Antifungal activities of essential oils and their constituents from indigenous cinnamon (Cinnamomum osmophloeum) leaves against wood decay fungi

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1 Bioresource Technology 96 (2005) Antifungl ctivities of essentil oils nd their constituents from indigenous cinnmon (Cinnmomum osmophloeum) leves ginst wood decy fungi Sheng-Yng Wng, Pin-Fun Chen b, Shng-Tzen Chng b, * Deprtment of Forestry, Ntionl Chung-Hsing University, Tichung 402, Tiwn b School of Forestry nd Resource Conservtion, Ntionl Tiwn University, No. 1, Section 4, Rossevelt Rod, Tipei 106, Tiwn Received 1 June 2003; received in revised form 12 July 2004; ccepted 16 July 2004 Avilble online 18 September 2004 Abstrct Cinnmomum osmophloeum Kneh is one of the hrdwood species indigenous to Tiwn tht possesses significnt ntifungl ctivity. To exmine the ntifungl ctivity of lef essentil oils nd dominnt constituents from C. osmophloeum, the essentil oils of leves from three clones (A, B, nd C) collected from Hw-Lin experimentl forest were extrcted nd their components nlyzed by gs chromtogrphy. Results from the ntifungl tests demonstrted tht the essentil oils of both B nd C leves hd strong inhibitory effects. The ntifungl indices of these two lef oils t 100ppm ginst five strins of white rot fungi nd four strins of brown rot fungi were ll 100%. Cinnmldehyde, the mjor compound in C. osmophloeum lef essentil oils, possessed the strongest ntifungl ctivities compred with the other components. Its ntifungl indices ginst both Coriolus versicolor nd Letiporus sulphureus were 100%. The MIC (minimum inhibitory concentrtion) of cinnmldehyde ginst C. versicolor nd L. sulphureus ws 50 nd 75 ppm, respectively. In ddition, comprisons of the ntifungl indices of cinnmldehydeõs congeners proved tht cinnmldehyde exhibited the strongest ntifungl ctivities. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Cinnmomum osmophloeum; Lef; Essentil oil; Cinnmldehyde; White rot fungi; Brown rot fungi; Antifungl ctivity 1. Introduction People hve relied on trees for shelter, building mterils, flvors/frgrnces, nd not lest, medicines, since ncient times. The specil properties of wood, including its esthetic ppernce, low density, low therml expnsion, nd desirble mechnicl strength, hve led to indoor nd outdoor pplictions. Durbility is one of the most importnt considertions for the use of wood in construction. Poor durbility hs often been recognized s one of woodõs disdvntges in certin * Corresponding uthor. Tel.: /3196; fx: E-mil ddress: peter@ntu.edu.tw (S.-T. Chng). ppliction. Developing methods to prolong the service life of wood hs lwys been the interest of wood industry reserchers. From the environmentl perspective, finding nturlly existent constituents in highly durble tree species nd understnding their mechnisms re the most pproprite pproches to chieve wood protection while preserving the environment (Chng et l., 2000). Owing to the unique ecosystem, there re mny vluble tree species endemic to Tiwn. In the pst few yers, we hve consistently studied the reltionship between the properties nd chemicl constituents of endemic trees in Tiwn. According to the results obtined from our previous screening ssy of endemic wood durbility ginst fungi, we hve found tht Cinnmomum osmophloeum Kneh (Lurcee), nmed s /$ - see front mtter Ó 2004 Elsevier Ltd. All rights reserved. doi: /j.biortech

2 814 S.-Y. Wng et l. / Bioresource Technology 96 (2005) Indigenous cinnmon tree, is one of the hrdwood species tht possesses significnt ntifungl ctivity. C. osmophloeum is n endemic tree tht grows t middle elevtions in TiwnÕs nturl hrdwood forest (Liu et l., 1988). Chemicl constituents of its lef essentil oils re similr to those of the fmous Cinnmomum cssi brk oil (Chng et l., 2001), so the potentil utiliztions of C. osmophloeum leves re worthy of explortion. Leves my be source of effective pesticidl compounds nd my come to be regrded s n inexhustible source of friendly pesticides hving low humn nd environment toxicity nd being esily biodegrdble. Although there hve been few studies tht discuss the ntimicrobil nd ntitermitic ctivities of C. osmophloeum (Chng et l., 2001; Chng nd Cheng, 2002), to our knowledge there is no literture concerning the ntifungl properties of C. osmophloeum leves ginst wood decy fungi. In this study, we distilled the lef oils from three C. osmophloeum trees. After gs chromtogrphy nlysis nd ntifungl ssys, the chemicl constituents nd the ntifungl performnces of these essentil oils were investigted. The results re reported here. In ddition, the ntifungl ctivity of cinnmldehyde congeners ws subsequently exmined to help to understnd the effect of chemicl structure on the ntifungl ctivity. 2. Methods 2.1. Collection of C. osmophloeum leves nd their essentil oils The leves of three twenty-yer-old C. osmophloeum clones, nmed s tree A, tree B, nd tree C, were collected in My 2000 from the Hw-Lin Experimentl Forest locted in Tipei county. The species ws identified, nd the voucher specimens (COA01, COB01, nd COC03) were deposited t the lbortory of wood chemistry, School of Forestry nd Resource Conservtion, Ntionl Tiwn University. The fresh lef oils of C. osmophloeum were obtined by using wter distilltion for 6 h nd their constituents determined by gs chromtogrphy Gs chromtogrphy (GC) nlysis GC ws performed using Shimdzu model-14b equipped with n FID. The column (Crbowx column) used ws 50m long by 0.22mm i.d. glss cpillry coted with silic. GC ws progrmmed from 60 to 220 C t 2 C/min. N 2 ws the crrier gs t the flow rte of 30 ml/min. Identifiction of the mjor components of indigenous cinnmon lef oils ws confirmed by comprison with stndrds, s well s by spiking. The quntity of compounds ws obtined by integrting the pek re of the chromtogrms. References stndrds were obtined from Acros (Belgium) Congeners of cinnmldehyde For the structure ctivity reltionship study of cinnmldehydeõs derivtives, the congeners of cinnmldehyde, including 4-hydroxybenzldehyde, cinnmic cid, cinnmyl lcohol, nd 3-phenylpropionldehyde, were purchsed from Acros (Belgium) Fungl strins In this study, five white rot fungi (Coriolus versicolor, Lenzites betulin, Pycnoporus coccineus, Trichptum bietinum, nd Oligoporus lowei) nd four brown rot fungi (Letiporus sulphureus, Antrodi tx, Fomitopsis pinicol,ndpheolus schweinitzii) were used in the ntifungl ssy. All of these fungl strins were gifts provided by Dr. Tun-Tschu Chng (Tiwn Forestry Reserch Institute) Antifungl ssys Antifungl ssys were performed bsed on the methods used in our previous studies (Chng et l., 1999, 2000) with slight modifictions. Briefly, 100 ppm of essentil oils or compounds ws dded to sterilize potto dextrose gr (PDA). After trnsferring the mycelium of fungi, the test pltes were incubted t 27 C. When the mycelium of fungi reched the edges of the control plte (without dded essentil oils or compounds), the ntifungl index ws clculted s follows: ntifungl index = (1 D/Db) 100, where D: the dimeter of growth zone in the test plte. Db: the dimeter of growth zone in the control plte. Ech experiment ws performed three times, nd the dt were verged Sttisticl nlyses The Scheffe method ws used to evlute differences in ntifungl index in ntifungl tests. Results with P < 0.05 were considered sttisticlly significnt. 3. Results nd discussion 3.1. Yields nd chemicl compositions of essentil oils from different clones The yields of lef oils from the three C. osmophloeum trees (A, B, nd C) were 6.02, 6.92, nd 7.93mL/kg of leves, respectively. Tble 1 shows the min components nd their reltive contents of lef essentil oils distilled from C. osmophloeum. The reltive mounts of ech component were determined by the GC nlysis. Cinn-

3 S.-Y. Wng et l. / Bioresource Technology 96 (2005) Tble 1 Min components nd their reltive contents (%) of Cinnmonmum osmophloeum lef essentil oils Compounds RT (min) Reltive percentges (%) Tree A Tree B Tree C 1,8-Cineol b Benzldehyde Linlool Borneol Terpineol Nerl Gernyl cette Gerniol Cinnmldehyde Eugenol Cinnmyl cette Coumrin RT: retention time, the nlysis conditions s described in Section 2. b : trce. mldehyde is the mjor compound in the essentil oils of both indigenous cinnmon tree B (81.08%) nd indigenous cinnmon tree C (76%). In contrst, no dominnt compound in the essentil oil of indigenous cinnmon tree A ws isolted, the mounts of ech compound detected re lower thn 13% (Tble 1). According to the results obtined by Hu et l. (1985), there re nine different types of C. osmophloeum s clssified by their chemicl constituents, nmely cssi type, cinnmldehyde type, coumrin type, linlool type, eugenol type, cmphor type, terpineol-4ol type, linlool terpineol, nd mixed type, respectively. On the bsis of this clssifiction, indigenous cinnmon tree A, which ws nlyzed in this study, belongs to the mixed type. On the other hnd, indigenous cinnmon trees B nd C belong to the cinnmldehyde type Antifungl ctivity of essentil oils It hs been proven tht cinnmon oil hs inhibitory effects ginst severl microorgnisms, such s met spoilge orgnisms (Outtr et l., 1997) nd fungi involved in respirtory trct mycoses (Singh et l., 1995). To the best of our knowledge, there hs not been relevnt study investigting the effectiveness of indigenous cinnmon lef oil nd its constituents ginst wood decy fungi. To evlute the ntifungl ctivity of C. osmophloeum lef essentil oils, t first we selected two typicl fungi, Coriolus versicolor (C.v., white rot fungus) nd Letiporus sulphureus (L.s., brown rot fungus), s test strins. Fig. 1 shows the ntifungl ctivities of the lef essentil oils of C. osmophloeum clones t concentrtion of 100ppm. Both brown rot fungus (L.s.) nd white rot fungus (C.v.) were completely inhibited t doses of 100 ppm. Indigenous cinnmon trees B nd C produced essentil oils with much higher ntifungl ctivities thn those possessed by indigenous Antifungl index (%) c b Tree A Tree B Tree C Fig. 1. Antifungl ctivity of three indigenous cinnmon lef oils (100ppm) ginst the white rot fungus Coriolus versicolor nd the brown rot fungus Letiporus sulphureus. White br: C. versicolor; blck br: L. sulphureus. Ech experiment ws performed three times, nd the dt verged (n = 3). Numbers followed by different letters ( c) re significntly different t the level of P < 0.05 ccording to the Scheffe test. cinnmon tree A. The ntifungl indices of indigenous cinnmon tree A were 18.37% nd 34.47% ginst C.v. nd L.s., respectively. The min constituent of essentil oils from indigenous cinnmon trees B nd C is cinnmldehyde (pproximtely 80%), wheres there is only 8.35% cinnmldehyde in the essentil oils from indigenous cinnmon tree A. Singh et l. (2000) hve proven tht cinnmldehyde is strong ntifungl gent ginst humn pthogens. According to their results, cinnmldehyde my be potentil led compound for the development of ntifungl drugs through the control b-(1,3)-glucn nd chitin synthesis in yests nd molds (Bng et l., 2000). Thus, we suspected tht cinnmldehyde ws the principl ntifungl gent of C. osmophloeum lef oil.

4 816 S.-Y. Wng et l. / Bioresource Technology 96 (2005) Antifungl effects of the min constituents of oils Tests were crried out to discover which of the eight min constituents of C. osmophloeum lef essentil oils possessed ntifungl ctivity. As shown in Fig. 2, cinnmldehyde nd eugenol exhibited the strongest ctivity ginst both C.v. nd L.s. mongst the eight compounds tested. Both cinnmldehyde nd eugenol totlly inhibited the growth of L.s. (ntifungl index = 100%) t 100 ppm. Concerning C.v., however, eugenol (ntifungl index = 71.19%) showed lower ctivity thn cinnmldehyde (ntifungl index = 100%). The effectiveness of ll these compounds in terms of ntifungl ctivity ginst C.v. t 100 ppm is rnked s cinnmldehyde (100%) > eugenol (71.19%) > cinnmyl lcohol (25.68%) > coumrin (17.41%) > gerniol (15.73%) > linlool (13.69%) > benzldehyde (11.37%) > -terpineol (6%). On the other hnd, the order of ntifungl indices ginst L.s. is cinnmldehyde (100%) = eugenol (100%) > cinnmyl lcohol (50.71%) > coumrin (37.88%) > gerniol (33.53%) > benzldehyde (28%) > linnool (0%) = -terpineol (0%). Although both cinnmldehyde nd eugenol possess stronger ntifungl ctivity, eugenol exists in only minor quntities in the lef oils of trees B nd C (less thn 1%). The results obtined in this study point to cinnmldehyde being the principl ntifungl constituent of C. osmophloeum. Since cinnmldehyde possessed significnt ntifungl ctivities ginst both white rot fungus (C.v.) nd brown rot fungus (L.s.), to further exmine its ntifungl ctivity we selected nother seven strins of wood Coumrin Cinnmyl lcohol Eugenol Gernil -Terpineol Linlool Benzldehyde Cinnmldehyde h h c, d e, f, g f, f, d, e, f c, d, e d, e, f f, b, c Antifungl index (%) Fig. 2. Antifungl ctivity of the components (100ppm) of indigenous cinnmon lef oil ginst the white rot fungus Coriolus versicolor (white br) nd the brown rot fungus Letiporus sulphureus (blck br). Ech experiment ws performed three times, nd the dt verged (n = 3). Numbers followed by different letters ( h) re significntly different t the level of P < 0.05 ccording to the Scheffe test. b Tble 2 Antifungl indices of cinnmldehyde (100ppm) ginst wood rot fungi Fungi Antifungl index (%) White rot fungi Coriolus versicolor 100 Lenzites betulin 100 Pycnoporus coccineus 100 Trichptum bietinum 100 Oligoporus lowei Brown rot fungi Antrodi tx 100 Fomitopsis pinicol 100 Letiporus sulphureus 100 Pheolus schweinitzii 100 Antifungl index see Section % = complete growth inhibition of mycelium. decy fungi, which re commonly found in the forestry of Tiwn. Tble 2 shows the ntifungl indices of cinnmldehyde ginst wood decy fungi t doses of 100ppm. All of the selected fungi used in this study were totlly inhibited by cinnmldehyde except for Oligoporus lowei (white rot fungi ctegory). The ntifungl index of cinnmldehyde ws 71.45% ginst O. lowei. Over ll the ntifungl ctivity of cinnmldehyde is excellent ginst wood decy fungi Structure ctivity reltionship study of cinnmldehyde s derivtives According to the results obtined in this study, cinnmldehyde is the strongest of ntifungl compounds in the lef oils of C. osmophloeum. To exmine the structure ctivity reltionships of cinnmldehyde, four compounds whose chemicl structures re similr to cinnmldehyde were selected for study. These four compounds were 4-hydroxybenzldehyde, cinnmic cid, cinnmyl lcohol, nd 3-phenylpropionldehyde, nd their chemicl structures re shown in Fig. 3. Fig. 4 presents the results of ntifungl ctivity of these compounds ginst both white rot fungus (C.v.) nd brown rot fungus (L.s.) t doses of 100ppm. It is obvious tht cinnmldehyde still possesses the strongest ntifungl ctivity compred with the other lef oil derivtives. When the ntifungl ctivity of the cinnmldehyde congeners hving both benzyl ring nd conjugted double bond ws rnked: cinnmldehyde (100% nd 100%; ntifungl ctivity ginst C.v. nd L.s.) > cinnmyl cid (75.68% nd 81.34%) > cinnmyl lcohol (25.68% nd 50.71%) > cinnmyl cette (22.35% nd 34.24%). It is cler tht compounds with n ldehyde group hve the best ntifungl ctivity. On the other hnd, compring with the compounds bering with n ldehyde group, the ntifungl indices

5 S.-Y. Wng et l. / Bioresource Technology 96 (2005) HO 1 2 CHO CHO 5 6 COOH O O CH 3 O tionship in potentil ntitumor promoting benzlcetone derivtives, s ssyed by the Epstein Brr virus erly ntigen ctivtion. Bsed upon their results obtined from EBV-EA ctivtion inhibition ssy model, the unsturted bonded-crbonyl system is necessry for inhibition of mutgenesis ctivity in cinnmldehyde derivtives. 3 4 O OH H Fig. 3. Structures of cinnmldehydeõs congeners. (1) Benzldehyde, (2) 4-hydroxybenzldehyde, (3) cinnmyl lcohol, (4) cinnmldehyde, (5) cinnmic cid, (6) cinnmyl cette, nd (7) 3- phenylpropionldehyde. of these ctegories were 100%, 69.56%, 12.47%, nd 11.73% for cinnmldehyde, 3-phenylpropionldehyde, 4-hydroxybenzldehyde, nd benzldehyde, respectively. These results suggest tht compound hving conjugted double bond nd long CH chin outside the ring, i.e. cinnmldehyde, possesses much stronger ntifungl ctivity. A similr observtion ws noted in our previous study on the ntitermitic ctivity of C. osmophloeum (Chng nd Cheng, 2002). Amongst congeners of cinnmldehyde, cinnmldehyde exhibited the strongest termiticidl property. A similr finding ws observed in other bioctivity ssy. Motohshi et l. (2000) hve evluted the structure ctivity rel- 7 H 4. Conclusions To develop methods to prolong the service life of wood is one of the chllenges of wood utiliztion reserchers. With regrd to environmentl protection, finding friendly preservtives from highly durble tree species is n pproprite pproch to chieve protection of the wood without polluting the environment. In this study, we investigted the ntifungl ctivities of lef essentil oils from indigenous cinnmon tree (C. osmophloeum) ginst wood decy fungi, which hs not been previously reported. Lef essentil oils of C. osmophloeum were proved to hve strong effects on the growth inhibition of fungi. Cinnmldehyde, the mjor compound in the lef essentil oils, possessed the strongest ntifungl ctivities compred with the other components. Moreover, there re some specil indigenous cinnmon tree clones (trees B nd C), which contin high mount of cinnmldehyde in the essentil oils of leves. Tht is to sy, to obtin high yield of nturl bioctive essentil oils or cinnmldehyde people could simply hrvest the renewble leves without logging or debrking the trees. We, therefore, suggest tht the essentil oils from indigenous cinnmon leves my be further explored s potentil led compound for the 3-Phenylpropionldehyde c, d, e b, c, d Cinnmyl cette e, f Cinnmic cid, b, c, b Cinnmyl lcohol d, e, f f, g 4-Hydroxybenzldehyde h Benzldehyde f, g Cinnmldehyde Antifungl index (%) Fig. 4. Antifungl ctivity of the congeners of cinnmldehyde t doses of 100ppm. White br: ginst C. versicolor; blck br: ginst L. sulphureus. Ech experiment ws performed three times, nd the dt verged (n = 3). Numbers followed by different letters ( h) re significntly different t the level of P < 0.05 ccording to the Scheffe test.

6 818 S.-Y. Wng et l. / Bioresource Technology 96 (2005) development of wood decy preservtion gents or fumignts in the ner future. References Bng, K.H., Lee, D.W., Prk, H.M., Rhee, Y.H., Inhibition of fungl cell wll synthesizing enzymes by trns-cinnmldehyde. Biosci. Biotech. Biochem. 64, Chng, S.T., Cheng, S.S., Antitermitic ctivity of lef essentil oils nd components from Cinnmomum osmophloeum. J. Agric. Food Chem. 50, Chng, S.T., Chen, P.F., Chng, S.C., Antibcteril ctivity of lef essentil oils nd their constituents from Cinnmomum osmophloeum. J. Ethnophrmcol. 77, Chng, S.T., Wng, S.Y., Wu, C.L., Su, Y.C, Kuo, Y.H., Antifungl compounds in the ethyl cette soluble frction of the extrctives of Tiwni (Tiwni cryptomerioides Hyt) hertwood. Holzforschung 53, Chng, S.T., Wng, S.Y., Wu, C.L., Chen, P.F., Kuo, Y.H., Comprison of the ntifungl ctivity of cdinne skeletl sesquiterpenoids from Tiwni (Tiwni cryptomerioides Hyt) hertwood. Holzforschung 54, Hu, T.W., Lin, Y.T., Ho, C.K., Nturl vrition of chemicl components of the lef oil of Cinnmomum osmophloeum Kneh. Bull. Tiwn Res. Inst. Eng. 78, 18. Liu, Y.C., Lu, F.Y., Ou, C.H., Trees of Tiwn; Monogrphic Public no. 7, College of Agriculture, Ntionl Chung-Hshing University, Tichung, Tiwn, p Motohshi, N., Ymgmi, C., Tokud, H., Okud, Y., Ichiishi, E., Mukink, T., Nishino, H., Sito, Y., Structure ctivity reltionship in potentilly nti-tumor promoting benzlcetone derivtives, s ssyed by the Epstein Brr virus erly ntigen ctivtion. Mutt. Res.-Genet. Toxicol. Environ. Mutt. 464, Outtr, B., Simrd, R.E., Holley, R.A., Piette, G.J.P., Begin, A., Antibcteril ctivity of selected ftty cids nd essentil oils ginst six met spoilge orgnisms. Int. J. Food Microbiol. 37, Singh, H.B., Srivstv, M., Singh, A.B., Srivstv, A.K., Cinnmon brk oil, potent fungitoxicnt ginst fungi cusing respirtory trct mycoses. Allergy 50,