Secondary metabolites from Tectona philippinensis

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Secondary metabolites from Tectona philippinensis

Consolacion Y. Ragasa ab; Maria Claribel Lapina a; Jadz Jevz Lee a; Emelina H. Mandia bc; John A. Rideout d a Chemistry Department, De La Salle University, 1004 Manila b Center for Natural Sciences and Ecological Research, De La Salle University, 1004 Manila c Biology Department, De La Salle University, 1004 Manila d School of Chemical and Biomedical Sciences, Central Queensland University Rockhampton, Queensland, 4702 Australia Online Publication Date: 01 June 2008 To cite this Article: Ragasa, Consolacion Y., Lapina, Maria Claribel, Lee, Jadz Jevz, Mandia, Emelina H. and Rideout, John A. (2008) 'Secondary metabolites from Tectona philippinensis', Natural Product Research, 22:9, 820 — 824 To link to this article: DOI: 10.1080/14786410701640551 URL: http://dx.doi.org/10.1080/14786410701640551

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Natural Product Research, Vol. 22, No. 9, 15 June 2008, 820–824

Secondary metabolites from Tectona philippinensis CONSOLACION Y. RAGASA*yz, MARIA CLARIBEL LAPINAy, JADZ JEVZ LEEy, EMELINA H. MANDIAzx and JOHN A. RIDEOUT{ yChemistry Department, De La Salle University, 2401 Taft Avenue, 1004 Manila zCenter for Natural Sciences and Ecological Research, De La Salle University, 2401 Taft Avenue, 1004 Manila xBiology Department, De La Salle University, 2401 Taft Avenue, 1004 Manila {School of Chemical and Biomedical Sciences, Central Queensland University Rockhampton, Queensland, 4702 Australia (Received 27 May 2006; in final form 12 February 2007) The air-dried leaves of Tectona philippinensis, an endemic and endangered Philippine medicinal plant, afforded 5-hydroxy-3,7,40 -trimethoxyflavone (1), 5,40 -dihydroxy-3,7-dimethoxyflavone (2), squalene (3), a mixture of lupeol (4a) and -amyrin (4b), chlorophyllide a (5), and hydrocarbons. Antimicrobial tests on 1 and 2 indicated low antifungal activity against the fungi, Candida albicans and Trichophyton mentagrophytes. Compound 1 was also found to have low antibacterial activity against Escherichia coli and Pseudomonas aeruginosa. Keywords: Tectona philippinensis; Verbenaceae; 5-Hydroxy-3,7,40 -trimethoxyflavone; 5,40 -Dihydroxy-3,7-dimethoxyflavone; Squalene; Lupeol; -Amyrin; Chlorophyllide

1. Introduction Tectona philippinensis is an endemic and endangered Philippine medicinal plant, which can only be found in Batangas and Occidental Mindoro. There are about 2500 trees found in the different municipalities of Batangas and Mindoro, which are now undergoing conservation efforts by the Philippine government [1]. The charred wood soaked in poppy juice and made into a paste was used to relieve the swelling of eyelids, while a decoction of the leaves is a cure to blood related disorders like menstruation [2]. To date, there is no reported chemical study on the plant. We report here the isolation and identification of 5-hydroxy-3,7,40 -trimethoxyflavone (1), 5,40 -dihydroxy-3,7-dimethoxyflavone (2), squalene (3), a mixture of lupeol (4a) and -amyrin (4b), chlorophyllide a (5), and hydrocarbons from T. philippinensis.

*Corresponding author. Email: [email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410701640551

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Secondary metabolites from T. philippinensis

821

OCH3 H3CO

O

OH H3CO

O

OCH3 OH

OCH3

O

OH

O 2

1

HO

3

4a

N

N Mg

N

N

O

HO

O O O

4b

HO 5

2. Results and discussion The dichloromethane extract of T. philippinensis afforded 5-hydroxy-3,7,40 -trimethoxyflavone (1), 5,40 -dihydroxy-3,7-dimethoxyflavone (2), squalene (3), lupeol (4a), -amyrin (4b), chlorophyllide a (5), and hydrocarbons. Their structures were identified by NMR spectroscopy and comparison of their NMR spectral data with those found in the literature. The 1H NMR spectrum of 1 indicated resonances for aromatic protons at  8.08 (2H, dd, J ¼ 4, 8 Hz), 7.03 (dd, J ¼ 4, 8 Hz), 6.45 (d, J ¼ 4 Hz), and 6.36 (d, J ¼ 4 Hz). Thus, the proton at  8.08 is ortho coupled to  7.03, while the resonance at  6.45 is

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meta coupled to  6.36. Three methoxy groups were deduced from the resonances at  3.90, 3.88, and 3.86. A chelated hydroxyl was also observed at  12.70. The 13C NMR spectrum of 1 indicated resonances for aromatic carbons at  165.38, 162.01, 161.65, 130.12, 114.2, 156.73, 156.1, 139.0, 123.2, and 106.0. The deshielded carbon resonances ( 165.38–139.0) are oxygenated, while the shielded resonances ( 123.2–106.0) are non-oxygenated. Protonated aromatic carbons were found at  97.75, 92.14, 130.5 (2C), and 114.4 (2C). A conjugated carbonyl carbon was deduced from the resonance at  179.1, while three methoxy groups were found at  55.74, 55.38, and 60.09. The 1H and 13C NMR resonances of 1 are characteristics of a flavone with one hydroxyl and three methoxy groups. Literature search revealed that 1 is 5-hydroxy3,7,40 -trimethoxyflavone as evidenced by similar 1H and 13C NMR spectral data [3]. Compound 2 gave similar 1H NMR spectral data to 1. The ortho aromatic protons at  7.83 (2H) and 6.82 (2H), the meta aromatic protons at  6.28 and 6.12, the chelated hydroxyl at  12.43, and two methoxy groups at  3.66 and 3.69 remained in 2. The only difference is that there are only two methoxy groups in 2, while 1 contained three methoxy groups. An additional hydroxyl was found in 2 at  8.53. This indicated that one of the three methoxy groups in 1 was a converted to a hydroxyl in 2. Literature search revealed that 2 is 5,40 -dihydroxy-3,7-dimethoxyflavone as evidenced by similar 1 H NMR resonances [3]. Compound 3 gave resonances for olefinic protons at  5.10–5.15, allylic methyl groups at  160 and 1.68, and allylic methylene protons at  1.96–2.09. These are characteristic resonances for squalene [4]. Isolate 4 is a mixture of two triterpenes (4a and 4b) in a 2 : 1 ratio, which was deduced from the disparity in single hydrogen peaks in the 1H NMR spectrum. The major compound 4a gave resonances for olefinic methylene protons at  4.68 and 4.69, a carbinyl proton at  3.18, an allylic methyl singlet at  1.68, and six methyl singlets at  0.97, 0.76, 0.83, 1.03, 0.97 and 0.79. This was identified as lupeol [5]. The presence of -amyrin (4b) in the mixture was deduced from the olefinic proton at  5.13, carbinyl proton at  3.20 and methyl singlets at  0.79, 0.83, 0.87, 0.95, 0.97, and 1.00. These resonances are similar to those reported for -amyrin [6]. Compound 5 was obtained as a blue violet crystal. It was isolated from the dark green fractions obtained in the initial chromatography of the crude extract. Literature search revealed that 5 had similar 1H NMR resonances to chlorophyll a [7]. The difference is in the side chain. Whereas, chlorophyll a has a phytyl side chain, 5 has a carboxylic acid with no esterified side chain. Compound 5 was identified as chlorophyllide a, which we also isolated from Macaranga tanarius. The structure of chlorophyllide a from M. tanarius was elucidated by extensive 1D and 2D NMR spectroscopy and comparison with the 13C NMR data of chlorophyll a [8]. A mixture of hydrocarbons was deduced from the resonances at  1.25 for methylene protons and  0.87 for the terminal methyl groups. As part of our continuing search for possible antimicrobial compounds from Philippine medicinal plants, 1 and 2 were tested for their antimicrobial potential against seven microorganisms. Results of the study (table 1) indicated that 1 is active against the fungus, Candia albicans with an activity index of 0.3 at a concentration of 30 mg. It was also found to be slightly active against the fungus, Trichophyton mentagrophytes and the bacteria, Escherichia coli and Pseudomonas aeruginosa with an activity index of 0.2 at the same concentration. It was inactive against Staphylococcus aureus, Bacillus subtilis, and Aspergillus niger. On the other hand, 2 has slight activity against

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Secondary metabolites from T. philippinensis Table 1.

823

Antimicrobial test results on 1 and 2. Clearing zone (mm)

Organism E. coli P. aeruginosa S. aureus B. subtilis C. albicans T. mentagrophytes A. niger

a

Sample (30 mg)

Replicate 1

Replicate 2

Replicate 3

1 2 Chloramphenicol 1 2 Chloramphenicol 1 2 Chloramphenicol 1 2 Chloramphenicol 1 2 Canesten, 0.2 ga 1 2 Canesten, 0.2 ga 1 2 Canesten, 0.2 ga

12 – 23 12 – 14 – – 25 – – 20 13 12 18 12 12 55 – – 23

12 –

12 –

12 –

13 –

– –

– –

– –

– –

13 12

13 12

12 12

12 12

– –

– –

Antimicrobial index (AI) 0.2 0 2.8 0.2 0 1.3 0 0 3.2 0 0 2.3 0.3 0.2 0.8 0.2 0.2 4.5 0 0 1.3

Contains 1% chlotrimazole.

the fungi, C. albicans and T. mentagrophytes with an activity index of 0.2 at a concentration of 30 mg. It was inactive against P. aeruginosa, E. coli, S. aureus, B. subtilis, and A. niger.

3. Experimental 3.1. General experimental procedures NMR spectra were recorded on a Bruker Avance 400 in CDCl3 at 400 MHz for H NMR and 100 MHz for 13C NMR Spectra. 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 sulfuric acid and warming.

1

3.2. Sample collection The plant material was collected from La Luz Beach Resort in San Juan, Batangas in September, 2005. It was identified as T. philippinensis by Emelina Mandia of the Biology Department at De La Salle University, Manila. 3.3. Isolation The air-dried leaves of T. philippinensis (800 g) was extracted with dichloromethane to afford a crude extract (79.0 g). The extract was chromatographed on silica gel with

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C. Y. Ragasa et al.

increasing proportions of acetone in dichloromethane (10% increments) as eluents. The DCM fraction was rechromatographed by gradient elution technique. It was first eluted with petroleum ether to afford hydrocarbons (colourless solid, 100 mg), followed by 2.5% ethyl acetate in petroleum ether to afford 3 (colourless oil, 15 mg). The 20% acetone in DCM fraction was rechromatographed in 10% ethyl acetate in petroleum ether. The more polar fractions afforded 1 (needle-like yellowish crystals, 15 mg). The less polar fractions were rechromatographed in 5% ethyl acetate in petroleum ether, followed by 2.5% ethyl acetate in petroleum ether to afford a mixture of 4a and 4b (colourless solid, 10 mg). The 30% acetone in DCM fraction was rechromatographed in 1 : 1 : 8 (acetonitrile : diethyl ether : DCM), followed by 0.5 : 0.5 : 9 (acetonitrile : diethyl ether : DCM) to afford 2 (yellowish powder, 25 mg). The 60% acetone in DCM fraction was rechromatographed in DCM, followed by 0.5 : 0.5 : 9 (acetonitrile : diethyl ether : DCM) to afford 5 (blue violet crystals, 14 mg) after washing with petroleum ether, then diethyl ether.

3.4. Antimicrobial tests The microorganisms used 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. mentagrophytes (UPCC 4193) and A. niger (UPCC 3701). Compound 2 was dissolved in 95% ethanol. The antimicrobial assay reported in the literature was employed [9].

Acknowledgements A research grant from the URCO of De La Salle University is gratefully acknowledged. The antimicrobial test was conducted at the University of the Philippines-Natural Sciences Research Institute.

References [1] A. Caringal. 2002. First national summit on Philippine teak. Inventory of Philippine teak. Lobo Batangas. Available online at: http://www.biocrawler.com/encyclopedia/Teak (Accessed 2 July 2005). [2] Y.T. Castaneto, E.T. Castaneto. Int. Forest. Rev., 7, 615 (2005). [3] G. Saltan. 2002. Tu¨rkiye’de yetien Ballota L. tu¨rlerinin diterpen ve flavonoit ic¸eriklerinin deôerlendirilmesi. Available online at: http://papirus.ankara.edu.tr/arastirma/2002/a2002_17/proje.pdf (Accessed 1 April 2006). [4] C.Y. Ragasa, J.A. Rideout, V.M.L. Inte. Asia Life Sci., 7, 11 (1998). [5] Y. Aratanechemuge, H. Hibasami, K. Sanpin, H. Katsuzaki, K. Imai, T. Komiya. Oncol. Rep., 11, 289 (2003). [6] T. Kushiro, M. Shibuya, Y. Ebizuka. Eur. J. Biochem., 256, 238 (1998). [7] J. Heja. 2000. Structural analysis of natural clorin derivatives utilizing NMR spectroscopy and molecular modelling. Available online at: http://ethesis.helsinki.fi/julkaisut/mat/kemia/vk/helajaj/structur.pdf# search¼0 nmr%20spectra%20of%20chlorophyll%20A (Accessed 17 April 2006). [8] C.E. Brown, R.B. Spencer, V.T. Burger, J.J. Katz. Proc. Natl. Acad. Sci. USA, 81, 641 (1984). [9] B.Q. Guevara, B.V. Recio. Acta Manilana, Supplements, UST Research Center, Manila (1985).

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