Bol. Soc. Chil. Quím., 47, 091-098 (2002)

ANTIBACTERIAL ACTIVITY OF 13-EPI-SCLAREOL, A LABDANE
TYPE DITERPENE ISOLATED FROM PSEUDOGNAPHALIUM
HETEROTRICHIUM AND P. CHEIRANTHIFOLIUM (ASTERACEAE)

L. MENDOZA^a *, L.TAPIA^b, M. WILKENS ^b, A. URZUA^c

^aDepartamento de Química de los Materiales,^b Departamento Ciencias Biológicas,
^cLaboratorio de Química Ecológica. Facultad de Química y Biología, Universidad de
Santiago de Chile, casilla 40, correo 33, Santiago, Chile
(Received: August 27, 2001 - Accepted: December 26, 2001)

ABSTRACT

The antibacterial activity of 13-epi-sclareol, a labdane type diterpene, isolated from the resinous exudate of Pseudognaphalium heterotrichium and P. cheiranthifolium was tested against four Gram positive and three Gram negative bacteria. The antimicrobial activity was determined using three different methods: solid medium, liquid medium and a liquid medium micro-method. The bacterial viability and growth curve were also studied. Gram positive bacteria were selectively affected by 13-epi-sclareol. At concentrations of 30 µg/mL, the compound produced the lysis of Bacillus cereus and B. subtilis.

KEYWORDS:: Pseudognaphalium heterotrichium; P. cheiranthifolium; Asteraceae; Resinous exudate; 13-epi-sclareol; Diterpenoid; Antibacterial activity.

RESUMEN

La actividad antibacteriana del 13-epi-esclareol, un diterpeno del grupo del labdano, aislado del exudado resinoso de las especies Pseudognaphalium heterotrichium y P. cheiranthifolium se estudió frente a cuatro bacterias Gram positivas y tres Gram negativas. La actividad antimicrobiana se determinó usando tres métodos diferentes: medio sólido, medio líquido y un micro-método en medio líquido. Adicionalmente se evaluó el efecto del compuesto sobre las bacterias en una curva de crecimiento y a través de la viabilidad bacteriana. El 13-epi-esclareol presentó actividad selectiva frente a las bacterias Gram positivas y a concentraciones de 30 µg/mL, produjo la lisis de Bacillus cereus y B. subtilis.

PALABRAS CLAVES: Pseudognaphalium heterotrichium; P. cheiranthifolium; Asteraceae; Exudado resinoso; 13-epi-esclareol; Diterpeno; Actividad antibacteriana.

INTRODUCTION

Pseudognaphalium spp. (Asteraceae) Anderberg make up an important group of medicinal plants used in Chilean folk medicine. They are known under the vernacular name ¨vira vira¨ (1-5). Different preparations of ¨vira vira¨ are used as wound-healing antiseptics, in the treatment of the common cold and influenza, and of different bronchial illnesses (2-4). In a previous study (6), the antimicrobial properties of the resinous exudates, diterpenoids and flavonoids isolated from four Chilean species of Pseudognaphalium (P. vira vira, P. robustum, P. heterotrichium and P. cheiranthifolium) against Gram negative and Gram positive bacteria were reported. The observed antibacterial activity against Gram positive bacteria was correlated with the presence in the extract of kaurane and labdane diterpenoids. In a preliminary analysis using the agar overlay method, 13-epi-sclareol [1], showed a selective antibacterial activity against Gram positive bacteria (6, 7).

In the present communication our preliminary results were expanded to include an evaluation of the antimicrobial activity of 13-epi-sclareol [1] against Gram positive and negative bacteria in liquid media using a macro and micro-method. In addition, the effects of the compound on the bacterial viability and growth curve were also studied.

Plant material

Pseudognaphalium cheiranthifolium (SGO 133618-99) and P. heterotrichium (SGO 133621-99) were collected during the flowering season (October 1999) between Zapallar and Papudo (V Región, Chile, 32 30' S, 71 30'W). Voucher specimens were deposited in the Herbarium of the Museo Nacional de Historia Natural, Santiago, Chile.

Extraction of the resinous exudates and pure compounds

The resinous exudates were obtained by immersion of the fresh plant materials in CH₂Cl₂ for 15-20 s and removal of the solvent. The CH₂Cl₂ extracts of P. cheiranthifolium (24 g) and P. heterotrichium (22 g) were fractionated by CC (silica gel) using a hexane - EtOAc step gradient to afford crude 13-epi-sclareol [1] (8.3% from P. cheiranthifolium exudate and 4.5% from P. heterotrichium exudate).

13- epi - sclareol

Repeated crystallization of crude 13-epi-sclareol from EtOH, yielded a pure compound mp 130-132 C [a]_D +7 (c: 2.02, CHCl₃); (lit. (8)), mp 130-132 C, [a]_D + 7.2 (c: 2, CHCl₃), IR n _max (KBr) 3326, 1642 cm^-1; MS m/z (relative intensity in %) 290(6); 204 (1); 189 (3); 177(22); 192(5); 137 (8); 71(44). ¹H NMR (200 MHz, CDCl₃) d ppm: 5.87(dd,1H, J=11 Hz, J=17 Hz); 5.22 (dd,1H, J=1.5 Hz, J=17 Hz); 5.07 (dd,1H, J=1.5 Hz, J=11 Hz); 1.25 (s,3H); 1.14 (s,3H); 0.86 (s,3H); 0.77 (s,6H). ¹³C NMR (50 MHz, CDCl₃) d ppm: 15.2; 18.4; 19.1; 20.5; 21.5; 24.5; 29.0; 33.2; 33.4; 39.3; 39.6; 42.0; 44.1; 44.7; 56.0; 61.8; 74.1; 75.0; 111.8; 145.2. ¹³C NMR DEPT-135 (200 MHz, CDCl₃) d ppm: 15.3 (C-20-CH₃); 18.4 (C-2-CH₂); 19.1 (C-11-CH₂); 20.5 (C-6-CH₂);21.5 (C-18-CH₃); 24.5 (C-17-CH₃); 29.0 (C-16-CH₃); 33.4 (C-19-CH₃); 39.6 (C-1-CH₂); 42.0 (C-3-CH₂); 44.1(C-7-CH₂); 56.0 (C-5-CH); 61.2 (C-9-CH); 111.8 (C-15-CH₂); 145.2 (C-14-CH). The spectroscopic and physical properties were identical to previously published data for 13-epi-sclareol (8) [1].

Antibacterial activity determination

Bacterial strains

The antimicrobial activity of 13-epi-sclareol [1] was evaluated against Bacillus cereus (NAS569), B. subtilis (ATCC6633), Staphylococcus aureus (ATCC 6538p), Escherichia coli (ATCC25922), Klebsiella pneumoniae (ATCC13883) Enterobacter cloacae and B. coagulans (Laboratorio de Bioinformática y Microbiología Molecular).

Solid medium

The antimicrobial activity was determined by the agar overlay method (7). Bacteria grown overnight in LB-broth (9) were diluted to McFarland 0.5-1.0 (1.5 - 3 x 10⁸ cells/mL) (10) and 100 mL of this dilution were mixed with 3 mL of melted soft agar (0.7%) at 50C. The soft agar was poured over Petri dishes containing 20 mL of 1.5% agar. Two-fold dilutions of 13-epi-sclareol (5 mL) in methanol were deposited over solidified agar, starting at 1,000 mg/mL down to 2 mg/mL. After 18 h of incubation at 37C, the diameter of the inhibition zone was determined. Control measurements were carried out with methanol. The minimum inhibitory concentration (MIC) corresponded to the minimum concentration of the dilution that showed a transparent halo of growth inhibition. The MIC determinations were carried out in three independent experiments.

Liquid medium: macro-method

Ten mL of B. cereus and B. subtilis cultures at McFarland 1.0 (3 x 10⁸ cells/mL) (10) were added to 2 mL of LB-broth containing different concentrations of 13-epi-sclareol [1]. The tubes were prepared in duplicate and incubated at 37C without agitation. After 12 h, the viability of each sample was determined by spreading one loop on agar plates, which were incubated for 12 h at 37C. The MIC was defined as the lowest concentration which prevented bacterial growth.

Liquid medium: micro-method

Sterile 96-well microtiter plates were used for this purpose. Different concentrations of 13-epi-sclareol [1] were prepared in LB-broth and 2 mL of an inoculum of B. cereus or B. subtilis at McFarland 1.0 (3 x 10⁸ cells/mL) was added in a final volume of 200 mL. Tests were perfomed in triplicate for each concentration. The control included inoculated growth medium without the compound. All plates were incubated at 37C for 12 h, and the growth was estimated spectrophotometrically (540 nm) with an ELISA detector. The percentage of inhibition was determined with relation to the control without compound.

Effect of 13-epi-sclareol on the growth curve of B. cereus, B. subtilis, S. aureus and E. coli

B. cereus, B. subtilis, S. aureus and E. coli were grown to OD_{540 nm} 0.5 in LB-broth and aliquots of 10 mL were distributed in several 125 mL flasks. Different concentrations of 13-epi-sclareol [1] were added to each flask and the absorbance was recorded. At the same time, 100 mL were removed, serially diluted, and plated to determine the number of viable cells. The colonies were counted after 18 h incubation at 37C. Tests were performed in triplicate for each concentration. The control included inoculated growth medium with methanol.

RESULTS

The resinous exudates of P. heterotrichium and P. cheiranthifolium yielded respectively 8.3% and 4.5% of crude 13-epi-sclareol [1]. The compound was identified spectroscopically by comparison with previously reported data (8).

13-epi-sclareol [1] is a stereoisomer of the general labdane skeleton 14-labdene-8,13-diol (11). Taking into account only the C-8 and C-13 hydroxylated stereogenic carbons, the compound 14-labdane-8,13-diol may exist in four stereochemical forms. The spectroscopic properties and [a]_D of the known stereoisomers (8a, 13R; 8a, 13S; ent-8b,13S) are closely similar (11). Although 13-epi-sclareol [1] is a known compound, to avoid any confusion its spectroscopic data were included in this communication.

The antimicrobial activity of 13-epi-sclareol was specific for Gram positive bacteria as judged by the results in solid media shown in Table 1. However, in static liquid cultures, the compound was inactive against S. aureus, but was active against the other Gram positive bacteria with MIC values ranging from 64 to 256 mg/mL (Table I).

Table I. Minimum inhibitory concentration of 13-epi-sclareol in solid and liquid medium

The inhibition of bacterial growth by 13-epi-sclareol [1] determined by the microdilution method is shown in Figure 1. At the concentration of 500 mg/mL complete growth inhibition of B. cereus, B. subtilis and B. coagulans was observed. At the same concentration, S. aureus growth was only partially inhibited (80%). On the other hand, 200 mg/mL of the diterpene produced only 70% of growth inhibition of Gram negative bacteria, this activity remaining with increasing concentrations.

Fig. 1. Determination of the percentage of inhibition of Gram positive bacteria (B. subtilis, B cereus, B coagulans, S. aureus) and Gram negative bacteria (E. coli, E.cloacae, K. pneumoniae) by 13-epi-sclareol. in sterile 96-well microtiter plates (micromethod) 200 ml of growth medium with different concentrations of 13-epi-sclareol was inoculated with 2 ml of tbacteria at Mc Farland 1.0. The topical density (O.D. 540 mm) were determined after 18 h of incubation at 37C. The percentage of inhibition was determined with relation to the control without compound. Each curve represents the average of three experiments.

Figures 2A, C and E show the effect of different concentrations of 13-epi-sclareol [1] on the growth of B. cereus, B. subtilis and S. aureus in liquid medium, as determined by the macro-method monitoring optical density at 540 nm. At a concentration of 30 mg/mL, the compound produced the lysis of B. cereus and B. subtilis. However, at the same concentration, it produced only a slow decline of the O.D. of S. aureus. The effect on the bacterial viability is also presented in figures 2B, D and F, showing a bactericidal activity of the compound at the highest concentration (30 mg/mL). On the contrary, at 100 mg/mL 13-epi-sclareol [1] is inactive against E. coli as shown by the growth curve in Figure 3. The same results were obtained with the other Gram negative bacteria tested (Klebsiella pneumoniae and Enterobacter cloacae).

Fig. 2. Antimicrobial activity of 13-epi-sclareol againts B. subtilis, B. cereus and S. aureus. Ten mL of bacterial cultures at O.D. _{540 nm} 0.5 were treated with 13-epi-sclareol at final concentrations of 10 (!); 20 (¨); and 30 (%) ug/mL; methanol (·); control (&). In A, C and E the optical density was followed and B, D and F the cell viability was determined.

Fig. 3. Effect of 13-epi-sclareol on the growth of E. coli. Ten mL of bacterial culture at O.D. _540nm 0.5 were treated with 13-epi-sclareol at final concentrations of 50 (¨) and 100 (%) ug/mL, methanol (·), control without compound (&).

DISCUSSION

The results of the antimicrobial activity study of 13-epi-sclareol [1] in solid and liquid media showed that the compound is slightly more active against B. cereus, B. subtilis and B. coagulans than against S. aureus. These results are in agreement with the fact that B. cereus, B. subtilis and B. coagulans form large and separate chains. This spatial arrangement would facilitate their interaction with 13-epi-sclareol [1]. On the contrary, S. aureus forms a globular association that would only allow the interaction of 13-epi-sclareol [1] with the surface of the external bacterial cells of those macro-structures.

The high selectivity of 13-epi-sclareol [1] against Gram positive bacteria would suggest that the cell wall of Gram negative bacteria acts as a barrier preventing the compound from approaching the cytoplasmic membrane. This cell wall is constituted by a thin layer of peptidoglycan and an outer membrane, a structure which is not present in Gram positive bacteria. The negative charge of the outer membrane and the hydrophobicity of 13-epi-sclareol [1] would prevent its diffusion through the Gram negative wall.

The antibacterial activity of 13-epi-sclareol [1] is in agreement with other data collected by us for labdane (12), kaurane (6,13) and clerodane (14) diterpenoids, which showed interesting activities against Gram positive, but not Gram negative bacteria.

The structural similarities of all of these diterpenoids, have resulted in a general model with a major structural requirement for activity: the presence of a hydrophobic skeleton, common to all three series, provided by a substituted decalin system with a hydrophilic site, capable of interactions with hydrogen-bond-acceptor groups of a putative receptor in the Gram positive bacteria.

Finally, the fact that this readily available compound has also proved to be antibacterial at relatively low concentrations clearly justifies our interest in pursuing in the future biological studies of its derivatives.

ACKNOWLEDGEMENTS

This work was supported by FONDECYT Grant N: 1990209 and DICYT (Universidad de Santiago de Chile).

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