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Sesquiterpenes from the Endophyte Glomerella cingulata Yunbao Liu, Yong Li, Zhen Liu, Li Li, Jing Qu, Shuanggang Ma, Ridao Chen, Jungui Dai, and Shishan Yu* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China S Supporting Information *

ABSTRACT: From the cultured endophytic fungus Glomerella cingulata isolated from a toxic plant, Gelsemium elegans, one new phenanthrene (1), four new sesquiterpenes (2−5), and three known sesquiterpenes (6−8) were isolated. Their structures were elucidated using spectroscopic methods. Based on the ECD calculations, the absolute configurations of the new compounds were determined. Compounds 2, 4, and 5 inhibited lipopolysaccharide (LPS)-induced NO production in BV2 cells by 50.6, 36.1, and 29.4%, respectively, at 1 μM (positive control curcumin, IC50 = 4.0 μM).

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penes named glomeremophilanes A−D (2−5), and three known metabolites, eremofortin B (6),15 eremofortin D (7),16,17 and PR toxin (8).15 The purified compounds were evaluated for their inhibitory activity of LPS-induced NO production and cytotoxicity. Herein, we report the structure elucidation and bioactivity of 1−8.

he characteristics of neuroinflammation include microglial and astroglial activation, and activated glia release massive inflammatory factors, which lead to neuronal death.1 Nitric oxide (NO), an endogenously synthesized free radical, exerts many biological regulatory roles in the nervous system.2 NO participates in the progress of neuroinflammation caused by other important inflammatory mediators, such as reactive oxygen species and tumor necrosis factor.3,4 Considerable evidence gained over the past decade has revealed that neuroinflammation plays an important role in the pathogenesis of neurodegenerative diseases, such an Alzheimer’s disease and Parkinson’s disease. 5,6 A number of compounds with antineuroinflammatory effects, as revealed by inhibiting the production of NO, may protect against neurodegenerative diseases.7−11 Modulating neuroinflammation may be an effective treatment for neurodegenerative diseases, and the effect on NO production has been widely used to evaluate the potential of compounds to inhibit neuroinflammation. Previously, we have reported a serious of eremophilane sesquiterpenes with NO production inhibitory bioactivities produced by an endophytic fungus, Guignardia mangiferae, residing in the toxic plant Gelsemium elegans.12 Glomerella cingulata is an endophytic fungus isolated from the same host plant. Only a few secondary metabolites have been reported from the genus Glomerella.13,14 Chemical screening of an extract of G. cingulata showed that the main secondary metabolites were sesquiterpenes. The same extract inhibited lipopolysaccharide (LPS)-induced NO production in BV2 cells by 28.5% at 10 μM. The systematic study of the bioactive endophytic fungus G. cingulata led to the isolation of a new phenanthrene named glomephenanthrene A (1), four new eremophilane sesquiter© 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The fungus G. cingulata was cultured in a solid rice medium for 30 days and was then extracted with EtOAc. The EtOAc extract was subjected to column chromatography over silica gel and Sephadex LH-20 followed by semipreparative HPLC to afford eight compounds (1−8). Received: January 18, 2017 Published: October 16, 2017 2609

DOI: 10.1021/acs.jnatprod.7b00054 J. Nat. Prod. 2017, 80, 2609−2614

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Partial structures I and II accounted for 7 out of the 17 degrees of unsaturation and 14 carbons. Therefore, the remaining 14 sp2 carbons, 2 methoxyls, and 10 degrees of unsaturation led to the assignment of partial structure III to be a methoxyl-substituted phenanthrene moiety. The 1H NMR data revealed that the phenanthrene moiety contained one singlet proton at δH 8.35 (s, H-9), two broad singlet protons at δH 8.08 (1H, brs, H-2) and 7.70 (1H, brs, H-4), three protons with ABX splitting pattern at δH 7.88 (1H, d, J = 8.7 Hz, H-5), 7.45 (1H, dd, J = 8.7, 2.3 Hz, H-6), and 8.45 (1H, d, J = 2.3 Hz, H-8), and two methoxyl groups at δH 4.12 and 4.02. The substitution pattern of partial structure III was determined based on HMBC and NOESY correlations (Figures 1 and 2).

Glomephenanthrene A (1) was determined to have a molecular formula of C30H29NO6, based on HRESIMS and interpretation of NMR data. The 1H NMR spectral data (Table 1) revealed a number of signals attributable to aromatic rings Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) Data of Compound 1 (in pyridine-d5) no.

δC, type

1 2

150.9, C 106.4, CH

3

125.6, C

4 4a 5

128.0, CH 127.7, C 130.8, CH

5a 6

126.7, C 118.8, CH

7 8 8a 9 10 10a

158.9, 106.2, 132.3, 105.3, 150.2, 125.6,

C CH C CH C C

δH (J in Hz) 8.08, brs

7.70, brs 7.88, d, 8.7

7.45, dd, 8.7, 2.3 8.45, d, 2.3 8.35, brs

no.

δC, type

2′ 3′

59.6, CH 23.4, CH2

4′

30.4, CH2

5′ 6′ 7′

175.2, C 173.2, C 45.4, CH2

1″ 2″

71.0, CH2 138.0, C

3″/7″ 4″/6″ 5″ OMe-1 OMe-10 OMe-6′

128.3, CH 129.1, CH 129.6, CH 56.2, CH3 56.4, CH3 52.1, CH3

δH (J in Hz) 4.07, 2.04, 1.92, 2.58, 2.39,

m m m m m

5.73, d, 14.5 4.74, d, 14.5 5.37, s

7.63, 7.41, 7.37, 4.12, 4.02, 3.52,

d, 7.4 t, 7.4 t, 7.4 s s s

(δH 7.37−8.45) and three methoxyl groups (δH 4.12, 4.02, and 3.52). The 13C NMR and DEPT data showed that 1 contained 20 aromatic carbons, two downfield signals of two carbonyls (δC 175.2 and 173.2), three methoxyls (δC 56.4, 56.2, and 52.1), four methylenes (δC 71.0, 45.4, 30.4, and 23.4), and one methine (δC 59.6). A detailed analysis of the 1D and 2D NMR data allowed the assignment of substructures I−III (Figure 1) for 1.

Figure 2. Key NOE correlations of compounds 1, 3, and 4.

The HMBC correlations from H-5 to C-7/C-8a/C-4a, from H6 to C-8/C-5a, from H-8 to C-9/C-5a, from H-9 to C-10a/C5a, from H-2 to C-10a/C-4, from H-4 to C-5a, from OMe-10 to C-10, and from OMe-1 to C-1 allowed for the assignment of partial structure III. The proposed structure III was further confirmed by the NOESY correlations between H-2/H-4 and H-7′, between H-4 and H-5, between H-8 and H-9, between OMe-1 and H-2, and between OMe-10 and H-9 (Figure 2). The connection of partial structures I and III via C-7′ and C3 was confirmed by the HMBC correlation from H-7′ to C-3. Similarly, partial structures II and III were determined to be connected through C1″−O−C7 according to the HMBC correlation from H-1″ to C-7. Thus, the planar structure of 1 was fully established and determined to possess an amino acid moiety, a phenanthrene moiety, and a benzyl moiety. Compound 1 contains one chiral carbon at C-2′. The absolute configuration of C-2′ was determined by the comparison of the experimental and calculated electronic circular dichroism (ECD) spectra.18 The theoretical ECD spectrum of 1a was consistent with the experimental ECD spectrum of 1 with an R configuration (Figure 3). Thus, the structure of compound 1 was established as shown and named gomephenanthrene A. Compound 2 was determined to have a molecular formula of C16H18O4 based on the HRESIMS data. Its 1H NMR data (Table 2) displayed three methyls [two singlets (δH 1.47 and 1.43) and one doublet (δH 1.44, d, J = 7.2 Hz)], one methoxyl (δH 3.67, s)], two olefinic protons (δH 6.30, d, J = 10 Hz and 7.09, d, J = 10 Hz), and two aromatic protons (δH 7.44 and 7.43, each 1H, s). The 13C NMR data (Table 2) of 2 contained 16 signals: one ketone (δC 187.0), one ester carbonyl carbon (δC 176.9), two olefinic carbons (δC 160.8 and 126.5), six aromatic carbons (δC 143.4, 127.9, 135.8, 154.9, 111.9, and

Figure 1. 1H−1H COSY (bold lines) and key HMBC (→) correlations of compounds 1 and 2.

The partial structure I (N-1′ through C-7′) was assigned as the methyl ester of 5′-oxopyrroline-2′-carboxylate with an additional methylene substituent in the nitrogen position. Based on the 1H−1H COSY and HSQC spectra, the spin system C2′−C3′−C4′ was elucidated. The key NMR correlations for the structural assignment of I included HMBC correlations from H-2′ (δH 4.07) to C-5′ (δC 175.2)/ C-6′ (δC 173.2), from H-7′ to C-5′, and from OMe-6′ (δH 3.52, 3H, s) to C-6′. Partial structure II was readily assigned as a benzyl group according to the splitting pattern of the 1H NMR data [δH 7.63 (2H, d, J = 7.4 Hz, H-3″/H-7″), 7.41 (2H, t, J = 7.4 Hz, H-4″/H-6″), and 7.37 (1H, t, J = 7.4 Hz, H-5″)] and the HMBC correlations from H-1″ (δH 5.37, 2H, s) to C-3″/C7″. 2610

DOI: 10.1021/acs.jnatprod.7b00054 J. Nat. Prod. 2017, 80, 2609−2614

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Figure 3. Experimental and calculated ECD of compounds 1−4.

Table 2. 1H NMR and 13C NMR Data of Compounds 2−5 2a no.

δC, type

1 2 3 4 5 6

187.0, C 126.5, CH 160.8, CH 38.5, C 143.4, C 127.9, CH

7 8 9 10 11 12 13 14 15

135.8, C 154.9, C 111.9, CH 131.2, C 41.5, CH 176.9, C 17.4, CH3 29.7, CH3 29.7, CH3 52.4, 12-OMe

3a δH (J in Hz) 6.30, d, 10.0 7.09, d, 10.0

7.43, s

7.44, s 4.07, q, 7.2 1.44, 1.43, 1.47, 3.67,

d, 7.2 s s s

δC, type 199.1, C 129.6, CH 155.8, CH 45.9, CH 48.0 C 37.2, CH2 157.4, C 105.6, C 82.0, CH 57.8, CH 126.4, C 174.2, C 8.2, CH3 14.5, CH3 13.9, CH3 62.0, 8-OMe

4a δH (J in Hz) 6.00, dd, 10, 3 6.70, dd, 10, 2 2.75, m 2.77, d, 13 2.35, d, 13

156.7, C 103.3, C 128.2, CH 147.9, C 127.3, C 172.5, C 8.3, CH3 19.4, CH3 14.2, CH3 51.0, 8-OMe

4.12, d, 3.0 2.89, d, 3.0

1.83, 0.83, 1.17, 3.24,

δC, type 188.7, C 129.1, CH 157.1, CH 43.4, CH 47.8 C 36.2, CH2

s s d, 7.5 s

5a δH (J in Hz)

6.15, dd, 10.0, 3.0 6.86, dd, 10.0, 2.0 2.90, m 3.03, d, 12.6 2.55, d, 12.6

6.68, s

1.93, 0.97, 1.28, 3.24,

s s d, 7.5 s

δC, type 189.0, C 129.6, CH 157.1, CH 43.4, CH 47.6 C 33.2, CH2 158.6, C 100.7, C 129.1, CH 143.5, C 124.8, C 173.3, C 8.2, CH3 19.4, CH3 14.2, CH3

δH (J in Hz) 6.13, dd, 10.0, 3.0 6.84, dd, 10.0, 2.0 2.88, m 3.01, d, 12.6 2.66, d, 12.6

6.70, s

1.87, s 0.95, s 1.26, d, 7.5

a

Data were recorded at 600 MHz for proton and at 150 MHz for carbon in methanol-d4. bData were recorded at 500 MHz for proton and at 125 MHz for carbon in methanol-d4.

A was a quinoid ketone. The chemical shifts and splitting patterns of the aromatic protons at δH 7.43 (1H, s, H-6) and 7.44 (1H, s, H-9) indicated the presence of one para tetrasubstituted aromatic ring (ring B). The HMBC correlations from H-6 to C-4/C-5/C-10/C-8/C-11, from H-9 to C-1/ C-5/C-7/C-10, and from H-11 to C-6/C-8 determined the fusing positions of rings A and B (C-5 and C-10). The above assigned spin system C11−C13 and the HMBC correlations from H-13 to C-12, from OMe-12 to C-12, from H-13 to C-7, and from H-6 to C-11 led to the assignment of the side chain [C12(OMe)−C11-C13] attached at C-7.

131.2), four methyls [δC 52.4 (O-methyl), 29.7 (two overlapped methyls), and 17.4], one sp3 methine (δC 41.5), and one sp3 quaternary carbon (δC 38.5). The analysis of 1H−1H COSY and HSQC confirmed the presence of two spin systems: C(2)H−C(3)H and C(11)H− C(13)H3 (Figure 1). The HMBC correlations (Figure 1) from H-3 to a ketone carbon (C-1, δC 187.0) and the assigned spin system C(2)H−C(3)H allowed for the establishment of an α,βunsaturated ketone in the structure. Moreover, the HMBC correlations from H-2 to C-10/C-4, from H-3 to C-1/C-5/C14/C-15, and from H-14/H-15 to C-3/C-5 indicated that ring 2611

DOI: 10.1021/acs.jnatprod.7b00054 J. Nat. Prod. 2017, 80, 2609−2614

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correlations from H-2 to C-4/C-10, from H-3 to C-1/C-5/C15, from H-4 to C-2/C-10, and from Me-15 to C-3/C-5 permitted the complete elucidation of ring A. The HMBC correlations from H-6 to C-8/C-10/C-4/C-14 and from H-9 to C-1/C-5/C-7 allowed the elucidation of ring B. Rings A and B established above, three double bonds, and two carbonyl groups accounted for seven of the eight degrees of unsaturation, indicating the presence of an additional ring in the structure. The analysis of the degrees of unsaturation and the HMBC correlations from Me-13 to C-7/C-12 revealed the presence of an α,β-unsaturated lactone (ring C), as shown in Figure 1. The methoxyl group at δH 3.24 (s) showed an HMBC correlation with C-8, indicating the linkage position at C-8. Thus, compound 4 was established as an eremophilane sesquiterpene (Figure 1). Structurally, compounds 3 and 4 possess an identical skeleton. The differences were that C-9 and C-10 in 3 were reduced to a double bond in 4. The relative configuration of 4 was established by analyses of the NOE correlations in the same manner as described for 3 (Figure 2). The NOE correlations of OMe-8/H-6a, Me-14/H-6a, and Me-15/H-6a showed that OMe-8, Me-14, and Me-15 were in the same direction with β-orientations. Then, the two possible absolute configurations of 4 were proposed to be 4a (4S, 5R, 8S) and 4b (4R, 5S, 8R) according to the established relative configuration. The calculated ECD spectrum of 4a displayed a CD curve similar to the experimental spectrum of 4 (Figure 3). Thus, the structure of 4 was elucidated and named glomeremophilane C. The molecular formula of 5 was assigned as C15H16O4 based on HR-ESIMS, with eight degrees of unsaturation. The 1H NMR data (Table 2) indicated the presence of three methyl signals at δH 0.95 (s), 1.26 (d, J = 7.5 Hz), and 1.87 (s) and three olefinic signals at δH 6.13 (dd, J = 10 and 3 Hz), 6.84 (dd, J = 10 and 2 Hz), and 6.70 (s). The 13C NMR data (Table 2) displayed 15 signals including signals from two carbonyls (δC 189.0, 173.3), six olefinic carbons (δC 157.1, 158.6, 143.5, 129.1, 129.6, and 124.8), three methyls (δC 19.4, 14.2, and 8.2), one methylene (δC 33.2), one methine (δC 43.4), one quaternary carbon (δC 47.6), and one hemiacetal carbon (δC 100.7). The comparison of the NMR (Table 2) and HR-ESIMS data with those of compound 4 revealed that the structures were similar except for an additional hydroxy group in 5 and the absence of a methoxyl group at C-8 in 4. Therefore, the planar structure of 5 was proposed as shown. The 1H−1H COSY, HSQC, and HMBC spectroscopic data further verified the structure of 5 as proposed (Figure 1). The identical spin system C(2)H−C(3)H−C(4)H−C(15)H3 was determined by 1 H−1H COSY and HSQC spectra as described in 4. Similar to 4, HMBC correlations from H-2 to C-4/C-10, from H-3 to C1/C-5/C-15, from H-4 to C-2/C-10, from Me-15 to C-3/C-5, from H2-6 to C-8/C-10/C-4/C-14, from H-9 to C-1/C-5/C-7/ C-8, and from Me-13 to C-7/C-11/C-12 were observed in 5. The relative configuration of 5 was determined to be identical to that of 4 based on the NOE correlations. The CD spectrum of 5 (Figure S62) was similar to that of 4, which indicated that compound 5 contained the same absolute configuration (4S, 5R, 8S) as 4. Thus, the structure of compound 5 was established, and it is named glomeremophilane D. In addition to the five new compounds (1−5) described above, three known compounds, eremofortin B (6),15 eremofortin D (7),16,17 and PR toxin (8),15 were identified by comparison with published data.

Compound 2 contains one chiral carbon at C-11. The absolute configuration of C-11 was determined to be an S configuration by the comparison of the experimental and calculated ECD spectra (Figure 3).18 Thus, the structure of 2 was established as shown and named glomeremophilane A. Compound 3 was assigned the molecular formula C16H20O5 based on HRESIMS, indicative of eight degrees of unsaturation. The 1H NMR data (Table 2) displayed three methyl groups with two singlets (δH 0.83 and 1.83), one doublet (δH 1.17, J = 7.5 Hz), one methoxyl group (δH 3.24), and two olefinic signals (δH 6.00 dd, J = 10.0, 3.0 Hz and 6.70, dd, J = 10.0, 2.0 Hz). The 13C NMR data (Table 2) displayed 16 signals, including signals from two carbonyls (δC 199.1, 174.2), four olefinic carbons (δC 157.4, 155.8, 129.6, and 126.4), three methyls (δC 14.5, 13.9, and 8.2), one methoxy (δC 62.0), one methylene (δC 37.2), three methines (δC 82.0, 57.8, and 45.9), one quaternary carbon (δC 48.0), and one hemiacetal carbon (δC 105.6). Based on the 1H−1H COSY and HSQC data, two spin systems C(2)H−C(3)H−C(4)H−C(15)H3 and C(9)H− C(10)H were established as shown in Figure 1. The assigned spin systems and HMBC correlations from H-2 to C-4, from H-3 to C-1/C-5/C-15, from H-4 to C-2/C-10, from Me-15 to C-3/C-5, from H-6 to C-8/C-10/C-4/C-14, from Me-13 to C7/C-11/C-12, and from H-9 (4.13, d, J = 3.0 Hz) to C-1/C-5/ C-7/C-8 led to the establishment of ring systems A−C. The HMBC correlations from OMe-8 (3.24, s) to C-8 confirmed that C-8 was substituted with a methoxy group. Thus, the planar structure of 3 was proposed as shown in Figure 1. The relative configuration of 3 was determined based on NOE correlations (Figure 2). The NOE correlation between H-9 and Me-15 revealed that both were in an axial orientation. Me-15 showed an NOE correlation with Me-14, indicating that Me-14 was β-orientated. The small coupling constants of axial H-9 (δH 4.12, d, J = 3.0 Hz) and H-10 (δH 2.89, d, J = 3.0 Hz) indicated that H-10 was an equatorial proton in ring B. Me-13 showed an NOE correlation with axial H-6a (δH 2.77, d, J = 13.0 Hz), which allowed for the assignment that ring C was underneath ring B and O-8 was a β-orientated bond. Finally, the relative correlations of 3 were unambiguously determined. The calculated ECD spectrum of 3a with a stereochemistry of 4S, 5R, 8R, 9S, and 10S displayed a CD curve inconsistent with the experimental spectrum of 3 (Figure 3). Thus, the absolute configuration of 3 was determined to be 4S, 5R, 8R, 9S, and 10S. Compound 3 was named glomeremophilane B. Compound 4 was obtained as a white powder. Its molecular formula, C16H18O4, was determined by HR-ESIMS, indicative of eight degrees of unsaturation. The IR spectrum showed absorption bands for a hydroxy (3394 cm−1), an enone carbonyl (1671 cm−1), and an α,β-unsaturated γ-lactone (1769 cm−1). The 1H NMR data (Table 2) indicated the presence of three methyl signals at δH 0.97 (s), 1.28 (d, J = 7.5 Hz), and 1.93 (s), one methoxyl signal at δH 3.24 (s), and three olefinic signals at δH 6.15 (dd, J = 10.0 and 3.0 Hz), 6.86 (dd, J = 10.0 and 2.0 Hz), and 6.68 (s). The 13C NMR data (Table 2) displayed 16 signals, including signals from two carbonyls (δC 188.7 and 172.5), six olefinic carbons (δC 157.1, 156.7, 147.9, 129.1, 128.2, and 127.3), three methyls (δC 19.4, 14.2, and 8.3), one methoxyl (δC 51.0), one methylene (δC 36.2), one methine (δC 43.4), one quaternary carbon (δC 47.8), and one hemiacetal carbon (δC 103.3). The inspection of the 1H−1H COSY and HSQC spectra led to the assignment of a C(2)H2−C(3)H2−C(4)H−C(15)H3 unit. The assigned spin system, together with the HMBC 2612

DOI: 10.1021/acs.jnatprod.7b00054 J. Nat. Prod. 2017, 80, 2609−2614

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(−18), 253 (+9), 285 (−2), 366 (+2.8) nm; IR νmax 3278, 2977, 1739, 1651, 1601 cm−1; HRESIMS m/z 275.1281 [M + H]+ (C16H19O4, calcd [M + H]+ 275.1278); 1H and 13C NMR (Table 2). Compound 3: white powder; [α]20D −21.2 (c 0.01, MeOH); UV (MeOH) λmax 228 nm; CD (MeOH) λmax (Δε) 232 (+4), 255 (−3); IR νmax 3219, 2933, 1750, 1659 cm−1; HRESIMS m/z 293.1385 [M + H]+ (C16H21O5, calcd [M + H]+ 293.1384); 1H and 13C NMR (Table 2). Compound 4: white powder; [α]20D +10.2 (c 0.1, MeOH); UV (MeOH) λmax 223, 253, nm; CD (MeOH) λmax (Δε) 228 (−18), 254 (+9); IR νmax 3394, 2923, 1769, 1671 cm−1; HRESIMS m/z 275.1282 [M + H]+ (C16H19O4, calcd [M + H]+ 275.1278); 1H and 13C NMR (Table 2). Compound 5: yellow powder; [α]20D +30.5 (c 0.12, MeOH); UV (MeOH) λmax 225, 250 nm; CD (MeOH) λmax (Δε) 230 (−3), 254 (+2); IR νmax 3363, 2921, 1764, 1669 cm−1; HRESIMS m/z 261.1119 [M + H]+ (C15H17O4, calcd [M + H]+ 261.1121); 1H and 13C NMR (Table 2). Anti-inflammatory Activity. The BV2 macrophage cell line was obtained from the Cell Culture Center at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. LPS (from Escherichia coli 055:B5) was obtained from Sigma-Aldrich. After preincubation for 24 h in a 96-well plate, the cells were treated with the test compounds, followed by stimulation with LPS for 24 h. The concentration of nitric oxide was assayed using a colorimetric reaction with the Griess reagent. The culture supernatant (100 μL) was incubated with Griess reagent (100 μL) at room temperature for 5 min in the dark. The absorbance at 540 nm was determined using a microplate reader (Bio-Tek μQuant). Curcumin was used as a positive control.

All the compounds were assayed for their anti-inflammatory and cytotoxic activities. Compounds 2, 4, and 5 exhibited antiinflammatory activities indirectly by suppressing the LPSinduced NO production in BV2 cells by 50.6, 36.1 and 29.4%, respectively, at 1 μM. Meanwhile, curcumin (positive control) gave 50% inhibition at 4.0 μM. All test compounds were inactive to the HCT-8, HCT-116, Bel-7402, BGC-823, A549, and A2780 cell lines in an MTT assay. The bioassay results indicated that 2, 4, and 5 have the potential to inhibit neural inflammation and possible applications to treating Parkinson’s disease (PD) and Alzheimer’s disease (AD).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a JASCO P-2000 automatic digital polarimeter. CD spectra were recorded on a JASCO J-815 spectropolarimeter. IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer. NMR spectra were recorded on an INOVA SX-600 or Bruker 800 spectrometer. HR-ESIMS data were recorded on an Agilent Technologies 6250 Accurate-Mass Q-TOF LC/MS spectrometer. Preparative HPLC was performed on a Shimadzu LC-6AD instrument with an SPD-10A detector, using a YMC-Pack ODS-A column (250 × 20 mm, 5 μm). Sephadex LH-20 (Amersham Pharmacia Biotech AB, Sweden), ODS (45−70 μm, Merck), a macroporous adsorptive resin (XAD-D101, Tianjin Nankai Chemical Inc. China), and silica gel (200−300 mesh, Qingdao Marine Chemical Inc., China) were used for column chromatography (CC). TLC was conducted on glass precoated with silica gel GF254 (Qingdao Marine Chemical Inc. China). Plant Material. Fresh leaves of G. elegans were collected in Guangxi Province, China, in May 2013. The plant was identified by Prof. Song-Ji Wei, Guangxi College of Chinese Traditional Medicine. A voucher specimen (specimen No. S2505) was deposited in the herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, China. Fungus. The fungus was identified as G. cingulata according to a molecular biological protocol by DNA amplification and the sequencing of the ITS region. The sequence data were submitted to GenBank with the accession number EU859960. The fungal strain (No. 2013Z22) was kept in Prof. Yu’s lab. Fermentation, Extraction, and Isolation. The fermentation was performed in six Erlenmeyer flasks (2.5 L each) on solid rice medium, which was prepared by mixing rice (250 g) and demineralized water (250 mL), followed by autoclaving (121 °C, 20 min). The fungus grown on a Petri dish was inoculated onto sterile rice medium and was allowed to grow (20 °C) under static conditions. After 30 days, the fermentation was stopped by adding 1000 mL of EtOAc to each flask, and then utrasonic extraction was carried out for 30 min three times. The EtOAc solution was evaporated to dryness to give a brown extract (11 g). Of this, 10 g was applied to a silica gel column, eluted with CHCl3−MeOH (20:1−1:1), to afford five fractions (A: 1.2 g; B: 3.4 g, C: 0.9 g, D: 0.8 g; E: 0.6 g). Fraction B (3.2 g) was chromatographed over an ODS column, eluted with a gradient of increasing methanol in water (5−100%), to afford five subfractions (B1: 0.2 g, B2: 0.7 g, B3: 1.1 g, B4: 0.3 g; B5: 0.6 g). Fr. B5 was purified by RP-HPLC using 45% acetonitrile in water to afford 1 (16 mg). Fr. B3 was separated by RP-HPLC using 35% acetonitrile in water to afford 2 (8.7 mg), 3 (20.5 mg), 6 (12.0 mg), and 7 (9.6 mg). Fraction B2 was purified by RPHPLC [solvent system: CH3CN−H2O (4:1)] to afford compounds 4 (17.1 mg), 5 (9.5 mg), and 8 (11 mg). Compound 1: pale yellow powder; [α]20D +4.3 (c 0.03, MeOH); UV (MeOH) λmax 214, 263, 324, 396 nm; CD (MeOH) λmax (Δε) 225 (+5.6) nm; IR νmax 3360, 2921, 1739, 1661, 1633 cm−1; HRESIMS m/ z 500.2080 [M + H]+ (C30H30NO6, calcd [M + H]+ 500.2068); 1H and 13C NMR (Table 1). Compound 2: colorless gum; [α]20D +25 (c 0.01, MeOH); UV (MeOH) λmax 222, 232, 268, 335 nm; CD (MeOH) λmax (Δε) 229

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00054. MS, IR, 1D and 2D NMR, and CD spectra for 1−5 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-10-63165324. Fax: +86-10-63017757. ORCID

Yunbao Liu: 0000-0002-1338-0271 Jungui Dai: 0000-0003-2989-9016 Shishan Yu: 0000-0003-4608-1486 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (No. 21572276) and the National Science and Technology Project of China (No. 2012ZX09301002-002). The authors are grateful to the Department of Instrumental Analysis in our institute for the measurement of the UV, IR, NMR, and MS spectra.

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REFERENCES

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