Antifungal activity and synergistic effect of cinnamaldehyde combined with antioxidants against wood decay fungi

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IRG/WP 07-30445 THE INTERNATIONAL RESEARCH GROUP ON WOOD PROTECTION Section 3 Wood protecting chemicals Antifungal activity and synergistic effect of cinnamaldehyde combined with antioxidants against

1 IRG/WP THE INTERNATIONAL RESEARCH GROUP ON WOOD PROTECTION Section 3 Wood protecting chemicals Antifungal activity and synergistic effect of cinnamaldehyde combined with antioxidants against wood decay fungi Fu-Lan Hsu 1, Tsair-Bor Yen 2, Hui- Ting Chang 3 and Shang-Tzen Chang 4 * 1 Division of Forestry Chemistry, Taiwan Forestry Research Institute No.53, Nan-Hai Road, Taipei, 100, Taiwan 2 Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology No.1, Hseuh-Fu Rd., Nei-Pu, Pingtung 912, Taiwan 3, 4 School of Forestry and Resource Conservation, National Taiwan University No.1, Sec. 4, Roosevelt Rd., Da-an District, Taipei City 106, Taiwan *Corresponding author Paper prepared for the joint IRG IUFRO Regional Research Symposium International Union of Forest Research Organizations All Division 5 Conference Taipei, Taiwan 29 October 2 November 2007 IRG SECRETARIAT Box 5609 SE Stockholm Sweden

2 Antifungal activity and synergistic effect of cinnamaldehyde combined with antioxidants against wood decay fungi Fu-Lan Hsu 1, Tsair-Bor Yen 2, Hui- Ting Chang 3 and Shang-Tzen Chang 4* 1 No.53, Nan-Hai Road, Taipei, 100, Taiwan. 2 No.1, Hseuh-Fu Rd., Nei-Pu, Pingtung 912, Taiwan 3 No.1, Sec. 4, Roosevelt Rd., Da-an District, Taipei City 106, Taiwan 4 No.1, Sec. 4, Roosevelt Rd., Da-an District, Taipei City 106, Taiwan ABSTRACT The objective of this study was to investigate the antifungal activity and synergistic effect of cinnamaldehyde combined with antioxidants against wood decay fungi. Five antioxidants, propyl gallate, octyl gallate, quercetin, eugenol and catechin were tested against various wood decay fungi. Octyl gallate and eugenol were found to be the only two antioxidants processed antifungal activities. IC 50 values of octyl gallate were 0.47 and 0.04 mm against L. betulina and L. sulphureus, respectively. The IC 50 values of eugenol were 0.37 and 0.25 mm against L. betulina and L. sulphureus, respectively. The synergistic effects were also found on the combinations of octyl gallate-cinnamaldehyde and eugenol-cinnamaldehyde. The combination of either using octyl gallate with cinnamaldehyde or eugenol with cinnamaldehyde greatly reduced the concentrations to achieve the inhibitory effect that a higher concentration was needed by octyl gallate, eugenol or cinnamaldehyde alone. The antifungal action of octyl gallate could be attributed to its pyrogallol group functioning as an attached moiety to the hydrophilic portion of the membrane surface and the octyl moiety interfering with the hydrophobic interior surfaces of the membrane. Meanwhile, the synergism of cinnamaldehyde with octyl gallate or eugenol could be due to the interference of fungal cell wall synthesis and destruction on cell wall and membrane plus the additional radical scavenging effect. Results also suggested that antioxidant with fungicidal effect might be a better candidate than pure antioxidant for the system of fungicide/antioxidant. Keywords: Antifungal activity; Antioxidants; Cinnamaldehyde; Synergistic effect; Wood decay fungi. 1. INTRODUCTION Wood is mainly composed of cellulose, hemicelluloses and lignin. It is susceptible to degradation caused by many organisms especially fungi, resulting in tremendous economic and resource losses. To extend the service life, wood products are often treated with preservatives. A major problem of the wood preservatives such as creosote and CCA is that they pose a serious threat to the environment. Because of this, environmentally benign organic preservatives for wood are urgently needed. Wood can be classified to different classes like very durable, durable and non-durable. Natural durable wood will be an ideal model to develop such preservatives. Natural compounds extracted from various woods have been proven to have antifungal properties. Examples of such compounds are cinnamaldehyde (Wang et al. 2005, Cheng et al. 2006), α-cadinol (Chang et al. 1999), carvacreol (Kai 1991), T-muurolol, T-cadinol, γ-cadinene (Kondo and Imamura 1986), 1

3 cryptomeridiol (Morita et al. 1997), tropolones (Hart 1989), pinosylvin (Schultz et al. 1990), oxyresveratrol, dihydromorin (Schultz et al. 1995), gallic acid (Kishino et al. 1995) and ferruginol (Rudman 1965), among many others. Indigenous cinnamon (Cinnamomum osmophloeum kaneh) is an endemic tree that grows in Taiwan s natural hardwood forest at elevations between 400 and 1500 m. C. osmophloeum has been of interest to researchers because the chemical constituents of its leaf essential oil are similar to those of Cinnamomum cassia bark oil. Cinnamon oil is commonly used in the food industry because of its special aroma. In addition, its antimicrobial properties has drawn great attention from many researchers (Hili et al. 1997, Azumi et al. 1997). Chang and co-workers found that the leaf essential oils of C. osmophloeum have an excellent inhibitory effect against bacteria (Chang et al. 2001) and fungi (Wang et al. 2005). Cinnamaldehyde was found the major ingredient in leaf essential oil of some C. osmophloeum (Hu et al. 1985) and was therefore selected as natural antifungal compound in this study. It has been suggested that wood degradation by fungi takes place through oxidative reactions involving metals (Hammel 1997; Kerem et al. 1999). Consequently, antioxidants may be a promising additive for the development of novel and environmentally benign preservative systems. In this study, five antioxidants, propyl gallate, octyl gallate, quercetin, eugenol and catechin are selected because they are quite safe in use. For example, propyl gallate and octyl gallate are currently permitted for use as antioxidant additives in food and cosmetic products (Aruoma et al. 1993). In the present study, the antifungal activity of propyl gallate, octyl gallate, quercetin, eugenol and catechin and cinnamaldehyde against wood decay fungi were evaluated. The synergistic effects of the combination using natural antifungal compound cinnamaldehyde and antioxidants were also studied. 2. EXPERIMENTAL METHODS 2.1. Fungal strains The wood decay fungi were obtained from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (Taiwan). The fungal strains used in experiments were as follows: white-rot fungi [Lenzites betulina (BCRC 35296)] and brown-rot fungi [Laetiporus sulphureus (BCRC 35305)] Chemicals Propyl gallate and octyl gallate were purchased from Tokyo Kasei Kogyo Co. (Japan) and 1- diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma Chemical Co. (America). Cinnamaldehyde, eugenol, catechin and quercetin were purchased from ACROS (Belgium). Commercial fungicide propiconazole was used as a positive control Free radical scavenging activity The scavenging activity of DPPH free radical was determined using the method reported by Gyamfi et al. (1999) and Chang et al. (2001). Antioxidants over a range of concentrations dissolved in ethanol (50 μl) were mixed with 1000 μl of 0.1 mm DPPH-ethanol solution and 450 μl of 50 mm Tris-HCl buffer (ph 7.4). Pure ethanol (50 μl) was used as the control for this experiment. After 30 min of incubation at ambient temperature in the dark, the reduction of the DPPH free radical was measured by reading the absorbance at 517 nm. Ascorbic acid was used as the positive control and the percent inhibition ratio was calculated using Eq. 1: % Inhibition = [(absorbance of control - absorbance of test sample)/absorbance of control] 100 (1) 2

4 2.4. Antifungal assays Antifungal assays were performed based on the methods used in our previous studies (Chang et al. 1999, 2000) with slight modifications. Propyl gallate, octyl gallate, quercetin, eugenol, catechin, cinnamaldehyde and propiconazole were dissolved in ethanol separately. Solutions with different concentrations of chemicals were mixed with sterilized potato dextrose agar (PDA) in 9 cm Petri dish and after transferring the mycelium of four fungal strains, the testing dishes were incubated in the dark at 26 ± 2 C and 70% relative humidity. When the mycelium of fungi reached the edges of the control dishes, the average antifungal indices were calculated. Each test was repeated three times. The formula to calculate antifungal index was shown as Eq. 2: Antifungal index (AI, %) = (1- Da/Db) 100 (2) where Da is the diameter of growth zone in the experimental dish (cm) and Db is the diameter of growth zone in the control dish (cm). The IC 50 values (the concentration in that inhibited 50% of the mycelium growth) were calculated by probit analysis Statistical analyses All results were expressed as mean ± SD (n = 3). The significance of difference was calculated using SAS Scheffe s test and results with P < 0.05 were considered to be statistically significant. 3. RESULTS AND DISCUSSION 3.1. Free radical scavenging activity The free radical has been proposed to be one of the decay mechanisms by fungi (Schultz and Nicholas 2002). Therefore, the DPPH assay was used to evaluate the relationship between radical scavenging and antifungal activities and the results were shown in Table 1. Both propyl gallate and octyl gallate showed stronger free radical scavenging activities than the control using (-)-ascorbic acid. Catechin showed only slightly less activity than (-)-ascorbic acid. On the other hand, cinnamaldehyde had the lowest free radical scavenging activity Table 1: Free radical scavenging activity of test compounds by the DPPH assay Compounds EC 50 (μg/ml) Propyl gallate 1.6 Octyl gallate 2.1 Eugenol 10.3 Quercetin 31.5 Catechin 5.4 Cinnamaldehyde > 500 (-)-Ascorbic 2.8 Results are mean ± SD (n = 3) Antifungal activity of individual compound The antifungal activities of test compounds were first examined at the concentration of 100 µg/ml and the results are shown in Table 2. Among all compounds tested, the commercial fungicide, propiconazole, was the most effective and completely inhibited the growth of L. betulina and L. sulphureus at the concentration of 1 µg/ml. Cinnamaldehyde and eugenol also 3

5 exhibited strong antifungal activities with antifungal index of 100% against both L. betulina and L. sulphureus, followed by octyl gallate, while propyl gallate, catechin and quercetin did not express antifungal activities at the same concentration. Table 2: Antifungal index (%) of test compounds against wood decay fungi at the concentration of 100 µg/ml Compounds Fungal strain L. betulina L. sulphureus Propyl gallate 1 ± ± 0.0 Octyl gallate 43 ± ± 0.0 Eugenol 100 ± ± 0.0 Quercetin 0 ± ± 0.0 Catechin 3 ± ± 1.8 Cinnamaldehyde 100 ± ± 0.0 Propiconazole* 100 ± ± 0.0 * Concentration of propiconazole was 1 µg/ml as a positive control. Results are mean ± SE (n = 5). The IC 50 and IC 90 values for individual compound were further determined and the results are shown in Table 3. It was found that eugenol and octyl gallate were the only two antioxidants showed similar antifungal activities as those of cinnamaldehyde against L. betulina and L. sulphurous. Octyl gallate even showed better antifungal activity than cinnamaldehyde against L. sulphurous. On the contrary, catechin and quercetin revealed very limited inhibitory effects against L. betulina and L. sulphureus. As for IC 90, the similar results were found. Although catechin and quercetin have been reported to have antifungal effects against Candida albicans (Hirasawa and Takada 2004), our results indicated that the antifungal activity of quecertin or catechin alone can not compare with eugenol and octyl gallate against wood decay fungi. Based on the results, we found that there was no correlation between the free radical scavenging capabilities and their antifungal properties, indicating that the radical scavenging properties of test compounds might not directly contribute to their antifungal activities Combined effects of cinnamaldehyde with antioxidants Cinnamaldehyde combined with antioxidants were studied to determine whether the combination has interactions or enhanced actions against wood decay fungi. The interaction was evaluated by comparing the isoeffective concentrations (IC 50 and IC 90 ) of test compounds and designated combinations. It is considered synergy when the isoeffective concentration of combination was significantly lower than those of compounds acting alone (Tallarida 2001). Cinnamaldehyde with antioxidants were prepared at 1:1 ratio in molarities with serial concentrations for assaying, and the values of IC 50 and IC 90 were given in Table 3. Significant synergy was observed on the combination of cinnamaldehyde with eugenol against L. sulphureus. The antifungal index of cinnamaldehyde against L. sulphureus at the concentration of 0.17 mm was 41%, and that of eugenol at the same concentration was 24%, while the antifungal index of combination using cinnamaldehyde and eugenol against L. sulphureus dramatically increased to 90%, indicating portent of synergistic effect. The synergy was further confirmed by comparing their isoeffective concentrations. The values of IC 50 and IC 90 for the combination of cinnamaldehyde with eugenol against L. sulphureus were 0.18 and 0.37 mm, respectively, which were significantly lower than those of using either cinnamaldehyde or eugenol alone. However, only additive effect was found on the combination of cinnamaldehyde and eugenol against L. betulina with IC 50 (0.38 mm) and IC 90 (0.63 mm). In addition, the combinations of cinnamaldehyde with catechin or quercetin against L. sulphureus also exhibited additive effects, but both combinations showed marked antagonistic effects against L. betulina. The values of IC 50 and IC 90 for the combination of cinnamaldehyde with catechin against L. betulina were 1.22 and 1.40 mm, and against L. sulphureus were 0.23 and 0.52 mm, 4

6 respectively. Among all samples tested, the strongest antagonistic effect was discovered on the combination of cinnamaldehyde and quercetin against L. betulina with IC 50 (1.44 mm) and IC 90 (1.65 mm) which were significantly higher than those of cinnamaldehyde alone. Table 3: IC 50 and IC 90 values of test compounds and in combinations with cinnamaldehyde against wood decay fungi Compounds L. betulina L. sulphureus IC 50 (mm) IC 90 (mm) IC 50 (mm) IC 90 (mm) Propyl gallate Octyl gallate Eugenol Quercetin > 100 > Catechin > 100 > > 100 Cinnamaldehyde Cin. + eugenol Cin. + catechin Cin. + quercetin Cin.: cinnamaldehyde. Significant synergy was also observed on the combination of cinnamaldehyde with octyl gallate against L. sulphureus. The antifungal index of cinnamaldehyde against L. betulina at the concentration of 50 μg/ml was 6% and that of octyl gallate against L. betulina at the concentration of 25 and 100 μg/ml was 16 and 43%, respectively. The antifungal index for the treatment using the combination of cinnamaldehyde with octyl gallate was found to be greatly increased to 64 and 100%, respectively, indicating that the cinnamaldehyde/octyl gallate combination had significant synergism against L. betulina. The same synergistic effect was also observed for G. trabeum (data not shown). 4. CONCLUSIONS In this study, we compared the antifungal activities of five antioxidants (propyl gallate, octyl gallate, quercetin, eugenol and catechin), a natural antifungal compound (cinnamaldehyde), and a commercial fungicide (propiconazole). Among the five antioxidants, octyl gallate and eugenol shows good antifungal properties even better than cinnamaldehyde in terms of antifungal index, IC 50 or IC 90. Potent synergistic effects were observed on the combination of cinnamaldehyde with either eugenol or octyl gallate againt wood decay fungi in vitro, suggesting these two combinations have highly potential to be developed as an environmentally-benign wood preservative. 5. REFERENCES Aruoma, O.I., Murcia, A., Butler, J., Halliwell, B.J. (1993) Evaluation of the antioxidant and prooxidant actions of gallic acid and its derivatives J. Agric. Food Chem. 41: Azumi, S., Tanimura, A., Tanamoto, K. I. (1997) A novel inhibitor of bacterial endotoxin derived from cinnamon bark. Biochem. Biophys. Res. Commun. 234: Chang, S. T., Chen, P. F., Chang, S. C. (2001) Antibacterial activity of leaf essential oils and their constituents from Cinnamomum osmophloeum. J. Ethnopharmacol. 77: Chang, S.T., Wang, S.Y., Wu, C.L., Chen, P.F., Kuo, Y.H. (2000) Comparison of the antifungal activity of cadinane skeletal sesquiterpenoids from Taiwania (Taiwania cryptomerioides Hayata) heartwood. Holzforschung 54:

7 Chang, S.T., Wang, S.Y, Wu, C.L., Su, Y.C., Kuo, Y.H. (1999) Antifungal compounds in the ethyl acetate soluble fraction of the extractives of Taiwania (Taiwania cryptomerioides Hayata) heartwood. Holzforschung 53: Chang, S.T., Wu, J.H., Wang, S.Y., Kang, P.L., Yang, N.S., Shyur, L.F. (2001). Antioxidant activity of extracts from Acacia confusa bark and heartwood. J. Agric. Food Chem. 49: Cheng, S.S., Liu, J.Y., Hsui, Y.R., Chang, S.T. (2006) Chemical polymorphism and antifungal activity of essential oils from leaves of different provenances of indigenous cinnamon (Cinnamomum osmophloeum). Bioresour. Technol. 97: Gyamfi, M.A., Yonamine, M., Aniya, Y. (1999) Free-radical scavenging action of medicinal herbs from Ghana: Thonningia sanguineaon experimentally induced liver injuries. Gen. Pharmacol. 32: Hammel, K.E. (1997) Fungal degradation of lignin, In: Cadisch, G., Giller, K.E. (Eds.), International Driven by Nature: Plant Litter Quality and Decomposition. CAB, pp Hart, J.H. (1989) The role of wood exudates and extractives in protecting wood from decay, In: Rowe, J.W. (Ed.), Natural Products of Woody Plants. Springer-Verlag Co., Berlin, pp Hili, P., Evans, C. S., Veness, R. G. (1997) Antimicrobial action of essential oils: the effect of dimethylsulphoxide on the activity of cinnamon oil. Lett. Appl. Microbiol. 24: Hirasawa, M., Takada, K. (2004). Multiple effects of green tea catechin on the antifungal activity of antimycotics against Candida albicans. J. Antimicrob. Chemother. 53: Hu, T. W., Lin, Y. T., Ho, C. K. (1985) Natural variation of chemical components of the leaf oil of Cinnamomum osmophloeum Kaneh. Bull. Taiwan For. Res. Inst. Eng. 78: 18. Kai, Y. (1991) Chemistry of extractives. In: Hon, D.N.S., Shiraish, N. (Eds.), Wood and Cellulosic Chemistry. Marcell Dekker Inc., New York, pp Kerem, Z., Jensen, K.A., Hammel, K.E. (1999) Biodegradative mechanism of the brown rot basidomycete Gloephyllum trabeum: Evidence for an extracellular hydroquinone-driven Fenton reaction. FEBS Lett. 446: Kishino, M., Ohi, H., Yamaguchi, A. (1995) Characteristics of methanol extractives from Chengal wood and their antifungal properties. Mokuzai Gakkaishi 41: Kondo, R., Imamura, H. (1986) Antifungal compounds in heartwood extractives of hinoki (Chamaecyparis obtusa Endl.). Mokuzai Gakkaishi 32: Morita, S.I., Hidaka, T., Yatagai, M. (1997) Antifungal compounds of the extractives of Yakusugi (Crypromeria japonica D. Don). Wood Preservation 23: Rudman, P. (1965) The causes of natural durability in timber. XVIII. Further notes on the fungi toxicity of wood extractives. Holzforschung 19:

8 Schultz, T.P., Harms, W.B., Fisher, T.H., McMurtrey, K.D., Minn, J., Nicholas., D.D.(1995) Durability of angisperm heartwood: The importance of extractives. Holzforschung 49: Schultz, T.P., Hubbard, T.F., Jin, J.L. Fisher, T.H., Nicholas., D.D.(1990) Role of stilbene in the natural durability of wood: Fungicidal structure-activity relationships. Phytochemistry 29: Schultz, T.P., Nicholas, D.D. (2002) Development of environmentally-benign wood preservatives based on the combination of organic biocides with antioxidants and metal chelators. Phytochemistry 61: Tallarida, R.J. (2001) Drug synergism: its detection and applications. J. 335 Pharmacol. Exp. Ther. 298: Wang, S.Y., Chen, P.F., Chang, S.T. (2005) Antifungal activities of essential oils and their constituents from indigenous cinnamon (Cinnamomum osmophloeum) leaves against wood decay fungi. Bioresour. Technol. 96:

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IRG/WP 09-30501 THE INTERNATIONAL RESEARCH GROUP ON WOOD PROTECTION Section 3 Wood protecting chemicals Antifungal Activities of Three Supercritical Fluid Extracted Cedar Oils Tianchuan Du 1, Todd F. Shupe