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Aza-Polycyclic Aromatic Hydrocarbons from Saruma henryi by Yangmin Ma* a ) b ), Qiangqiang Jia a ), Pengfei Wang a ), Xiaowei Cheng a ), and Yongxiang Kang c ) a
) Key Laboratory of Auxiliary Chemistry & Technology for Chemical Industry, Ministry of Education, Shaanxi University of Science & Technology, Xian 710021, P. R. China (phone/fax: þ 86-29-86168312; e-mail: [email protected]) b ) Shaanxi Research Institute of Agricultural Products Processing Technology, Shaanxi University of Science & Technology, Xian 710021, P. R. China c ) College of Forestry, Northwest A & F University, Yangling Shaanxi 712100, P. R. China
A new azafluoranthene alkaloid, named sarumine (1), along with six known N-containing derivatives of phenanthrenes, 2 – 7, were isolated from the whole herb of Saruma henryi. Their structures were elucidated on the basis of extensive spectroscopic analysis. Moreover, antimicrobial activities of all compounds were evaluated.
Introduction. – Saruma henryi (Aristolochiaceae), the only species of genus Saruma, is a peculiar plant distributed only in China. Its root and rhizome have been used as folk medicine for the treatment of stomachache and arthralgia [1]. However, there are few reports on phytochemisty of S. henryi [2] [3], and the biologically active components have not been identified. Aiming at discovering chemical constituents with significant bioactivities, we conducted the phytochemical investigation of the whole herb of S. henryi, which led to the isolation of a new azafluoranthene alkaloid (1) and six known N-containing derivatives of phenanthrene, 2 – 7 (Fig.). Herein, the isolation, structure elucidation, and antimicrobial activities of all compounds are described.
Figure. Structures of compounds 1 – 7
Results and Discussion. – In addition to six known compounds, 2 – 7, a new one was obtained by repeated chromatography on SiO2 and Sephadex LH-20 from the EtOH extract of the air-dried whole herb of Saruma henryi. 2015 Verlag Helvetica Chimica Acta AG, Zrich
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Compound 1 was obtained as yellow crystals with a molecular formula C18H13NO4 , deduced from HR-ESI-MS (m/z 308.0930 ([M þ H] þ , C18H14NO þ4 ; calc. 308.0923)). The IR spectrum evidenced the presence of OH (3424 cm 1) and C¼O (1704 cm 1) groups, and aromatic rings (1603, 1514, 1459 cm 1). The 1H-NMR spectra (Table 1) displayed six aromatic H-atom signals (d(H) 7.48 (s), 8.53 (d, J ¼ 1.36), 8.08 (dd, J ¼ 7.84, 1.36), 8.13 (d, J ¼ 7.84), 8.67 (d, J ¼ 5.72), and 7.73 (d, J ¼ 5.72)), and two MeO Hatom signals (d(H) 4.06 (s) and 4.12 (s)). These 1H-NMR data were similar to those of the known compound telitoxine [4] [5]. However, a discernible C¼O signal at d(C) 167.1 of compound 1 appeared, compared with telitoxine, in the 13C-NMR spectrum. Furthermore, bromocresol green test of compound 1 gave a positive result. These observations indicated that there is a COOH group in the structure of compound 1, instead of a OH group. The position of the COOH group was confirmed by the correlations of the carboxy C-atom to both HC(7) and HC(9) in HMBC spectra. Finally, the compound 1 was unambiguously confirmed to be 5,6-dimethoxyindeno[1,2,3-ij]isoquinoline-8-carboxylic acid, an azafluoranthene alkaloid. So far, the 13C-NMR data for azafluoranthene-type alkaloids were reported only once in literature [6], presumably because of the long relaxation time of quaternary Catom. Therefore, the data of 13C-NMR of 1 provide a practical significance for the structure elucidation of azafluoranthenes. The discovery of sarumine (1) supports a certain relationship between Aristolochiaceae and Menispermaceae, with 1 being a significant evolutionary marker of the Aristolochiaceae. The hypothesis corresponds to the idea of the English botanist J. Hutchinson that Aristolochiaceae were the most closely related family to the Menispermaceae (Ranunculales) on the basis of the similarity of their stem structure [7]. Compound 2 was readily identified as aristololactam IV by comparing the melting point, and MS and 1H-NMR data with those in [8] [9]. The 1H-NMR spectrum (Table 2) Table 1. 1H- and 13C-NMR Data (in ( D6 )DMSO) of 1. d in ppm, J in Hz. Position
d( H )
d(C )
HMBC
2 3 3a 4 5 6 6a 6b 7 8 9 10 10a 10b 10c COOH 5-MeO 6-MeO
8.67 (d, J ¼ 5.72, 1 H ) 7.73 (d, J ¼ 5.72, 1 H )
145.9 118.3 129.9 106.4 158.7 148.0 123.7 137.4 124.8 129.9 130.4 121.4 142.2 156.4 123.0 167.1 56.7 61.3
C(3a), C(10b) C(4), C(10c)
7.48 (s, 1 H )
8.53 (d, J ¼ 1.36, 1 H ) 8.08 (dd, J ¼ 7.84, 1.36, 1 H ) 8.13 (d, J ¼ 7.84, 1 H )
4.06 (s, 3 H ) 4.12 (s, 3 H )
C(3), C(6), C(10c)
C(6a), C(9), C(10a) C(7), C(10a), COOH C(6b), C(8), C(10b)
C(5) C(6)
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Table 2. 1H- and 13C-NMR Data (in ( D6 )DMSO) of 2. d in ppm, J in Hz. Trivial atom numbering as indicated in the Figure. Position
d( H)
[9] 1 2 3 4 4a 4b 5 6 7 8 8a 9 10 10a C¼O 6-MeO 8-MeO OCH2O NH a
d(C )
Ref.
HMBC
[10]
7.68 (s, 1 H ) a )
7.52
7.62
7.65 (d, J ¼ 2.1, 1 H ) a )
7.60
7.65
6.85 (d, J ¼ 2.1, 1 H )
6.77
6.82
7.29 (s, 1 H )
7.27
7.28
3.91 (s, 3 H ) 3.99 (s, 3 H ) 6.49 (s, 2 H ) 10.70 (s, 1 H )
3.91 3.98 6.40 10.60
3.88 3.94 6.40 10.60
119.8 106.4 149.0 147.6 111.3 126.1 101.1 158.3 99.1 156.8 118.9 98.8 133.1 126.1 168.4 55.8 56.5 103.7
C(4), C¼O, C(10a)
C(4a), C(7), C(8a) C(6), C(8), C(8a), C(5)
C(4b), C(8), C(10a)
C(6) C(8) C(3), C(4)
) The assignments of HC(2) and HC(5) were corrected.
displayed four aromatic H-atom signals (d(H) 7.29 (s, 1 H), 7.68 (s, 1 H), 6.85 (d, J ¼ 2.1, 1 H) and 7.65 (d, J ¼ 2.1, 1 H), two singlets corresponding to HC(2) and HC(9), and two doublets corresponding to HC(5) and HC(7). In the HMBC spectrum, the correlation of d(H) 7.68 (s) with d(C) 168.4 (CONH) evidenced that the signal at d(H) 7.68 corresponded to HC(2); another singlet at d(H) 7.29 was the signal of HC(9), as deduced from the correlation of HC(9) with C(8). The correlations of the signal at d(H) 6.85 to those of both C(6) and C(8) indicated that the signal at d(H) 6.85 corresponded to HC(7); therefore the last doublet d(H) 7.65 was attributed to HC(5). The 13C-NMR data were obtained for the first time, which provide some practical significance for the structure elucidation of aristololactams. The isolated known compounds were identified as 7-methoxyaristololactam IV (3) [10], aristololactam AII (4) [11], aristololactam BII (5) [12], aristolochic acid IV (6) [13], and aristolochic acid I (7) [14] on the basis of spectroscopic and physicochemical comparison with literature data. Among the known compounds, compounds 2, 5, and 6 were isolated from the genus Saruma for the first time. Since aristolochic acids and aristololactams are considered to be the characteristic constituents of Aristolochiaceae plants [2], the result of this investigation provides a new chemotaxonomic evidence for the Aristolochiaceae family. The screening data of the antibacterial activities revealed that compound 1 exhibited potent in vitro antibacterial activity, especially against Gram-positive bacteria with a MIC value of 7.81 mg/ml, which was similar to that of penicillin sodium.
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Table 3. Antimicrobial Activities of Compounds 1 – 7 Compound
1 2 3 4 5 6 7 Penicillin G sodium Streptomycin sulfate Ketoconazole
MIC [mg/ml] S. aureus
B. subtilis
P. aeruginosa
E. coli
C. gloeosporioides
V. mali
A. alternata
A. brassicae
7.81 31.3 31.3 31.3 62.5 62.5 31.3 7.81 – –
7.81 62.5 62.5 31.3 62.5 62.5 62.5 7.81 – –
31.3 62.5 62.5 62.5 31.3 125 62.5 – 7.81 –
31.3 62.5 62.5 62.5 31.3 62.5 62.5 – 15.6 –
125 250 250 250 500 500 250 – – 15.6
125 250 125 125 125 125 500 – – 15.6
250 125 250 250 500 500 250 – – 15.6
250 250 250 125 125 125 250 – – 15.6
Compounds 2 – 7 also showed satisfactory antibacterial activities with MIC values in the range of 31.3 – 62.5 mg/ml against the four bacteria. All compounds showed certain antifungal activities with MIC values ranging from 125 to 250 mg/ml. These results, together with those of known antibiotics as standards, are compiled in Table 3. This work was financially supported by the National Forestry Public Service Specific Research of China (200904004) and the Specialized Research Fund for the Doctoral Program of Higher Education (20116125110001). Experimental Part General. All the solvents were purchased from Tianjin Hongyan Chemical Reagents Factory (P. R. China). (D6 )DMSO was from Beijing Boya Dabei Technological Development (P. R. China). The bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa, and fungi Colletotirchum gloeosporioides, Valsamali mali, and Alternaria alternata, Alternaria brassicae were provided by the College of Life Science & Engineering, Shaanxi University of Science & Technology (Xian, P. R. China). Column chromatography (CC): silica gel (SiO2 , 200 – 300 mesh; Tsingtao Marine Chemical Factory, P. R. China). M.p.: X-6 micro-melting point apparatus; uncorrected. UV Spectra: DR 5000 spectrophotometer; lmax (log e) in nm. IR Spectra: Bruker VECTOR-22 FT-IR spectrometer; ˜n in cm 1. 1D- and 2D-NMR spectra: Bruker AVANCE III-400 spectrometer; d in ppm rel. to Me4Si as internal standard, J in Hz. HR-ESI-MS: Bruker maXis 4G UHR-TOF, in the positive-ion mode; in m/z. Plant Material. The whole herb of S. henryi, collected from the Taibai Mountains, Shaannxi Province, P. R. China, on August 2011, was identified by Prof. Y. K. of Northwest A & F University. A voucher specimen (Kang008-NWAFU) was deposited with Northwest A & F University Herbaria. Extraction and Isolation. The air-dried whole herb of S. henryi (8.5 kg) were powdered and extracted repeatedly with 95% EtOH at r.t. for 48 h (3 50 l). The EtOH extract was evaporated to leave a viscous residue (450 g), which was then suspended in H2O (3 l) and extracted with AcOEt to afford the AcOEt extract (187 g), which was subjected to CC (SiO2 ; petroleum ether (PE)/AcOEt 1 : 0, 20 : 1, 10 : 1, 5 : 1, 2 : 1, 1 : 1, 0 : 1) to give seven fractions, Frs. A – G. Fr. D (15 g) was separated by CC (SiO2 ; with a gradient of AcOEt in PE) to give five subfractions Frs. D1 – D5, Fr. D4 (4 g; eluted by PE/AcOEt 5 : 1) was separated by repeated CC (SiO2 ) to yield pure compounds 3 (20 mg) and 5 (30 mg). Fr. E (20 g) was separated by CC (SiO2 ; with a gradient of AcOEt in PE) to give five subfractions, Frs. E1 – E5. Fr. E3 (8 g; eluted by PE/AcOEt 5 : 2) was separated by repeated CC (SiO2 and Sephadex LH-20; AcOEt/ MeOH 1 : 1) to yield compounds 1 (4 mg), 2 (10 mg) and 4 (20 mg). Fr. E4 (6 g, eluted by PE/AcOEt 2 : 1) was separated by repeated CC (SiO2 ) to afford compounds 6 (10 mg) and 7 (40 mg).
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Sarumine ( ¼ 5,6-Dimethoxyindeno[1,2,3-ij]isoquinoline-8-carboxylic Acid; 1). Yellow crystals. M.p. > 3008. UV (EtOH): 229.0 (3.36). IR (KBr): 3424, 2923, 1704, 1662, 1633, 1603, 1514, 1459. 1H- and 13 C-NMR: see Table 1. HR-ESI-MS: 308.0930 ([M þ H] þ , C18H14NO þ4 ; calc. 308.0923). Aristololactam IV ( ¼ 8,10-Dimethoxy[1,3]benzodioxolo[6,5,4-cd]benzo[f]indol-5(6H)-one; 2). Yellow crystals. M.p. 344 – 3468. 1H- (400 MHz, (D6 )DMSO) and 13C-NMR: see Table 2. HR-ESI-MS: 324.0879 ([M þ H] þ , C18H14NO þ5 ; calc. 324.0872). Biological Assay. The antimicrobial activities were evaluated as minimum inhibitory concentrations (MICs) values. Both antibacterial and antifungal activities were determined by the continuous dilution method [15]. Each compound was set at a concentration of 500, 250, 125, 62.5, 31.3, 15.6, 7.81, and 3.91 mg/ ml by the continuous dilution method, while the tested strains were incubated in the liquid mediums at the set temps. For bacteria, the beef extract peptone medium was made up of beef extract (5.0 g/l), peptone (10.0 g/l), NaCl (5.0 g/l), pH 7.2 – 7.4; the culture temp. was 378. For fungi, the potato-glucose medium was made up of percolate of 200 g of potato under boiling for 30 min, glucose (20 g), and constant volume of 1 l by H2O, the culture temp. was 288. The in vitro antimicrobial activities were tested against two Gram-positive (G þ ) bacteria (S. aureus and B. subtilis), two Gram-negative (G ) bacteria (E. coli and P. aeruginosa), and four plant pathogenic fungi (C. gloeosporioides, V. mali, A. alternata, A. brassicae). The activities were determined at different concentrations of the compounds 1 – 7 (3.91 – 500 mg/ml) in DMSO, and the results were compared with lower concentrations of known antibiotics including penicillin G sodium against G þ bacteria, streptomycin sulfate against G bacteria, and ketoconazole against fungi.
REFERENCES [1] Chinese Academy of Science Flora of China Editorial Board, Flora of China, Science Press, Beijing, 1988, Vol. 72, p. 160. [2] S. W. Dong, M. Y. Shang, X. Wang, S. X. Zhang, C. Li, S. Q. Cai, J. Chin. Pharm. Sci. 2009, 18, 146. [3] T. Iwashina, W. Marubashi, T. Suzuki, Biochem. Syst. Ecol. 2002, 30, 1101. [4] M. D. Menachery, M. P. Cava, J. Nat. Prod. 1981, 44, 320. [5] M. D. Menachery, C. D. Muthler, J. Nat. Prod. 1987, 50, 726. [6] H. Morita, K. Matsumoto, K. Takeya, H. Itokawa, Chem. Pharm. Bull. 1993, 41, 1307. [7] J. Hutchinson, The Families of Flowering Plants, Clarendon Press, Oxford, 1973, 3rd edn., p. 510. [8] S. M. Kupchan, J. J. Merianos, J. Org. Chem. 1968, 33, 3735. [9] H. A. Priestap, Phytochemistry 1985, 24, 849. [10] B. B. Xie, M. Y. Shang, X. Wang, S. Q. Cai, K. H. Lee, Acta. Pharm. Sin. 2011, 46, 188. [11] H. A. Priestap, Magn. Reson. Chem. 1989, 27, 460. [12] K. V. Rao, G. C. S. Reddy, J. Nat. Prod. 1990, 53, 309. [13] L. Pistelli, E. Nieri, A. R. Bilia, A. Marsil, J. Nat. Prod. 1993, 56, 1605. [14] J. Zhang, Y. S. Xiao, J. T. Feng, S. L. Wu, X. Y. Xue, X. L. Zhang, X. M. Liang, J. Pharmaceut. Biomed. 2010, 52, 446. [15] M. Zhang, W. L. Wang, Y. C. Fang, T. J. Zhu, Q. Q. Gu, W. M. Zhu. J. Nat. Prod. 2008, 71, 985. Received February 4, 2014
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Aza-Polycyclic Aromatic Hydrocarbons from Saruma henryi by Yangmin Ma* a ) b ), Qiangqiang Jia a ), Pengfei Wang a ), Xiaowei Cheng a ), and Yongxiang Kang c ) a
) Key Laboratory of Auxiliary Chemistry & Technology for Chemical Industry, Ministry of Education, Shaanxi University of Science & Technology, Xian 710021, P. R. China (phone/fax: þ 86-29-86168312; e-mail: [email protected]) b ) Shaanxi Research Institute of Agricultural Products Processing Technology, Shaanxi University of Science & Technology, Xian 710021, P. R. China c ) College of Forestry, Northwest A & F University, Yangling Shaanxi 712100, P. R. China
A new azafluoranthene alkaloid, named sarumine (1), along with six known N-containing derivatives of phenanthrenes, 2 – 7, were isolated from the whole herb of Saruma henryi. Their structures were elucidated on the basis of extensive spectroscopic analysis. Moreover, antimicrobial activities of all compounds were evaluated.
Introduction. – Saruma henryi (Aristolochiaceae), the only species of genus Saruma, is a peculiar plant distributed only in China. Its root and rhizome have been used as folk medicine for the treatment of stomachache and arthralgia [1]. However, there are few reports on phytochemisty of S. henryi [2] [3], and the biologically active components have not been identified. Aiming at discovering chemical constituents with significant bioactivities, we conducted the phytochemical investigation of the whole herb of S. henryi, which led to the isolation of a new azafluoranthene alkaloid (1) and six known N-containing derivatives of phenanthrene, 2 – 7 (Fig.). Herein, the isolation, structure elucidation, and antimicrobial activities of all compounds are described.
Figure. Structures of compounds 1 – 7
Results and Discussion. – In addition to six known compounds, 2 – 7, a new one was obtained by repeated chromatography on SiO2 and Sephadex LH-20 from the EtOH extract of the air-dried whole herb of Saruma henryi. 2015 Verlag Helvetica Chimica Acta AG, Zrich
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Compound 1 was obtained as yellow crystals with a molecular formula C18H13NO4 , deduced from HR-ESI-MS (m/z 308.0930 ([M þ H] þ , C18H14NO þ4 ; calc. 308.0923)). The IR spectrum evidenced the presence of OH (3424 cm 1) and C¼O (1704 cm 1) groups, and aromatic rings (1603, 1514, 1459 cm 1). The 1H-NMR spectra (Table 1) displayed six aromatic H-atom signals (d(H) 7.48 (s), 8.53 (d, J ¼ 1.36), 8.08 (dd, J ¼ 7.84, 1.36), 8.13 (d, J ¼ 7.84), 8.67 (d, J ¼ 5.72), and 7.73 (d, J ¼ 5.72)), and two MeO Hatom signals (d(H) 4.06 (s) and 4.12 (s)). These 1H-NMR data were similar to those of the known compound telitoxine [4] [5]. However, a discernible C¼O signal at d(C) 167.1 of compound 1 appeared, compared with telitoxine, in the 13C-NMR spectrum. Furthermore, bromocresol green test of compound 1 gave a positive result. These observations indicated that there is a COOH group in the structure of compound 1, instead of a OH group. The position of the COOH group was confirmed by the correlations of the carboxy C-atom to both HC(7) and HC(9) in HMBC spectra. Finally, the compound 1 was unambiguously confirmed to be 5,6-dimethoxyindeno[1,2,3-ij]isoquinoline-8-carboxylic acid, an azafluoranthene alkaloid. So far, the 13C-NMR data for azafluoranthene-type alkaloids were reported only once in literature [6], presumably because of the long relaxation time of quaternary Catom. Therefore, the data of 13C-NMR of 1 provide a practical significance for the structure elucidation of azafluoranthenes. The discovery of sarumine (1) supports a certain relationship between Aristolochiaceae and Menispermaceae, with 1 being a significant evolutionary marker of the Aristolochiaceae. The hypothesis corresponds to the idea of the English botanist J. Hutchinson that Aristolochiaceae were the most closely related family to the Menispermaceae (Ranunculales) on the basis of the similarity of their stem structure [7]. Compound 2 was readily identified as aristololactam IV by comparing the melting point, and MS and 1H-NMR data with those in [8] [9]. The 1H-NMR spectrum (Table 2) Table 1. 1H- and 13C-NMR Data (in ( D6 )DMSO) of 1. d in ppm, J in Hz. Position
d( H )
d(C )
HMBC
2 3 3a 4 5 6 6a 6b 7 8 9 10 10a 10b 10c COOH 5-MeO 6-MeO
8.67 (d, J ¼ 5.72, 1 H ) 7.73 (d, J ¼ 5.72, 1 H )
145.9 118.3 129.9 106.4 158.7 148.0 123.7 137.4 124.8 129.9 130.4 121.4 142.2 156.4 123.0 167.1 56.7 61.3
C(3a), C(10b) C(4), C(10c)
7.48 (s, 1 H )
8.53 (d, J ¼ 1.36, 1 H ) 8.08 (dd, J ¼ 7.84, 1.36, 1 H ) 8.13 (d, J ¼ 7.84, 1 H )
4.06 (s, 3 H ) 4.12 (s, 3 H )
C(3), C(6), C(10c)
C(6a), C(9), C(10a) C(7), C(10a), COOH C(6b), C(8), C(10b)
C(5) C(6)
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Table 2. 1H- and 13C-NMR Data (in ( D6 )DMSO) of 2. d in ppm, J in Hz. Trivial atom numbering as indicated in the Figure. Position
d( H)
[9] 1 2 3 4 4a 4b 5 6 7 8 8a 9 10 10a C¼O 6-MeO 8-MeO OCH2O NH a
d(C )
Ref.
HMBC
[10]
7.68 (s, 1 H ) a )
7.52
7.62
7.65 (d, J ¼ 2.1, 1 H ) a )
7.60
7.65
6.85 (d, J ¼ 2.1, 1 H )
6.77
6.82
7.29 (s, 1 H )
7.27
7.28
3.91 (s, 3 H ) 3.99 (s, 3 H ) 6.49 (s, 2 H ) 10.70 (s, 1 H )
3.91 3.98 6.40 10.60
3.88 3.94 6.40 10.60
119.8 106.4 149.0 147.6 111.3 126.1 101.1 158.3 99.1 156.8 118.9 98.8 133.1 126.1 168.4 55.8 56.5 103.7
C(4), C¼O, C(10a)
C(4a), C(7), C(8a) C(6), C(8), C(8a), C(5)
C(4b), C(8), C(10a)
C(6) C(8) C(3), C(4)
) The assignments of HC(2) and HC(5) were corrected.
displayed four aromatic H-atom signals (d(H) 7.29 (s, 1 H), 7.68 (s, 1 H), 6.85 (d, J ¼ 2.1, 1 H) and 7.65 (d, J ¼ 2.1, 1 H), two singlets corresponding to HC(2) and HC(9), and two doublets corresponding to HC(5) and HC(7). In the HMBC spectrum, the correlation of d(H) 7.68 (s) with d(C) 168.4 (CONH) evidenced that the signal at d(H) 7.68 corresponded to HC(2); another singlet at d(H) 7.29 was the signal of HC(9), as deduced from the correlation of HC(9) with C(8). The correlations of the signal at d(H) 6.85 to those of both C(6) and C(8) indicated that the signal at d(H) 6.85 corresponded to HC(7); therefore the last doublet d(H) 7.65 was attributed to HC(5). The 13C-NMR data were obtained for the first time, which provide some practical significance for the structure elucidation of aristololactams. The isolated known compounds were identified as 7-methoxyaristololactam IV (3) [10], aristololactam AII (4) [11], aristololactam BII (5) [12], aristolochic acid IV (6) [13], and aristolochic acid I (7) [14] on the basis of spectroscopic and physicochemical comparison with literature data. Among the known compounds, compounds 2, 5, and 6 were isolated from the genus Saruma for the first time. Since aristolochic acids and aristololactams are considered to be the characteristic constituents of Aristolochiaceae plants [2], the result of this investigation provides a new chemotaxonomic evidence for the Aristolochiaceae family. The screening data of the antibacterial activities revealed that compound 1 exhibited potent in vitro antibacterial activity, especially against Gram-positive bacteria with a MIC value of 7.81 mg/ml, which was similar to that of penicillin sodium.
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Table 3. Antimicrobial Activities of Compounds 1 – 7 Compound
1 2 3 4 5 6 7 Penicillin G sodium Streptomycin sulfate Ketoconazole
MIC [mg/ml] S. aureus
B. subtilis
P. aeruginosa
E. coli
C. gloeosporioides
V. mali
A. alternata
A. brassicae
7.81 31.3 31.3 31.3 62.5 62.5 31.3 7.81 – –
7.81 62.5 62.5 31.3 62.5 62.5 62.5 7.81 – –
31.3 62.5 62.5 62.5 31.3 125 62.5 – 7.81 –
31.3 62.5 62.5 62.5 31.3 62.5 62.5 – 15.6 –
125 250 250 250 500 500 250 – – 15.6
125 250 125 125 125 125 500 – – 15.6
250 125 250 250 500 500 250 – – 15.6
250 250 250 125 125 125 250 – – 15.6
Compounds 2 – 7 also showed satisfactory antibacterial activities with MIC values in the range of 31.3 – 62.5 mg/ml against the four bacteria. All compounds showed certain antifungal activities with MIC values ranging from 125 to 250 mg/ml. These results, together with those of known antibiotics as standards, are compiled in Table 3. This work was financially supported by the National Forestry Public Service Specific Research of China (200904004) and the Specialized Research Fund for the Doctoral Program of Higher Education (20116125110001). Experimental Part General. All the solvents were purchased from Tianjin Hongyan Chemical Reagents Factory (P. R. China). (D6 )DMSO was from Beijing Boya Dabei Technological Development (P. R. China). The bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa, and fungi Colletotirchum gloeosporioides, Valsamali mali, and Alternaria alternata, Alternaria brassicae were provided by the College of Life Science & Engineering, Shaanxi University of Science & Technology (Xian, P. R. China). Column chromatography (CC): silica gel (SiO2 , 200 – 300 mesh; Tsingtao Marine Chemical Factory, P. R. China). M.p.: X-6 micro-melting point apparatus; uncorrected. UV Spectra: DR 5000 spectrophotometer; lmax (log e) in nm. IR Spectra: Bruker VECTOR-22 FT-IR spectrometer; ˜n in cm 1. 1D- and 2D-NMR spectra: Bruker AVANCE III-400 spectrometer; d in ppm rel. to Me4Si as internal standard, J in Hz. HR-ESI-MS: Bruker maXis 4G UHR-TOF, in the positive-ion mode; in m/z. Plant Material. The whole herb of S. henryi, collected from the Taibai Mountains, Shaannxi Province, P. R. China, on August 2011, was identified by Prof. Y. K. of Northwest A & F University. A voucher specimen (Kang008-NWAFU) was deposited with Northwest A & F University Herbaria. Extraction and Isolation. The air-dried whole herb of S. henryi (8.5 kg) were powdered and extracted repeatedly with 95% EtOH at r.t. for 48 h (3 50 l). The EtOH extract was evaporated to leave a viscous residue (450 g), which was then suspended in H2O (3 l) and extracted with AcOEt to afford the AcOEt extract (187 g), which was subjected to CC (SiO2 ; petroleum ether (PE)/AcOEt 1 : 0, 20 : 1, 10 : 1, 5 : 1, 2 : 1, 1 : 1, 0 : 1) to give seven fractions, Frs. A – G. Fr. D (15 g) was separated by CC (SiO2 ; with a gradient of AcOEt in PE) to give five subfractions Frs. D1 – D5, Fr. D4 (4 g; eluted by PE/AcOEt 5 : 1) was separated by repeated CC (SiO2 ) to yield pure compounds 3 (20 mg) and 5 (30 mg). Fr. E (20 g) was separated by CC (SiO2 ; with a gradient of AcOEt in PE) to give five subfractions, Frs. E1 – E5. Fr. E3 (8 g; eluted by PE/AcOEt 5 : 2) was separated by repeated CC (SiO2 and Sephadex LH-20; AcOEt/ MeOH 1 : 1) to yield compounds 1 (4 mg), 2 (10 mg) and 4 (20 mg). Fr. E4 (6 g, eluted by PE/AcOEt 2 : 1) was separated by repeated CC (SiO2 ) to afford compounds 6 (10 mg) and 7 (40 mg).
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Sarumine ( ¼ 5,6-Dimethoxyindeno[1,2,3-ij]isoquinoline-8-carboxylic Acid; 1). Yellow crystals. M.p. > 3008. UV (EtOH): 229.0 (3.36). IR (KBr): 3424, 2923, 1704, 1662, 1633, 1603, 1514, 1459. 1H- and 13 C-NMR: see Table 1. HR-ESI-MS: 308.0930 ([M þ H] þ , C18H14NO þ4 ; calc. 308.0923). Aristololactam IV ( ¼ 8,10-Dimethoxy[1,3]benzodioxolo[6,5,4-cd]benzo[f]indol-5(6H)-one; 2). Yellow crystals. M.p. 344 – 3468. 1H- (400 MHz, (D6 )DMSO) and 13C-NMR: see Table 2. HR-ESI-MS: 324.0879 ([M þ H] þ , C18H14NO þ5 ; calc. 324.0872). Biological Assay. The antimicrobial activities were evaluated as minimum inhibitory concentrations (MICs) values. Both antibacterial and antifungal activities were determined by the continuous dilution method [15]. Each compound was set at a concentration of 500, 250, 125, 62.5, 31.3, 15.6, 7.81, and 3.91 mg/ ml by the continuous dilution method, while the tested strains were incubated in the liquid mediums at the set temps. For bacteria, the beef extract peptone medium was made up of beef extract (5.0 g/l), peptone (10.0 g/l), NaCl (5.0 g/l), pH 7.2 – 7.4; the culture temp. was 378. For fungi, the potato-glucose medium was made up of percolate of 200 g of potato under boiling for 30 min, glucose (20 g), and constant volume of 1 l by H2O, the culture temp. was 288. The in vitro antimicrobial activities were tested against two Gram-positive (G þ ) bacteria (S. aureus and B. subtilis), two Gram-negative (G ) bacteria (E. coli and P. aeruginosa), and four plant pathogenic fungi (C. gloeosporioides, V. mali, A. alternata, A. brassicae). The activities were determined at different concentrations of the compounds 1 – 7 (3.91 – 500 mg/ml) in DMSO, and the results were compared with lower concentrations of known antibiotics including penicillin G sodium against G þ bacteria, streptomycin sulfate against G bacteria, and ketoconazole against fungi.
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