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New enantiomeric isoquinoline alkaloids from Coptis chinensis
Phytochemistry Letters 7 (2014) 89–92
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New enantiomeric isoquinoline alkaloids from Coptis chinensis Lei Wang a,b,1, Sheng-Yuan Zhang a,1, Liang Chen a, Xiao-Jun Huang a,b, Qing-Wen Zhang c,**, Ren-Wang Jiang a, Fen Yao d,**, Wen-Cai Ye a,b,* a
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China JNU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research, Jinan University, Guangzhou 510632, PR China c Institute of Chinese Medical Sciences, University of Macau, Macau, PR China d Department of Pharmacology, Medical College, Shantou University, Shantou 515041, PR China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 June 2013 Received in revised form 5 September 2013 Accepted 1 October 2013 Available online 22 October 2013
A pair of new enantiomeric isoquinoline alkaloids, (+)- and ()-5-hydroxyl-8-oxyberberine (1), along with nine known analogs (2–10) were isolated from the rhizoma of Coptis chinensis. Their structures with absolute configurations were elucidated on the basis of spectroscopic methods, X-ray diffraction analysis, and quantum chemical calculation. The antibacterial activities of 1–10 were also evaluated. Berberine (8) displayed synergism against methicillin-resistant Staphylococcus aureus (MRSA) in combination with ceftazidime and cefepiem. ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Ranunculaceae Coptis chinensis Isoquinoline alkaloid
1. Introduction The plant Coptis chinensis (Ranunculaceae) is mainly distributed in western China, such as Sichuan and Shanxi provinces. The rhizome of this plant has been widely used as a traditional Chinese medicine in the treatment of diarrhea, fever and eczema (Tang et al., 2009). Previous phytochemical studies of this plant had led to the isolation of more than thirty alkaloids (Wang et al., 2007; Zhao et al., 2010; Liu et al., 2011), which demonstrated antibacterial, antiviral, anti-inflammatory and antitumor activities (Kuo et al., 2005; Hwang et al., 2006; Tang et al., 2009; Chin et al., 2010). Berberine, a major constituent in C. chinensis, was found to have synergistic effect against methicillin-resistant Staphylococcus aureus (MRSA) in combination with azithromycin and levofloxacin (Zuo et al., 2012). In the course of searching for biologically interesting and structural unique alkaloids from Chinese herbal medicines (Zhao et al., 2012; Ouyang et al., 2011; Wang et al., 2009), a pair of new enantiomeric isoquinoline alkaloids, (+)- and ()-5-hydroxyl-8-oxyberberine [(+)-1 and ()-1], along with nine known alkaloids (2–10) were isolated from the rhizoma of C. chinensis (Fig. 1). In this paper, the chiral separation and structure
* Corresponding author at: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China. Tel.: +86 20 8522 0936; fax: +86 20 8522 1559. ** Corresponding authors. Tel.: +86 20 8522 0936; fax: +86 20 8522 1559. E-mail addresses: [email protected] (Q.-W. Zhang), [email protected] (F. Yao), [email protected] (W.-C. Ye). 1 These authors contributed equally to this work.
elucidation of 1 were described. The antibacterial activities and synergism of 1–10 with ceftazidime and cefepiem were also evaluated. 2. Results and discussion Compound (1) was obtained as yellow needle crystal. The HRESIMS spectrum of 1 exhibited a quasi-molecular ion at m/z 368.1114 [M+H]+ (calcd for C20H18NO6, 368.1129), consistent with the molecular formula C20H17NO6. The UV spectrum showed absorption maxima at 225 and 340 nm. The IR spectrum of 1 suggested the presence of hydroxyl (3441 cm1), carbonyl (1643 cm1) and aromatic ring (1595, 1510, 1493 cm1). The 1H NMR spectrum of 1 showed signals for a pair of ortho-coupled aromatic protons [dH 7.52 (1H, d, J = 8.7 Hz), 7.41 (1H, d, J = 8.7 Hz)], three isolated aromatic protons [dH 7.50, 7.13, 7.01 (each 1H, s)], a methylenedioxyl group [dH 6.07 (2H, s)], three alkyl protons [dH 4.74 (1H, m), 4.26 (1H, dd, J = 13.6, 6.0 Hz), and 3.96 (1H, dd, J = 13.6, 3.4 Hz)], and two methoxyl groups [dH 3.87 and 3.78 (each 3H, s)]. The 13C NMR and DEPT spectra displayed twenty signals including two methyls, two methylenes, six methines, and ten quaternary carbons. The above NMR data suggested that 1 was a protoberberine type alkaloid. With the aid of 1H–1H COSY, HSQC, HMBC and ROESY experiments, all the 1H and 13C NMR signals of 1 could be assigned as shown in Table 1. The comparison of the NMR data of 1 with those of the known compound 8-oxyberberine (2) revealed that their signals were similar, except for the appearance of the signals for a oxygenated methenyl [dH 4.74 (1H, m); dC 64.1] in 1. Thus, 1 was proposed to be
1874-3900/$ – see front matter ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2013.10.007
L. Wang et al. / Phytochemistry Letters 7 (2014) 89–92
90
Fig. 1. Chemical structures of compounds 1–10.
a 5-hydroxyl-substituted derivative of 2, which was supported by the HMBC correlations between H-4 (dH 7.01)/H-6a (dH 4.26) and C-5 (dc 64.1), as well as the ROESY correlation between H-5 (dH 4.74) and H-4 (dH 7.01) (Fig. 2). Therefore, 1 was identified as 5hydroxyl-8-oxyberberine.
Table 1 1 H and 13C NMR data for compound 1 (DMSO-d6, J in Hz). No.
dH
dC
1 2 3 4 4a 5 6
7.50 (1H, s) – – 7.01 (1H, s) – 4. 74 (1H, m) 4.26 (1H, dd, 13.6, 6.0, H-6a) 3.96 (1H, dd, 13.6, 3.4, H-6b) – – – – 7.52 (1H, d, 8.7) 7.41 (1H, d, 8.7) – 7.13 (1H, s) – – 6.07 (2H, br s) 3.78 (3H, s) 3.87 (3H, s) 5.59 (1H, d, 4.5)
104.5 148.1 147.8 106.8 132.3 64.1 45.4
8 8a 9 10 11 12 12a 13 13a 13b –OCH2O– 9-OCH3 10-OCH3 5-OH
159.2 118.6 148.3 151.0 119.1 122.7 131.8 101.0 134.5 122.5 101.5 60.8 56.3 –
The chemical structure of 1 was further confirmed by an X-ray diffraction analysis (Fig. 3). The space group P21/n of the crystal together with the lack of optical activity indicated that 1 was a racemate. Chiral separation of 1 by HPLC led to the isolation of two enantiomers [(+)-1 and ()-1]. To determine the absolute configuration of each enantiomer, a comparison between the experimental and calculated CD spectra using the time-dependent DFT method was performed. The measured CD spectrum of (+)-1 showed positive Cotton effect at 341.5 nm (De + 2.5) and negative one at 220.1 nm (De 6.2), which was consistent with the calculated CD spectrum for S isomer (Fig. 4). Whereas, the CD spectrum of ()-1 displayed reverse Cotton effects at the similar wavelengths, which corresponded to R isomer (Fig. 4). These results revealed that (+)-1 and ()-1 corresponded to (5S)-1 and (5R)-1, respectively. The nine known compounds were identified as 8-oxyberberine (2), 8-oxocoptisine (3), tetrahydroberberine (4), berbithine (5), 6([1,3] dioxolo[4,5-g] isoquinoline-5-carbonyl)-2,3-dimethoxybenzoic acid methyl ester (6), palmatine (7), berberine (8), epiberberine (9), and coptisine (10) by comparison of their NMR and MS data with those reported in the literatures (Wang et al., 2007; Jung et al., 2008; Zhao et al., 2010; Liu et al., 2011). All isolated compounds (1–10) were tested for their antibacterial effects. No inhibition zone was found with compounds 1–10 (25.6 mg per paper disc), indicating all compounds have no direct effect against S. aureus, Escherichia coli and Pseudomonas aeruginosa. Inhibition zone of ceftazidime against multi-resistanct clinical strains EC1 and PA1 were both zero, while inhibition zone of cefepime against EC1 and PA1 were 11 mm and 8 mm,
L. Wang et al. / Phytochemistry Letters 7 (2014) 89–92
91
Fig. 2. Key HMBC and ROESY correlations of 1.
respectively. When combined with compounds 1–10 respectively, the diameters of inhibition zones remained unchanged, indicating that these compounds displayed no synergism effect with ceftazidime and cefepime against EC1 and PA1. However, in the anti-MRSA1 test, the inhibition zone diameter of ceftazidime and cefepime discs enlarged from 0 to 9 mm when combined with compound 8. These results revealed the synergism of compound 8 against MRSA in combination with ceftazidime and cefepime. 3. Experimental 3.1. General experimental procedures Fig. 4. Calculated and experimental CD spectra of (+)-1 and ()-1.
Optical rotations were measured by a Jasco P-1020 digital polarimeter at room temperature. UV spectra were recorded on a Jasco-V-550 UV-vis spectrophotometer. IR spectra were conducted on a Jasco FT/IR-480 plus Fourier transform infrared spectrometer. HRESIMS were performed on an Agilent 6210 ESI/TOF mass spectrometer. 1D (1H, 13C, and DEPT) and 2D (1H–1H COSY, HSQC, HMBC and ROESY) NMR spectra were recorded on a Bruker AV-300 spectrometer with TMS as internal standard, and chemical shifts were expressed in d values (ppm). Silica gel (200–300 mesh) (Qingdao, PR China), Sephadex LH-20 (Pharmacia Biotec AB), and RP-18 (Merck, Darmstadt, Germany) were used for column chromatographies (CC). Preparative high-performance liquid chromatography (HPLC) was carried on an Agilent chromatograph 1260 equipped with a G1310B pump and a G1365D UV detector using a C18 reversed-phase column (Cosmosil, 30 mm 250 mm,
5 mm). Chiral HPLC was carried on an Agilent chromatograph 1260 equipped with a G1311C pump and a G1315D UV detector using a Lux Cellulose-2 column (4.6 mm 250 mm, 5 mm, Phenomenex, Torrance, CA). All solvents used in column chromatography were analytical grade (Tianjin Damao Chemical Plant, Tianjin, PR China). 3.2. Plant material The rhizoma of C. chinensis were purchased from Nanjing City, Jiangsu Province of China, in August of 2009. The plant was authenticated by Prof. Guang-Xiong Zhou of the Jinan University. A voucher specimen (No. 2009092001) was deposited in the Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, PR China. 3.3. Extraction and isolation
Fig. 3. X-ray crystal structure of 1.
The air-dried rhizoma of C. chinensis (20 kg) were pulverized and extracted with 90% EtOH under reflux (three times, each 1.5 h). The extract was concentrated, suspended in H2O, and then successively extracted with EtOAc. The EtOAc solution was concentrated to afford a residue (230 g), which was separated by silica gel column chromatography eluted with CHCl3–MeOH (100:0 ! 100:5, v/v) to obtain ten fractions (A–J). Fraction C (20.3 g) was separated by silica gel eluting with petroleum etheracetone (12:1 ! 1:1, v/v) to afford compounds 2 (9.5 mg) and 3 (6.5 mg). Fraction D (16.5 g) was subjected to silica gel and eluted with petroleum ether-acetone (10:1 ! 1:1, v/v) to afford compound 6 (15.5 mg). Fraction F (2.1 g) was purified by Sephadex LH20 column chromatography (2.0 cm 80.0 cm, CHCl3: MeOH, 1:1, 300 ml) to give compounds 1 (12.3 mg), 4 (8.4 mg) and 5 (12.3 mg), respectively. Compound 1 was further separated by HPLC with a Phenomenex Lux Cellulose-2 chiral column (CH3CN:H2O, 40:60, 0.9 mL/min, 230 nm, 25 8C), which led to the isolation of (+)-5-hydroxyl-8-oxyberberine ((+)-1, 3.8 mg,
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L. Wang et al. / Phytochemistry Letters 7 (2014) 89–92
tR = 24.08 min) and ()-5-hydroxyl-8-oxyberberine (()-1, 3.5 mg, tR = 26.29 min). The aqueous layer was subjected to macroporous resin D101 column (8.0 cm 120.0 cm, 3 kg) eluting with H2O–EtOH (100:0, 30:70, 50:50, 30:70, 0:100, each 30 L) to give five corresponding fractions (Fr.1–Fr.5). Fr.2 (3.0 g) was further separated on ODS column (3.5 cm 80.0 cm, 100 g) eluting with a gradient of MeOH–H2O (0:100, 20:80, 40:60, 60:40, 100:0) to yield seven subfractions (Fr.2a–Fr.2e). Fr.2b was purified on Sephadex LH-20 to give compound 7 (22.3 mg). Fr.2c was separated by semi-preparative HPLC using MeOH–H2O (30:70, v/ v) at 3 mL/min to yield 8 (7.0 mg, tR = 8.21 min), 9 (10.2 mg, tR = 11.82 min), and 10 (16.1 mg, tR = 15.26 min), respectively. 3.4. 5-Hydroxyl-8-oxyberberine (1) Yellow needle crystal; UV (CH3OH) lmax (log e) 225.6 (4.04), 340.4 (3.80) nm; IR (KBr) nmax 3441, 2922, 1643, 1595, 1510, 1493, 1276, 1066 cm1; HRESIMS m/z 368.1114 [M+H]+ (calcd for C20H18NO6, 368.1129), 1H and 13C NMR data: see Table 1. (+)-1: yellow needle crystal, mp 310–311 8C; [a]D25 + 5.58 (c 0.66, CH3OH); CD (CH3CN, c = 4.1 104 mol/L) lmax (De): 341.5 (2.5), 220.1 (6.2) nm. ()-1: yellow needle crystal, mp 309– 310 8C; [a]D25 5.98 (c 0.81, CH3OH); CD (CH3CN, c = 4.8 104 mol/L) lmax (De): 342.1 (1.7), 220.3 (5.5) nm. X-ray crystal data for 1: C20H18NO6, MW = 367.35, monoclinic. Space group: P2(1)/n, a = 7.7032(3) A˚, b = 10.6089(4) A˚, c = 19.4070(7) A˚, a = g = 908, b = 92.084(3)8, V = 1584.94(10) A˚3, Z = 4, Dc = 1.539 Mg/m3, F(0 0 0) = 768 and absorption coefficient = 0.961 mm1. Data collection and reduction: crystal size 0.32 mm 0.27 mm 0.22 mm. Absorption correction: Semiempirical from equivalents. Refinement method: full-matrix leastsquares on F2. A total of 8876 reflections were collected (2481 unique, Rint = 0.0370) in the range of 4.56–62.558 of u. The final refinement gave R1 = 0.0631 (wR2 = 0.1430) for 2481 observed reflections [with I > 2s(I)] and 253 variable parameters, and R1 = 0.0649 (wR2 = 0.1439) for all unique reflections. The space group is P2(1)/n, a centrosymmetric space group. Thus the absolute configuration could not be determined. CCDC contains the supplementary crystallographic data for this paper (CCDC945697). These data can be obtained free of charge via http:// www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; email: [email protected]). 3.5. Bioassay for antimicrobial activity Antimicrobial susceptibility testing was performed by Kirby– Bauer (KB) disk diffusion method. Microorganisms included S. aureus (American Type Culture Collection (ATCC) No. 25923 and MRSA1), E. coli (ATCC 25922 and EC1) and Pseudomonas aeruginosa (ATCC 27853 and PA1). Multi-resistant clinical strains MRSA1, EC1 and PA1 were obtained from the Clinical Microbiology Laboratory of the First Affiliated Hospital of Shantou University, Shantou, PR China. Briefly, a suspension of the tested microorganism (0.1 mL of 108 cells/mL) was spread on solid media plates. Filter paper discs (6 mm in diameter) were impregnated with 5 ml of compounds
1–10 (contained 25.6 mg). In case of synergistic effect, 5 ml of each compound (contained 25.6 mg) was added into ceftazidime and cefepime discs, respectively. The plates were incubated at 37 8C for 20 h. The antibacterial activity was assessed by measuring the inhibition zone diameter (mm) around the paper disk. Synergism was considered when combination exhibited enlargement of inhibition zone size by 5 mm (Ahmad and Aqil, 2007). Acknowledgements This work was supported by National Natural Science Foundation of China (Nos. 81273391 and 81273562), Program for Changjiang Scholars and Innovative Research Team in University (IRT0965), Ministry of Science and Technology of China (Nos. 2013BAI11B05 and 2013DFM30080), and China Postdoctoral Science Foundation (No. 201104369). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2013.10.007. References Ahmad, I., Aqil, F., 2007. In vitro efficacy of bioactive extracts of 15 medicinal plants against ESB L-producing multidrug-resistant enteric bacteria. Microbiol. Res. 162, 264–275. Chin, L.W., Cheng, Y.W., Lin, S.S., Lai, Y.Y., Lin, L.Y., Chou, M.Y., Chou, M.C., Yang, C.C., 2010. Anti-herpes simplex virus effects of berberine from Coptidis rhizoma, a major component of a Chinese herbal medicine, Ching-Wei-San. Arch. Virol. 155, 1933–1941. Hwang, J.M., Kuo, H.C., Tseng, T.H., Liu, J.Y., Chu, C.Y., 2006. Berberine induces apoptosis through a mitochondria/caspases pathway in human hepatoma cells. Arch. Toxicol. 80, 62–73. Jung, H.A., Yoon, N.Y., Bae, H.J., Min, B.S., Choi, J.S., 2008. Inhibitory activities of the alkaloids from Coptidis rhizoma against aldose reductase. Arch. Pharm. Res. 31, 1405–1412. Kuo, C.L., Chi, C.W., Liu, T.Y., 2005. Modulation of apoptosis by berberine through inhibition of cyclooxygenase-2 and Mcl-1 expression in oral cancer cells. In Vivo 19, 247–252. Liu, Q.X., Qiu, S.Y., Yu, H.K., X., Y., Y., J., Liang, X.M., 2011. Selective separation of structure-related alkaloids in Rhizoma coptidis with ‘‘click’’ binaphthyl stationary phase and their structural elucidation with liquid chromatography–mass spectrometry. Analyst 136, 4357–4365. Ouyang, S., Wang, L., Zhang, Q.W., Wang, G.C., Wang, Y., Huang, X.J., Zhang, X.Q., Jiang, R.W., Yao, X.S., Che, C.T., Ye, W.C., 2011. Six new monoterpenoid indole alkaloids from the aerial part of Gelsemium elegans. Tetrahedron 67, 4807–4813. Tang, J., Feng, Y.B., Tsao, S.Y., Wang, N., Curtain, R., Wang, Y.W., 2009. Berberine and Coptidis rhizoma as novel antineoplastic agents: a review of traditional use and biomedical investigations. J. Ethnopharmacol. 126, 5–17. Wang, L., Zhang, X.Q., Yin, Z.Q., Wang, Y., Ye, W.C., 2009. Two new amaryllidaceae alkaloids from the bulbs of Lycoris radiata. Chem. Pharm. Bull. 57, 610–611. Wang, W., Zhang, Q.W., Ye, W.C., Wang, Y.T., 2007. Isoquinoline alkaloids from the rhizoma of Coptis chinensis. Chin. J. Nat. Med. 15, 348–350. Zhao, B.X., Wang, Y., Zhang, D.M., Huang, X.J., Bai, L.L., Yan, Y., Chen, J.M., Lu, T.B., Wang, Y.T., Zhang, Q.W., Ye, W.C., 2012. Virosaines A and B, two new birdcageshaped Securinega alkaloids with an unprecedented skeleton from Flueggea virosa. Org. Lett. 14, 3096–3099. Zhao, M., Xian, Y.F., Ip, S.P., Fong, H.H.S., Che, C.T., 2010. A new and weakly antispasmodic protoberberine alkaloid from Rhizoma Coptidis. Phytother. Res. 24, 1414–1416. Zuo, G.Y., Li, Y., Han, J., Wang, G.C., Zhang, Y.L., Bian, Z.Q., 2012. Antibacterial and synergy of berberines with antibacterial agents against clinical multi-drug resistant isolates of methicillin-resistant Staphylococcus aureus (MRSA). Molecules 17, 10322–10330.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
New enantiomeric isoquinoline alkaloids from Coptis chinensis Lei Wang a,b,1, Sheng-Yuan Zhang a,1, Liang Chen a, Xiao-Jun Huang a,b, Qing-Wen Zhang c,**, Ren-Wang Jiang a, Fen Yao d,**, Wen-Cai Ye a,b,* a
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China JNU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research, Jinan University, Guangzhou 510632, PR China c Institute of Chinese Medical Sciences, University of Macau, Macau, PR China d Department of Pharmacology, Medical College, Shantou University, Shantou 515041, PR China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 June 2013 Received in revised form 5 September 2013 Accepted 1 October 2013 Available online 22 October 2013
A pair of new enantiomeric isoquinoline alkaloids, (+)- and ()-5-hydroxyl-8-oxyberberine (1), along with nine known analogs (2–10) were isolated from the rhizoma of Coptis chinensis. Their structures with absolute configurations were elucidated on the basis of spectroscopic methods, X-ray diffraction analysis, and quantum chemical calculation. The antibacterial activities of 1–10 were also evaluated. Berberine (8) displayed synergism against methicillin-resistant Staphylococcus aureus (MRSA) in combination with ceftazidime and cefepiem. ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Ranunculaceae Coptis chinensis Isoquinoline alkaloid
1. Introduction The plant Coptis chinensis (Ranunculaceae) is mainly distributed in western China, such as Sichuan and Shanxi provinces. The rhizome of this plant has been widely used as a traditional Chinese medicine in the treatment of diarrhea, fever and eczema (Tang et al., 2009). Previous phytochemical studies of this plant had led to the isolation of more than thirty alkaloids (Wang et al., 2007; Zhao et al., 2010; Liu et al., 2011), which demonstrated antibacterial, antiviral, anti-inflammatory and antitumor activities (Kuo et al., 2005; Hwang et al., 2006; Tang et al., 2009; Chin et al., 2010). Berberine, a major constituent in C. chinensis, was found to have synergistic effect against methicillin-resistant Staphylococcus aureus (MRSA) in combination with azithromycin and levofloxacin (Zuo et al., 2012). In the course of searching for biologically interesting and structural unique alkaloids from Chinese herbal medicines (Zhao et al., 2012; Ouyang et al., 2011; Wang et al., 2009), a pair of new enantiomeric isoquinoline alkaloids, (+)- and ()-5-hydroxyl-8-oxyberberine [(+)-1 and ()-1], along with nine known alkaloids (2–10) were isolated from the rhizoma of C. chinensis (Fig. 1). In this paper, the chiral separation and structure
* Corresponding author at: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, PR China. Tel.: +86 20 8522 0936; fax: +86 20 8522 1559. ** Corresponding authors. Tel.: +86 20 8522 0936; fax: +86 20 8522 1559. E-mail addresses: [email protected] (Q.-W. Zhang), [email protected] (F. Yao), [email protected] (W.-C. Ye). 1 These authors contributed equally to this work.
elucidation of 1 were described. The antibacterial activities and synergism of 1–10 with ceftazidime and cefepiem were also evaluated. 2. Results and discussion Compound (1) was obtained as yellow needle crystal. The HRESIMS spectrum of 1 exhibited a quasi-molecular ion at m/z 368.1114 [M+H]+ (calcd for C20H18NO6, 368.1129), consistent with the molecular formula C20H17NO6. The UV spectrum showed absorption maxima at 225 and 340 nm. The IR spectrum of 1 suggested the presence of hydroxyl (3441 cm1), carbonyl (1643 cm1) and aromatic ring (1595, 1510, 1493 cm1). The 1H NMR spectrum of 1 showed signals for a pair of ortho-coupled aromatic protons [dH 7.52 (1H, d, J = 8.7 Hz), 7.41 (1H, d, J = 8.7 Hz)], three isolated aromatic protons [dH 7.50, 7.13, 7.01 (each 1H, s)], a methylenedioxyl group [dH 6.07 (2H, s)], three alkyl protons [dH 4.74 (1H, m), 4.26 (1H, dd, J = 13.6, 6.0 Hz), and 3.96 (1H, dd, J = 13.6, 3.4 Hz)], and two methoxyl groups [dH 3.87 and 3.78 (each 3H, s)]. The 13C NMR and DEPT spectra displayed twenty signals including two methyls, two methylenes, six methines, and ten quaternary carbons. The above NMR data suggested that 1 was a protoberberine type alkaloid. With the aid of 1H–1H COSY, HSQC, HMBC and ROESY experiments, all the 1H and 13C NMR signals of 1 could be assigned as shown in Table 1. The comparison of the NMR data of 1 with those of the known compound 8-oxyberberine (2) revealed that their signals were similar, except for the appearance of the signals for a oxygenated methenyl [dH 4.74 (1H, m); dC 64.1] in 1. Thus, 1 was proposed to be
1874-3900/$ – see front matter ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2013.10.007
L. Wang et al. / Phytochemistry Letters 7 (2014) 89–92
90
Fig. 1. Chemical structures of compounds 1–10.
a 5-hydroxyl-substituted derivative of 2, which was supported by the HMBC correlations between H-4 (dH 7.01)/H-6a (dH 4.26) and C-5 (dc 64.1), as well as the ROESY correlation between H-5 (dH 4.74) and H-4 (dH 7.01) (Fig. 2). Therefore, 1 was identified as 5hydroxyl-8-oxyberberine.
Table 1 1 H and 13C NMR data for compound 1 (DMSO-d6, J in Hz). No.
dH
dC
1 2 3 4 4a 5 6
7.50 (1H, s) – – 7.01 (1H, s) – 4. 74 (1H, m) 4.26 (1H, dd, 13.6, 6.0, H-6a) 3.96 (1H, dd, 13.6, 3.4, H-6b) – – – – 7.52 (1H, d, 8.7) 7.41 (1H, d, 8.7) – 7.13 (1H, s) – – 6.07 (2H, br s) 3.78 (3H, s) 3.87 (3H, s) 5.59 (1H, d, 4.5)
104.5 148.1 147.8 106.8 132.3 64.1 45.4
8 8a 9 10 11 12 12a 13 13a 13b –OCH2O– 9-OCH3 10-OCH3 5-OH
159.2 118.6 148.3 151.0 119.1 122.7 131.8 101.0 134.5 122.5 101.5 60.8 56.3 –
The chemical structure of 1 was further confirmed by an X-ray diffraction analysis (Fig. 3). The space group P21/n of the crystal together with the lack of optical activity indicated that 1 was a racemate. Chiral separation of 1 by HPLC led to the isolation of two enantiomers [(+)-1 and ()-1]. To determine the absolute configuration of each enantiomer, a comparison between the experimental and calculated CD spectra using the time-dependent DFT method was performed. The measured CD spectrum of (+)-1 showed positive Cotton effect at 341.5 nm (De + 2.5) and negative one at 220.1 nm (De 6.2), which was consistent with the calculated CD spectrum for S isomer (Fig. 4). Whereas, the CD spectrum of ()-1 displayed reverse Cotton effects at the similar wavelengths, which corresponded to R isomer (Fig. 4). These results revealed that (+)-1 and ()-1 corresponded to (5S)-1 and (5R)-1, respectively. The nine known compounds were identified as 8-oxyberberine (2), 8-oxocoptisine (3), tetrahydroberberine (4), berbithine (5), 6([1,3] dioxolo[4,5-g] isoquinoline-5-carbonyl)-2,3-dimethoxybenzoic acid methyl ester (6), palmatine (7), berberine (8), epiberberine (9), and coptisine (10) by comparison of their NMR and MS data with those reported in the literatures (Wang et al., 2007; Jung et al., 2008; Zhao et al., 2010; Liu et al., 2011). All isolated compounds (1–10) were tested for their antibacterial effects. No inhibition zone was found with compounds 1–10 (25.6 mg per paper disc), indicating all compounds have no direct effect against S. aureus, Escherichia coli and Pseudomonas aeruginosa. Inhibition zone of ceftazidime against multi-resistanct clinical strains EC1 and PA1 were both zero, while inhibition zone of cefepime against EC1 and PA1 were 11 mm and 8 mm,
L. Wang et al. / Phytochemistry Letters 7 (2014) 89–92
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Fig. 2. Key HMBC and ROESY correlations of 1.
respectively. When combined with compounds 1–10 respectively, the diameters of inhibition zones remained unchanged, indicating that these compounds displayed no synergism effect with ceftazidime and cefepime against EC1 and PA1. However, in the anti-MRSA1 test, the inhibition zone diameter of ceftazidime and cefepime discs enlarged from 0 to 9 mm when combined with compound 8. These results revealed the synergism of compound 8 against MRSA in combination with ceftazidime and cefepime. 3. Experimental 3.1. General experimental procedures Fig. 4. Calculated and experimental CD spectra of (+)-1 and ()-1.
Optical rotations were measured by a Jasco P-1020 digital polarimeter at room temperature. UV spectra were recorded on a Jasco-V-550 UV-vis spectrophotometer. IR spectra were conducted on a Jasco FT/IR-480 plus Fourier transform infrared spectrometer. HRESIMS were performed on an Agilent 6210 ESI/TOF mass spectrometer. 1D (1H, 13C, and DEPT) and 2D (1H–1H COSY, HSQC, HMBC and ROESY) NMR spectra were recorded on a Bruker AV-300 spectrometer with TMS as internal standard, and chemical shifts were expressed in d values (ppm). Silica gel (200–300 mesh) (Qingdao, PR China), Sephadex LH-20 (Pharmacia Biotec AB), and RP-18 (Merck, Darmstadt, Germany) were used for column chromatographies (CC). Preparative high-performance liquid chromatography (HPLC) was carried on an Agilent chromatograph 1260 equipped with a G1310B pump and a G1365D UV detector using a C18 reversed-phase column (Cosmosil, 30 mm 250 mm,
5 mm). Chiral HPLC was carried on an Agilent chromatograph 1260 equipped with a G1311C pump and a G1315D UV detector using a Lux Cellulose-2 column (4.6 mm 250 mm, 5 mm, Phenomenex, Torrance, CA). All solvents used in column chromatography were analytical grade (Tianjin Damao Chemical Plant, Tianjin, PR China). 3.2. Plant material The rhizoma of C. chinensis were purchased from Nanjing City, Jiangsu Province of China, in August of 2009. The plant was authenticated by Prof. Guang-Xiong Zhou of the Jinan University. A voucher specimen (No. 2009092001) was deposited in the Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, PR China. 3.3. Extraction and isolation
Fig. 3. X-ray crystal structure of 1.
The air-dried rhizoma of C. chinensis (20 kg) were pulverized and extracted with 90% EtOH under reflux (three times, each 1.5 h). The extract was concentrated, suspended in H2O, and then successively extracted with EtOAc. The EtOAc solution was concentrated to afford a residue (230 g), which was separated by silica gel column chromatography eluted with CHCl3–MeOH (100:0 ! 100:5, v/v) to obtain ten fractions (A–J). Fraction C (20.3 g) was separated by silica gel eluting with petroleum etheracetone (12:1 ! 1:1, v/v) to afford compounds 2 (9.5 mg) and 3 (6.5 mg). Fraction D (16.5 g) was subjected to silica gel and eluted with petroleum ether-acetone (10:1 ! 1:1, v/v) to afford compound 6 (15.5 mg). Fraction F (2.1 g) was purified by Sephadex LH20 column chromatography (2.0 cm 80.0 cm, CHCl3: MeOH, 1:1, 300 ml) to give compounds 1 (12.3 mg), 4 (8.4 mg) and 5 (12.3 mg), respectively. Compound 1 was further separated by HPLC with a Phenomenex Lux Cellulose-2 chiral column (CH3CN:H2O, 40:60, 0.9 mL/min, 230 nm, 25 8C), which led to the isolation of (+)-5-hydroxyl-8-oxyberberine ((+)-1, 3.8 mg,
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tR = 24.08 min) and ()-5-hydroxyl-8-oxyberberine (()-1, 3.5 mg, tR = 26.29 min). The aqueous layer was subjected to macroporous resin D101 column (8.0 cm 120.0 cm, 3 kg) eluting with H2O–EtOH (100:0, 30:70, 50:50, 30:70, 0:100, each 30 L) to give five corresponding fractions (Fr.1–Fr.5). Fr.2 (3.0 g) was further separated on ODS column (3.5 cm 80.0 cm, 100 g) eluting with a gradient of MeOH–H2O (0:100, 20:80, 40:60, 60:40, 100:0) to yield seven subfractions (Fr.2a–Fr.2e). Fr.2b was purified on Sephadex LH-20 to give compound 7 (22.3 mg). Fr.2c was separated by semi-preparative HPLC using MeOH–H2O (30:70, v/ v) at 3 mL/min to yield 8 (7.0 mg, tR = 8.21 min), 9 (10.2 mg, tR = 11.82 min), and 10 (16.1 mg, tR = 15.26 min), respectively. 3.4. 5-Hydroxyl-8-oxyberberine (1) Yellow needle crystal; UV (CH3OH) lmax (log e) 225.6 (4.04), 340.4 (3.80) nm; IR (KBr) nmax 3441, 2922, 1643, 1595, 1510, 1493, 1276, 1066 cm1; HRESIMS m/z 368.1114 [M+H]+ (calcd for C20H18NO6, 368.1129), 1H and 13C NMR data: see Table 1. (+)-1: yellow needle crystal, mp 310–311 8C; [a]D25 + 5.58 (c 0.66, CH3OH); CD (CH3CN, c = 4.1 104 mol/L) lmax (De): 341.5 (2.5), 220.1 (6.2) nm. ()-1: yellow needle crystal, mp 309– 310 8C; [a]D25 5.98 (c 0.81, CH3OH); CD (CH3CN, c = 4.8 104 mol/L) lmax (De): 342.1 (1.7), 220.3 (5.5) nm. X-ray crystal data for 1: C20H18NO6, MW = 367.35, monoclinic. Space group: P2(1)/n, a = 7.7032(3) A˚, b = 10.6089(4) A˚, c = 19.4070(7) A˚, a = g = 908, b = 92.084(3)8, V = 1584.94(10) A˚3, Z = 4, Dc = 1.539 Mg/m3, F(0 0 0) = 768 and absorption coefficient = 0.961 mm1. Data collection and reduction: crystal size 0.32 mm 0.27 mm 0.22 mm. Absorption correction: Semiempirical from equivalents. Refinement method: full-matrix leastsquares on F2. A total of 8876 reflections were collected (2481 unique, Rint = 0.0370) in the range of 4.56–62.558 of u. The final refinement gave R1 = 0.0631 (wR2 = 0.1430) for 2481 observed reflections [with I > 2s(I)] and 253 variable parameters, and R1 = 0.0649 (wR2 = 0.1439) for all unique reflections. The space group is P2(1)/n, a centrosymmetric space group. Thus the absolute configuration could not be determined. CCDC contains the supplementary crystallographic data for this paper (CCDC945697). These data can be obtained free of charge via http:// www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; email: [email protected]). 3.5. Bioassay for antimicrobial activity Antimicrobial susceptibility testing was performed by Kirby– Bauer (KB) disk diffusion method. Microorganisms included S. aureus (American Type Culture Collection (ATCC) No. 25923 and MRSA1), E. coli (ATCC 25922 and EC1) and Pseudomonas aeruginosa (ATCC 27853 and PA1). Multi-resistant clinical strains MRSA1, EC1 and PA1 were obtained from the Clinical Microbiology Laboratory of the First Affiliated Hospital of Shantou University, Shantou, PR China. Briefly, a suspension of the tested microorganism (0.1 mL of 108 cells/mL) was spread on solid media plates. Filter paper discs (6 mm in diameter) were impregnated with 5 ml of compounds
1–10 (contained 25.6 mg). In case of synergistic effect, 5 ml of each compound (contained 25.6 mg) was added into ceftazidime and cefepime discs, respectively. The plates were incubated at 37 8C for 20 h. The antibacterial activity was assessed by measuring the inhibition zone diameter (mm) around the paper disk. Synergism was considered when combination exhibited enlargement of inhibition zone size by 5 mm (Ahmad and Aqil, 2007). Acknowledgements This work was supported by National Natural Science Foundation of China (Nos. 81273391 and 81273562), Program for Changjiang Scholars and Innovative Research Team in University (IRT0965), Ministry of Science and Technology of China (Nos. 2013BAI11B05 and 2013DFM30080), and China Postdoctoral Science Foundation (No. 201104369). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2013.10.007. References Ahmad, I., Aqil, F., 2007. In vitro efficacy of bioactive extracts of 15 medicinal plants against ESB L-producing multidrug-resistant enteric bacteria. Microbiol. Res. 162, 264–275. Chin, L.W., Cheng, Y.W., Lin, S.S., Lai, Y.Y., Lin, L.Y., Chou, M.Y., Chou, M.C., Yang, C.C., 2010. Anti-herpes simplex virus effects of berberine from Coptidis rhizoma, a major component of a Chinese herbal medicine, Ching-Wei-San. Arch. Virol. 155, 1933–1941. Hwang, J.M., Kuo, H.C., Tseng, T.H., Liu, J.Y., Chu, C.Y., 2006. Berberine induces apoptosis through a mitochondria/caspases pathway in human hepatoma cells. Arch. Toxicol. 80, 62–73. Jung, H.A., Yoon, N.Y., Bae, H.J., Min, B.S., Choi, J.S., 2008. Inhibitory activities of the alkaloids from Coptidis rhizoma against aldose reductase. Arch. Pharm. Res. 31, 1405–1412. Kuo, C.L., Chi, C.W., Liu, T.Y., 2005. Modulation of apoptosis by berberine through inhibition of cyclooxygenase-2 and Mcl-1 expression in oral cancer cells. In Vivo 19, 247–252. Liu, Q.X., Qiu, S.Y., Yu, H.K., X., Y., Y., J., Liang, X.M., 2011. Selective separation of structure-related alkaloids in Rhizoma coptidis with ‘‘click’’ binaphthyl stationary phase and their structural elucidation with liquid chromatography–mass spectrometry. Analyst 136, 4357–4365. Ouyang, S., Wang, L., Zhang, Q.W., Wang, G.C., Wang, Y., Huang, X.J., Zhang, X.Q., Jiang, R.W., Yao, X.S., Che, C.T., Ye, W.C., 2011. Six new monoterpenoid indole alkaloids from the aerial part of Gelsemium elegans. Tetrahedron 67, 4807–4813. Tang, J., Feng, Y.B., Tsao, S.Y., Wang, N., Curtain, R., Wang, Y.W., 2009. Berberine and Coptidis rhizoma as novel antineoplastic agents: a review of traditional use and biomedical investigations. J. Ethnopharmacol. 126, 5–17. Wang, L., Zhang, X.Q., Yin, Z.Q., Wang, Y., Ye, W.C., 2009. Two new amaryllidaceae alkaloids from the bulbs of Lycoris radiata. Chem. Pharm. Bull. 57, 610–611. Wang, W., Zhang, Q.W., Ye, W.C., Wang, Y.T., 2007. Isoquinoline alkaloids from the rhizoma of Coptis chinensis. Chin. J. Nat. Med. 15, 348–350. Zhao, B.X., Wang, Y., Zhang, D.M., Huang, X.J., Bai, L.L., Yan, Y., Chen, J.M., Lu, T.B., Wang, Y.T., Zhang, Q.W., Ye, W.C., 2012. Virosaines A and B, two new birdcageshaped Securinega alkaloids with an unprecedented skeleton from Flueggea virosa. Org. Lett. 14, 3096–3099. Zhao, M., Xian, Y.F., Ip, S.P., Fong, H.H.S., Che, C.T., 2010. A new and weakly antispasmodic protoberberine alkaloid from Rhizoma Coptidis. Phytother. Res. 24, 1414–1416. Zuo, G.Y., Li, Y., Han, J., Wang, G.C., Zhang, Y.L., Bian, Z.Q., 2012. Antibacterial and synergy of berberines with antibacterial agents against clinical multi-drug resistant isolates of methicillin-resistant Staphylococcus aureus (MRSA). Molecules 17, 10322–10330.