sp3 methines [δC 94.4 1 ), 47.2 1 (C-3′)], methylenes [δ32.5 C 66.3 (C-6′), 34.4 (C-5), 32.4[δ (C-4), 31.81 (C-4′), and 31.4 (C-5′)], and two 81.0 (C-1five (C-21 ), and (C-31 )], five methylenes C 66.3 (C-6 ), 34.4 (C-5), 32.4 (C-4), 31.8 (C-4 ), 1 1 methyls [δ C 19.0 (C-7′) and 18.3 (C-7)]. As two of the five degrees of unsaturation were accounted for and 31.4 (C-5 )], and two methyls [δC 19.0 (C-7 ) and 18.3 (C-7)]. As two of the five degrees of by a double bond a carbonyl group, the remaining degrees of unsaturation that 1 unsaturation wereand accounted for by a double bond and athree carbonyl group, the remainingrequired three degrees was tricyclic. of unsaturation required that 1 was tricyclic. 13C (125 MHz)-NMR data of 1−2 in CDCl3 (J in Hz, δ in ppm). Table1.1.11H H(500 (500MHz) MHz)and and13 Table C (125 MHz)-NMR data of 1´2 in CDCl3 (J in Hz, δ in ppm).
Position
Position
1 21 2 3 3
44 55 66 7 7 11 1′21 2′31 3′41 4′51 5′61 71
6′ 7′
1 δH
1
δH
2.52 2.52(1H, (1H,m) m) 2.41 2.41(1H, (1H,m) m) 2.36(1H, (1H,m) m) 2.36 5.36(1H, (1H,s)s) 5.36 2.31 (3H, s) 2.31 4.25(3H, (1H,s)m) 4.25 1.18(1H, (1H,m) ddd, 10.7, 4.5, 3.0) 2.42(1H, (1H,ddd, m) 10.7, 4.5, 3.0) 1.18 2.12(1H, (1H,m) m) 2.42 1.25 (1H, m) 2.12 (1H, m) 1.70 (1H, ddd, 14.3, 9.1, 4.9) 1.25 1.87(1H, (1H,m) m) 1.70 14.3, 9.1, 4.9) 4.04(1H, (1H,ddd, d, 11.8) 4.07 (1H, dd, 11.8, 3.0) 1.87 (1H, m) 1.04 (3H, d, 6.7) 4.04 (1H, d, 11.8) 4.07 (1H, dd, 11.8, 3.0) 1.04 (3H, d, 6.7)
δC 206.8 206.8 137.4 137.4 177.8 δC
δH
2
2
δH
177.8
δC 207.1 207.1 136.0 136.0 177.5 δC
177.5
32.4 2.55 2.55 (2H, ddd, 32.5 32.4 (2H, ddd, 5.6, 5.6, 2.3, 2.3, 1.1) 1.1) 32.5 34.4 34.4 94.4 94.4 18.3 18.3 81.0 81.0 47.2 32.5 47.2
2.37 (1H, m) m) 2.37 (1H, 5.80 (1H, s) s) 5.80 (1H, 2.25 (3H, s) 2.25 (3H, s) 1.27 (3H, d, 6.1) 1.27 (3H, 6.1) 3.82 (1H, dd,d, 7.8, 6.1) 3.70 (1H, dd,dd, 7.8,7.8, 6.0) 6.1) 3.82 (1H,
34.5 34.5 95.9 95.9 17.8 17.8 17.3 17.3 78.6 80.4 78.6
32.5 1.36 3.70 (1H, dd, 7.8, 6.0) 31.8 (3H, d, 6.0)
80.4 16.5
31.8 31.4 66.3 31.4 19.0
66.3 19.0
1.36 (3H, d, 6.0)
16.5
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(HSQC, HMBC the Detailed and afforded Detailed Detailed 2D-NMR 2D-NMR 2D-NMR studies studies studies (HSQC, (HSQC, 1111HH-111H-COSY, H-COSY, and and HMBC HMBC experiments) experiments) experiments) afforded afforded the the gross gross gross 1H-1H-COSY, and HMBC experiments) afforded the gross Detailed 2D-NMR studies (HSQC, 1Hstructures of two correlation H-4 and structures structuresof oftwo twosub-units sub-units sub-units(A (A (Aand and andB) B) B)as as asdepicted depicted depictedin in inFigure Figure Figure2.2. 2.111HH-1111H-COSY H-COSYcorrelation correlationbetween betweenH-4 H-4and and structures of two sub-units (A and B) as depicted in Figure 2. 1H-1H-COSY correlation between H-4 and H-5 33-7 C-1, H-5 from H-5 H-5and and andHMBC HMBC HMBCcorrelations correlations correlationsfrom from fromHH H -7to to toC-2/C-3/C-4, C-2/C-3/C-4, C-2/C-3/C-4, from H-5 to toC-1, C-1,and and andfrom from fromH-6 H-6 H-6toto toC-1/C-2/C-3, C-1/C-2/C-3, C-1/C-2/C-3, 33-7 H-5 and HMBC correlations from H3-7 to C-2/C-3/C-4, from H-5 to C-1, and from H-6 to C-1/C-2/C-3, revealed unit A was which revealedunit unitA Awas washighly highly highlysimilar similar similarto to to2-hydroxymethyl-3-methylcyclopent-2-enone 2-hydroxymethyl-3-methylcyclopent-2-enone 2-hydroxymethyl-3-methylcyclopent-2-enone(4) (4) (4)(Figure (Figure (Figure2), 2), 2),which which revealed revealed unit A was highly similar to 2-hydroxymethyl-3-methylcyclopent-2-enone (4) (Figure 2), which was by of their data. As wasfurther further furtherconfirmed confirmed confirmed by bycomparison comparison comparison of of their their1D-NMR 1D-NMR 1D-NMR data. data. As Asfor for forunit unit unitB, B, B,the the thestructural structural structuralfragments fragments fragments was was further confirmed by comparison of their 1D-NMR data. As for unit1 B, the1 structural fragments 1 ÑC-71 ) were 1 H1 H-COSY 1 /C-1 1, 111H111H-COSY (C-1′→C-7′) (C-1′→C-7′) were established by the HH-COSY correlations correlations of of C-1′/C-2′/C-3′/C-4′/C-5′/C-1′, C-1′/C-2′/C-3′/C-4′/C-5′/C-1′, and and C-2′/ C-2′/ (C-1 wereestablished establishedby bythe the correlations of C-1 /C-2 /C-31 /C-41 /C-5 (C-1′→C-7′) were established by the 1H-1H-COSY correlations of C-1′/C-2′/C-3′/C-4′/C-5′/C-1′, and C-2′/ 1 1 1 1 C-6′ C-6′ and and C-3′/C-7′. C-3′/C-7′. The connectivities of oftwo twosub-units sub-units were weremainly mainly mainlyachieved achieved achieved by by HMBC HMBC interactions interactions from from and C-2 /C-6 andThe C-3connectivities /C-7 . The connectivities ofwere two sub-units were by mainly achieved by from HMBC C-6′ and C-3′/C-7′. The connectivities of two sub-units HMBC interactions 1 1 H-6 to C-1′ and C-6′. The relative configuration of 1 was determined by a NOESY experiment. NOESY H-6 relative of11was was determinedby a NOESY experiment. NOESY interactions from H-6The to C-1 and configuration C-6 . The relative configuration ofby 1a was determined by NOESY a NOESY H-6totoC-1′ C-1′and andC-6′. C-6′. The relative configuration of determined NOESY experiment. 1 /H-2 1 /H-31 that 1 /H-6 1 indicated correlations correlations of of H-6/H-1′/H-2′/H-3′ H-6/H-1′/H-2′/H-3′ and and Me-7′/H-6′ Me-7′/H-6′ indicated indicated that H-6, H-6, H-1′, H-1′, H-2′, H-2′, and and H-3′ H-3′ were were cofacial cofacial experiment. NOESY correlations of H-6/H-1 and Me-7 that H-6, H-11 , correlations of H-6/H-1′/H-2′/H-3′ and Me-7′/H-6′ indicated that H-6, H-1′, H-2′, and H-3′ were cofacial 1 1 and and designated as α-oriented (Figure S6). S6).Thus, Thus,compound compound compound was was determined determined as as depicted depicted and and given given the the H-2 ,designated and H-3 as were cofacial(Figure and designated as α-oriented (Figure S6).asThus, compound 1the was and designated asα-oriented α-oriented (Figure S6). Thus, 111was determined depicted and given trivial trivial name name endomeketal endomeketal A. A. determined as depicted and trivial name endomeketal A. given the trivial name endomeketal A.
1H-COSY ( Figure 2. Key1111 1HFigure 2. Figure2. 2. Key Key HH-1111H-COSY H-COSY ((
), HMBC ( ),), HMBC HMBC ((
), and NOE ( ),), and and NOE NOE ((
) correlations of compounds 1–2. correlations of compounds 1–2. )))correlations correlationsof ofcompounds compounds1–2. 1–2.
HRESIMS analysis of 2 revealed an adduct ion [M + Na]+ ++consistent with a molecular formula HRESIMS HRESIMS analysis analysis analysis of of of222revealed revealed revealedan an anadduct adduct adduction ion ion[M [M [M+++Na] Na] Na]++ consistent consistent consistent with with withaaamolecular molecular molecularformula formula formula HRESIMS (C11H16O3) requiring four double bond equivalents (Figure S14). The 1D-NMR data of 2 were similar 11 16 (C (C 11 11H H 16 16O O 333)) requiring requiring four four double double bond bond equivalents equivalents (Figure (Figure S14). S14). The The 1D-NMR 1D-NMR data data of of 2 2 were were similar (Cto ) requiring four equivalents (Figure S14). The 1D-NMR of 2 wereofsimilar similar 11 H 16 O3of those 1 except for thedouble absencebond of signals for the saturated pentacyclic unit anddata the presence the to tosignals those thoseof of 11except except for for the absence absence of of signals for for the the saturated saturated pentacyclic pentacyclic unit unit and and the theS9). presence presence of ofthe the to those of except forthe the ofsignals signals for the saturated pentacyclic unit presence bearing heteroatoms (Table 1, Figures S8and and the The gross of for1two methyls andabsence two sp3 methines 333methines methines bearing bearing heteroatoms heteroatoms (Table (Table 1, 1, Figures Figures S8 S8 and and S9). S9). The The gross gross signals signals for for two two methyls methyls and and two two sp sp 3 thestructure signals of for2two andbytwo sp methines bearingdata heteroatoms (Table 1,COSY Figures S8 and S9). wasmethyls established analyses of its 2D-NMR (Figures S10–S13). correlations structure structure of was established by byC-1′ analyses analyses of ofso its its 2D-NMR 2D-NMR data (Figures (Figures COSY COSY correlations correlations revealed a 212Hwas spinestablished system from to C-4′, that theof sub-structure B data in S10–S13). 1S10–S13). was substituted in 2 by a The grossof structure of 2 was established by analyses itsdata 2D-NMR (Figures S10–S13). COSY 111H revealed revealed a a H spin spin system system from from C-1′ C-1′ to to C-4′, C-4′, so so that that the the sub-structure sub-structure B B in in 1 1 was was substituted substituted in in 2 2 by 1 1 1 butane-2,3-diyl residue. assigned unit A in 2 wereBnearly identical toby aa correlations revealed a H The spincarbon systemchemical from C-1shifts to C-4 , so thattothe sub-structure in 1 was substituted butane-2,3-diyl butane-2,3-diyl residue. residue. The The carbon carbon chemical chemical shifts shifts assigned assigned to to unit unit A A in in 2 2 were were nearly nearly identical identical to carbon chemical assigned to shifts this fragment The A HMBC interactions from to in the 2 bycorresponding a butane-2,3-diyl residue. The shifts carbon chemical assignedinto1.unit in 2 were nearly identical the corresponding carbon chemical shifts assigned to this fragment in 1. The HMBC interactions from the corresponding carbon chemical shifts to this fragment 1.in The HMBC interactions from H-6 to C-1, C-2, and C-3, and from H-2′ to assigned C-6assigned connected Bfragment to unitinA. As of the four degrees to the corresponding carbon chemical shifts tounit this 1.three The HMBC interactions H-6 H-6 to to C-1, C-1, C-2, C-2, and and C-3, C-3, and and from from H-2′ H-2′ to to C-6 C-6 connected connected unit unit B B to to unit unit A. A. As As three three of of the the four four degrees degrees 1 of unsaturation were accounted for by the unsaturated pentacyclic unit, the remaining double bond from H-6 to C-1, C-2, and C-3, and from H-2 to C-6 connected unit B to unit A. As three of the four of ofequivalent unsaturation unsaturation were were accounted accounted for by byunit the the unsaturated unsaturated pentacyclic pentacyclic unit, unit, the the remaining remaining double double bond bond required that unit Bfor and inthe 2 unsaturated were connected via a 1,3-dioxolane. The relative degrees of unsaturation were accounted for A by pentacyclic unit, the remaining double configuration ofrequired 2 was a unit NOESY NOESY correlations of H-6/H-3′ and equivalent equivalent required required that thatdetermined unit unitunit BB and and unit unit A A in in in 22experiment. were connected via via aaa1,3-dioxolane. 1,3-dioxolane. The The relative relative relative bond equivalent that B by and A 2were wereconnected connected via 1,3-dioxolane. The H-3′/H3-1′ indicated that H3-1′, H-3′, and H-6 were cofacial and designated as α-oriented, while configuration configuration of of 2 2 was was determined determined by by a a NOESY NOESY experiment. experiment. NOESY NOESY correlations correlations of of H-6/H-3′ H-6/H-3′ and and configuration of 2 was determined by a NOESY experiment. NOESY correlations of H-6/H-31 and correlation of H-2′/H 3 -4′ assigned H-2′ and H 3 -4′ as β. Thus, compound 2 was assigned as depicted 3 3 H-3′/H H-3′/H 3 3 -1′ -1′ indicated indicated that that H H 3 3 -1′, -1′, H-3′, H-3′, and and H-6 H-6 were were cofacial cofacial and and designated designated as as α-oriented, α-oriented, while while H-31 /H3 -11 indicated that H3 -11 , H-31 , and H-6 were cofacial and designated as α-oriented, while and named B. correlation correlation of ofendomeketal H-2′/H H-2′/H -4′1assigned assigned H-2′ H-2′1 and and H H333-4′ -4′1as as β. β. Thus, Thus, compound compound 22 was was assigned assigned as as depicted depicted 1 /H333-4′ correlation of H-2 3 -4 assigned H-2 and H3 -4 as β. Thus, compound 2 was assigned as depicted By comparison of their spectroscopic data (Figures S15–S18) with those reported [16–18], and and named named endomeketal endomeketal B. B. and named endomeketal B. theBy structures of compounds 3 and 4 were assigned as 2,3-dimethylcyclopent-2-en-1-one (3)[16–18], and comparison of their spectroscopic data (Figures S15–S18) with those reported By comparison of their their spectroscopic spectroscopic data (Figures (Figures S15–S18) with with those those reported reported [16–18], By comparison of data S15–S18) [16–18], 2-hydroxymethyl-3-methylcyclopent-2-enone (4), respectively. Although compound 3 was previously the the structures structures structures of of compounds compounds compounds 333 and and and 444were were were assigned assigned as as 2,3-dimethylcyclopent-2-en-1-one 2,3-dimethylcyclopent-2-en-1-one (3) (3) and and the reported as a of synthetic compound, this is the assigned first reportasof2,3-dimethylcyclopent-2-en-1-one its isolation from a natural source. (3) and 2-hydroxymethyl-3-methylcyclopent-2-enone 2-hydroxymethyl-3-methylcyclopent-2-enone (4), (4), respectively. respectively. Although Although compound compound 3 3 was was previously previously 2-hydroxymethyl-3-methylcyclopent-2-enone (4), respectively. Although compound 3 was previously reported reportedas as asaaasynthetic synthetic syntheticcompound, compound, compound,this this thisis isisthe the thefirst first firstreport report reportof of ofits its itsisolation isolation isolationfrom from fromaaanatural natural naturalsource. source. source. reported 2.2. Cytotoxicity Assay The in vitro cytotoxicities of compounds 1–4 were evaluated against four tumor cell lines, 2.2. Assay 2.2. Cytotoxicity Cytotoxicity Assay 2.2. Assay including SF-268 (human glioblastoma cell line, ATCC HTB-14), MCF-7 (human breast adenocarcinoma The in against four tumor cell lines, The in vitro vitro cytotoxicities cytotoxicities of of compounds compounds compounds 1–4 1–4 1–4 were were were evaluated evaluated against four tumor tumor cell celland lines, vitro cytotoxicities of evaluated against four lines, cellThe line,in ATCC HTB-22), NCI-H460 (human non-small cell lung cancer cell line, ATCC HTB-177), including including SF-268 SF-268 (human (human glioblastoma glioblastoma cell cell line, line, ATCC ATCC HTB-14), HTB-14), MCF-7 MCF-7 (human (human breast breast adenocarcinoma adenocarcinoma HepG-2 (human hepatocellular carcinoma cell line, ATCC HB-8065). However, no compound showed cell cell line, line,ATCC ATCC HTB-22), HTB-22), NCI-H460 NCI-H460 (human (human non-small non-small cell cell lung lung cancer cancer cell cellcell line, line,line, ATCC ATCC HTB-177), HTB-177), and and cell line, HTB-22), NCI-H460 (human non-small cell lung cancer ATCC HTB-177), obvious cytotoxic activity against these human tumor cell lines at the concentration of 100 µM. HepG-2 HepG-2 (human (human hepatocellular hepatocellular carcinoma carcinoma cell cellline, line, ATCC HB-8065). HB-8065). However, However, no nocompound compound showed showed and HepG-2 (human hepatocellular carcinoma cellATCC line, ATCC HB-8065). However, no compound obvious obviousobvious cytotoxic cytotoxic activity activityagainst againstthese these human human tumor cell lines linescell at atthe the concentration concentration of of100 100µM. µM. showed cytotoxic activity against thesetumor humancell tumor lines at the concentration of 100 µM.
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3. Materials and Methods 3.1. General Experimental Procedures Optical rotation was measured on an Anton Paar MCP-500 spectropolarimeter (Anton Paar, Vienna, Austria). The IR spectrum was recorded on an IRAffinity-1 spectrophotometer in cm´1 (Shimadzu Corporation, Kyoto, Japan). UV spectra were measured on a SHIMADZU UV-2600 UV-VIS spectrophotometer (Shimadzu Corporation). 1D- and 2D-NMR spectra were recorded on a Bruker Avance-600 spectrometer with TMS as internal standard (Bruker BioSpin International, Geneva, Switzerland), δ in ppm, J in Hz. HRESIMS was measured on a Thermo MAT95XP high-resolution mass spectrometer. A Shimadzu LC-20 AT (Shimadzu Corporation) equipped with an SPD-M20A PDA detector (Shimadzu Corporation) was used for HPLC, a YMC-pack ODS-A column (250 m ˆ 10 mm, 5 µm, 12 nm) was used for semi-preparative HPLC separation. Silica gel (200–300 mesh) was used for column chromatography, and precoated silica gel GF254 plates (Qingdao Marine Chemical Inc., Qingdao, China) were used for TLC spotting. All solvents were analytical grade (Guangzhou Chemical Reagents Company, Ltd., Guangzhou, China). 3.2. Fungal Material The endophytic fungal strain A326 was isolated from the twigs of Ficus hirta, which was collected at Luofu Mountain Nature Reserve, Huizhou, Guangdong province of China, in October 2010. The isolated strain was identified as Endomelanconiopsis endophytica based on a morphological study, and sequence analysis of rDNA ITS (internal transcribed spacer), showing 100% similarity to the strain of E. endophytica (Accession No. EU687005). The strain is preserved at the Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology. 3.3. Extraction and Isolation E. endophytica A326 was grown on potato-dextrose agar (PDA) medium at 28 ˝ C for five days and then inoculated into five flasks (500 mL) containing potato-dextrose (PD) medium (250 mL). After five days of incubation at 28 ˝ C on a rotary shaker at 120 r/min, a portion of the liquid culture was aseptically transferred into each of a total of 200 flasks (500 mL) containing potato-dextrose (PD) medium (250 mL). Following seven days of cultivation at 28 ˝ C and 120 r/min on a rotary shaker, the culture (a total of 50 L) was filtered to give the filtrate and mycelia. The broth was exhaustively extracted with ethyl acetate (EtOAc) for four times, and then the EtOAc layers were combined and evaporated under reduced pressure at a temperature not exceeding 40 ˝ C to yield a dark brown gum (11.7 g). The crude extract was subjected to silica gel column chromatography with a CH2 Cl2 /MeOH gradient (9:1Ñ1:9) to afford six fractions (Fr. I–VI). Fr. II (1.1 g) was subjected to column chromatography on silica gel using n-hexane as the first eluent, and then EtOAc of increasing polarity, to give nine sub-fractions (Fr. IIa ´Fr. IIi ). Fr.IId was further separated by RP-HPLC equipped with a semi-preparative column (CH3 CN, 3 mL/min) to afford 2 (5 mg, tR 7.6 min) and 3 (19 mg, tR 9.1 min). Fr. IIe was chromatographed by RP-HPLC equipped with a semi-preparative column (MeOH/H2 O, 70:30, 3 mL/min) to afford 4 (36 mg, tR 7.2 min) and 1 (24 mg, tR 13.0 min). 3.4. Spectroscopic Data Endomeketal A (1): white, amorphous powder (MeOH); rαs25 D +33.8 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 229 (3.83) nm; IR (KBr) νmax = 1693, 1435, 1409, 1259, 1092, 1018 cm´1 ; HRESIMS m/z 259.1308 [M + Na]+ (calcd for C14 H20 O3 Na, 259.1310, Figure S7); 1 H- and 13 C-NMR data, see Table 1. Endomeketal B (2): white, amorphous powder (MeOH); rαs25 D +23.0 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 222 (3.56) nm; IR (KBr) νmax = 1691, 1381, 1257, 1087, 1022 cm´1 ; HRESIMS m/z 219.1002 [M + Na]+ (calcd for C11 H16 O3 Na, 219.0997, Figure S14); 1 H- and 13 C-NMR data, see Table 1.
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3.5. Cytotoxicity Assay The cell growth inhibitory activities of compounds 1–4 against human tumor cell lines SF-268, MCF-7, NCI-H460, and HepG-2, were tested using the previously-published method [19]. 4. Conclusions Fungal endophytes live inside plant tissues for all, or part, of their life without causing apparent disease symptoms. Endophytic fungi have been found within host tissues at high density and diversity in all tropical and temperate ecosystems where endophytes have been sought [20]. Endomelanconiopsis is a new anamorph genus in the Botryosphaeriaceae, and usually lives inside plant tissues [21]. Previous investigation onto E. endophytica A326 in our research group led to the isolation of a series of xyloketals, which were the first chemical constituents of the genus Endomelanconiopsis [22]. As the fermentation products of the fungus E. endophytica displayed considerable structural diversity, continued investigation of this fungus has resulted in the isolation of two further new secondary metabolites, which were named as endomeketals A and B. The structures were determined by spectroscopic analysis. All of the isolates were evaluated for in vitro cytotoxicity against SF-268, MCF-7, NCI-H460, and HepG-2 cell lines. This study not only enriches the chemical diversity of the genus Endomelanconiopsis, but also implies that this endophytic fungus may be a potential source for producing a series of ketals. Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/ 21/7/943/s1. Acknowledgments: This work was supported financially by the National Basic Research Program of China (973 Program, 2014CB460613), the Natural Science Foundation of Guangdong Province (2015A030313710), and the Guangdong Provincial Project for Science and Technology (2014A030304050, 2015A030302060). Author Contributions: Z.-H.S. and H.-H.L. elucidated structures and wrote the paper. F.-L.L. fractionated the extract, isolated the compounds. Y.-C.C. performed the bioassays. H.-X.L., S.-N.L. and G.-H.T. performed the experiments and analyzed the data. W.-M.Z. designed and coordinated the study and reviewed the manuscript. Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–4 are available from the authors. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
