Philippine Journal of Science 133 (2): 97-101, December 2004 ISSN 0031 - 7683
Antimicrobial Compounds from Artocarpus heterophyllus Consolacion Y. Ragasa*1, Karen Jorvina1 and John A. Rideout2 1
Chemistry Department, De La Salle University 2401 Taft Avenue, Manila 1004, Philippines 2 School of Chemical and Biomedical Sciences Central Queensland University, Queensland 4701, Queensland, Australia The freeze-dried unripe fruit of Artocarpus heterophyllus Lam., common name: langka, afforded cycloartenone 1, cycloartenol 2, and a diastereomeric mixture of 2,3-butanediols 3a and 3b in a 3:1 ratio. Antimicrobial tests on 1-3 indicated that 1 has low activity against E. coli, P. aeruginosa, and T. mentagrophytes, moderate activity against C. albicans and A. niger and inactive against S. aureus and B. subtilis. 2 has no antibacterial activity and only low antifungal activity. The diastereomeric mixture of 3 exhibited high activity against P. aeruginosa, moderate activity against C. albicans, and low activity against S. aureus, T. mentagrophytes, and A. niger. 3 has the same activity as the standard antibiotic against P. aeruginosa. Keywords: Artocarpus heterophyllus Lam., Moraceae, langka, cycloartenone, cycloartenol, 2,3-butanediol, antimicrobial
introduction
heterophyllus. The antimicrobial test results on 1-3 are also reported. To the best of our knowledge this is the first report on the isolation of 3a and 3b from A. heterophyllus, and their antimicrobial properties.
Artocarpus heterophyllus Lam., commonly known as langka, is cultivated throughout the Philippines for its fruit which is eaten fresh or made into preserves and other sweets. The unripe fruit is used as an astringent, while the ripe fruit is used as demulcent, nutritive and laxative (Quisumbing 1978). A number of studies were conducted on A. heterophyllus which reported the isolation of cycloartenone, cycloartenol, and other tetracyclic triterpenoids (Baric et al. 1994), flavonoids (Lu & Lin 1994; Lu et al. 1995; Radhakrishnan et al. 1965; Chung et al. 1995), and Diels-Alder-type adducts (Shinomiya et al. 1995; Aida et al. 1990).
18 12
19 1 18
R 12
20
17 28 29 H This paper reports the isolation of cycloartenone 19 14 1, cycloartenol 2, and a diastereomeric mixture of 1 R = O 24 1 9 2,3-butanediol 3a and 3b from the unripe fruit of A. 2 R = H, b-OH 30 5 R
*Corresponding author:
[email protected]
H 28
29
27
CH3
CH3 H
24
H
30
5
H
17 14
H
9
20
27 CH3
OH
HO
OH
H
H OH
CH3
CH3
3aCH 3
3b
H
OH
HO
H
OH
H
H OH
CH3
CH3
3a
3b
97
Ragasa et al.
Materials and Methods General Experimental Procedures NMR spectra were recorded on a Bruker Avance 400 in CDCl3 at 400 MHz for 1H and 100 MHz for 13C. Column chromatography was performed with silica gel 60 (70-230 mesh), while the TLC was performed with plastic backed plates coated with silica gel F254. The plates were visualized with vanillin-H2SO4 and warming. Sample Collection Freeze-dried fruits of Artocarpus heterophyllus were collected from Las Piñas, Philippines in October 2003. It was identified as Artocarpus heterophyllus Lam. at the Philippine National Museum and a voucher specimen # 73 is kept at the Chemistry Department, De La Salle University, Manila, Philippines. Isolation The freeze-dried fruits (1.3 kg) of A. heterophyllus were ground in an osterizer, soaked in dichloromethane for three days, then filtered. The filtrate was concentrated under vacuum to afford a crude extract. The treated extract was chromatographed on silica gel with increasing proportions of acetone in dichloromethane (10% increments). The 30%-40% acetone in dichloromethane fractions were rechromatographed in 5% ethyl acetate in petroleum ether, then 2.5% ethyl acetate in petroleum ether to produce 1 (12.5 mg). The 40%-50% acetone in dichloromethane fractions were rechromatographed (2x) in dichloromethane to produce 2 (9.7 mg). The acetone fraction was rechromatographed in acetonitrile:diethyl ether:dichloromethane (0.5:0.5:9) to produce a mixture of 3a and 3b (colorless oil, 8.6 mg).
28.1 (C-7), 48.0 (C-8), 20.1 (C-9), 26.1 (C-10), 26.0 (C-11), 37.3 (C-12), 45.3 (C-13), 48.8 (C-14), 32.9 (C-15), 26.5 (C-16), 52.3 (C-17), 18.0 (C-18), 29.9 (C19), 35.9 (C-20), 18.2 (C-21), 36.4 (C-22), 25.0 (C-23), 125.3 (C-24), 130.9 (C-25), 17.6 (C-26), 25.7 (C-27), 19.3 (C-28), 14.0 (C-29), 25.4 (C-30).
Results and Discussion Silica gel chromatography of the dichloromethane extract from the freeze-dried unripe fruit of A. heterophyllus produced cycloartenone 1, cycloartenol 2, and a diastereomeric mixture of 2,3-butanediol 3a and 3b. The structure of 1 was elucidated by extensive 1D and 2D NMR spectroscopy and confirmed by mass spectrometry, while 2 was elucidated by comparison of its 1H and 13C NMR spectral data with those of 1. The structures of the diastereomeric mixture of 3a and 3b were deduced by NMR spectroscopy and their 1 H NMR compared with those found in the literature (Pouchert 1983). The 1H NMR spectrum of 1 (Table 1) indicated resonances for an olefinic proton, δ 5.10 (H-24) allylically coupled to two methyl groups at δ 1.61 (H-26) and 1.69 (H-27); four methyl singlets at δ 1.10 (H-28), 1.05 (H-29), 1.00 (H-18), 0.91 (H-30), and a methyl doublet at δ 0.89 (H-21). The doublets at δ 0.57 (H-19a) and 0.79 (H-19b) indicated a cyclopropyl ring. These
18
Antimicrobial Tests The microorganisms used in these tests were obtained from the University of the Philippines Culture Collection (UPCC) Diliman. These are Pseudomonas aeruginosa UPCC 1244, Bacillus subtilis UPCC 1149, Escherichia coli UPCC 1195, Staphylococcus aureus UPCC 1143, Candida albicans UPCC 2168, Trichophyton mentagrophytes UPCC 4193 and Aspergillus niger UPCC 3701. The test compound was dissolved in 95% ethanol. The antimicrobial assay procedure reported in the literature (Guevara et al. 1985) was employed. Cycloartenone 1: 1H and 13C NMR (see Table 1). Cycloartenol 2: 1H NMR: 3.25 (H-3), 0.45 (H-19), 0.65 (H-19'), 5.10 (H-24), 13C NMR: 32.0 (C-1), 30.40 (C-2), 78.9 (C-3), 40.5 (C-4), 47.1 (C-5), 21.1 (C-6),
98
12
19 1
H
9
17 14
20 23
5
O
27
H
Figure 1. 1H- 1H COSY for 1.
Fig. 1.
1 H- 1 H COSY for
1
resonances are characteristic of cycloartenone. The COSY (Figure 1) showed four isolated spin systems, H2-1/H2-2, H-5/H2-6/H-7/ H-8, H2-12/H2-13, H2-15/H2-16/H-17/H-20/H-3-21/H2-22/H2-23/H-24/H26/H-27. The 1H and
13
C assignments of 1 (Table 1) were
Artocarpus heterophyllus
Table 1. 400 MHz 1H NMR and 100MHz 13C NMR, HMBC and NOESY correlations of 1 Position
δC
δH mult.* (J Hz)
HMBC Correlations
1
33.4
1.56, 1.86
H2-19, H-5, H2-2
2
37.6
2.28 ddd (2.8, 4.4, 14.0) 2.70 dt (6.4, 14.0)
H2-1
3
216.5
H2-1, H2-2, H3-28, H3-29
4
50.2
H-2a, H-5, H3-28, H3-29
5
48.5
1.70
H2-2, H2-6, H2-7, H2-19, H3-28, H3-29
6
21.5
1.55, 0.95
H-5
7
28.1
1.30, 1.90
8
47.9
1.59
9
21.1
10
27.0
11
26.8
1.15, 2.05
H-8, H2-19
12
32.8
1.65
13
45.3
14
48.7
H-11, H-17, H3-18 H-1b, H-7b, H2-12, H-15a, H3-18, H3-30 H-15, H2-16, H-17, H3-18, H3-30
15
25.9
1.10, 1.40
H-17
16
35.6
1.30
17
52.3
1.60
H3-18, H3-21
18
17.6
19
29.6
H2-2, H-5, H-8, H2-11
20
35.9
1.00 s, Me 0.57 (4.4 ) 0.79 (4.4) 1.40
21
19.3
0.89 d (7.6), Me
H-17, H-20
22
36.3
1.05, 1.45
H-24
23
25.0
1.90, 2.10
H-24
24
125.2
5.10
H2-22, H2-23, H3-28, H-29
25
130.9
26
18.2
1.61 s, Me
H-24
H-24, H-25
27
25.7
1.69 s, Me
H-24
H-24
28
20.8
1.10 s, Me
H3-29
29
22.2
1.05 s, Me
H3-28
H-5
30
18.1
0.91 s, Me
H-7, H-15
H-15b
NOESY Correlations
H-2b, H2-1 H-2a, H-1a
H2-11, H2-19, H3-30
H-7b, H-29
H-11b, H-18, H-19b, H-28
H-5, H-8, H2-11, H2-19 H-5, H-19
H-17 H2-16, H3-21, H2-22, H-23
H-11b H-1b, H-11a, H-12, H3-30
H-8, H-17, H-20 H-1b, H-8, H-2b, H3-28, H3-18 H-8, H3-18, H-19a, H3-28
H3-27
H2-23, H3-26, H3-27
*Multiplet unless otherwise indicated.
18 12
19
H
9
1
17
20 23
14
5
O
H 1
27
13
Figure 2. Key H- C long-range correlation for 1
Fig. 2. Key 1 H- 13 C long-range correlations for 1
verified by HSQC and their connectivities were verified by HMBC (Table 1 and Figure 1). The carbonyl was placed at C-3 due to long-range correlations between this carbon and H2-1, H2-2, H-28, and H-29. The cyclopropyl was placed at C-9 and C-10 due to long-range correlations between H2-19 and C-1, C-9, C-10, and C-11. The olefinic carbons (C-24 and C-25) were assigned on the basis of long-range correlations of these carbons to the allylic methyls (H3-26 and H3-27). The relative stereochemistry of 1 was deduced from NOESY spectral data interpretation. The cyclopropyl proton (H-19a) was close to H-1, H-2', H-29, and H-8 which was
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Ragasa et al.
The 1H NMR spectrum of 3 indicated resonances for a mixture of diastereomers in a 3:1 ratio. Based on resonance intensities, the methyl doublet at δ 1.12 (H-1, H-4, J = 6.3 Hz) and the carbinyl proton δ 3.78 (H-2, H-3) were attributed to the major compound 3a, while the methyl doublet at δ 1.16 (H-1, H-4, J = 6.0 Hz) and the carbinyl proton at δ 3.50 (H-2, H-3) were attributed to the minor compound 3b. These resonances were compared with those reported in the literature (Pouchert 1983) for the meso isomer and the optically active isomer of 2,3-butanediol, respectively. The 13C NMR spectrum of 3 gave resonances for carbinyl carbon at δ 70.87 and methyl carbon at δ 16.93 for 3a, while the resonances at δ 72.51 and δ 19.29 were attributed to 3b. These compounds were reported as constituents of the Malay rose apple, Syzygium malaccense (L.) Merr. and Perryl (Pinoja et al. 2004) and Cystoseira crinita Bory (Kamenarska 2002). They could also be produced by fermentation of glucose or xylose (Voloch et al. 1983).
in turn close to H3-18, H-17, and H-20. This indicated that they are at the same side of the molecule. On the other side of the molecule is H-5 which is close to H-14. The molecular formula of 1 was confirmed by high resolution electron impact mass spectrometry (HREIMS). A molecular ion was observed at 424.3697 [M+], while the calculated value for a compound with molecular formula C30H48O is 424.3705. Thus, 1 is cycloartenone. The 1H NMR spectrum of 2 indicated a similar structure to that of 1. The differences are the appearance of a carbinyl proton (δ 3.25, H-3) in 2 and the absence in 2 of the methylene protons α to the carbonyl (δ 2.70 and 2.28) in 1. The 13C NMR spectrum of 2 indicated a carbinyl at δ 78.9 and the absence of the carbonyl at δ 216.5. This indicated that the carbonyl in 1 was converted to a carbinyl in 2. Thus, 2 is cycloartenol. Confirmatory evidence is the 1H NMR data of 2 and cycloartenol (De Pascual et al. 1987 ). They match in all essential respects.
Table 2. Antimicrobial test results on 2, and 3a and 3b from A. heterophyllus fruit SAMPLE
Concn. (µg)
Staphylococcus aureus
Escherichia coli
Pseudomonas aeruginosa
Bacillus subtilis
Candida albicans Aspergillus niger
Trichophyton mentagrophytes
C.Z.* (mm)
A.I.
C. Z.* (mm)
A.I.
C. Z.* (mm)
A.I.
C.Z.* (mm)
A.I.
C.Z.* (mm)
A.I.
C.Z.* (mm)
A.I.
C.Z.* (mm)
A.I.
1 2 3a and 3b
30 30 30
13
0 0 0.3
11 -
0.1 0 0
11 13
0.1 0.3
-
0 0 0
14 12 13
0.4 0.2 0.3
13 12 11
0.3 0.2 0.1
14 12 12
0.4 0.2 0.2
Standard
30
25
3.2
23
2.8
8
0.3
20
2.3
10
0.7
10
0.7
50
7.3
Antibiotic
Chloramphenicola Chloramphenicola Chloramphenicola Chloramphenicola Chlortrimazoleb Chlortrimazoleb Chlortrimazoleb
CZ - clear zone, *Average of three trials, AI - activity index, achloramphenicol at 30 mg, bchlotrimazole at 50 mg
Since the unripe fruit is used as an astringent, 1-3 were tested for possible antimicrobial activities. Results of the study (Table 2) indicated that 1 has low activity against E. coli, P. aeruginosa, and T. mentagrophytes, moderate activity against C. albicans and A. niger and inactive against S. aureus and B. subtilis. Compound 2 had no antibacterial activity and low antifungal activity against C. albicans, T. mentagrophytes, and A. niger. The diastereomeric mixture of 3a and 3b indicated same activity as the standard antibiotic against P. aeruginosa, moderate activity against C. albicans, and low activity against S. aureus, T. mentagrophytes and A. niger.
Acknowledgments The antimicrobial tests were conducted at the University of the Philippines-Natural Sciences Research Institute (UP-NSRI). A research faculty grant from the De La Salle University is gratefully acknowledged.
References Aida M, Hano Y, & Nomura T. 1990. Journal of Natural Products 53(2):391-395. Baric BR, Bhaumik AK, Dey AK & Kundu AB. 1994. Phytochemistry 35(4):1001-1004. Chung MI, Lu CM, Lin CN & Huang PL. 1995. Phytochemistry 40(4):1279-1282.
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De Pascual TJ, Urones JG, Marcos IS, Basabe P, Cuadrado, MJS & Moro RF. 1987. Phytochemistry 26:1767-1776. Guevara BQ & Recio BV. 1985. Acta Manilana Supplements. Manila: UST Research Center. Kamenarska Z, Yalcin FN, Ersoz T, Calis I, Stefanov K & Popov S. 2002. Z. Naturforsch 57C:584-590. Lu CM & Lin CN. 1994. Phytochemistry 35(3): 781-783. Lu CM, Lin CN & Huang PL. 1995. Phytochemistry. 39(6):1447-1451. Pinoja JA, Marbot R, Rosado A & Vasquez C. 2004. Flavour and Fragrance Journal 19(1):32-35. Pouchert CJ. 1983. The Aldrich Library of NMR Spectra. 1:123D-124A. 2nd Ed. USA: The Aldrich Chemical Company, Inc. Quisumbing E. 1978. Medicinal Plants of the Philippines. Manila: Bureau of Printing. Radhakrishnan AV, Rama Rao AV & Venkataraman K. 1965. Tetrahedron Letters 11:663-667. Shinomiya K, Aida M, Hano Y & Nomura T. 1995. Phytochemistry 40(4):1317-1319. Voloch M, Ladisch MR, Rodwell VW & Tsao GT. 1983. Biotechnol Bioeng 25:173-183.
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