antifungal metabolites from blumea balsamifera

Natural Product Research, Vol. 19, No. 3, April 2005, pp. 231–237

ANTIFUNGAL METABOLITES FROM BLUMEA BALSAMIFERA CONSOLACION Y. RAGASAa,*, ANGEL LYN KRISTIN C. COa and JOHN A. RIDEOUTb a

Chemistry Department, De La Salle University, 2401 Taft Avenue, Manila 1004, Philippines; School of Chemical and Biomedical Sciences, Central Queensland University, Rockhampton, Queensland, 4702 Australia

b

(Received 28 October 2003; In final form 10 March 2004)

The leaves of Blumea balsamifera afforded icthyothereol acetate, cryptomeridiol, lutein, and -carotene. The structures of icthyothereol acetate (1) and cryptomeridiol (2) were elucidated by extensive 1D and 2D NMR spectroscopy, while those of lutein and -carotene were identified by comparison with literature data. Antimicrobial tests indicated that 1 has moderate activity against the fungi Aspergillus niger, Trichophyton mentagrophytes, and Candida albicans, while 2 has low activity against A. niger, T. mentagrophytes, and C. albicans. Both compounds have no antimicrobial activity against Psuedomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, and Escherichia coli. Keywords: Blumea balsamifera; Compositae; Icthyothereol acetate; Cryptomeridiol; Lutein; -Carotene

INTRODUCTION Blumea balsamifera is a common medicinal plant found throughout the Philippines. The leaves are applied to the forehead to relieve headache. Leaf decoctions are used as an antidiarrhetic and antigastralgic, for stomach pains, and for aromatic baths in rheumatism. An infusion is used as a bath for women in child birth [1]. Earlier studies reported the essential oil constituents of B. balsamifera as follows: myrcene, cineole, and camphene [2], L-borneol, -caryophyllene, camphor, eudesmol, gurjunene, linalool, and others [3]. (2R,3R)-7,50 -Dimethoxy-3,5,20 -trihydroxyflavanone [4], (2R,3R)dihydroquerce tin-40 -methyl ether and (2R,3R)-dihydroquercetin-40 ,7-dimethyl ether [5] and other flavonoids [6,7], sesquiterpene lactones [8], blumealactones A, B, and C [9], and cryptomeridiol [10] were also reported from the plant. The blumealactones are antitumor agents [9], while cryptomeridiol is antispasmodic [10].

*Corresponding author. Tel./Fax: þ63 2 5360230. E-mail: [email protected] Natural Product Research ISSN 1478-6419 print: ISSN 1478-6427 online ß 2005 Taylor & Francis Ltd http://www.tandf.co.uk/journals DOI: 10.1080/14786410410001709773

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We now report the isolation, structure elucidation, and antifungal activities of icthyothereol acetate (1) and cryptomeridiol (2) from the dichloromethane extract of the leaves of B. balsamifera. Lutein and -carotene were also isolated from the plant. This is the first report on the isolation of 1 from B. balsamifera, the full structure elucidation of 1 and 2 by NMR spectroscopy, and their antifungal activities. 15

O

1 10

1

3'

1'

6'

3

9'

OCOCH3

HO

H

7

4

12 11

H

OH

14

1

13

2

RESULTS AND DISCUSSION The air-dried leaves of B. balsamifera afforded icthyothereol acetate (1), cryptomeridiol (2), lutein, and -carotene. The structures of 1 and 2 were elucidated by extensive 1D and 2D NMR spectroscopy as follows. The 1H NMR spectral data of 1 (Table I) indicated resonances for carbinyl methine protons at 3.78 and 4.49, and carbinyl methylene protons at 3.39 and 3.94; olefinic protons at 6.30 and 5.79; methylene protons at 1.47, 2.20, and 1.70 (2H); and two methyl singlets at 1.98 and 2.04. The large coupling constant between the carbinyl protons at 3.78 and 4.49 (J ¼ 9.4 Hz) indicated that they are both in axial positions. The large coupling constant (J ¼ 16 Hz) between the olefinic protons indicated trans coupling. The 13C NMR spectral data of 1 (Table I) gave resonances for 16 carbons with the following functionalities: two methine and one methylene carbinyl carbons at 78.8, TABLE I 300 MHz 1H and 75 MHz Position 1 2 3 4 5 10 20 30 40 50 60 70 80 90 OAc

C 78.8 71.5 29.3 24.7 67.5 144.1 111.4 73.5 75.6 58.9 67.4 64.9 78.2 4.6 169.9 21.1

H mult. (J Hz) 3.78 ddd (1.6, 5.3, 9.4) 4.49 ddd (4.7, 9.3, 10.6) 1.47, 2.20 1.70 (2H) 3.39 dt (3.8, 11.4), 3.94 m 6.30 dd (5.4, 15.9) 5.79 dd (1.7, 16.0)

13

C NMR spectral data of 1

HMBC correlations 0

H-1 , H-2 , H-5 H-1, H-3a H2-4, H-5a H-3b H-1, H-20 H-1 H-10 H-20 H-90 H-90 H-90 H-90

1.98 s OAc 2.04 s

0

NOESY correlations H-5a, H-3a, H-10 , H-20 H-3b, H-10 , H-4 H-3a, H-5a, H-1 H-2, H-4, H-30 H-2, H-1, H-5a, H-3b H-4, H-5b, H-1, H-3a H-1, H-2 H-1

ANTIFUNGAL METABOLITES FROM BLUMEA BALSAMIFERA

233

OCOCH3 4 1

: COSY : HMBC

O 1' 3'

6'

9' FIGURE 1

1

H-1H COSY and key 1H-13C long-range correlations for 1.

71.5, and 67.5; two methylenes at 29.3 and 24.7; two olefinic carbons at 144.1 and 111.4; a carbonyl at 169.9; and two methyl carbons at 21.1 and 4.6. The shielded methyl at 4.6 indicated that it is bonded to a conjugated triyne system at 58.9, 64.9, 67.4, 73.5, 75.6, and 78.2. An acetate was deduced from the resonances at 169.9 and 21.1. The COSY 2D NMR spectrum of 1 indicated the following spin system: H2-5/H2-4/ H2-3/H-2/H-1/H-10 /H-20 (Fig. 1). The 1H and 13C assignments of 1 (Table I) were verified by HSQC and connectivity was verified by HMBC (Table I and Fig. 1). The olefin was placed at C-1 due to longrange correlations between H-1, and C-10 , C-20 . The correlations between C-1 and H-5a indicated that C-1 and C-5 are joined through an oxygen to form a ring system. The second carbinyl was placed at C-2 due to long-range correlations of this carbon to H-1 and H-3a. Three conjugated triple bonds were bonded to C-20 . The molecule ended with a methyl singlet at 1.98 which was long-range correlated to four of the triple bond carbons at 78.2, 64.9, 67.4, and 58.9. All long-range correlations observed were consistent with the structure of 1. The NOESY spectrum did not indicate correlations between H-1 and H-2; and H-10 and H-20 . Hence, H-1 and H-2, and H-10 and H-20 are on the opposite faces of the molecule. This is supported by the coupling constants which indicate that H-1 and H-2 are in the axial position, while H-10 and H-20 are trans to each other. Literature search revealed that 1 is icthyothereol acetate which was previously reported as constituent of Ichthyothere terminalis [11]. Chemical Abstracts also lists a compound with the same relative stereochemistry as 1 under the separate name of cunaniol acetate. The 1H NMR spectral data of 2 (Table II) indicated resonances for four methyl singlets at 0.87, 1.12, and 1.20 (2CH3), and methylene and methine resonances in the shielded region ( 1.02–1.93). The 13C NMR spectral data (Table II) gave resonances for 15 carbons broken down as follows: four methyl, six methylene, two methine, and three quaternary (two oxygenated at 72.3 and 73.0) carbons. Assuming two alcohol functionalities, the molecular formula of 2 is C15H28O2. These are characteristic resonances for a bicyclic sesquiterpene with two quaternary alcohols. The COSY 2D NMR spectrum of 2 showed correlations of two spin systems as follows: H2-1/H2-2/H2-3 and H-5/H2-6/H-7/H2-8/H2-9 (Fig. 2).

234

C.Y. RAGASA et al. TABLE II 300 MHz 1H and 75 MHz C

H mult. (J Hz)

1

41.0

2 3

20.2 43.5

4 5

72.3 54.8

1.10, 1.39 1.57 (2H) 1.36, 1.79 – 1.22 dd

6

21.5

7 8

49.9 22.5

9

44.6

10 11 12 13 14 15

34.5 73.0 27.1 27.3 22.6 18.7

Position

1.05, 1.93 1.38 1.30, 1.60 1.18, 1.45 dt (2.8, 12.4) – – 1.20 s (Me) 1.20 s (Me) 1.12 s (Me) 0.87 s (Me)

FIGURE 2

13

C NMR spectral data of 2

HMBC correlations H-2, H-30 , H-9, H-15 H-1, H-10 , H-3, H-30 H-1, H-10 , H-2, H-5, H-15, H-2 H-1, H-2, H-3, H-30 , H-5, H-14 H-1, H-3, H-30 , H-6, H-60 , H-7, H-90 , H-14, H-15 H-5, H-8 H-5, H-6, H-8, H-12, H-13 H-6, H-7, H-9, H-90 H-8, H-15 H-1, H-2, H-5, H-6, H-15 H-6, H-12, H-13 H-13, H-6, H-7 H-12, H-6, H-7 H-3, H-5 H-1, H-5, H-6, H-9

NOESY correlations H-10 , H-30 , H-15 H-1 H-30 , H-15 H-1, H-2, H-3, H-60 , H-80 H-2, H-6, H-6 H-80 H-8, H-6, H-9,

H-60 , H-7, H-15 H-5 H-9 H-90 H-8

H-6, H-7 H-6, H-7 H-15 H-2, H-14

1

H-1H COSY and key 1H-13C long-range correlations for 2.

The 1H and 13C assignments (Table II) were verified by HSQC and connectivity was verified by HMBC (Table II and Fig. 2). One of the hydroxyls was attached to C-4 due to long-range correlation between this carbon and H-1, H-2, H-3, H-30 , H-5, and H-14. The second hydroxyl was attched to C-11 since long-range correlations were observed between C-11, H-6, H-12, and H-13. The H-15 methyl was attached to C-10 due to long-range correlations between C-10, H-1, H-2, H-5, H-6, and H-15. All long-range correlations observed were consistent with the structure of 2. The NOESY spectral data (Table II) indicated the following correlations. The methyl singlet at 1.12 (H-14) is close in space to the methyl singlet at 0.87 (H-15), which is in turn close to the methine proton at 1.38 (H-7). Thus, they are in the same face of the molecule. The methine proton at 1.22 (H-5) is in the opposite face of the molecule since it does not correlate with any of the aforementioned protons. Literature search revealed that 2 is cryptomeridiol which was previously reported as constituent of B. balsamifera [10]. Lutein [12] and -carotene [13] were identified by comparison of their 1H NMR spectral data with those found in the literature. Compounds 1 and 2 were tested for possible antimicrobial activity by the agar cup method. Results of the study (Table III) indicated that 1 has moderate activity

Sample

Conc. (mg)

Staphylococcus aureus CZ* (mm)

1 2 Standard Antibiotic

30 30 30

AI

– 0 – 0 25 3.2 Chloramphenicol

Escherichia coli CZ* (mm)

Pseudomonas aeruginosa AI


CZ: Clear zone; AI: activity index. *Average of three trials.

CZ* (mm)

AI


Bacillus subtilis CZ* (mm)

Candida albicans AI


CZ* (mm)

Aspergillus niger AI

13 0.3 12 0.2 10 0.7 Chlotrimazole

CZ* (mm)

Trichophyton mentagrophytes AI

14 0.4 12 0.2 10 0.7 Chlotrimazole

CZ* (mm)

AI

18 0.8 13 0.3 50 7.3 Chlotrimazole


TABLE III Antimicrobial test results on 1 and 2

235

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C.Y. RAGASA et al.

against Candida albicans with AI of 0.3, Trichophyton mentagrophytes with AI of 0.8, and Aspergillus niger with AI of 0.4 at a mass of 30 mg. Compound 2 has low activity against C. albicans with AI of 0.2, T. mentagrophytes with AI of 0.3, and A. niger with AI of 0.2 at the same mass. Both compounds were inactive against the bacteria, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis.

MATERIALS AND METHODS General Experimental Procedures NMR spectra were recorded on a Bruker Avance 400 NMR spectrometer in CDCl3 (400 MHz for 1H NMR and 100 MHz for 13C NMR). Column chromatography was performed with silica gel 60 (70–230 mesh). TLC was performed with plastic backed plates coated with silica gel F254; plates were visualized by spraying with vanillin– H2SO4 and warming. Sample Collection The samples were obtained from Altermed Corporation in Sta. Rosa, Nueva Ecija in October 2002. It was identified as Blumea balsamifera at the Philippine National Museum and voucher specimen # 070 was deposited at the Chemistry Department of De La Salle University. Extraction and Isolation Air-dried leaves (1 kg) of B. balsamifera were extracted with ethyl acetate to afford a crude extract (12 g), which was fractionated by gravity column chromatography using increasing proportions of acetone in dichloromethane (10% increment) as eluents. The dichloromethane fraction was rechromatographed in petroleum ether (b.pt. 35–60 C), then washed with petroleum ether, followed by diethyl ether to afford -carotene (15 mg). The 10 and 20% acetone in dichloromethane fractions were rechromatographed in 5% ethyl acetate in petroleum ether, followed by 2.5% ethyl acetate in petroleum ether, then petroleum ether, and finally 5% ethyl acetate in petroleum ether to afford 1 (12 mg). The 60% acetone in dichloromethane fraction was rechromatographed in CH3CN : Et2O : CH2Cl2 (1.5 : 1.5 : 7) to afford an orange solid which was washed with petroleum ether, followed by diethyl ether. The orange solid was rechromatographed in CH3CN : Et2O : CH2Cl2 (0.5 : 0.5 : 9) to afford lutein (20 mg) after washing with diethyl ether. The 90% acetone in dichloromethane fraction was rechromatographed (4X) in CH3CN : Et2O : CH2Cl2 (1.5 : 1.5 : 7) to afford 2 (9 mg). Antimicrobial Tests The microorganisms used in these tests were obtained from the University of the Philippines Culture Collection (UPCC). These are P. aeruginosa UPCC 1244, B. subtilis UPCC 1149, E. coli UPCC 1195, S. aureus UPCC 1143, C. albicans UPCC 2168, T.


237

mentagrophytes UPCC 4193 and A. niger UPCC 3701. The antimicrobial assay procedure reported in the literature was employed [14]. Acknowledgments The antimicrobial tests were conducted at the University of the Philippines-Natural Sciences Research Institute (UP-NSRI). A research grant from the College Research Fund and University Research Coordination Office of De La Salle University is gratefully acknowledged. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]

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