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Boletín de la Sociedad Chilena de Química

versión impresa ISSN 0366-1644

Bol. Soc. Chil. Quím. v.47 n.2 Concepción jun. 2002

http://dx.doi.org/10.4067/S0366-16442002000200011 

Bol. Soc. Chil. Quím., 47, 151-157 (2002)

 

ANTIFUNGAL AND ANTIBACTERIAL ACTIVITY OF DITERPENES
ISOLATED FROM WOOD EXTRACTABLES OF CHILEAN
PODOCARPACEAE.

J. Becerra, C. Flores, J. Mena, P. Aqueveque, J. Alarcón *, M. Bittner,
V. Hernández, M. Hoeneisen, E. Ruiz & M. Silva

Laboratorio de Química de Productos Naturales. Departamento de Botánica.
Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Chile.
*Departamento de Ciencias Básicas. Facultad de Ciencias. Universidad del Bío Bío, Chile.
(Received: July 25, 2001 - Accepted: February 28, 2002)

ABSTRACT

Ten diterpenos, ferruginol (1a), hinokiol (1b), hinokione (1c), totarol (2a), totarodiol (2b), totarolone (2c), abietatriene (3), 6,7-dehydroferruginol (4), isopimarol (5) and acetylferruginol (6), were isolated from wood and bark of four Chilean species of Podocarpaceae. The structures of these compounds were determined by spectroscopic methods. The antimicrobial activities of seven of these compounds against the bacteria: Citrobacter sp., Bacilus subtillis, B. brevis, Escherichia coli, Micrococcus luteus, Shigella sp., Streptococcus pyogenes, Enterococcus faecalis, Providencia sp., Staphylococcus aureus, Proteus mirabilis, Salmonella thyphymurium, Pseudomonas sp. and on the fungi Aspergillus sp., Fusarium fujikuroi, F. ciliatum, Mucor miehei, Nematospora coryli, Paecilomyces variotii and Penicillium notatum , were determined. Six of these diterpenes exhibit antibacterial activity and five antifungal activities.

KEYWORDS: Gymnospermae, Podocarpaceae, extractives, diterpenes, antifungal and antibacterial activities.

RESUMEN

Diez diterpenos, ferruginol (1a), hinokiol (1b), hinokione (1c), totarol (2a), totarodiol (2b), totarolone (2c), abietatriene (3), 6,7-dehydroferruginol (4), isopimarol (5) and acetylferruginol (6), fueron aislados desde la madera y corteza de cuatro especies de la familia Podocarpaceae. La estructura de estos compuestos fue determinada mediante métodos espectroscópicos. Se determinó la actividad antimicrobiana de siete de estos compuestos frente a las bacterias: Bacillus brevis, B. subtilis, Escherichia coli, Micrococcus luteus, Providencia sp., Pseudomonas sp., Shigella sp., Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes y sobre los hongos Aspergillus sp., Fusarium fujikuroi, F. ciliatum, Mucor miehei, Nematospora coryli, Paecilomyces variotii and Penicillium notatum. Seis de estos diterpenos exhibieron actividad antibacteriana y cinco antifúngica.

PALABRAS CLAVES: Gymnospermae, Podocarpaceae, extraíbles, diterpenos, actividad antifungica y antibacteriana.

INTRODUCTION

The Podocarpaceae family belonging to the Coniferophyta group is represented by 17 genera and about 125 species that are mainly distributed in the subtropical mountains of the Southern Hemisphere. In Chile five native species grow: Podocarpus nubigena Lind, Podocarpus saligna D. Don, Prumnopitys andina (Poepp. ex Endl.) de Laub., Saxegothaea conspicua Lindl, Lipidothamnus fonckii Phil., from which the first four of them are trees with forestal significance and the latter is a shrub-like 1). These species exhibit a high endemism and grow in quite restricted areas such as the Andes mountain ranges from the VII to the VIII Regions of the country. They also grow in humid areas of the Coastal mountain range 2).

The wood from Podocarpaceae is of excellent quality because its high biodegradation resistance and natural durability. These properties give to them a great commercial value in the construction of houses, furniture and ships 3).

These plants produce secondary metabolites corresponding to the extractives of the woods. Many years ago, the scientists called them, cellular waste products, but later they realised that these compounds were bioactive principles which are directly involved in complex interactions such as symbiosis, resistance and defences against plagues or diseases 4).

The terms "wood extractives" include a wide number of compounds that can be obtained from these plants by solvent extraction. In a narrow sense, the extractives are those compounds soluble in organic solvents that can be of a neutral or acidic character. However, the water-soluble sugars and compounds can also be extractive 5).

Though the chemical composition of the extractives changes among botanical species, in many cases, factors such as the geographical area, the tree's age, growing conditions and seasonal variation influence their composition. In the wood these compounds are found in the resiniferous channels, in the xylem rays and at a cellular level they can be found as part of the middle lamella, intercellular spaces, tracheid walls and bast fibres 6).

Many specialists in natural products chemistry have carried out diverse studies to know the composition of the extractives from the Angiospermae and Gymnospermae and they have found that these extractives exhibit series of compounds of diverse structures that have been classified in diverse based on their structural characteristics and economic significance. Thus, we can distinguish fatty acids, hydrocarbons, alkaloids, flavonoids, lignans, phenols, terpenes, steroids, tannins, quinones and resins 7).

Studies on the chemical structure of the secondary metabolites, from wood and bark extractives of some Coniferophyta, the Cupressaceae and Podocarpaceae families in particular, show a series of compounds as monoterpenes, sesquiterpenes, sesquiterpenlactones, diterpenelactones, diterpenes, triterpenes, biflavones and lignans 8-10). In some cases those compounds would be responsible for the resistance of woods to the action of insects, fungi and bacteria. Thus, these compounds could have applications as pesticides in wood preservation 11- 12).

The species of the Podocarpaceae family have been studied particularly in Australia, Brazil, Japan, New Zealand and Chile 13). In Chile have been studied Prumnopitys andina, Podocarpus nubigena y P. saligna. These species have shown to contain diterpenes, triterpenes, diterpenelactones, norditerpenedilactones, bisnorditerpendilactones, steroids and moulting hormones. Some of those compounds exhibit a significant biological activity as herbicide, insecticide, cytotoxic, and antitumoral activity 9, 14 - 16). The resistance to termites of the wood of Podocarpus species is due to the presence of nagilactone C and inumakilactone A 11).

In order to contribute to the knowledge of these native woods, this study had as a fundamental objective to evaluate the antibacterial and antifungal activity of the diterpenes that are part of the wood and bark extractives of the arboreal species of the Chilean Podocarpaceae family and to relate this characteristic to the natural durability of woods.

EXPERIMENTAL

Wood samples: The samples of wood were collected in native forest in different ecological areas of Chile, Podocarpus nubigena was collected in the Cordillera Pelada, X Region, and in the Cordillera de Nahuelbuta, VIII Region, Chile in November 1999; P. saligna in The Termas de Chillán and Cordillera de Nahuelbuta, VIII Region, in May 2000; Prumnopytis andina in Termas de Chillán, VIII Region, in March 2000 and Saxegothaea conspicua in the Cordillera Pelada, X Region, in April 2000. Specialist of the Department of Botany of the Universidad de Concepcion identified the samples of those plants.

Sample processing: The wood (10 Kg) and bark (1 Kg) were separated mechanical and finely chopped.

Extraction and fractionation: The wood and bark were extracted with methanol at 40 C for 48 hours for three times, and the crude extracts were evaporated to dryness under vacuum. In order to determine the biological activity of the total extracts, samples of 10 mg/ml were dissolved in methanol and aliquots of 10 ul were used to test antimicrobial activities. The total woods or barks extracts of the studied species were fractionated by fast cc [(sil gel 60, 0.063-0.200 mm particle size) 1:20 relation (1g extract: 20g silica-gel) ]. The column was eluted with increasing polarity solvents mixtures to obtain a total of seven fractions: 90% cyclohexane - 10% ethyl acetate; 80% cyclohexane - 20% ethyl acetate; 70% cyclohexane - 30% ethyl acetate; 50% cyclohexane - 50% ethyl acetate; 100% ethyl acetate; 50% ethyl acetate - 50% methanol and 100% methanol.

Isolation and purification of diterpenes: Each one of the seven fractions obtained from the fast column was analysed by different chromatographic techniques: TLC, HPLC, GLC and GLC-Mass. The purification of the compounds was carried out using different chromatographic techniques CC, TLC and HPLC The purity of the compounds was controlled by HPLC with diodes arrangement LC 10 A-VP detector.

Diterpenes identification: The isolated and purified compounds were characterised and identified following analytical techniques: melting point: were determined on a Kofler block. The infrared spectra were determined in an IR-408 SHIMADZU spectrophotometer. The ultraviolet spectra were determined with an UV-160 SHIMADZU spectrometer. The NMR spectra (1H and 13 C) were recorded in an AM-400 BRUKER spectrometer (250 MHz) and mass spectra were carried out in a 5972 series Hewlett Packard mass spectrometer. The technique CG/EM (MS detection at 70 eV) was performed using the following conditions: Column HP-5, 30m x 0.25mm x 0.25um. Temperature: 100C isothermal for 5 minutes, with a 10 degrees increase by minute up to 275C, keeping this temperature for 20 minutes. Split injection 100:1. Injector temperature 275C. Detector temperature 300C and Helium Carrier

Compound 1a (ferruginol). Mp 175°C, MS m/z (rel. int.): 286 (M)+ (100), 271 (89), 255 (2), 229 (28), 215 (18), 201 (55), 189 (81), 175 (87), 159 (25), 149 (36), 133 (20), 115 (20), 91 (14), 69 (72), 55 (33). The 1H- NMR spectrum showed the presence of two downfield protons as sharp singlets, indicating H-11 at d 6.62 (1H,s) and H-14 at d 6.83 (1H,s). The isopropenyl group was observed at - d 3.20 (1H, septet, J=7 Hz, H-18), together with methyl signals at d 1.23 (3H, d, J=7 Hz), d 1.25 (3H, d, J=7Hz) (Me-16 and Me-17), three other methyl signals were at d 1.16 (3H,s,Me -18), d 0.9 (3H,s, Me-19) and d 0.88 (3H,s, Me-20). The 13C-NMR (DEPT) spectrum showed the presence of five methyl, five methylene, four methine and six quaternary carbon signals for 20 carbon atoms. The protons bearing carbon were unambiguously assigned by HETCOR experiment.

Compound 1b (hinokiol). Mp 240-242 °, MS m/z (rel. int.): 302 (M)+ (90), 287 (34), 269 (100), 255 (60), 227 (34), 213 (38), 189 (69), 175 (67), 147 (54), 133 (27), 115 (23), 91 (15), 55 (30).

Compound 1c (hinokione). Mp 192-195°, MS m/z (rel. int.): 300 (M)+ (100), 285 (30), 267 (15), 257 (22), 225 (19), 189 (52), 175 (38), 159 (22), 147 (39), 115 (17), 91 (17), 55 (41).

Compound 2a (totarol). Mp 128-130°, MS m/z (rel. int.): 286 (M)+ (44), 271 (100), 255 (2), 243 (7), 229 (7), 215 (7), 201 (52), 189 (31), 175 (83), 159 (16), 133 (11), 115 (10), 91 (7), 69 (31), 55 (18). The 1H- NMR spectrum showed the presence of two protons as sharp singlets, indicating H-12 at d 6.70 (1H,d, J=8Hz) and H-13 at d 6.83 (1H,d, J=8Hz). The isopropenyl group was observed at d 3.10 (1H, septet, J=7 Hz, H-18), together with methyl signals at d 1.28 (3H, d, J=7 Hz), d 1.20 (3H, d, J=7Hz) (Me-16 and Me-17), three other methyl signals were at d 1.16 (3H,s, Me-18), d 0.9 (3H,s, Me-19) and d 0.88 (3H,s, Me-20). The 13C- NMR (DEPT) spectrum showed the presence of five methyl, five methylene, four methine and six quaternary carbon signals for 20 carbon atoms.

Compound 2b (totaradiol) Mp 179-182 °C, MS m/z (rel. int.): 302 (M)+ (49), 287 (82), 269 (15), 241 (4), 227 (15), 201 (22), 189 (19), 175 (100), 159 (15), 133 (20), 115 (10), 91 (8), 67 (4), 55 (13).

Compound 2c (totarolone) Mp 186-188 °C, MS m/z (rel. int.): 300 (M)+ (28), 285 (8), 257 (23), 225 (3), 201 (35), 175 (100), 159 (10), 133 (11), 115 (10), 91 (5), 55 (4).

Compound 3 (abietatriene) Mp 175 °C, MS m/z (rel. int.): 270 (M)+ (32), 255 (100), 227 (7), 213 (11), 185 (64), 173 (71), 159 (78), 143 (35), 128 (23), 117 (20), 91 (16), 69 (61), 56 (13). The 1H- NMR Spectrum showed the presence of three downfield protons two doublet and one singlet, indicating H-11 at d 7.16 (1H, d), H-12 at d 7.00 (1H, d) and H-14 at 6.80 (1H, s). The isopropenyl group was observed at 2.86 (1H, septet. J= 6.9 Hz, H-18), together with methyl signals. The 13C-NMR (DEPT) spectrum showed the presence of five methyl, five methyl, five methine and five quaternary carbon signal for 20 carbons atoms. The proton bearing carbons were unambiguously assigned by HETCOR experiment.

Compound 4 (6,7 dehydroferruginol). The main difference in the 1H-NMR spectrum between ferruginol and 6,7 dehydroferruginol was the observation of the protons H-11 at d 6.81 (1H,s) and H-14 at d 6.92 (1H,s), H 7 at d 5.78 (1H,m) and H8 at d 6.10 (1H,m).

Compound 5 (isopimarol) present a 1H-NMR spectrum signal at d 5.7-5.8 (1H, d, J=17.5, 11.0 Hz, H-15), 5.46 (1H,s, H-7), 4.8-4-9 (2H, m. H-16cis and H-16 trans ), 3.36 (1H,d,J=12,H-19), 3.12( 1H,d, J=12, H-19), 1.07 (3H,s, H-18), 0.84 (3H,s, H-17), 0.80 (3H,s, H20). The 13C-NMR - DEPT spectrum showed three methyl, nine methylene, four methine and four quaternary carbon signals for 20 carbon atoms. The protons bearing carbons were unambiguously assigned by HETCOR experiment.

Compound 6 (acetyl ferruginol). The 1H-NMR signals were similar to those of compound 1a. The main difference between ferruginol and acetyl ferruginol was the observation of the presence of an acetyl group at d 2.05.

Evaluation of the biological activity: The antibacterial and antifungal activities of seven of ten diterpenes obtained from the wood and bark of the Podocarpus nubigena, P. saligna, Prumnopytis andina and Saxegothaea conspicua species were determined. Testing paper disc (6mm Whatman) were impregnated with a solution of 10 ul with 100 ug concentration of each compound to perform the test against the Citrobacter sp., Bacilus subtillis, B. brevis, Escherichia coli, Micrococcus luteus, Shigella sp., Streptococcus pyogenes, Enterococcus faecalis, Providencia sp, Staphylococcus aureus, Proteus mirabilis, Salmonella thyphymurium, Pseudomonas sp., and the fungi Penicillium notatum, Paecilomyces variotii, Mucor miehei, Nematospora coryli Aspergillus sp., Fusarium fujikuroi and F. ciliatum strains.

RESULTS AND DISCUSSION

From the bark and wood of the Chilean Podocarpaceae species: Podocarpus, nubigena, P. saligna, Prumnopitys andina y Saxegothaea conspicua, were isolated and identified ten diterpenos: ferruginol (1a), hinokiol (1b), hinokione (1c), totarol (2a), totarodiol (2b), totarolone (2c), abietatriene (3), 6,7 dehydroferruginol (4), isopimarol (5), acetylferruginol (6) [Fig 1, see Table I for % of pure compounds from extractables]

A comparative analysis of the diterpenes present in the wood and bark of the four species studied was carried out by GLC/EM. P. nubigena y P. saligna showed five diterpenes in common in wood and two in bark. Totarol was the major in both species, followed by totarodiol in wood. Totarol and totarodiol are also present in S. conspicua, but the main compound in the wood of this plant is ferruginol followed by hinokiol in de former and totarol in the last. A different composition of diterpenes was found in the wood and bark of Prumnopitys andina, since totarol and its derivatives were not found. However, it contains as S. conspicua ferruginol and hinokiol in the wood and ferruginol in the bark.

Seven diterpenes were evaluated for their antibacterial activity showing activity against six of these species tested and the results are shown in Table II. It is important to note that six of these diterpenes studied exhibit activity against S. aureus y Pseudomonas sp. The diterpenes with highest activity are totarol, ferruginol, dehydroferruginol and acetylferruginol. As in the research done by Kubo et al., in our investigation antimicrobial activity of totarol against gram positive and gram negative bacteria was evidenced. Similar activity is observed in other diterpens derivatives, such as ferruginol, 6,7 dehydroferruginol, and acetylferruginol. Those seven diterpenes were tested against Aspergillus sp., Fusarium fujikuroi, F. ciliatum, Mucor miehei, Nematospora coryli, Penicillium notatum and Paecilomyces variotii, six of them exhibit activities. From these diterpenes, totarol and ferruginol are the most active. The results are shown in Table III.

This research suggest that the wood resistance to fungi and bacteria of Chilean Podocarpaceae is due to the high concentration of phenolic diterpenes. These results also agree with the observations made by Cambie et al. on the resistance to fungi of the Fijian Podocarpaceae woods 6).

R
R
1a
1b
1c
- H
- OH
= O
2a
2b
2c
- H
- OH
= O

3

4

5
6

Figura 1. DITERPENES ISOLATED FROM CHILEAN PODOCARPACEAE

TABLE I. Diterpenes present in the wood and bark of the Chilean Podocarpaceae
(% of pure compound from extractables)

TABLE II. Antibacterial activity (mm diameter of inhibition zone) of the isolated diterpenos.

TABLE III. Antifungal activity (mm diameter of inhibition zone) of the isolated diterpenes

ACKNOWLEDGEMENTS

We acknowledge the financial support of FONDECYT, Grant number 1990444, Escuela de Graduados Universidad de Concepción and the Dirección de Investigación of the Universidad de Concepción PI 201.111.026-1.3.

We also thank Professors Drs. Heidrum Anke and Timm Anke of the Department of Biotechnology of the University of Kaiserslautern of Germany, for their valuable help on the determination of the biological activity and to Mr Zenón Rosas for his collaboration in the project.

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