Arch. Pharm. Res. DOI 10.1007/s12272-014-0513-3
RESEARCH ARTICLE
Penipyrols A–B and peniamidones A–D from the mangrove derived Penicillium solitum GWQ-143 Wenqiang Guo • Xianglan Kong • Tianjiao Zhu Qianqun Gu • Dehai Li
•
Received: 6 August 2014 / Accepted: 28 October 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Chemical investigation the extract of Penicillium solitum GWQ-143 led to yield four new compounds penipyrols A–B (1–2) and peniamidones A–B (3–4), together with peniamidones C–D (5–6), which had been previously described as synthetic intermediates, not obtained from natural resource. The structures of those new compounds were established through extensive spectroscopic analysis. Compounds 1–6 exhibited great radical scavenging activities against DPPH with IC50 values ranged from 4.7 to 15.0 lM. Keywords Mangrove Penicillium solitum Penipyrol Peniamidone Radical scavenging activity
Introduction Mangrove commonly known as the salt tolerant, oligotrophic forest ecosystem mainly distributed between the tropical and subtropical intertidal regions around the world (Bandaranayake 2002). Occurring at the interface of terrestrial and marine ecosystems, it represented a rich biological diversity of bacterium, fungus, etc. (Thatoi et al. 2013). Fungi from the mangrove plant (endogenous or rhizosphere soil) attracted broad attentions, and were considered as a great source of novel natural products,
Electronic supplementary material The online version of this article (doi:10.1007/s12272-014-0513-3) contains supplementary material, which is available to authorized users. W. Guo X. Kong T. Zhu Q. Gu D. Li (&) Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China e-mail: [email protected]
which generally exhibited various biological activities such as antioxidant (Zhang et al. 2014), antimicrobial (Ai et al. 2014), anticancer (Meng et al. 2013), antiviral (Fan et al. 2013), etc. In the course of our investigation on bioactive secondary metabolites from mangrove derived fungi (Peng et al. 2013; Gao et al. 2013a; Gao et al. 2013b; Gao et al. 2012; Zhou et al. 2011; Zhang et al. 2011), the extract of Penicillium solitum GWQ-143, isolated from the rhizosphere soil of a mangrove plant Rhizophora stylosa, was selected for the TLC expansions and characteristic UV absorption observed in the HPLC–UV profile. The chemical explorations for the organic extract led to the isolation of two a-pyrone derivatives penipyrols A–B (1–2) and four dihydroxybenzoic acids derivatives peniamidones A–D (3–6). Penipyrol A (1) had the distinctive hexadienoic acid chain, and those new compounds firstly reported the radical scavenging activities. Herein, we report the isolation, structural elucidation and biological activities of the new compounds.
Materials and methods General Specific rotation was obtained on a JASCOP-1020 digital polarimeter. UV spectra were recorded on Waters 2487. IR spectra were recorded on a NICOLET NEXUS 470 spectrophotometer in KBr discs. 1H NMR, 13C NMR, DEPT and 2D NMR spectra were recorded on JEOL JNM-ECP 600 spectrometer. HRESIMS and ESIMS data were obtained using a Thermo Scientific LTQ Orbitrap XL mass spectrometer. Column chromatography (CC) were performed on silica gel (100–400 mesh, Qingdao Marine Chemical Factory), Sephadex LH-20 (Amersham
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W. Guo et al.
Biosciences) and ODS resin (50 mm, Merck). Preparative HPLC collection used a C18 column [YMC-Pack ODS-A, 10 9 250 mm, 5 lm, 3 mL/min].
The fungus isolated from the rhizosphere soil of a mangrove plant R. stylosa, and identified as P. solitum according to the DNA sequences of the ITS region with 100 % similarity to P. solitum. The sequence data have been submitted to GenBank with the accession number KJ577599.
fractions (Fractions 1–9). The Fractions seven and eight were further separated on a Sephadex LH-20 column with MeOH to provide eight fractions. The Fraction 7-7 was isolated by semi-preparative HPLC employing isocratic elution with CH3CN–H2O (45:55) to obtain compound 1 (7.5 mg, tR = 16.8 min) and 2 (7.8 mg, tR = 20.6 min), with MeOH–H2O (60:40) to obtain compound 3 (14.0 mg, tR = 12.8 min) and 4 (13.0 mg, tR = 14.8 min). The Fraction 8-6 applied isocratic elution with MeOH–H2O (65:35) to yield compound 5 (9.0 mg, tR = 18.5 min) and 6 (10.0 mg, tR = 19.6 min).
Fermentation and extraction
Biological assay
Erlenmeyer flasks (1,000 mL) containing 300 mL fermentation media were directly inoculated with spores. The media contained yeast powder (1 g), peptone (5 g), glucose (20 g) and malt extract (3 g) dissolved in 1 L naturallycollected seawater, and sterilized in 121 °C for 21 min. The flasks were cultured in static condition at 28 °C for 30 days. The whole fermentation broth (60 L) was filtered through cheese cloth to separate the supernatant from the mycelia. The supernatant was extracted with EtOAc, and the mycelia was macerated and extracted with acetone. All extracts were evaporated under reduced pressure and concentrated to give a crude extract (17.0 g).
In the DPPH scavenging assay (Chen et al. 1999), samples to be tested were dissolved in MeOH and the solution (160 lL) was dispensed into wells of a 96-well micro-titer tray. Forty micro-liters of the DPPH solution in MeOH were added to each well. The mixture was shaken and left to stand for 30 min. After the reaction, absorbance was measured at 510 nm, and the percent inhibition was calculated. IC50 values denoted the concentration of sample required to scavenge 50 % of the DPPH free radicals. Compounds 1–6 were evaluated for radical scavenging activities against DPPH with IC50 values ranged from 4.7 to 15.0 lM (Table 3).
Strain identification
Penipyrol A (1) Purification Yellow oil; [a]20 D = -43.5 (c 0.1, CH2Cl2); UV (MeOH): 220 (3.50), 250 (3.35) nm; IR (KBr) mmax 3268, 2955, 1740, 1650, 1450, 1360, 980 cm-1; HRESIMS [M?Na]? m/z
The extracted material was separated by ODS in the gradient elution step of MeOH–H2O (5–100 %) to give nine Table 1 1H NMR (600 MHz) and 13C NMR (150 MHz) data of compounds 1–2 in DMSO-d6
123
Position
1
2
d(C)
d(H) (J in Hz)
d(C)
d(H) (J in Hz)
1
162.6
–
162.8
–
2
123.3
–
123.7
–
3
141.2
7.37, d (J = 6.6, 1H)
141.3
7.46, d (J = 7.2, 1H)
4
105.9
6.36, d (J = 6.6, 1H)
105.7
6.67, d (J = 7.2, 1H)
5
157.6
–
156.5
–
6
119.2
6.30, d (J = 15.4, 1H)
125.4
–
7
137.2
6.93, d (J = 15.4, 1H)
132.5
7.26 (s, 1H)
8
135.3
–
137.6
–
9
136.1
5.79, d (J = 9.4, 1H)
144.0
–
10
46.9
11 12
175.0 26.1
3.27 (m, 1H)
136.4
–
– 1.73 (m, 1H); 1.52 (m, 1H)
127.4 169.7
7.35 (s, 1H) –
13
12.0
0.86, t (J = 7.14, 3H)
21.0
2.33 (s, 3H)
14
13.0
1.83 (s, 3H)
23.1
2.47 (m, 2H)
15
16.9
2.00 (s, 3H)
14.7
1.12, t (J = 7.7, 3H)
16
–
–
20.9
2.30 (s, 3H)
17
–
–
16.7
2.03 (s, 3H)
Penipyrols A–B and peniamidones A–D Table 2 1H NMR and Position
13
C NMR data of compounds 3–6
a
4b
3
5a
6b
d(C)
d(H) (J in Hz)
d(C)
d(H) (J in Hz)
d(C)
d(H) (J in Hz)
d(C)
d(H) (J in Hz)
1
148.7
–
145.0
–
148.8
–
147.9
–
2
145.3
–
148.7
–
145.4
–
144.1
–
3
115.6
7.26 (brs, 1H)
114.4
7.27 (brs, 1H)
115.6
7.26 (brs, 1H)
113.7
7.27, d (J = 2.2, 1H)
4
126.6
–
125.9
–
126.4
–
125.0
–
5
119.4
7.16, dd (J = 8.3, 1.6, 1H)
119.2
7.19, dd (J = 8.2, 1.6, 1H)
119.4
7.16, d (J = 8.3, 1H)
118.4
7.19, dd (J = 8.3, 2.2, 1H)
6
115.3
6.74, d (J = 8.3, 1H)
114.5
6.79, d (J = 8.2, 1H)
115.4
6.74, d (J = 8.3, 1H)
113.6
6.79, d (J = 8.3, 1H)
7
166.5
–
169.0
–
166.7
–
–
–
8-NH
–
8.11, t (J = 5.5, 1H)
–
–
–
8.10 (brs, 1H)
–
–
9
40.0
3.20 (m, 2H)
39.1
3.35, t (J = 6.3, 2H)
37.0
3.25 (m, 2H)
38.2
3.37 (m, 2H)
10
30.6
1.50 (m, 2H)
25.8
1.67 (m, 2H)
33.1
1.63 (m, 2H)
30.3
1.88 (m, 2H)
11
26.5
1.43 (m, 2H)
25.9
1.67 (m, 2H)
59.2
3.44 (m, 2H)
23.8
2.37, t (J = 7.4, 2H)
12
61.1
3.40 (m, 2H)
64.1
4.09, t (J = 6.1, 2H)
–
–
175.0
–
13
–
–
–
–
–
–
–
14
–
–
–
–
–
–
171.7 19.5
2.02 (s, 3H)
a
1
Spectra were recorded at 600 MHz for H NMR and at 150 MHz for
13
b
Spectra were recorded at 600 MHz for 1H NMR and at 150 MHz for
13
C NMR using DMSO as solvent and TMS as internal standard
285.1100 (calculated for C15H18O4Na, 285.1097); 1H NMR and 13C NMR (see Table 1). Penipyrol B (2) Pale-yellow oil; UV (MeOH): 215 (3.23), 255 (3.42) nm; IR (KBr) mmax 3045, 2940, 1660, 1620, 1560, 1505, 1455, 1160, 870 cm-1; HRESIMS [M ? H]? m/z 289.1049 (calculated for C17H19O4, 287.1278); 1H NMR and 13C NMR (see Table 1). Peniamidone A (3) Brown oil; UV (MeOH): 210 (2.72), 266 (3.25) nm; IR (KBr) mmax 3440, 3170, 2965, 1680, 1650, 1610, 1545, 1455, 960 cm-1; HRESIMS [M?H]? m/z 226.1078 (calculated for C11H16O4N, 226.1074); 1H NMR and 13C NMR (see Table 2).
C NMR using CD3OD as solvent and TMS as internal standard
Peniamidone D (6) Brown oil; UV (MeOH): 212 (2.66), 265 (3.20) nm; IR (KBr) mmax 3445, 3350, 2978, 1720, 1660, 1630, 1545, 1510, 955 cm-1; 1H NMR and 13C NMR (see Table 2).
Results and discussion The fungal strain P. solitum GWQ-143 was cultured in static condition at 28 °C for 30 days. The whole extract was injected to repeated silica gel and LH-20 column chromatography and semi-preparative HPLC collection to yield the new compounds 1 (7.5 mg), 2 (7.8 mg), 3 (12.8 mg) and 4 (13.0 mg) (Fig. 1). Structure elucidation
Peniamidone B (4) Brown oil; UV (MeOH): 210 (2.82), 260 (3.05) nm; IR (KBr) mmax 3450, 2968, 1680, 1650, 1610, 1535, 1500, 1445, 990 cm-1; HRESIMS [M?H]? m/z 268.1187 (calculated for C13H18O5N, 268.1179); 1H NMR and 13C NMR (see Table 2). Peniamidone C (5) Brown oil; UV (MeOH): 215 (3.16), 265 (3.48) nm; IR (KBr) mmax 3535, 3170, 2960, 1675, 1650, 1545, 1505, 1440, 948 cm-1; 1H NMR and 13C NMR (see Table 2).
Penipyrol A (1) was obtained as yellow oil. The molecular formula was determined as C15H18O4 on the basis of the sodiated HRESIMS peak at m/z 285.1100, indicating seven degrees of unsaturation. The 1H, 13C NMR and DEPT spectroscopic data (Table 1) showed two olefinic methyl singlets (dC 13.0, dH 1.83; dC 16.9, dH 2.00) and eight sp2 hybrid carbons included five olefinic methines (dC 142.1, dH 7.37; dC 137.2, dH 6.93; dC 105.9, dH 6.36; dC 119.2, dH 6.30; dC 136.1, dH 5.79). The planar structure of 1 was identification from interpretations of COSY and HMBC spectroscopic analysis
123
W. Guo et al. Fig. 1 Structures of 1–6
13
14
O
1
O
11
5 7
15
9
10
OH
O
1
3
HO
O
3
HO
N H
7 5
1
11
9
R
16 9
11
O
1
O
13
12
O
5
14
7 15
O
3
3
R=
4
R=
OH O
6
R=COOH
16
Fig. 2 Selected 2D-NMR correlations of compounds 1–4
13
14
O
1
O
5 9
7
11
OH
10
11
O
O
1
9
15
2
1 3
HO HO
O 7
1
N H
9
11
12
OH
3
HO HO
5
1
O 7
5
N H
9
11
12
O
13
14
O
4
3 COSY
(Fig. 2). The HMBC correlations from H-3 to C-1/C-5 and from H-4 to C-5, and the COSY correlation between H-3 and H-4 together with the chemical shift of C-1 (dC 162.6) indicated the existence of a-pyrone moiety, which also supported by the similar NMR data to those of the reported gibepyrone A (Barrero et al. 1993). The additional COSY correlations (H-6/H-7, and H-9/H-10/H2-12/H3-13) together with the observed HMBC correlations from H-6 to C-8, from H-9 to C-7/C-8, from H-10 to C-8/C-11 and from H3-13 to C-10 were constructed the presence of hexadienoic acid chain. The connection between C-5 and C-6 was evidenced by the HMBC correlations from H-6 to C-4/C-5 and from H-7 to C-5. Finally, the gross structure of 1 was deduced by attaching the methyl groups to C-2/C-8 based on the HMBC correlations from H3-14 to C-7/C-8/C-9 and from H3-15 to C-1/C-2/C-3. The E configuration of the double bond (C-6/C-7) was evident from the coupling constant (15.4 Hz), and the geometry of C-8/C-9 was tentatively proposed as E suggested by the chemical shifts of H-9 (dH 5.79) and H3-14 (dH 1.83) (for E geometry the responding protons were at 5.76 and 1.82 ppm, while for Z at 5.49 and 2.05 ppm, respectively) (Garnier et al. 2007).
12
14
7
3
15
13
O O
5
O
3
15
123
R=OH
O
15
2
5
HMBC
The absolute configuration of C-10 was determined as R by comparing the optical rotation of 1 (-43.5) to the previously reported (R,E)-2-methyl-4-phenylpent-3-enoic acid (-53.1) (Garnier et al. 2007). Penipyrol B (2) was isolated as pale-yellow oil with the molecular formula of C17H18O4, requiring nine degrees of unsaturation. The presence of carbonyl group was deduced by the IR absorption at 1,660 cm-1. Analysis of the 1D NMR data (Table 2) established that 2 possessed four methyls, one methylene (dH 2.47, dC 23.1), two ester carbonyls (dC 169.7, 162.8), and five sp2 hybrid quaternary carbons. Detailed analysis of the 2D NMR spectroscopic data allowed to establish the planar structure of 2. Analysis the NMR chemical shifts (Table 1), COSY and HMBC correlations (Fig. 2) between 1 and 2 suggested they shared the same a-pyrone fragment. The HMBC correlations from H-7 to C-6/C-9/C-11 and from H-11 to C-6/C-7/C-9 confirmed the presence of tetrasubstituted phenyl. Further HMBC correlations from H2-14 to C-7/C-8/C-9, from H315 to C-8, from H3-13 to C-12, from H3-16 to C-9/C-10/C11 and the chemical shift (C-9, dC 144.0) located the
Penipyrols A–B and peniamidones A–D Table 3 DPPH radical scavenging activity of compounds 1–6 Compound
IC50 (lM)
Compound
IC50 (lM)
1
15.0
4
4.7
2
14.0
5
6.0
5.6
6
5.2
3 a
AA a
15.6
AA = ascorbic acid (positive control)
methyl, ethyl and acetyl to C-10/C-8/C-9, respectively. Finally, the structure of 2 was established by connecting the substituted phenyl to the a-pyrone at C-5 according to the HMBC correlations from H-11 to C-5/C-6 (Fig. 2). Peniamidones A–B (3–4) were purified as brown oil. The 1H, 13C NMR spectroscopic data of 3 (Table 2) showed three olefinic methines singlets (dC 119.4, dH 7.16; dC 115.6, dH 7.26; dC 115.3, dH 6.74) and three sp2 hybrid quaternary carbons (dC 148.7, 145.3, 126.6), which gave evidence of a dihydroxybenzoic acid. The additional COSY correlations between H-8/H2-9/H2-10/H2-11/H2-12 established the spin system of aminobutan, and confirmed by the HMBC correlations (Fig. 2). Finally, the gross structure of 3 was determined by the connection of the two moieties basing on the HMBC correlation from H2-9 to C-7, as well as the consideration of molecular formula. Analysis the NMR spectroscopic data of 4 (Table 2) showed 4 was almost identical to 3, except an acetyl group in 4 (dC 171.7, 19.5). Peniamidones C–D (5–6) were obtained as brown oil. Comparison of the 1H NMR data between 5, 6 and reported benzamide (Ohi et al. 1986), butanoic acid (Matyus et al. 1994) suggested they shared same structural features, which were intermediates in the semi-synthesis of b-lactam antibiotics and N-benzoylamino acid derivatives. In the present research, the 13C NMR, DEPT data were presented for those first isolated new natural products (Table 2). Biological effect and discussion The radical scavenging activities of the new compounds 1–6 were evaluated against DPPH for radical scavenging activity. Compounds 1–6 exhibited well activities with the IC50 values ranged from 4.7 to 15.0 lM (Table 3). In summary, under the guidance of characteristic UV absorption tracking method, six new compounds were isolated from the P. solitum GWQ-143. Penipyrol A (1) had the distinctive hexadienoic acid chain, and the great radical scavenging activities of those new compounds were firstly reported in the present research. Actually, autoxidation had been comprehensive researched by the autocatalytic free radical reaction leading to the aging of cardiovascular and cell injury (Gordon 1996), which was the
primary inducements of human disease processes such as cancer, atherosclerosis, rheumatoid arthritis and chronic inflammation (Ani et al. 2006). The antioxidant mechanism for these new compounds remained unclear and needed further investigation. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Nos. 21372208 and 41176120), the National High Technology Research and Development Program of China (No. 2013AA092901) and the State Key Laboratory of Bio-organic and Natural Products Chemistry (SKLBNPC12331).
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RESEARCH ARTICLE
Penipyrols A–B and peniamidones A–D from the mangrove derived Penicillium solitum GWQ-143 Wenqiang Guo • Xianglan Kong • Tianjiao Zhu Qianqun Gu • Dehai Li
•
Received: 6 August 2014 / Accepted: 28 October 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Chemical investigation the extract of Penicillium solitum GWQ-143 led to yield four new compounds penipyrols A–B (1–2) and peniamidones A–B (3–4), together with peniamidones C–D (5–6), which had been previously described as synthetic intermediates, not obtained from natural resource. The structures of those new compounds were established through extensive spectroscopic analysis. Compounds 1–6 exhibited great radical scavenging activities against DPPH with IC50 values ranged from 4.7 to 15.0 lM. Keywords Mangrove Penicillium solitum Penipyrol Peniamidone Radical scavenging activity
Introduction Mangrove commonly known as the salt tolerant, oligotrophic forest ecosystem mainly distributed between the tropical and subtropical intertidal regions around the world (Bandaranayake 2002). Occurring at the interface of terrestrial and marine ecosystems, it represented a rich biological diversity of bacterium, fungus, etc. (Thatoi et al. 2013). Fungi from the mangrove plant (endogenous or rhizosphere soil) attracted broad attentions, and were considered as a great source of novel natural products,
Electronic supplementary material The online version of this article (doi:10.1007/s12272-014-0513-3) contains supplementary material, which is available to authorized users. W. Guo X. Kong T. Zhu Q. Gu D. Li (&) Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China e-mail: [email protected]
which generally exhibited various biological activities such as antioxidant (Zhang et al. 2014), antimicrobial (Ai et al. 2014), anticancer (Meng et al. 2013), antiviral (Fan et al. 2013), etc. In the course of our investigation on bioactive secondary metabolites from mangrove derived fungi (Peng et al. 2013; Gao et al. 2013a; Gao et al. 2013b; Gao et al. 2012; Zhou et al. 2011; Zhang et al. 2011), the extract of Penicillium solitum GWQ-143, isolated from the rhizosphere soil of a mangrove plant Rhizophora stylosa, was selected for the TLC expansions and characteristic UV absorption observed in the HPLC–UV profile. The chemical explorations for the organic extract led to the isolation of two a-pyrone derivatives penipyrols A–B (1–2) and four dihydroxybenzoic acids derivatives peniamidones A–D (3–6). Penipyrol A (1) had the distinctive hexadienoic acid chain, and those new compounds firstly reported the radical scavenging activities. Herein, we report the isolation, structural elucidation and biological activities of the new compounds.
Materials and methods General Specific rotation was obtained on a JASCOP-1020 digital polarimeter. UV spectra were recorded on Waters 2487. IR spectra were recorded on a NICOLET NEXUS 470 spectrophotometer in KBr discs. 1H NMR, 13C NMR, DEPT and 2D NMR spectra were recorded on JEOL JNM-ECP 600 spectrometer. HRESIMS and ESIMS data were obtained using a Thermo Scientific LTQ Orbitrap XL mass spectrometer. Column chromatography (CC) were performed on silica gel (100–400 mesh, Qingdao Marine Chemical Factory), Sephadex LH-20 (Amersham
123
W. Guo et al.
Biosciences) and ODS resin (50 mm, Merck). Preparative HPLC collection used a C18 column [YMC-Pack ODS-A, 10 9 250 mm, 5 lm, 3 mL/min].
The fungus isolated from the rhizosphere soil of a mangrove plant R. stylosa, and identified as P. solitum according to the DNA sequences of the ITS region with 100 % similarity to P. solitum. The sequence data have been submitted to GenBank with the accession number KJ577599.
fractions (Fractions 1–9). The Fractions seven and eight were further separated on a Sephadex LH-20 column with MeOH to provide eight fractions. The Fraction 7-7 was isolated by semi-preparative HPLC employing isocratic elution with CH3CN–H2O (45:55) to obtain compound 1 (7.5 mg, tR = 16.8 min) and 2 (7.8 mg, tR = 20.6 min), with MeOH–H2O (60:40) to obtain compound 3 (14.0 mg, tR = 12.8 min) and 4 (13.0 mg, tR = 14.8 min). The Fraction 8-6 applied isocratic elution with MeOH–H2O (65:35) to yield compound 5 (9.0 mg, tR = 18.5 min) and 6 (10.0 mg, tR = 19.6 min).
Fermentation and extraction
Biological assay
Erlenmeyer flasks (1,000 mL) containing 300 mL fermentation media were directly inoculated with spores. The media contained yeast powder (1 g), peptone (5 g), glucose (20 g) and malt extract (3 g) dissolved in 1 L naturallycollected seawater, and sterilized in 121 °C for 21 min. The flasks were cultured in static condition at 28 °C for 30 days. The whole fermentation broth (60 L) was filtered through cheese cloth to separate the supernatant from the mycelia. The supernatant was extracted with EtOAc, and the mycelia was macerated and extracted with acetone. All extracts were evaporated under reduced pressure and concentrated to give a crude extract (17.0 g).
In the DPPH scavenging assay (Chen et al. 1999), samples to be tested were dissolved in MeOH and the solution (160 lL) was dispensed into wells of a 96-well micro-titer tray. Forty micro-liters of the DPPH solution in MeOH were added to each well. The mixture was shaken and left to stand for 30 min. After the reaction, absorbance was measured at 510 nm, and the percent inhibition was calculated. IC50 values denoted the concentration of sample required to scavenge 50 % of the DPPH free radicals. Compounds 1–6 were evaluated for radical scavenging activities against DPPH with IC50 values ranged from 4.7 to 15.0 lM (Table 3).
Strain identification
Penipyrol A (1) Purification Yellow oil; [a]20 D = -43.5 (c 0.1, CH2Cl2); UV (MeOH): 220 (3.50), 250 (3.35) nm; IR (KBr) mmax 3268, 2955, 1740, 1650, 1450, 1360, 980 cm-1; HRESIMS [M?Na]? m/z
The extracted material was separated by ODS in the gradient elution step of MeOH–H2O (5–100 %) to give nine Table 1 1H NMR (600 MHz) and 13C NMR (150 MHz) data of compounds 1–2 in DMSO-d6
123
Position
1
2
d(C)
d(H) (J in Hz)
d(C)
d(H) (J in Hz)
1
162.6
–
162.8
–
2
123.3
–
123.7
–
3
141.2
7.37, d (J = 6.6, 1H)
141.3
7.46, d (J = 7.2, 1H)
4
105.9
6.36, d (J = 6.6, 1H)
105.7
6.67, d (J = 7.2, 1H)
5
157.6
–
156.5
–
6
119.2
6.30, d (J = 15.4, 1H)
125.4
–
7
137.2
6.93, d (J = 15.4, 1H)
132.5
7.26 (s, 1H)
8
135.3
–
137.6
–
9
136.1
5.79, d (J = 9.4, 1H)
144.0
–
10
46.9
11 12
175.0 26.1
3.27 (m, 1H)
136.4
–
– 1.73 (m, 1H); 1.52 (m, 1H)
127.4 169.7
7.35 (s, 1H) –
13
12.0
0.86, t (J = 7.14, 3H)
21.0
2.33 (s, 3H)
14
13.0
1.83 (s, 3H)
23.1
2.47 (m, 2H)
15
16.9
2.00 (s, 3H)
14.7
1.12, t (J = 7.7, 3H)
16
–
–
20.9
2.30 (s, 3H)
17
–
–
16.7
2.03 (s, 3H)
Penipyrols A–B and peniamidones A–D Table 2 1H NMR and Position
13
C NMR data of compounds 3–6
a
4b
3
5a
6b
d(C)
d(H) (J in Hz)
d(C)
d(H) (J in Hz)
d(C)
d(H) (J in Hz)
d(C)
d(H) (J in Hz)
1
148.7
–
145.0
–
148.8
–
147.9
–
2
145.3
–
148.7
–
145.4
–
144.1
–
3
115.6
7.26 (brs, 1H)
114.4
7.27 (brs, 1H)
115.6
7.26 (brs, 1H)
113.7
7.27, d (J = 2.2, 1H)
4
126.6
–
125.9
–
126.4
–
125.0
–
5
119.4
7.16, dd (J = 8.3, 1.6, 1H)
119.2
7.19, dd (J = 8.2, 1.6, 1H)
119.4
7.16, d (J = 8.3, 1H)
118.4
7.19, dd (J = 8.3, 2.2, 1H)
6
115.3
6.74, d (J = 8.3, 1H)
114.5
6.79, d (J = 8.2, 1H)
115.4
6.74, d (J = 8.3, 1H)
113.6
6.79, d (J = 8.3, 1H)
7
166.5
–
169.0
–
166.7
–
–
–
8-NH
–
8.11, t (J = 5.5, 1H)
–
–
–
8.10 (brs, 1H)
–
–
9
40.0
3.20 (m, 2H)
39.1
3.35, t (J = 6.3, 2H)
37.0
3.25 (m, 2H)
38.2
3.37 (m, 2H)
10
30.6
1.50 (m, 2H)
25.8
1.67 (m, 2H)
33.1
1.63 (m, 2H)
30.3
1.88 (m, 2H)
11
26.5
1.43 (m, 2H)
25.9
1.67 (m, 2H)
59.2
3.44 (m, 2H)
23.8
2.37, t (J = 7.4, 2H)
12
61.1
3.40 (m, 2H)
64.1
4.09, t (J = 6.1, 2H)
–
–
175.0
–
13
–
–
–
–
–
–
–
14
–
–
–
–
–
–
171.7 19.5
2.02 (s, 3H)
a
1
Spectra were recorded at 600 MHz for H NMR and at 150 MHz for
13
b
Spectra were recorded at 600 MHz for 1H NMR and at 150 MHz for
13
C NMR using DMSO as solvent and TMS as internal standard
285.1100 (calculated for C15H18O4Na, 285.1097); 1H NMR and 13C NMR (see Table 1). Penipyrol B (2) Pale-yellow oil; UV (MeOH): 215 (3.23), 255 (3.42) nm; IR (KBr) mmax 3045, 2940, 1660, 1620, 1560, 1505, 1455, 1160, 870 cm-1; HRESIMS [M ? H]? m/z 289.1049 (calculated for C17H19O4, 287.1278); 1H NMR and 13C NMR (see Table 1). Peniamidone A (3) Brown oil; UV (MeOH): 210 (2.72), 266 (3.25) nm; IR (KBr) mmax 3440, 3170, 2965, 1680, 1650, 1610, 1545, 1455, 960 cm-1; HRESIMS [M?H]? m/z 226.1078 (calculated for C11H16O4N, 226.1074); 1H NMR and 13C NMR (see Table 2).
C NMR using CD3OD as solvent and TMS as internal standard
Peniamidone D (6) Brown oil; UV (MeOH): 212 (2.66), 265 (3.20) nm; IR (KBr) mmax 3445, 3350, 2978, 1720, 1660, 1630, 1545, 1510, 955 cm-1; 1H NMR and 13C NMR (see Table 2).
Results and discussion The fungal strain P. solitum GWQ-143 was cultured in static condition at 28 °C for 30 days. The whole extract was injected to repeated silica gel and LH-20 column chromatography and semi-preparative HPLC collection to yield the new compounds 1 (7.5 mg), 2 (7.8 mg), 3 (12.8 mg) and 4 (13.0 mg) (Fig. 1). Structure elucidation
Peniamidone B (4) Brown oil; UV (MeOH): 210 (2.82), 260 (3.05) nm; IR (KBr) mmax 3450, 2968, 1680, 1650, 1610, 1535, 1500, 1445, 990 cm-1; HRESIMS [M?H]? m/z 268.1187 (calculated for C13H18O5N, 268.1179); 1H NMR and 13C NMR (see Table 2). Peniamidone C (5) Brown oil; UV (MeOH): 215 (3.16), 265 (3.48) nm; IR (KBr) mmax 3535, 3170, 2960, 1675, 1650, 1545, 1505, 1440, 948 cm-1; 1H NMR and 13C NMR (see Table 2).
Penipyrol A (1) was obtained as yellow oil. The molecular formula was determined as C15H18O4 on the basis of the sodiated HRESIMS peak at m/z 285.1100, indicating seven degrees of unsaturation. The 1H, 13C NMR and DEPT spectroscopic data (Table 1) showed two olefinic methyl singlets (dC 13.0, dH 1.83; dC 16.9, dH 2.00) and eight sp2 hybrid carbons included five olefinic methines (dC 142.1, dH 7.37; dC 137.2, dH 6.93; dC 105.9, dH 6.36; dC 119.2, dH 6.30; dC 136.1, dH 5.79). The planar structure of 1 was identification from interpretations of COSY and HMBC spectroscopic analysis
123
W. Guo et al. Fig. 1 Structures of 1–6
13
14
O
1
O
11
5 7
15
9
10
OH
O
1
3
HO
O
3
HO
N H
7 5
1
11
9
R
16 9
11
O
1
O
13
12
O
5
14
7 15
O
3
3
R=
4
R=
OH O
6
R=COOH
16
Fig. 2 Selected 2D-NMR correlations of compounds 1–4
13
14
O
1
O
5 9
7
11
OH
10
11
O
O
1
9
15
2
1 3
HO HO
O 7
1
N H
9
11
12
OH
3
HO HO
5
1
O 7
5
N H
9
11
12
O
13
14
O
4
3 COSY
(Fig. 2). The HMBC correlations from H-3 to C-1/C-5 and from H-4 to C-5, and the COSY correlation between H-3 and H-4 together with the chemical shift of C-1 (dC 162.6) indicated the existence of a-pyrone moiety, which also supported by the similar NMR data to those of the reported gibepyrone A (Barrero et al. 1993). The additional COSY correlations (H-6/H-7, and H-9/H-10/H2-12/H3-13) together with the observed HMBC correlations from H-6 to C-8, from H-9 to C-7/C-8, from H-10 to C-8/C-11 and from H3-13 to C-10 were constructed the presence of hexadienoic acid chain. The connection between C-5 and C-6 was evidenced by the HMBC correlations from H-6 to C-4/C-5 and from H-7 to C-5. Finally, the gross structure of 1 was deduced by attaching the methyl groups to C-2/C-8 based on the HMBC correlations from H3-14 to C-7/C-8/C-9 and from H3-15 to C-1/C-2/C-3. The E configuration of the double bond (C-6/C-7) was evident from the coupling constant (15.4 Hz), and the geometry of C-8/C-9 was tentatively proposed as E suggested by the chemical shifts of H-9 (dH 5.79) and H3-14 (dH 1.83) (for E geometry the responding protons were at 5.76 and 1.82 ppm, while for Z at 5.49 and 2.05 ppm, respectively) (Garnier et al. 2007).
12
14
7
3
15
13
O O
5
O
3
15
123
R=OH
O
15
2
5
HMBC
The absolute configuration of C-10 was determined as R by comparing the optical rotation of 1 (-43.5) to the previously reported (R,E)-2-methyl-4-phenylpent-3-enoic acid (-53.1) (Garnier et al. 2007). Penipyrol B (2) was isolated as pale-yellow oil with the molecular formula of C17H18O4, requiring nine degrees of unsaturation. The presence of carbonyl group was deduced by the IR absorption at 1,660 cm-1. Analysis of the 1D NMR data (Table 2) established that 2 possessed four methyls, one methylene (dH 2.47, dC 23.1), two ester carbonyls (dC 169.7, 162.8), and five sp2 hybrid quaternary carbons. Detailed analysis of the 2D NMR spectroscopic data allowed to establish the planar structure of 2. Analysis the NMR chemical shifts (Table 1), COSY and HMBC correlations (Fig. 2) between 1 and 2 suggested they shared the same a-pyrone fragment. The HMBC correlations from H-7 to C-6/C-9/C-11 and from H-11 to C-6/C-7/C-9 confirmed the presence of tetrasubstituted phenyl. Further HMBC correlations from H2-14 to C-7/C-8/C-9, from H315 to C-8, from H3-13 to C-12, from H3-16 to C-9/C-10/C11 and the chemical shift (C-9, dC 144.0) located the
Penipyrols A–B and peniamidones A–D Table 3 DPPH radical scavenging activity of compounds 1–6 Compound
IC50 (lM)
Compound
IC50 (lM)
1
15.0
4
4.7
2
14.0
5
6.0
5.6
6
5.2
3 a
AA a
15.6
AA = ascorbic acid (positive control)
methyl, ethyl and acetyl to C-10/C-8/C-9, respectively. Finally, the structure of 2 was established by connecting the substituted phenyl to the a-pyrone at C-5 according to the HMBC correlations from H-11 to C-5/C-6 (Fig. 2). Peniamidones A–B (3–4) were purified as brown oil. The 1H, 13C NMR spectroscopic data of 3 (Table 2) showed three olefinic methines singlets (dC 119.4, dH 7.16; dC 115.6, dH 7.26; dC 115.3, dH 6.74) and three sp2 hybrid quaternary carbons (dC 148.7, 145.3, 126.6), which gave evidence of a dihydroxybenzoic acid. The additional COSY correlations between H-8/H2-9/H2-10/H2-11/H2-12 established the spin system of aminobutan, and confirmed by the HMBC correlations (Fig. 2). Finally, the gross structure of 3 was determined by the connection of the two moieties basing on the HMBC correlation from H2-9 to C-7, as well as the consideration of molecular formula. Analysis the NMR spectroscopic data of 4 (Table 2) showed 4 was almost identical to 3, except an acetyl group in 4 (dC 171.7, 19.5). Peniamidones C–D (5–6) were obtained as brown oil. Comparison of the 1H NMR data between 5, 6 and reported benzamide (Ohi et al. 1986), butanoic acid (Matyus et al. 1994) suggested they shared same structural features, which were intermediates in the semi-synthesis of b-lactam antibiotics and N-benzoylamino acid derivatives. In the present research, the 13C NMR, DEPT data were presented for those first isolated new natural products (Table 2). Biological effect and discussion The radical scavenging activities of the new compounds 1–6 were evaluated against DPPH for radical scavenging activity. Compounds 1–6 exhibited well activities with the IC50 values ranged from 4.7 to 15.0 lM (Table 3). In summary, under the guidance of characteristic UV absorption tracking method, six new compounds were isolated from the P. solitum GWQ-143. Penipyrol A (1) had the distinctive hexadienoic acid chain, and the great radical scavenging activities of those new compounds were firstly reported in the present research. Actually, autoxidation had been comprehensive researched by the autocatalytic free radical reaction leading to the aging of cardiovascular and cell injury (Gordon 1996), which was the
primary inducements of human disease processes such as cancer, atherosclerosis, rheumatoid arthritis and chronic inflammation (Ani et al. 2006). The antioxidant mechanism for these new compounds remained unclear and needed further investigation. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Nos. 21372208 and 41176120), the National High Technology Research and Development Program of China (No. 2013AA092901) and the State Key Laboratory of Bio-organic and Natural Products Chemistry (SKLBNPC12331).
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