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A new anthraquinone from Morinda elliptica Ridl. a
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Kiedparinya Loonjang , David Duangjinda , Souwalak cd
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Phongpaichit , Nongyao Sawangjaroen , Suthida Rattanaburi & Wilawan Mahabusarakam
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Department of Chemistry, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand b
Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand c
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Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand d
Faculty of Science, Natural Products Research Center of Excellence, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Published online: 17 Feb 2015.
To cite this article: Kiedparinya Loonjang, David Duangjinda, Souwalak Phongpaichit, Nongyao Sawangjaroen, Suthida Rattanaburi & Wilawan Mahabusarakam (2015): A new anthraquinone from Morinda elliptica Ridl., Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1009062 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1009062
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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1009062
A new anthraquinone from Morinda elliptica Ridl.
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Kiedparinya Loonjanga, David Duangjindab, Souwalak Phongpaichitcd, Nongyao Sawangjaroencd, Suthida Rattanaburib and Wilawan Mahabusarakambd* a Department of Chemistry, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand; bDepartment of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand; cDepartment of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand; dFaculty of Science, Natural Products Research Center of Excellence, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
(Received 16 September 2014; final version received 12 January 2015)
A new anthraquinone, morinquinone, together with 18 known anthraquinones were isolated from the stems of Morinda elliptica Ridl. Their structures were elucidated on the basis of spectroscopic data. They each showed weak inhibitory activity against a susceptible strain of Staphylococcus aureus and a methicillin-resistant S. aureus. Damnacanthal was effective against Microsporum gypseum (MIC 1 mg/mL). Lucidin was active against Entamoeba histolytica (MIC 31.25 mg/mL) and Giardia intestinalis (MIC 7.8 mg/mL). Keywords: Morinda elliptica; Rubiaceae; anthraquinones; antimicrobial activity
1. Introduction Infectious diseases caused by antibiotic-resistant microbes pose an increasing threat to public health worldwide. This includes bacteria (Doyle et al. 2011; Van der Bij et al. 2011), fungi (Bueid et al. 2010; Pfaller et al. 2010) and protozoa (Abboud et al. 2001; Petri 2003). There is an urgent need to find novel sources of antimicrobial drugs or novel ways to treat diseases. Natural products from plants are still regarded as being the most important sources for discovering new drugs. Morinda elliptica Ridl. (Rubiaceae), locally called Yaw Pa, has been traditionally used for the treatment of several health problems, including loss of appetite, headaches, cholera, diarrhoea, fever and hemorrhoids. We have screened the ability of a crude extract from its stems for inhibition of pathogenic bacteria, fungi and protozoa. It displayed inhibition of the growth of Staphylococcus aureus (MIC 64 –128 mg/mL), Microsporum gypseum (MIC 32 –128 mg/mL) and Entamoeba histolytica (MIC 19 – 25 mg/mL). These results led us to further investigate and identify its antimicrobial substances (Figure 1). 2. Results and discussion Stems of M. elliptica were extracted with hexane and methylene chloride, successively. These extracts were fractionated by chromatographic methods to provide 19 pure anthraquinones. They
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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Figure 1. The structure of compounds from the stems of Morinda elliptica Ridl.
were identified as 1-hydroxy-2-methylanthraquinone (1), 5-hydroxy-2,2-dimethyl-4H-anthra [2,3-d ][1,3]dioxine-6,11-dione (2), tectoquinone (3), damnacanthal (4), nordamnacanthal (5), morinquinone (6), morindone-5-methyl ether (7), 1,7-dihydroxy-6-methoxy-2-methylanthraquinone (8), 1,3-dihydroxy-2-methoxyanthraquinone (9), alizarin-1-methyl ether (10), 1,5,15trimethylmorindol (11), soranjidiol (12), 1,6-dihydroxy-5-methoxy-2-methoxymethylanthraquinone (13), digiferruginol (14), 2-hydroxyanthraquinone (15), lucidin-v-methyl ether (16), lucidin (17), rubiadin (18) and rubiadin-1-methyl ether (19). Morinquinone (6) was a new compound. The structures of all compounds were identified by analysis of their spectroscopic data. The elucidation of the structures of the known compounds was also confirmed by comparing the spectroscopic data to the previously reported literature (Balakrishna et al. 1960; Burnett & Thomson 1968; Brisson & Brassard 1981; Inoue et al. 1981; Chang & Lee 1984; Chang & Lee 1985; Koumaglo et al. 1992; Sang et al. 2001; Takashima et al. 2007; Feng et al. 2012). Morinquinone (6) was a yellow solid, m.p. 198 –1998C. The molecular formula of C18H12O4 was determined by HR-MS ([Mþ] m/z 292.0785). The UV spectrum showed maximum absorption peaks at 242, 278 and 407 nm. The IR spectrum showed the stretching of a hydroxyl group at 3447 cm21 and a carbonyl group at 1671 and 1630 cm21. The 13C NMR spectrum showed the resonances of three carbonyl carbons (d 198.6, C-30 ; d 189.0, C-9; d 181.9, C-10), eight methine carbons (d 119.2, C-4; d 127.1, C-8; d 127.5, C-5; d 130.5, C-20 , d 134.4, C-3; d 135.0, C-6 and C-7), five quaternary carbons (d 116.4, C-9a; d 129.7, C-2; d 132.9, C-10a; d 133.5, C-8a; d 134.0, C-10a) and a methyl carbon (d 27.6, 30 -CH3). The 1H NMR spectrum exhibited the resonances of a chelated hydroxyl proton (d 13.45, 1-OH), ortho aromatic protons (d 7.94, d, H-3; d 7.88, d, H-4; J ¼ 8.0 Hz) and an AA0 BB0 type benzene ring (d 8.35, dd, J ¼ 8.0, 2.0 Hz, H-8; d 8.32, dd, J ¼ 8.0, 2.0 Hz, H-5; d 7.85, m, H-6, H-7). Proton H-4 showed HMBC correlation to C-10 (d 181.9), it was then placed peri to the carbonyl carbon C-10. Due to the downfield chemical shift values, the resonances at d 8.32 and d 8.35 were assigned for aromatic protons peri to carbonyl carbons. Unfortunately, the HMBC data could not specify which value belonged to H-5 or H-8. However, most of the previous reports for unsubstituted anthraquinones at C-ring such as 1,3-dihydroxy-2-methoxyanthraquinone, alizarin-1-methyl ether (Burnett & Thomson 1968), rubiadin (Inoue et al. 1981), damnacanthal (Koumaglo et al. 1992), 2-formyl-1hydroxyanthraquinone (Ismail et al. 1997), morindoquinone (Favre-Godal et al. 2014) reported that H-8 resonated at lower field than H-5. Therefore, the chemical shifts of d 8.35 and d 8.32 were tentatively assumed for H-8 and H-5, respectively. Nevertheless, some report on anthraquinone such as 1,2-diacetoxy-9,10-anthraquinone (Danielsen et al. 1995) showed inverse data.
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The side chain ZCHvCHCOCH3 was proposed from the resonances of two doublet signals of trans-olefinic protons (J ¼ 16.5 Hz) at d 7.92 (H-10 ) and at d 6.97 (H-20 ), and a singlet of acyl methyl protons at d 2.45 together with the HMBC correlation of H-10 to C-30 (d 198.8). This group was placed at C-2 according to the 3J correlation of H-10 to C-1 (d 161.4), C-3 (d 134.4) and of H-20 to C-2 (d 129.7). Therefore, 1-hydroxy-2-[(E)-10 -buten-30 -one]anthraquinone was proposed for morinquinone, a new compound. Some of the compounds from M. elliptica were tested against pathogenic bacteria, fungi and protozoa. Compounds 4, 16, 11, 17, 5, 9 and 10 showed antifungal activity against M. gyseum with MIC values ranged from 1 to 200 mg/mL, whereas no activity was shown against yeasts. Damnacanthal (4) exhibited the best antifungal activity (MIC 1 mg/mL), which was comparable with the standard antifungal drug miconazole (MIC 2 mg/mL). Previous reports of compound (4) revealed cytotoxic and immunomodulatory effects and a potential for cancer treatment (Noorjahan et al. 2010) and also a highly potent, selective inhibitor of p56lck tyrosine kinase activity (Faltynek et al. 1995). In this study, we found that this compound possessed a strong antifungal activity. Compounds 1, 6, 9, 10, 16 and 17 exhibited weak antibacterial activity against both strains of S. aureus with MIC values of 50–200 mg/mL, whereas no activity against Gram-negative bacteria. Only compounds 2, 17 and 19 were active against both E. histolytica and Giardia intestinalis (MIC 7.8–125 mg/mL). Notably, compound 17 showed the lowest MIC values (MIC ¼ 31.25 and 7.80 mg/mL, respectively) but were less active than the standard metronidazole (MIC ¼ 2.0 mg/ mL). It was interesting to note that compound 17 was active against all microorganisms tested, whereas 9, 10 and 16 exhibited both antibacterial and antifungal activity. 3. Experimental 3.1. General experimental procedures Melting points were recorded in 8C and were determined on a digital Electrothermal 9100 Melting Point Apparatus (Electrothermal, UK). The ultraviolet spectra were measured with a UV-160A spectrophotometer (Shimadzu, Kyoto, Japan). Principle bands (lmax) were recorded as wavelengths (nm) and log 1 in a methanol solution. Infrared spectra were obtained on a FTS165 FT-IR spectrophotometer (Perkin-Elmer, Shelton, USA) and were recorded in wave number (cm21). 1H and 13C-Nuclear magnetic resonance spectra were recorded on an FT-NMR Bruker Ultra ShieldTM 300 MHz (Bruker, Rheinstetten, Germany) or Varian UNITY INOVA spectrometer 500 MHz (Varian, Polo Alto, CA, USA). The high-resolution mass spectra were recorded on a MAT 95 XL (Thermo Finnigan, Bremen, Germany). Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60H (Merck, Darmstadt, Germany) and silica gel 60 (Merck, Darmstadt, Germany), respectively. Precoated plates of silica gel 60 GF254 were used for TLC analysis. 3.2. Plant material The stems of M. elliptica Ridl. were collected from Pattani province in the southern part of Thailand. Identification was made by Dr. Kitichate Sridith, Department of Biology, Faculty of Science, Prince of Songkla University. A voucher specimen (Herbarium No. 0012286) has been deposited in the Herbarium, Department of Biology, Faculty of Science, Prince of Songkla University, Thailand. 3.3. Extraction and isolation Chopped-dried stems of M. elliptica (5.5 kg) were immersed at room temperature in hexane (7 days) and CH2Cl2 (7 days), successively. After evaporation of the solvents, the brown solid of
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the hexane extract (6.3 g) and the viscous liquid of the CH2Cl2 extract (39.0 g) were obtained. The hexane extract (6.3 g) was subjected to QCC using silica gel as the stationary phase and eluted with hexane, hexane-CH2Cl2, CH2Cl2 and CH2Cl2-Me2CO. On the basis of their TLC characteristics, the collected fractions with the same major components were combined to give 10 fractions. Fraction 1 was rechromatographed on CC and eluted with CH2Cl2-hexane (1:9), resulting in yellow solids of 1 (19.8 mg) and 2 (27.5 mg). Fraction 2 was rechromatographed on CC and eluted with CH2Cl2-hexane (1:9) to afford 3 (6.0 mg) as a yellow solid. The CH2Cl2 extract (39.0 g) was subjected to a QCC using silica gel 60H as the stationary phase and eluted with hexane-CH2Cl2, CH2Cl2-MeOH. The collected fractions were combined on the basis of their TLC characteristics to give 21 fractions. Fraction 4 (2.7 g) was dissolved in hexane and then CH2Cl2 to give a hexane soluble and CH2Cl2 insoluble fractions. The soluble fraction was chromatographed on CC and eluted with CH2Cl2-hexane (3:7) to give a yellow solid 4 (250.0 mg) and an orange solid 5 (125.0 mg). Fraction 5 (1.2 g) was dissolved in hexane to give hexane soluble and insoluble fractions. Further chromatograph of the insoluble fraction by eluting with CH2Cl2-hexane (4:6) gave yellow solids 6 (34.0 mg) and 7 (20.0 mg). Fraction 7 (340.2 mg) was dissolved in hexane to give soluble and insoluble fractions. Each fraction was chromatographed and eluted with CH2Cl2-hexane (1:1) to afford yellow solids 8 (7.3 mg) and 9 (39.0 mg), and a brown solid 10 (22.4 mg). Fraction 8 (2.2 g) was dissolved in hexane to give soluble and insoluble fractions. The soluble fraction was chromatographed and eluted with CH2Cl2-hexane (8:2) to afford a yellow solid of 11 (17.5 mg). Fraction 11 (0.4 g) was dissolved in CH2Cl2 to give soluble and insoluble fractions. The soluble fraction was chromatographed and eluted with CH2Cl2-hexane (8:2) to give yellow solids of 12 (10.2 mg), 13 (15.3 mg) and 14 (7.5 mg). The insoluble fraction was chromatographed and eluted with CH2Cl2-hexane (9:1) to give 15 (20.4 mg) as a red viscous liquid. Fraction 19 (0.8 g) was chromatographed on CC and eluted with CH2Cl2-methanol (8:2) to give 16 (5.0 mg), 17 (15.2 mg), 18 (16.6 mg) and 19 (35.7 mg), each as a yellow solid. 3.3.1. Morinquinone (6) Yellow solid, m.p. 198 –1998C. UV (CH3OH) lmax nm (log 1): 242 (4.16), 278 (4.12), 407 (3.57). IR (KBr) n (cm21): 3447, 1671, 1630. EIMS m/z (% rel int.): 293 ([M þ 1] þ, 1), 292 ([M] þ, 4), 277 (7), 252 (1), 250 (39), 249 (100), 222 (1), 221 (4), 220 (2), 194 (1), 193 (4), 166 (2), 165 (11), 139 (2), 110 (2), 105 (4), 96 (2), 82 (4) 61 (9). HR-MS m/z: 292.0785 (calcd. for C18H4O12, 292.0735). 1H NMR spectral data (500 MHz, CDCl3): 13.45 (s, 1-OH), 8.35 (dd, J ¼ 8.0, 2.0 Hz, H-8)a, 8.32 (dd, J ¼ 8.0, 2.0 Hz, H-5)a, 7.94 (d, J ¼ 8.0 Hz, H-3), 7.92 (d, J ¼ 16.5 Hz, H-10 ), 7.88 (d, J ¼ 8.0 Hz, H-4), 7.85 (m, H-6, H-7), 6.97 (d, J ¼ 16.5 Hz, H-20 ), 2.45 (s, 30 -CH3). 13C NMR spectral data (125 MHz, CDCl3): 198.6 (C-30 ), 189.0 (C-9), 181.9 (C10), 161.4 (C-1), 135.7 (C-10 ), 135.0 (C-6, C-7), 134.4 (C-3), 134.0 (C-10a)b, 133.5 (C-8a)b, 132.9 (C-4a), 130.5 (C-20 ), 129.7 (C-2), 127.5 (C-5)a, 127.1 (C-8)a, 119.2 (C-4), 116.4 (C-9a), 27.6 (30 -CH3)a,b. Assignment with the same superscript may be interchanged. 3.4. Antibacterial activity All compounds at concentrations of 200 mg/mL were screened for antibacterial activity against S. aureus ATCC25923 and a clinical isolate of methicillin-resistant S. aureus (MRSA) SK1, Escherichia coli ATCC25922, and Pseudomonas aeruginosa ATCC27853 by the colorimetric microdilution method according to a modification of CLSI M7-A4 (Clinical and Laboratory Standards Institute [CLSI] 2000) Resazurin (0.18%) was used as the indicator of growth. After incubation, a blue or purple colour of the wells indicated inhibition of growth (positive result), whereas a pink colour meant that growth had occurred (negative result). The minimum
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inhibitory concentration (MIC) of active compounds was determined by the same method. The lowest concentration of the compound that inhibited growth (blue or purple colour) was recorded as the MIC. Vancomycin and gentamicin were used as standard antibacterial agents for the positive inhibitory controls. 3.5. Antifungal activity Each pure compound was screened for antifungal activity at a concentration of 200 mg/mL by a modification of the microbroth dilution CLSI M27-A2 (CLSI 2002a) against yeasts (Candida albicans ATCC90028, Cryptococcus neoformans ATCC90112) and a modification of the microbroth dilution CLSI M38-A (CLSI 2002b) against a clinical isolate of M. gypseum. Amphotericin B was used as a positive inhibitory control for yeasts and miconazole for M. gypseum. 3.6. Antiprotozoal assay Cells were harvested by chilling the tube on ice for 15 min to detach the monolayer then centrifuged at 300g for 5 min. The supernatant was decanted, and cells were resuspended in a fresh medium. The numbers of viable cells were calculated using a haemocytometer and 0.4% (w/v) trypan blue. The criteria for viability were motility and dye exclusion. For assays, trophozoites, 2 £ 105 cells/mL, were incubated in the presence of serial twofold dilutions of extract that ranged from 31.25 to 1000 mg/mL. Metronidazole, with concentrations ranged from 0.625 to 20 mg/mL and a complete medium with added DMSO were used as negative and positive controls, respectively. After 24 h of incubation at 378C under anaerobic conditions, the trophozoites from each well were examined and counted using an inverted microscope. The appearance and number of trophozoites were scored from 1 to 4 with 1 showing the most inhibition of growth and 4 showing no inhibition according to Upcroft and Upcroft, and the MIC was recorded (the lowest concentration at which . 90% of the trophozoites rounded up). Each concentration was tested in duplicate and at least three experiments were performed on separate occasions. 4. Conclusion Investigation of the active antimicrobial components from the stems of M. elliptica resulted in the isolation of 19 unsubstituted C-ring anthraquinones including a new anthraquinone (6). Eight of them (2, 3, 8, 9, 11, 13 –15) were first isolated from this plant. For antimicrobial activity, damnacanthal (4) showed strong activity against M. gypseum, whereas lucidin (17) exhibited strong antiprotozoal activity against G. lamblia. Supplementary material Tables S1 and S2 and Figures S1 – S3 relating to this article are available online. References Abboud P, Lemee V, Gargala G, Brasseur P, Ballet JJ, Borsa-Lebas F, Caron F, Favennec L. 2001. Successful treatment of metronidazole- and albendazole-resistant giardiasis with nitazoxanide in a patient with acquired immunodeficiency syndrome. Clin Infect Dis. 32:1792–1794. doi:10.1086/320751. Balakrishna S, Seshadri TR, Venkatarumani B. 1960. Special chemical components of commercial woods and related plant material IX moridonin, a new glycoside of morindone. J Sci Ind Res B. 19:433–436. Brisson C, Brassard P. 1981. Regiospecific reactions of some vinylogous ketene acetals with haloquinones and their regioselective formation by dienolization. J Org Chem. 46:1810–1814. doi:10.1021/jo00322a012.
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Bueid A, Howard SJ, Moore CB, Richardson MD, Harrison E, Bowyer P, Denning DW. 2010. Azole antifungal resistance in Aspergillus fumigatus: 2008 and 2009. J Antimicrob Chemother. 65:2116–2118, doi:10.1093/jac/ dkq279 Burnett AR, Thomson RH. 1968. Anthraquinones in Morinda umbellate L. Phytochemitry. 7:1421–1422. doi:10.1016/ S0031-9422(00)85651-4. Chang P, Lee KH. 1984. Cytotoxic antileukemic anthraquinones from Morida parvifolia. Phytochemistry. 23:1733–1736. doi:10.1016/S0031-9422(00)83480-9. Chang P, Lee KH. 1985. Antitumor agents, 75. Synthesis of cytotoxic anthraquinones digiferruginol and morindaparvinB. J Nat Prod. 48:948–951. doi:10.1021/np50042a011. Clinical and Laboratory Standards Institute. 2000. Reference method for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A4. Pennsylvania, PA: Clinical Laboratory Standards Institute. Clinical and Laboratory Standards Institute. 2002a. Reference method for broth dilution antifungal susceptibility testing of yeasts: approved standard-second edition. CLSI documents M27-A2. Pennsylvania, PA: Clinical Laboratory Standards Institute. Clinical and Laboratory Standards Institute. 2002b. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi: approved standard. CLSI documents M38-A. Pennsylvania, PA: Clinical Laboratory Standards Institute. Danielsen K, Aksnes DW, Francis GW. 1995. The complete assignment of the 1H and 13C chemical shift of 1,2diacetoxy-9,10-anthraquinone. Acta Chem Scand. 49:464–466. doi:10.3891/acta.chem.scand.49-0464. Doyle JS, Buising KL, Thursky KA, Worth LJ, Richards MJ. 2011. Epidemiology of infections acquired in intensive care units. Semin Respir Crit Care Med. 32:115– 138. doi:10.1055/s-0031-1275525. Faltynek CR, Schroeder J, Mauvais P, Miller D, Wang S, Murphy D, Lehr R, Kelley M, Maycock A. 1995. Damnacanthal is a highly potent, selective inhibitor of p56lck tyrosine kinase activity. Biochemistry. 34:12404– 12410. doi:10. 1021/bi00038a038. Favre-Godal Q, Dorsaz S, Queiroz EF, Conan C, Marcourt L, Wardojo BP, Voinesco F, Buchwalder A, Gindro K, Sanglard D, Wolfender JL. 2014. Comprehensive approach for the detection of antifungal compounds using a susceptible strain of Candida albicans and confirmation of in vivo activity with the Galleria mellonella model. Phytochemistry. 105:68–78. doi:10.1016/j.phytochem.2014.06.004. Feng Z, Sujuan W, Sheng L, Chenggen Z, Zhenggang Y, Yang Y, Bo L, Xiuli W, Yongchun Y, Yan L, Jiangong S. 2012. Natural and unnatural anthraquinones isolated from the ethanol extract of the roots of Knoxia valerianoides. Acta Pharm Sin B. 2(3):260–266. doi:10.1016/j.apsb.2012.03.004. Inoue K, Nayeshiro H, Inouye H, Zenk M. 1981. Anthraquinones in cell suspension cultures of Morinda citrifolia. Phytochemistry. 20:1693–1700. doi:10.1016/S0031-9422(00). Ismail NH, Ali AM, Aimi N, Kitajima M, Takayama H, Lajis NH. 1997. Anthraquinones from Morinda elliptica. Phytochemistry. 45:1723–1725. doi:10.1016/S0031-9422(97)00252-5. Koumaglo K, Gbeassor M, Nikabu O, Werner W. 1992. Effects of three compounds extracted from Morinda lucida on Plasmodium falciparum. Planta Med. 58:533– 534. doi:10.1055/s-2006-961543. Noorjahan BMA, Ali AM, Yeap SK, Suhaimi M, Lajis NH, Mashitoh AR, Ho WY, Ismail NH. 2010. Cytotoxicity and immunomodulatory effects of damnacanthal and nordamnacanthal isolated from roots of Morinda elliptica. J Agrobiotech. 1:29–42. Petri WA. 2003. Therapy of intestinal protozoa. Trends Parasitol. 19:523–526. doi:http://dx.doi.org/10.1016/j.pt.2003. 09.003 Pfaller MA, Castanheira M, Messer SA, Moet GJ, Jones RN. 2010. Variation in Candida spp. distribution and antifungal resistance rates among bloodstream infection isolates by patient age: report from the SENTRY Antimicrobial Surveillance Program (2008–2009). Diagn Microbiol Infect Dis. 68:278–283. doi:10.1016/j.diagmicrobio.2010. 06.015 Sang S, Cheng X, Zhu N, Stark RE, Badmaev V, Ghai G, Rosen RT, Ho CT. 2001. Iridoid glycosides from the leaves of Morinda citrifolia. J Agric Food Chem. 49:4478– 4481. doi:10.1021/np010011l. Takashima J, Ikeda Y, Komiyama K, Hayashi M, Kishida A, Ohsaki A. 2007. New constituents from the leaves of Morinda citrifolia. Chem Pharm Bull. 55:343–345. doi:10.1248/cpb.55.343. Van der Bij AK, Van Mansfeld R, Peirano G, Goessens WHF, Sevenn JA, Pitout JDD, Willems R, Van Westreenen M. 2011. First outbreak of VIM-2 metallo-beta-lactamase-producing Pseudomonas aeruginosa in the Netherlands: microbiology, epidemiology and clinical outcomes. Int J Antimicrob Agents. 37:513– 518. doi:10.1016/ j.ijantimicag.2011.02.010
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new anthraquinone from Morinda elliptica Ridl. a
b
Kiedparinya Loonjang , David Duangjinda , Souwalak cd
cd
b
Phongpaichit , Nongyao Sawangjaroen , Suthida Rattanaburi & Wilawan Mahabusarakam
bd
a
Department of Chemistry, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand b
Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand c
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Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand d
Faculty of Science, Natural Products Research Center of Excellence, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Published online: 17 Feb 2015.
To cite this article: Kiedparinya Loonjang, David Duangjinda, Souwalak Phongpaichit, Nongyao Sawangjaroen, Suthida Rattanaburi & Wilawan Mahabusarakam (2015): A new anthraquinone from Morinda elliptica Ridl., Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1009062 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1009062
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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1009062
A new anthraquinone from Morinda elliptica Ridl.
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Kiedparinya Loonjanga, David Duangjindab, Souwalak Phongpaichitcd, Nongyao Sawangjaroencd, Suthida Rattanaburib and Wilawan Mahabusarakambd* a Department of Chemistry, Faculty of Science and Technology, Prince of Songkla University, Pattani 94000, Thailand; bDepartment of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand; cDepartment of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand; dFaculty of Science, Natural Products Research Center of Excellence, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
(Received 16 September 2014; final version received 12 January 2015)
A new anthraquinone, morinquinone, together with 18 known anthraquinones were isolated from the stems of Morinda elliptica Ridl. Their structures were elucidated on the basis of spectroscopic data. They each showed weak inhibitory activity against a susceptible strain of Staphylococcus aureus and a methicillin-resistant S. aureus. Damnacanthal was effective against Microsporum gypseum (MIC 1 mg/mL). Lucidin was active against Entamoeba histolytica (MIC 31.25 mg/mL) and Giardia intestinalis (MIC 7.8 mg/mL). Keywords: Morinda elliptica; Rubiaceae; anthraquinones; antimicrobial activity
1. Introduction Infectious diseases caused by antibiotic-resistant microbes pose an increasing threat to public health worldwide. This includes bacteria (Doyle et al. 2011; Van der Bij et al. 2011), fungi (Bueid et al. 2010; Pfaller et al. 2010) and protozoa (Abboud et al. 2001; Petri 2003). There is an urgent need to find novel sources of antimicrobial drugs or novel ways to treat diseases. Natural products from plants are still regarded as being the most important sources for discovering new drugs. Morinda elliptica Ridl. (Rubiaceae), locally called Yaw Pa, has been traditionally used for the treatment of several health problems, including loss of appetite, headaches, cholera, diarrhoea, fever and hemorrhoids. We have screened the ability of a crude extract from its stems for inhibition of pathogenic bacteria, fungi and protozoa. It displayed inhibition of the growth of Staphylococcus aureus (MIC 64 –128 mg/mL), Microsporum gypseum (MIC 32 –128 mg/mL) and Entamoeba histolytica (MIC 19 – 25 mg/mL). These results led us to further investigate and identify its antimicrobial substances (Figure 1). 2. Results and discussion Stems of M. elliptica were extracted with hexane and methylene chloride, successively. These extracts were fractionated by chromatographic methods to provide 19 pure anthraquinones. They
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
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Figure 1. The structure of compounds from the stems of Morinda elliptica Ridl.
were identified as 1-hydroxy-2-methylanthraquinone (1), 5-hydroxy-2,2-dimethyl-4H-anthra [2,3-d ][1,3]dioxine-6,11-dione (2), tectoquinone (3), damnacanthal (4), nordamnacanthal (5), morinquinone (6), morindone-5-methyl ether (7), 1,7-dihydroxy-6-methoxy-2-methylanthraquinone (8), 1,3-dihydroxy-2-methoxyanthraquinone (9), alizarin-1-methyl ether (10), 1,5,15trimethylmorindol (11), soranjidiol (12), 1,6-dihydroxy-5-methoxy-2-methoxymethylanthraquinone (13), digiferruginol (14), 2-hydroxyanthraquinone (15), lucidin-v-methyl ether (16), lucidin (17), rubiadin (18) and rubiadin-1-methyl ether (19). Morinquinone (6) was a new compound. The structures of all compounds were identified by analysis of their spectroscopic data. The elucidation of the structures of the known compounds was also confirmed by comparing the spectroscopic data to the previously reported literature (Balakrishna et al. 1960; Burnett & Thomson 1968; Brisson & Brassard 1981; Inoue et al. 1981; Chang & Lee 1984; Chang & Lee 1985; Koumaglo et al. 1992; Sang et al. 2001; Takashima et al. 2007; Feng et al. 2012). Morinquinone (6) was a yellow solid, m.p. 198 –1998C. The molecular formula of C18H12O4 was determined by HR-MS ([Mþ] m/z 292.0785). The UV spectrum showed maximum absorption peaks at 242, 278 and 407 nm. The IR spectrum showed the stretching of a hydroxyl group at 3447 cm21 and a carbonyl group at 1671 and 1630 cm21. The 13C NMR spectrum showed the resonances of three carbonyl carbons (d 198.6, C-30 ; d 189.0, C-9; d 181.9, C-10), eight methine carbons (d 119.2, C-4; d 127.1, C-8; d 127.5, C-5; d 130.5, C-20 , d 134.4, C-3; d 135.0, C-6 and C-7), five quaternary carbons (d 116.4, C-9a; d 129.7, C-2; d 132.9, C-10a; d 133.5, C-8a; d 134.0, C-10a) and a methyl carbon (d 27.6, 30 -CH3). The 1H NMR spectrum exhibited the resonances of a chelated hydroxyl proton (d 13.45, 1-OH), ortho aromatic protons (d 7.94, d, H-3; d 7.88, d, H-4; J ¼ 8.0 Hz) and an AA0 BB0 type benzene ring (d 8.35, dd, J ¼ 8.0, 2.0 Hz, H-8; d 8.32, dd, J ¼ 8.0, 2.0 Hz, H-5; d 7.85, m, H-6, H-7). Proton H-4 showed HMBC correlation to C-10 (d 181.9), it was then placed peri to the carbonyl carbon C-10. Due to the downfield chemical shift values, the resonances at d 8.32 and d 8.35 were assigned for aromatic protons peri to carbonyl carbons. Unfortunately, the HMBC data could not specify which value belonged to H-5 or H-8. However, most of the previous reports for unsubstituted anthraquinones at C-ring such as 1,3-dihydroxy-2-methoxyanthraquinone, alizarin-1-methyl ether (Burnett & Thomson 1968), rubiadin (Inoue et al. 1981), damnacanthal (Koumaglo et al. 1992), 2-formyl-1hydroxyanthraquinone (Ismail et al. 1997), morindoquinone (Favre-Godal et al. 2014) reported that H-8 resonated at lower field than H-5. Therefore, the chemical shifts of d 8.35 and d 8.32 were tentatively assumed for H-8 and H-5, respectively. Nevertheless, some report on anthraquinone such as 1,2-diacetoxy-9,10-anthraquinone (Danielsen et al. 1995) showed inverse data.
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The side chain ZCHvCHCOCH3 was proposed from the resonances of two doublet signals of trans-olefinic protons (J ¼ 16.5 Hz) at d 7.92 (H-10 ) and at d 6.97 (H-20 ), and a singlet of acyl methyl protons at d 2.45 together with the HMBC correlation of H-10 to C-30 (d 198.8). This group was placed at C-2 according to the 3J correlation of H-10 to C-1 (d 161.4), C-3 (d 134.4) and of H-20 to C-2 (d 129.7). Therefore, 1-hydroxy-2-[(E)-10 -buten-30 -one]anthraquinone was proposed for morinquinone, a new compound. Some of the compounds from M. elliptica were tested against pathogenic bacteria, fungi and protozoa. Compounds 4, 16, 11, 17, 5, 9 and 10 showed antifungal activity against M. gyseum with MIC values ranged from 1 to 200 mg/mL, whereas no activity was shown against yeasts. Damnacanthal (4) exhibited the best antifungal activity (MIC 1 mg/mL), which was comparable with the standard antifungal drug miconazole (MIC 2 mg/mL). Previous reports of compound (4) revealed cytotoxic and immunomodulatory effects and a potential for cancer treatment (Noorjahan et al. 2010) and also a highly potent, selective inhibitor of p56lck tyrosine kinase activity (Faltynek et al. 1995). In this study, we found that this compound possessed a strong antifungal activity. Compounds 1, 6, 9, 10, 16 and 17 exhibited weak antibacterial activity against both strains of S. aureus with MIC values of 50–200 mg/mL, whereas no activity against Gram-negative bacteria. Only compounds 2, 17 and 19 were active against both E. histolytica and Giardia intestinalis (MIC 7.8–125 mg/mL). Notably, compound 17 showed the lowest MIC values (MIC ¼ 31.25 and 7.80 mg/mL, respectively) but were less active than the standard metronidazole (MIC ¼ 2.0 mg/ mL). It was interesting to note that compound 17 was active against all microorganisms tested, whereas 9, 10 and 16 exhibited both antibacterial and antifungal activity. 3. Experimental 3.1. General experimental procedures Melting points were recorded in 8C and were determined on a digital Electrothermal 9100 Melting Point Apparatus (Electrothermal, UK). The ultraviolet spectra were measured with a UV-160A spectrophotometer (Shimadzu, Kyoto, Japan). Principle bands (lmax) were recorded as wavelengths (nm) and log 1 in a methanol solution. Infrared spectra were obtained on a FTS165 FT-IR spectrophotometer (Perkin-Elmer, Shelton, USA) and were recorded in wave number (cm21). 1H and 13C-Nuclear magnetic resonance spectra were recorded on an FT-NMR Bruker Ultra ShieldTM 300 MHz (Bruker, Rheinstetten, Germany) or Varian UNITY INOVA spectrometer 500 MHz (Varian, Polo Alto, CA, USA). The high-resolution mass spectra were recorded on a MAT 95 XL (Thermo Finnigan, Bremen, Germany). Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60H (Merck, Darmstadt, Germany) and silica gel 60 (Merck, Darmstadt, Germany), respectively. Precoated plates of silica gel 60 GF254 were used for TLC analysis. 3.2. Plant material The stems of M. elliptica Ridl. were collected from Pattani province in the southern part of Thailand. Identification was made by Dr. Kitichate Sridith, Department of Biology, Faculty of Science, Prince of Songkla University. A voucher specimen (Herbarium No. 0012286) has been deposited in the Herbarium, Department of Biology, Faculty of Science, Prince of Songkla University, Thailand. 3.3. Extraction and isolation Chopped-dried stems of M. elliptica (5.5 kg) were immersed at room temperature in hexane (7 days) and CH2Cl2 (7 days), successively. After evaporation of the solvents, the brown solid of
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the hexane extract (6.3 g) and the viscous liquid of the CH2Cl2 extract (39.0 g) were obtained. The hexane extract (6.3 g) was subjected to QCC using silica gel as the stationary phase and eluted with hexane, hexane-CH2Cl2, CH2Cl2 and CH2Cl2-Me2CO. On the basis of their TLC characteristics, the collected fractions with the same major components were combined to give 10 fractions. Fraction 1 was rechromatographed on CC and eluted with CH2Cl2-hexane (1:9), resulting in yellow solids of 1 (19.8 mg) and 2 (27.5 mg). Fraction 2 was rechromatographed on CC and eluted with CH2Cl2-hexane (1:9) to afford 3 (6.0 mg) as a yellow solid. The CH2Cl2 extract (39.0 g) was subjected to a QCC using silica gel 60H as the stationary phase and eluted with hexane-CH2Cl2, CH2Cl2-MeOH. The collected fractions were combined on the basis of their TLC characteristics to give 21 fractions. Fraction 4 (2.7 g) was dissolved in hexane and then CH2Cl2 to give a hexane soluble and CH2Cl2 insoluble fractions. The soluble fraction was chromatographed on CC and eluted with CH2Cl2-hexane (3:7) to give a yellow solid 4 (250.0 mg) and an orange solid 5 (125.0 mg). Fraction 5 (1.2 g) was dissolved in hexane to give hexane soluble and insoluble fractions. Further chromatograph of the insoluble fraction by eluting with CH2Cl2-hexane (4:6) gave yellow solids 6 (34.0 mg) and 7 (20.0 mg). Fraction 7 (340.2 mg) was dissolved in hexane to give soluble and insoluble fractions. Each fraction was chromatographed and eluted with CH2Cl2-hexane (1:1) to afford yellow solids 8 (7.3 mg) and 9 (39.0 mg), and a brown solid 10 (22.4 mg). Fraction 8 (2.2 g) was dissolved in hexane to give soluble and insoluble fractions. The soluble fraction was chromatographed and eluted with CH2Cl2-hexane (8:2) to afford a yellow solid of 11 (17.5 mg). Fraction 11 (0.4 g) was dissolved in CH2Cl2 to give soluble and insoluble fractions. The soluble fraction was chromatographed and eluted with CH2Cl2-hexane (8:2) to give yellow solids of 12 (10.2 mg), 13 (15.3 mg) and 14 (7.5 mg). The insoluble fraction was chromatographed and eluted with CH2Cl2-hexane (9:1) to give 15 (20.4 mg) as a red viscous liquid. Fraction 19 (0.8 g) was chromatographed on CC and eluted with CH2Cl2-methanol (8:2) to give 16 (5.0 mg), 17 (15.2 mg), 18 (16.6 mg) and 19 (35.7 mg), each as a yellow solid. 3.3.1. Morinquinone (6) Yellow solid, m.p. 198 –1998C. UV (CH3OH) lmax nm (log 1): 242 (4.16), 278 (4.12), 407 (3.57). IR (KBr) n (cm21): 3447, 1671, 1630. EIMS m/z (% rel int.): 293 ([M þ 1] þ, 1), 292 ([M] þ, 4), 277 (7), 252 (1), 250 (39), 249 (100), 222 (1), 221 (4), 220 (2), 194 (1), 193 (4), 166 (2), 165 (11), 139 (2), 110 (2), 105 (4), 96 (2), 82 (4) 61 (9). HR-MS m/z: 292.0785 (calcd. for C18H4O12, 292.0735). 1H NMR spectral data (500 MHz, CDCl3): 13.45 (s, 1-OH), 8.35 (dd, J ¼ 8.0, 2.0 Hz, H-8)a, 8.32 (dd, J ¼ 8.0, 2.0 Hz, H-5)a, 7.94 (d, J ¼ 8.0 Hz, H-3), 7.92 (d, J ¼ 16.5 Hz, H-10 ), 7.88 (d, J ¼ 8.0 Hz, H-4), 7.85 (m, H-6, H-7), 6.97 (d, J ¼ 16.5 Hz, H-20 ), 2.45 (s, 30 -CH3). 13C NMR spectral data (125 MHz, CDCl3): 198.6 (C-30 ), 189.0 (C-9), 181.9 (C10), 161.4 (C-1), 135.7 (C-10 ), 135.0 (C-6, C-7), 134.4 (C-3), 134.0 (C-10a)b, 133.5 (C-8a)b, 132.9 (C-4a), 130.5 (C-20 ), 129.7 (C-2), 127.5 (C-5)a, 127.1 (C-8)a, 119.2 (C-4), 116.4 (C-9a), 27.6 (30 -CH3)a,b. Assignment with the same superscript may be interchanged. 3.4. Antibacterial activity All compounds at concentrations of 200 mg/mL were screened for antibacterial activity against S. aureus ATCC25923 and a clinical isolate of methicillin-resistant S. aureus (MRSA) SK1, Escherichia coli ATCC25922, and Pseudomonas aeruginosa ATCC27853 by the colorimetric microdilution method according to a modification of CLSI M7-A4 (Clinical and Laboratory Standards Institute [CLSI] 2000) Resazurin (0.18%) was used as the indicator of growth. After incubation, a blue or purple colour of the wells indicated inhibition of growth (positive result), whereas a pink colour meant that growth had occurred (negative result). The minimum
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inhibitory concentration (MIC) of active compounds was determined by the same method. The lowest concentration of the compound that inhibited growth (blue or purple colour) was recorded as the MIC. Vancomycin and gentamicin were used as standard antibacterial agents for the positive inhibitory controls. 3.5. Antifungal activity Each pure compound was screened for antifungal activity at a concentration of 200 mg/mL by a modification of the microbroth dilution CLSI M27-A2 (CLSI 2002a) against yeasts (Candida albicans ATCC90028, Cryptococcus neoformans ATCC90112) and a modification of the microbroth dilution CLSI M38-A (CLSI 2002b) against a clinical isolate of M. gypseum. Amphotericin B was used as a positive inhibitory control for yeasts and miconazole for M. gypseum. 3.6. Antiprotozoal assay Cells were harvested by chilling the tube on ice for 15 min to detach the monolayer then centrifuged at 300g for 5 min. The supernatant was decanted, and cells were resuspended in a fresh medium. The numbers of viable cells were calculated using a haemocytometer and 0.4% (w/v) trypan blue. The criteria for viability were motility and dye exclusion. For assays, trophozoites, 2 £ 105 cells/mL, were incubated in the presence of serial twofold dilutions of extract that ranged from 31.25 to 1000 mg/mL. Metronidazole, with concentrations ranged from 0.625 to 20 mg/mL and a complete medium with added DMSO were used as negative and positive controls, respectively. After 24 h of incubation at 378C under anaerobic conditions, the trophozoites from each well were examined and counted using an inverted microscope. The appearance and number of trophozoites were scored from 1 to 4 with 1 showing the most inhibition of growth and 4 showing no inhibition according to Upcroft and Upcroft, and the MIC was recorded (the lowest concentration at which . 90% of the trophozoites rounded up). Each concentration was tested in duplicate and at least three experiments were performed on separate occasions. 4. Conclusion Investigation of the active antimicrobial components from the stems of M. elliptica resulted in the isolation of 19 unsubstituted C-ring anthraquinones including a new anthraquinone (6). Eight of them (2, 3, 8, 9, 11, 13 –15) were first isolated from this plant. For antimicrobial activity, damnacanthal (4) showed strong activity against M. gypseum, whereas lucidin (17) exhibited strong antiprotozoal activity against G. lamblia. Supplementary material Tables S1 and S2 and Figures S1 – S3 relating to this article are available online. References Abboud P, Lemee V, Gargala G, Brasseur P, Ballet JJ, Borsa-Lebas F, Caron F, Favennec L. 2001. Successful treatment of metronidazole- and albendazole-resistant giardiasis with nitazoxanide in a patient with acquired immunodeficiency syndrome. Clin Infect Dis. 32:1792–1794. doi:10.1086/320751. Balakrishna S, Seshadri TR, Venkatarumani B. 1960. Special chemical components of commercial woods and related plant material IX moridonin, a new glycoside of morindone. J Sci Ind Res B. 19:433–436. Brisson C, Brassard P. 1981. Regiospecific reactions of some vinylogous ketene acetals with haloquinones and their regioselective formation by dienolization. J Org Chem. 46:1810–1814. doi:10.1021/jo00322a012.
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