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Neolignans from Callistemon lanceolatus
Phytochemistry Letters 5 (2012) 18–21
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Neolignans from Callistemon lanceolatus Suthida Rattanaburi a, Wilawan Mahabusarakam a,b,*, Souwalak Phongpaichit b,c, Anthony R. Carroll d a
Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Natural Product Research Center, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand c Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand d Griffith School of Environment (Gold Coast Campus), Griffith University, Queensland Q4222, Australia b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 May 2011 Received in revised form 15 August 2011 Accepted 23 August 2011 Available online 17 September 2011
Two neolignans, named callislignan A and B together with known C-methyl-flavonoids, a lignan and pentacyclic triterpenoid esters were isolated from the leaves of Callistemon lanceolatus. Their structures were characterized by spectroscopic methods. Callislignan A and B had antibacterial activity against Staphylococcus aureus ATCC25923 and MRSA SK1 with callislignan B having an MIC of 8 mg/mL. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Callistemon lanceolatus Myrtaceae Lignans C-methyl-flavonoids b-Triketones Triterpenoid esters
1. Introduction
2. Result and discussion
Callistemon lanceolatus DC., also known as Crimson Bottlebrush, is a shrub in the family Myrtaceae. Preparations from it have been used in folk medicine for the treatment of coughs and bronchitis (Marzouk, 2008). Previous chemical investigations of compounds from this family have revealed the presence of various types of secondary metabolites, including acylphloroglucinols (Lounasmaa et al., 1977), C-methyl flavonoids (Huq and Misra, 1997), tannins (Hanaa and Mohamed, 2002) and triterpenoids (Varma and Parthasarathy, 1975). The flowers of this plant have been reported to contain tannins with antioxidant and hepatoprotective activities (Hanaa and Mohamed, 2002). The essential oil from the leaves has also exhibited significant antimicrobial activities (Silva et al., 2010). The search for bioactive metabolites and chemical constituents from natural sources has been an ongoing project in our laboratory. We therefore tested fractions obtained from a crude extract of the leaves of C. lanceolatus for antibacterial activity. Positive results prompted us to further evaluate the chemical constituents of the leaves of C. lanceolatus.
Separation of a CH2Cl2 extract of the leaves of C. lanceolatus has resulted in the isolation of two new compounds, callislignan A and B (1, 2) together with nine known compounds (Fig. 1). The known compounds were identified as 5,40 -dihydroxy-7-methoxy-6,8dimethylflavanone (Wollenweber et al., 2000), nectandrin B (Le et al., 1980), 8-demethylsideroxylin (Cardona and Seoan, 1982), 5,40 -dihydroxy-7-methoxy-6-methylflavanone (Wollenweber et al., 2000), 5,40 -dihydroxy-7-methoxy-6,8-dimethylflavone (Hillis and Isoi, 1965), 5-hydroxy-7,40 -dimethoxy-6-methylflavone (Huq and Misra, 1997), 5-hydroxy-7,40 -dimethoxy-6,8-dimethylflavone (Huq and Misra, 1997), 3b-trans-feruloyloxy-2a-hydroxyurs-12-en-28-oic acid (Ito et al., 2001) and olean-12-en-28-oic acid (Siddiqui et al., 1997). This is the first time that all of the flavones except 5-hydroxy-7,40 -dimethoxy-6,8-dimethylflavone have been isolated from C. lanceolatus. All structures were determined from analyses of 1H, 13C NMR, COSY, HMQC and HMBC spectra. Callislignan A (1) was isolated as a yellowish gum. Its molecular formula of C19H20O4 was established from analysis of high resolution EI mass spectrometry (EI-MS) ([M]+ m/z 312.1362). The IR spectrum showed an absorption band for a hydroxyl group at 3392 cm1. Inspection of 1H NMR spectroscopic data in combination with COSY correlations indicated that 1 contained resonances assigned to two meta coupled aromatic protons (dH 6.67, H-4; dH 6.65, H-6), methine (dH 3.29, H-3) and oxy-methine protons (dH 4.97, d, H-2) that were trans coupled (9.6 Hz) (Li et al.,
* Corresponding author at: Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand. Tel.: +66 7428 8432; fax: +66 7455 8841. E-mail address: [email protected] (W. Mahabusarakam).
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2011.08.010
S. Rattanaburi et al. / Phytochemistry Letters 5 (2012) 18–21
HO 5"
OCH3 3"
OH 1''
O 7a
2
H 3C
3 3a
9
H 3C
6 4
2' 1'
CH3 3'
1
H3CO HO
3 4
A
1
8
O
7
OH
6
19
OH 4'
3'
B
8'
1'
6'
7'
CH3 9'
2
Fig. 1. Structure formula of callislignan A (1) and callislignan B (2).
1997), an aliphatic methyl doublet (dH 1.26, d, J = 6.6 Hz) that was vicinal to H-3, a (E)-propenyl group (dH 6.23, dq, H-10 ; dH 5.94, dq, H20 ; dH 1.72, dd, H-30 ), a 1,2,4-trisubstituted phenyl group (dH 6.76 d, J = 8.4, H-500 ; dH 6.84, dd, J = 8.4, 1.8, H-600 and dH 7.03, d, J = 1.8, H-200 ), an aromatic methoxyl group (dH 3.77, 300 -OCH3) and two phenolic protons (dH 7.85, 7-OH; dH 7.63, 400 -OH). The 13C NMR spectrum contained seven sp2 hybridised quaternary carbons, seven protonated sp2 hybridised carbons and five additional protonated carbons that resonated upfield of 100 ppm. This data suggested that 1 contained a 2,3-dihydrobenzofuran skeleton (Nascimento and Lopes, 1999; Achenbach et al., 1987; Li et al., 1997). HMBC correlations from 7-OH to C-6 (dC 113.3), C-7 (dC 140.9) and C-7a (dC 146.8), from H-4 to C-3 (dC 45.5), C-6 (dC 113.3), C-7a (dC 146.8) and C-10 (dC 131.2) and from H-6 to C-4 (dC 112.2), C-7 (dC 140.9), C7a (dC 146.8) and C-10 (dC 131.2) indicated that the hydroxyl group and the propenyl group were ortho and para to the heteroatom of the furan ring, respectively. The substituents on the furan ring were assigned to a methyl group at C-3 and a 3-methoxy-4-hydroxyphenyl group at C-2 since HMBC correlations were observed from 3-CH3 to C-2 (dC 93.1) and C-3a (dC 133.5), while H-2 correlated to C200 (dC 110.0) and C-600 (dC 119.5). The placement of the methoxyl group at C-300 was indicated by the NOE enhancement of the H-200 resonance upon irradiation of the methoxyl resonance (dH 3.77). This analysis in total suggested that 1 was 2,3-dihydro-2-(4-hydroxy-3methoxyphenyl)-7-hydroxy-3-methyl-5-(E)-propenylbenzofuran. The absolute configuration of the stereogenic centers, C-2 and C-3, in 1 were assigned as 2R, 3R from comparison of its optical rotation (+56.7) with that of the known compound (+)-licarin A, (2R, 3R)-2,3dihydro-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-3-methyl5-(E)-propenylbenzofuran ([a]D21 + 52; MeOH) (Achenbach et al., 1987). Callislignan A (1) was therefore the 7-demethyl derivative of (+)-licarin A. Callislignan B (2) was isolated as a yellowish gum, [a]D29 15.8 (c 4.55, MeOH). The molecular formula was determined as C19H22O5 from analysis of high resolution EI-MS ([M]+ m/z 330.1466). The IR spectrum exhibited a stretching band for a hydroxyl group at 3393 cm1. The 1H NMR spectrum (Table 2) contained resonances that were assigned to two 1,2,4-trisubstituted phenyl groups (ring A: dH 6.81 (d, H-5), dH 6.86 (dd, H-6), dH 6.93 (d, H-2), and ring B: dH 6.90 (d, H-50 ), dH 6.74 (dd, H-60 ), dH 6.96 (d, H-20 ). A doublet at dH 4.65, a quartet of doublets at dH 4.05 and a doublet at dH 1.09 were assigned to a benzylic oxymethine proton (H-7), an oxymethine proton (H-8) and a methyl proton (8-CH3), respectively. Analysis of COSY correlations and coupling constants indicated that H-7 was vicinal to H-8 and their mutual coupling constant of 8.4 Hz suggested that these two protons possessed a threo-relative configuration (lit. J = 8.0 Hz, threo; J = 3.0, erythro) (Conserva et al., 1990). The presence of a (E)-propenyl group was assigned from signals at dH 6.30 (dq, H-70 ), dH 6.86 (dq, H-80 ) and dH 1.85 (dd, H-90 ). Two 3JCH correlations from both H-20 and H-60 to C40 (dC 144.4) and C-70 (dC 130.4) revealed that the propenyl side chain was attached to ring B and was para to C-40 . An HMBC correlation between H-8 and C-40 (dC 144.4) and C-7 (dC 78.3) and between H-7 and C-2 (dC 113.2) and C-6 (dC 118.9) suggested that
an ether bond between C-8 and C-40 linked the two benzylpropanoid groups. A methoxyl group which resonated at dH 3.88 was attached at C-3 of ring A since HMBC correlations were observed between 3-OCH3, H-2 and H-5 and C-3 (dC 146.8). Accordingly, 2 was identified as threo-1-(4-hydroxy-3-methoxyphenyl)-2-(2hydroxy-4-(E)-propenylphenoxy)-1-propanol. All of the pure compounds were evaluated for their antibacterial activity against two strains of Staphylococcus aureus. S. aureus ATTCC25923 is sensitive to many commercially available antibiotics whereas MRSA SK1 is resistant to methicillin and other beta-lactam antibiotics as well as other classes of antibiotics. Only compounds 1 (callislignan A) and 2 (callislignan B) showed antibacterial activity against both strains of S. aureus with MIC values of 200 (1, ATTCC25923), 64 (1, MRSA SK1) and 8 (2, ATTCC25923), 8 (2, MRSA SK1) mg/mL. Compound 2 exhibited the best anti-S. aureus activity (MIC 8mg/mL) as compared to vancomycin, a standard antibiotic (MIC 1 mg/mL). It is worth noting that callislignan B inhibited both S. aureus strains with the same MIC. This result indicated that callislignan B may act on different bacterial targets and this compound may be useful for treating MRSA infections. 3. Experimental 3.1. General experimental procedures Melting points were recorded with a Fisher–Johns melting point apparatus and are uncorrected. The IR spectra were measured on a FTS 165 FT-IR Perkin-Elmer spectrophotometer. UV spectra were recorded on a SPECORD S100 spectrophotometer. 1 H and 13C nuclear magnetic resonance spectra were recorded in CDCl3 or acetone-d6 using either a FT-NMR Bruker Avance 300 MHz or 500 MHz spectrometers. Optical rotations were recorded in CHCl3 or MeOH solutions at the sodium D line (589 nm) on a JASCO P-1020 polarimeter. The EI-MS and HREIMS mass spectra were obtained using a MAT 95 XL mass spectrometer (Thermofinigan). Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60H (Merck) and silica gel 100 (Merck), respectively. Precoated plates of silica gel 60 GF254 were used for TLC analysis. 3.2. Plant material The leaves of C. lanceolatus (Myrtaceae) were collected from Amphur Mueang Nakhon Si Thammarat, Nakhon Si Thammarat Province in October 2007. The plant was identified by Mr. Ponlawat Pattarakulpisutti and a herbarium specimen (S. Rattanaburi 1) has been deposited at the Herbarium within the Department of Biology, Faculty of Science, Prince of Songkla University, Thailand. 3.3. Extraction and isolation Ground, dried leaves (4.5 kg) of C. lanceolatus were extracted with CH2Cl2 at room temperature for 3 days. The viscous extract (349.5 g), after removal of solvent, was sequentially dissolved in
S. Rattanaburi et al. / Phytochemistry Letters 5 (2012) 18–21
20
hexane and Me2CO to give hexane soluble [A] (107.6 g), Me2CO soluble (77.3 g) and Me2CO insoluble fractions [B] (138.20 g). The Me2CO soluble fraction was further partitioned with EtOAc/ 5%NaOH to give an EtOAc layer [C] (16.20 g), an aqueous layer [D] and a solid [E] (30.70 g) that was formed during partition. The aqueous layer [D] after neutralization with HCl (14.30 g) was dissolved in MeOH from which 5,40 -dihydroxy-7-methoxy-6,8dimethylflavone (70.1 mg) precipitated. The filtrate from fraction D (11.90 g) was further purified by CC using CH2Cl2–MeOH (9:1) as eluant. The fractions that contained similar components, on the basis of their TLC characteristics, were combined to give fractions D1–D6. Fraction D2 (2.16 g) was further subjected to CC and eluted with Me2CO–hexane (3:17) to Me2CO–hexane (1:5) to give 5,40 -dihydroxy-7-methoxy-6,8-dimethylflavanone (10.4 mg) and 1 (7.3 mg). Fraction D3 (1.24 g) was chromatographed on silica with Me2CO–hexane (1:19) followed by Me2CO–hexane (7:3) to give a white solid nectandrin B (4.3 mg). Fraction D4 (2.098 g) was purified by CC on silica with hexane–Me2CO (9:1) yielding 16 fractions (D4A–D4P). Crystallization of fraction D4F in hexane– Me2CO (9:1) yielded 8-demethylsideroxylin (3.2 mg). Fraction D4J (246.0 mg) was chromatographed on silica using CH2Cl2– MeOH (49:1) as an eluant to give 6 portions (D4J.1–D4J.6). A yellow solid, 5,40 -dihydroxy-7-methoxy-6-methylflavanone (0.9 mg) was obtained from fraction D4J.4. Fraction D4J.5 (34.1 mg) was further purified by PTLC using CH2Cl2–MeOH (99:1) as eluant yielding a yellowish gum 2 (10.2 mg). The EtOAc layer [C] (16.20 g) was separated by silica CC sequentially eluting with hexane–CH2Cl2, CH2Cl2 and CH2Cl2–MeOH respectively. On the basis of their TLC characteristic, the fractions which contained the same major components were combined to give fractions C1– C7. Fraction C2 (20.1 mg) was crystallized from hexane–Me2CO (9:1) to yield a white solid 5-hydroxy-7,40 -dimethoxy-6-methylflavone (4.0 mg). The filtrate (12.5 mg) was rechromatographed on silica CC using hexane–Me2CO (9:1) as eluant to give a yellow solid, 5-hydroxy-7,40 -dimethoxy-6,8-dimethylflavone (7.8 mg). Fraction C5 (1.38 g) was chromatographed on silica CC using hexane-Me2CO (9:1) as an eluant to give white solids 3b-transferuloyloxy-2a-hydroxyurs-12-en-28-oic acid (1.3 mg) and olean-12-en-28-oic acid (3.0 mg). 3.3.1. Callislignan A (1) A yellowish gum. [a]D29 + 56.7 (c 0.80, MeOH). HRMS m/z 312.1362 for C19H20O4 (calcd. 312.1362). EI-MS m/z (% rel int): 312 Table 1 13 C, 1H, and HMBC spectral data of 1 (in acetone-d6). No.
dC
dH (mult., JHz)
HMBC (H ! C)
2 3 3a 4 5 6 7 7a 10 20 30 100 200 300 400 500 600 3-CH3 7-OH 400 -OH 300 -OCH3
93.1 45.5 133.5 112.2 131.9 113.3 140.9 146.8 131.2 122.1 17.5 132.0 110.0 147.6 146.1 114.7 119.5 16.8
4.97 (d, 9.6) 3.29 (obscure)
C-3, C-100 , C-200 , C-600 , 3-CH3
6.67 (br s)
C-3, C-6, C-7a, C-10
6.65 (br s)
C-4, C-7, C-7a, C-10
6.23 (dq, 17.1, 1.5) 5.94 (dq, 17.1, 6.6) 1.72 (dd, 6.6, 1.5)
C-20 C-5, C-10 , C-30 C-10 , C-20
7.03 (d, 1.8)
C-2, C-300 , C-400 , C-600
6.76 6.84 1.26 7.85 7.63 3.77
C-100 , C-300 , C-400 C-200 , C-400 C-2, C-3, C-3a C-6, C-7, C-7a C-300 , C-400 , C-500 C-300
55.4
(d, 8.4) (dd, 8.4, 1.8) (d, 6.6) (s) (s) (s)
Table 2 13 C, 1H, and HMBC spectral data of 2 (in CDCl3). No.
dC
1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 90 3-OCH3
133.1 113.2 146.8 145.8 110.6 118.9 78.3 83.5 16.8 134.5 113.2 148.7 144.4 119.6 117.6 130.4 124.8 18.4 55.9
dH (mult., JHz)
HMBC (H ! C)
6.93 (d, 1.8)
C-7, C-1, C-3, C-4, C-6
6.81 6.86 4.65 4.05 1.09
C-1, C-7, C-2, C-7, C-7,
(d, 8.4) (dd, 8.4, 1.8) (d, 8.4) (qd, 8.4, 6.3) (d, 6.3)
C-3, C-4, C-6 C-1, C-2, C-4, C-5 C-6, C-1, C-8, C-9 C-40 C-8
6.96 (d, 2.1)
C-40 , C-60 , C-70
6.90 6.74 6.30 6.86 1.85 3.88
C-60 , C-40 , C-20 , C-10 , C-70 , C-3
(d, 8.4) (dd, 8.4, 2.1) (dq, 15.6, 1.5) (dq, 15.6, 6.6) (dd, 6.6, 1.5) (s)
C-10 , C-20 , C-60 , C-70 , C-80
C-30 , C-40 C-70 C-80 , C-90 C-90
([M]+, 100), 297 (16), 149 (18), 137 (22), 105 (48), 77 (19). UV (MeOH) lmax nm (log e): 207 (3.84), 231 (3.51), 280 (3.19). IR (neat)n (cm1): 3392. 1H NMR spectroscopic data (300 MHz, acetone-d6) and 13C NMR spectroscopic data (75 MHz, acetoned6): see Table 1. 3.3.2. Callislignan B (2) A yellowish gum. [a]D29 15.8 (c 4.55, MeOH). HRMS m/z 330.1466 for C19H22O5 (calcd. 330.1467). UV (MeOH) lmax nm (log e): 205 (4.00), 227 (3.65), 269 (3.30). IR (neat) n (cm1): 3393. EI-MS m/z (% rel int): 330 [M] +, (57), 312 (70), 180 (30), 164 (41), 151 (93), 150 (100), 149 (61), 133 (23), 108 (36), 105 (44), 65 (47). 1H NMR spectroscopic data (300 MHz, CDCl3) and 13 C NMR spectroscopic data (75 MHz, CDCl3): see Table 2. 3.4. Antibacterial activity The crude extract and purified compounds were screened for antibacterial activity at a concentration of 200 mg/mL by a broth microdilution method against four bacteria: S. aureus ATCC25923, a clinical isolate of methicillin-resistant S. aureus SK1, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853. Crude extracts and purified compounds having antibacterial activity were further determined for their minimum inhibitory concentrations (MIC) by broth microdilution methods according to a modification of the Clinical and Laboratory Standards Institute. Resazurin was added as viability indicator according to Drummond and Waigh (2000). Colorimetric MIC end-points were interpreted as the lowest concentration that remained blue (indicating no growth) or the first dilution that changed from blue to slightly purple (equivalent to prominent growth inhibition). Acknowledgments We are grateful for the financial support from the Royal Golden Jubilee Ph.D. Program (a scholarship to S. Rattanaburi, Grant No. PHD/0284/2550), the Center for Innovation in Chemistry (PERCHCIC), the National Research University Project of Thailand’s Office of the Higher Education Commission and the Graduate School, Prince of Songkla University for scholarship. The Scientific Equipment Center Prince of Songkla University is also gratefully acknowledged for MS data.
S. Rattanaburi et al. / Phytochemistry Letters 5 (2012) 18–21
References Achenbach, H., Grob, J., Dominguez, X.A., Cano, D.G., Star, J.V., Del, L., Brussolo, C., Munoz, G., Salgado, F., Lopez, L., 1987. Lignans neolignans and norneolignans from krameria cystisoides. Phytochemistry 26 (4), 1159–1166. Cardona, M.L., Seoan, E., 1982. Flavonoids and xanthonolignoids of Hypericum ericoides. Phytochemistry 21 (11), 2759–2760. Conserva, L.M., Silva, M.S.D., Filho, R.B., 1990. Lignans from Aristolochia birostris. Phytochemistry 29 (1), 257–260. Drummond, A.J., Waigh, R.D., 2000. The development of microbiological methods for phytochemical screening. Recent research in developmental. Phytochemistry 4, 143–152. Hanaa, H.A., Mohamed, S.A.M., 2002. Antioxidant and hepatoprotective activity of hydrolyzable tannins from Callistemon lanceolatus against CCL4-induced liver damage in rats. Bulletin of the Faculty of Pharmacy (Cairo University) 40 (2), 175–187. Hillis, W.E., Isoi, K., 1965. Variation in the chemical composition of Eucalyptus sideroxylon. Phytochemistry 4 (4), 541–550. Huq, F., Misra, L.N., 1997. An alkenol and C-methylated flavones from Callistemon lanceolatus leaves. Planta Medica 64 (4), 369–370. Ito, H., Kobayashi, E., Li, S.H., Hatano, T., Sugita, D., Kubo, N., Shimura, S., Itoh, Y., Yoshida, T., 2001. Megastigmane glycosides and an acylated triterpenoid from Eriobotrya japonica. Journal of Natural Products 64 (6), 737–740.
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Le, Q.P.W., Larrahondo, J.E., Raffauf, R.F., 1980. Antitumor plants. X. Constituents of Nectandra rigida. Journal of Natural Products 43 (3), 353–359. Li, S., Iliefski, T., Lundquist, K., Wallis, A.F.A., 1997. Reassignment of relative stereochemistry at C-7 and C-8 in arylcoumarin neolignans. Phytochemistry 46 (5), 929–934. Lounasmaa, M., Puri, H.S., Widen, C.J., 1977. Phloroglucinol derivatives of Callistemon lanceolatus leaves. Phytochemistry 16 (11), 1851–1852. Marzouk, M.S.A., 2008. An acylated flavonol glycoside and hydrolysable tannins from Callistemon lanceolatus flowers and leaves. Phytochemical Analysis 19 (6), 541–549. Nascimento, I.R., Lopes, L.M.X., 1999. 2,3-Dihydrobenzofuran neolignans from Aristolochia pubescens. Phytochemistry 52 (2), 345–350. Siddiqui, B.S., Farhat, Begum, S., Siddiqui, S., 1997. Isolation and structural elucidation of acylated pentacyclic triterpenoids from the leaves of Eucalyptus camaldulensis var. obtusa. Planta Medica 63 (1), 47–50. Silva, C.J., Barbosa, L.C.A., Demuner, A.J., Montanari, R.M., Pinheiro, A.L., Dias, I., Andrade, N.J., 2010. Chemical composition and antibacterial activities from the essential oils of Myrtaceae species planted in Brazil. Quimica Nova 33 (1), 104– 108. Varma, R.S., Parthasarathy, M.R., 1975. Triterpenoids of Callistemon lanceolatus leaves. Phytochemistry 14 (7), 1675–1676. Wollenweber, E., Wehde, R., Dorr, M., Lang, G., Stevens, J.F., 2000. C-methylflavonoids from the leaf waxes of some Myrtaceae. Phytochemistry 55 (8), 965–970.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Neolignans from Callistemon lanceolatus Suthida Rattanaburi a, Wilawan Mahabusarakam a,b,*, Souwalak Phongpaichit b,c, Anthony R. Carroll d a
Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Natural Product Research Center, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand c Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand d Griffith School of Environment (Gold Coast Campus), Griffith University, Queensland Q4222, Australia b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 May 2011 Received in revised form 15 August 2011 Accepted 23 August 2011 Available online 17 September 2011
Two neolignans, named callislignan A and B together with known C-methyl-flavonoids, a lignan and pentacyclic triterpenoid esters were isolated from the leaves of Callistemon lanceolatus. Their structures were characterized by spectroscopic methods. Callislignan A and B had antibacterial activity against Staphylococcus aureus ATCC25923 and MRSA SK1 with callislignan B having an MIC of 8 mg/mL. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Callistemon lanceolatus Myrtaceae Lignans C-methyl-flavonoids b-Triketones Triterpenoid esters
1. Introduction
2. Result and discussion
Callistemon lanceolatus DC., also known as Crimson Bottlebrush, is a shrub in the family Myrtaceae. Preparations from it have been used in folk medicine for the treatment of coughs and bronchitis (Marzouk, 2008). Previous chemical investigations of compounds from this family have revealed the presence of various types of secondary metabolites, including acylphloroglucinols (Lounasmaa et al., 1977), C-methyl flavonoids (Huq and Misra, 1997), tannins (Hanaa and Mohamed, 2002) and triterpenoids (Varma and Parthasarathy, 1975). The flowers of this plant have been reported to contain tannins with antioxidant and hepatoprotective activities (Hanaa and Mohamed, 2002). The essential oil from the leaves has also exhibited significant antimicrobial activities (Silva et al., 2010). The search for bioactive metabolites and chemical constituents from natural sources has been an ongoing project in our laboratory. We therefore tested fractions obtained from a crude extract of the leaves of C. lanceolatus for antibacterial activity. Positive results prompted us to further evaluate the chemical constituents of the leaves of C. lanceolatus.
Separation of a CH2Cl2 extract of the leaves of C. lanceolatus has resulted in the isolation of two new compounds, callislignan A and B (1, 2) together with nine known compounds (Fig. 1). The known compounds were identified as 5,40 -dihydroxy-7-methoxy-6,8dimethylflavanone (Wollenweber et al., 2000), nectandrin B (Le et al., 1980), 8-demethylsideroxylin (Cardona and Seoan, 1982), 5,40 -dihydroxy-7-methoxy-6-methylflavanone (Wollenweber et al., 2000), 5,40 -dihydroxy-7-methoxy-6,8-dimethylflavone (Hillis and Isoi, 1965), 5-hydroxy-7,40 -dimethoxy-6-methylflavone (Huq and Misra, 1997), 5-hydroxy-7,40 -dimethoxy-6,8-dimethylflavone (Huq and Misra, 1997), 3b-trans-feruloyloxy-2a-hydroxyurs-12-en-28-oic acid (Ito et al., 2001) and olean-12-en-28-oic acid (Siddiqui et al., 1997). This is the first time that all of the flavones except 5-hydroxy-7,40 -dimethoxy-6,8-dimethylflavone have been isolated from C. lanceolatus. All structures were determined from analyses of 1H, 13C NMR, COSY, HMQC and HMBC spectra. Callislignan A (1) was isolated as a yellowish gum. Its molecular formula of C19H20O4 was established from analysis of high resolution EI mass spectrometry (EI-MS) ([M]+ m/z 312.1362). The IR spectrum showed an absorption band for a hydroxyl group at 3392 cm1. Inspection of 1H NMR spectroscopic data in combination with COSY correlations indicated that 1 contained resonances assigned to two meta coupled aromatic protons (dH 6.67, H-4; dH 6.65, H-6), methine (dH 3.29, H-3) and oxy-methine protons (dH 4.97, d, H-2) that were trans coupled (9.6 Hz) (Li et al.,
* Corresponding author at: Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand. Tel.: +66 7428 8432; fax: +66 7455 8841. E-mail address: [email protected] (W. Mahabusarakam).
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2011.08.010
S. Rattanaburi et al. / Phytochemistry Letters 5 (2012) 18–21
HO 5"
OCH3 3"
OH 1''
O 7a
2
H 3C
3 3a
9
H 3C
6 4
2' 1'
CH3 3'
1
H3CO HO
3 4
A
1
8
O
7
OH
6
19
OH 4'
3'
B
8'
1'
6'
7'
CH3 9'
2
Fig. 1. Structure formula of callislignan A (1) and callislignan B (2).
1997), an aliphatic methyl doublet (dH 1.26, d, J = 6.6 Hz) that was vicinal to H-3, a (E)-propenyl group (dH 6.23, dq, H-10 ; dH 5.94, dq, H20 ; dH 1.72, dd, H-30 ), a 1,2,4-trisubstituted phenyl group (dH 6.76 d, J = 8.4, H-500 ; dH 6.84, dd, J = 8.4, 1.8, H-600 and dH 7.03, d, J = 1.8, H-200 ), an aromatic methoxyl group (dH 3.77, 300 -OCH3) and two phenolic protons (dH 7.85, 7-OH; dH 7.63, 400 -OH). The 13C NMR spectrum contained seven sp2 hybridised quaternary carbons, seven protonated sp2 hybridised carbons and five additional protonated carbons that resonated upfield of 100 ppm. This data suggested that 1 contained a 2,3-dihydrobenzofuran skeleton (Nascimento and Lopes, 1999; Achenbach et al., 1987; Li et al., 1997). HMBC correlations from 7-OH to C-6 (dC 113.3), C-7 (dC 140.9) and C-7a (dC 146.8), from H-4 to C-3 (dC 45.5), C-6 (dC 113.3), C-7a (dC 146.8) and C-10 (dC 131.2) and from H-6 to C-4 (dC 112.2), C-7 (dC 140.9), C7a (dC 146.8) and C-10 (dC 131.2) indicated that the hydroxyl group and the propenyl group were ortho and para to the heteroatom of the furan ring, respectively. The substituents on the furan ring were assigned to a methyl group at C-3 and a 3-methoxy-4-hydroxyphenyl group at C-2 since HMBC correlations were observed from 3-CH3 to C-2 (dC 93.1) and C-3a (dC 133.5), while H-2 correlated to C200 (dC 110.0) and C-600 (dC 119.5). The placement of the methoxyl group at C-300 was indicated by the NOE enhancement of the H-200 resonance upon irradiation of the methoxyl resonance (dH 3.77). This analysis in total suggested that 1 was 2,3-dihydro-2-(4-hydroxy-3methoxyphenyl)-7-hydroxy-3-methyl-5-(E)-propenylbenzofuran. The absolute configuration of the stereogenic centers, C-2 and C-3, in 1 were assigned as 2R, 3R from comparison of its optical rotation (+56.7) with that of the known compound (+)-licarin A, (2R, 3R)-2,3dihydro-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-3-methyl5-(E)-propenylbenzofuran ([a]D21 + 52; MeOH) (Achenbach et al., 1987). Callislignan A (1) was therefore the 7-demethyl derivative of (+)-licarin A. Callislignan B (2) was isolated as a yellowish gum, [a]D29 15.8 (c 4.55, MeOH). The molecular formula was determined as C19H22O5 from analysis of high resolution EI-MS ([M]+ m/z 330.1466). The IR spectrum exhibited a stretching band for a hydroxyl group at 3393 cm1. The 1H NMR spectrum (Table 2) contained resonances that were assigned to two 1,2,4-trisubstituted phenyl groups (ring A: dH 6.81 (d, H-5), dH 6.86 (dd, H-6), dH 6.93 (d, H-2), and ring B: dH 6.90 (d, H-50 ), dH 6.74 (dd, H-60 ), dH 6.96 (d, H-20 ). A doublet at dH 4.65, a quartet of doublets at dH 4.05 and a doublet at dH 1.09 were assigned to a benzylic oxymethine proton (H-7), an oxymethine proton (H-8) and a methyl proton (8-CH3), respectively. Analysis of COSY correlations and coupling constants indicated that H-7 was vicinal to H-8 and their mutual coupling constant of 8.4 Hz suggested that these two protons possessed a threo-relative configuration (lit. J = 8.0 Hz, threo; J = 3.0, erythro) (Conserva et al., 1990). The presence of a (E)-propenyl group was assigned from signals at dH 6.30 (dq, H-70 ), dH 6.86 (dq, H-80 ) and dH 1.85 (dd, H-90 ). Two 3JCH correlations from both H-20 and H-60 to C40 (dC 144.4) and C-70 (dC 130.4) revealed that the propenyl side chain was attached to ring B and was para to C-40 . An HMBC correlation between H-8 and C-40 (dC 144.4) and C-7 (dC 78.3) and between H-7 and C-2 (dC 113.2) and C-6 (dC 118.9) suggested that
an ether bond between C-8 and C-40 linked the two benzylpropanoid groups. A methoxyl group which resonated at dH 3.88 was attached at C-3 of ring A since HMBC correlations were observed between 3-OCH3, H-2 and H-5 and C-3 (dC 146.8). Accordingly, 2 was identified as threo-1-(4-hydroxy-3-methoxyphenyl)-2-(2hydroxy-4-(E)-propenylphenoxy)-1-propanol. All of the pure compounds were evaluated for their antibacterial activity against two strains of Staphylococcus aureus. S. aureus ATTCC25923 is sensitive to many commercially available antibiotics whereas MRSA SK1 is resistant to methicillin and other beta-lactam antibiotics as well as other classes of antibiotics. Only compounds 1 (callislignan A) and 2 (callislignan B) showed antibacterial activity against both strains of S. aureus with MIC values of 200 (1, ATTCC25923), 64 (1, MRSA SK1) and 8 (2, ATTCC25923), 8 (2, MRSA SK1) mg/mL. Compound 2 exhibited the best anti-S. aureus activity (MIC 8mg/mL) as compared to vancomycin, a standard antibiotic (MIC 1 mg/mL). It is worth noting that callislignan B inhibited both S. aureus strains with the same MIC. This result indicated that callislignan B may act on different bacterial targets and this compound may be useful for treating MRSA infections. 3. Experimental 3.1. General experimental procedures Melting points were recorded with a Fisher–Johns melting point apparatus and are uncorrected. The IR spectra were measured on a FTS 165 FT-IR Perkin-Elmer spectrophotometer. UV spectra were recorded on a SPECORD S100 spectrophotometer. 1 H and 13C nuclear magnetic resonance spectra were recorded in CDCl3 or acetone-d6 using either a FT-NMR Bruker Avance 300 MHz or 500 MHz spectrometers. Optical rotations were recorded in CHCl3 or MeOH solutions at the sodium D line (589 nm) on a JASCO P-1020 polarimeter. The EI-MS and HREIMS mass spectra were obtained using a MAT 95 XL mass spectrometer (Thermofinigan). Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60H (Merck) and silica gel 100 (Merck), respectively. Precoated plates of silica gel 60 GF254 were used for TLC analysis. 3.2. Plant material The leaves of C. lanceolatus (Myrtaceae) were collected from Amphur Mueang Nakhon Si Thammarat, Nakhon Si Thammarat Province in October 2007. The plant was identified by Mr. Ponlawat Pattarakulpisutti and a herbarium specimen (S. Rattanaburi 1) has been deposited at the Herbarium within the Department of Biology, Faculty of Science, Prince of Songkla University, Thailand. 3.3. Extraction and isolation Ground, dried leaves (4.5 kg) of C. lanceolatus were extracted with CH2Cl2 at room temperature for 3 days. The viscous extract (349.5 g), after removal of solvent, was sequentially dissolved in
S. Rattanaburi et al. / Phytochemistry Letters 5 (2012) 18–21
20
hexane and Me2CO to give hexane soluble [A] (107.6 g), Me2CO soluble (77.3 g) and Me2CO insoluble fractions [B] (138.20 g). The Me2CO soluble fraction was further partitioned with EtOAc/ 5%NaOH to give an EtOAc layer [C] (16.20 g), an aqueous layer [D] and a solid [E] (30.70 g) that was formed during partition. The aqueous layer [D] after neutralization with HCl (14.30 g) was dissolved in MeOH from which 5,40 -dihydroxy-7-methoxy-6,8dimethylflavone (70.1 mg) precipitated. The filtrate from fraction D (11.90 g) was further purified by CC using CH2Cl2–MeOH (9:1) as eluant. The fractions that contained similar components, on the basis of their TLC characteristics, were combined to give fractions D1–D6. Fraction D2 (2.16 g) was further subjected to CC and eluted with Me2CO–hexane (3:17) to Me2CO–hexane (1:5) to give 5,40 -dihydroxy-7-methoxy-6,8-dimethylflavanone (10.4 mg) and 1 (7.3 mg). Fraction D3 (1.24 g) was chromatographed on silica with Me2CO–hexane (1:19) followed by Me2CO–hexane (7:3) to give a white solid nectandrin B (4.3 mg). Fraction D4 (2.098 g) was purified by CC on silica with hexane–Me2CO (9:1) yielding 16 fractions (D4A–D4P). Crystallization of fraction D4F in hexane– Me2CO (9:1) yielded 8-demethylsideroxylin (3.2 mg). Fraction D4J (246.0 mg) was chromatographed on silica using CH2Cl2– MeOH (49:1) as an eluant to give 6 portions (D4J.1–D4J.6). A yellow solid, 5,40 -dihydroxy-7-methoxy-6-methylflavanone (0.9 mg) was obtained from fraction D4J.4. Fraction D4J.5 (34.1 mg) was further purified by PTLC using CH2Cl2–MeOH (99:1) as eluant yielding a yellowish gum 2 (10.2 mg). The EtOAc layer [C] (16.20 g) was separated by silica CC sequentially eluting with hexane–CH2Cl2, CH2Cl2 and CH2Cl2–MeOH respectively. On the basis of their TLC characteristic, the fractions which contained the same major components were combined to give fractions C1– C7. Fraction C2 (20.1 mg) was crystallized from hexane–Me2CO (9:1) to yield a white solid 5-hydroxy-7,40 -dimethoxy-6-methylflavone (4.0 mg). The filtrate (12.5 mg) was rechromatographed on silica CC using hexane–Me2CO (9:1) as eluant to give a yellow solid, 5-hydroxy-7,40 -dimethoxy-6,8-dimethylflavone (7.8 mg). Fraction C5 (1.38 g) was chromatographed on silica CC using hexane-Me2CO (9:1) as an eluant to give white solids 3b-transferuloyloxy-2a-hydroxyurs-12-en-28-oic acid (1.3 mg) and olean-12-en-28-oic acid (3.0 mg). 3.3.1. Callislignan A (1) A yellowish gum. [a]D29 + 56.7 (c 0.80, MeOH). HRMS m/z 312.1362 for C19H20O4 (calcd. 312.1362). EI-MS m/z (% rel int): 312 Table 1 13 C, 1H, and HMBC spectral data of 1 (in acetone-d6). No.
dC
dH (mult., JHz)
HMBC (H ! C)
2 3 3a 4 5 6 7 7a 10 20 30 100 200 300 400 500 600 3-CH3 7-OH 400 -OH 300 -OCH3
93.1 45.5 133.5 112.2 131.9 113.3 140.9 146.8 131.2 122.1 17.5 132.0 110.0 147.6 146.1 114.7 119.5 16.8
4.97 (d, 9.6) 3.29 (obscure)
C-3, C-100 , C-200 , C-600 , 3-CH3
6.67 (br s)
C-3, C-6, C-7a, C-10
6.65 (br s)
C-4, C-7, C-7a, C-10
6.23 (dq, 17.1, 1.5) 5.94 (dq, 17.1, 6.6) 1.72 (dd, 6.6, 1.5)
C-20 C-5, C-10 , C-30 C-10 , C-20
7.03 (d, 1.8)
C-2, C-300 , C-400 , C-600
6.76 6.84 1.26 7.85 7.63 3.77
C-100 , C-300 , C-400 C-200 , C-400 C-2, C-3, C-3a C-6, C-7, C-7a C-300 , C-400 , C-500 C-300
55.4
(d, 8.4) (dd, 8.4, 1.8) (d, 6.6) (s) (s) (s)
Table 2 13 C, 1H, and HMBC spectral data of 2 (in CDCl3). No.
dC
1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 90 3-OCH3
133.1 113.2 146.8 145.8 110.6 118.9 78.3 83.5 16.8 134.5 113.2 148.7 144.4 119.6 117.6 130.4 124.8 18.4 55.9
dH (mult., JHz)
HMBC (H ! C)
6.93 (d, 1.8)
C-7, C-1, C-3, C-4, C-6
6.81 6.86 4.65 4.05 1.09
C-1, C-7, C-2, C-7, C-7,
(d, 8.4) (dd, 8.4, 1.8) (d, 8.4) (qd, 8.4, 6.3) (d, 6.3)
C-3, C-4, C-6 C-1, C-2, C-4, C-5 C-6, C-1, C-8, C-9 C-40 C-8
6.96 (d, 2.1)
C-40 , C-60 , C-70
6.90 6.74 6.30 6.86 1.85 3.88
C-60 , C-40 , C-20 , C-10 , C-70 , C-3
(d, 8.4) (dd, 8.4, 2.1) (dq, 15.6, 1.5) (dq, 15.6, 6.6) (dd, 6.6, 1.5) (s)
C-10 , C-20 , C-60 , C-70 , C-80
C-30 , C-40 C-70 C-80 , C-90 C-90
([M]+, 100), 297 (16), 149 (18), 137 (22), 105 (48), 77 (19). UV (MeOH) lmax nm (log e): 207 (3.84), 231 (3.51), 280 (3.19). IR (neat)n (cm1): 3392. 1H NMR spectroscopic data (300 MHz, acetone-d6) and 13C NMR spectroscopic data (75 MHz, acetoned6): see Table 1. 3.3.2. Callislignan B (2) A yellowish gum. [a]D29 15.8 (c 4.55, MeOH). HRMS m/z 330.1466 for C19H22O5 (calcd. 330.1467). UV (MeOH) lmax nm (log e): 205 (4.00), 227 (3.65), 269 (3.30). IR (neat) n (cm1): 3393. EI-MS m/z (% rel int): 330 [M] +, (57), 312 (70), 180 (30), 164 (41), 151 (93), 150 (100), 149 (61), 133 (23), 108 (36), 105 (44), 65 (47). 1H NMR spectroscopic data (300 MHz, CDCl3) and 13 C NMR spectroscopic data (75 MHz, CDCl3): see Table 2. 3.4. Antibacterial activity The crude extract and purified compounds were screened for antibacterial activity at a concentration of 200 mg/mL by a broth microdilution method against four bacteria: S. aureus ATCC25923, a clinical isolate of methicillin-resistant S. aureus SK1, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853. Crude extracts and purified compounds having antibacterial activity were further determined for their minimum inhibitory concentrations (MIC) by broth microdilution methods according to a modification of the Clinical and Laboratory Standards Institute. Resazurin was added as viability indicator according to Drummond and Waigh (2000). Colorimetric MIC end-points were interpreted as the lowest concentration that remained blue (indicating no growth) or the first dilution that changed from blue to slightly purple (equivalent to prominent growth inhibition). Acknowledgments We are grateful for the financial support from the Royal Golden Jubilee Ph.D. Program (a scholarship to S. Rattanaburi, Grant No. PHD/0284/2550), the Center for Innovation in Chemistry (PERCHCIC), the National Research University Project of Thailand’s Office of the Higher Education Commission and the Graduate School, Prince of Songkla University for scholarship. The Scientific Equipment Center Prince of Songkla University is also gratefully acknowledged for MS data.
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