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Antifungal Activity of the Oil and its Major Sesquiterpene Compounds Izabela Jasicka-Misiaka,*, Jacek Lipoka, Ewa M. Nowakowskaa, Piotr P. Wieczoreka, Piotr Młynarzb, and Paweł Kafarskia a Institute of Chemistry, University of Opole, Oleska 48, 45-052 Opole, Poland. Fax: +4877444401. E-mail: [email protected] b Institute of Organic Chemistry, Biochemistry & Biotechnology, Wroclaw University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland * Author for correspondence and reprint requests Z. Naturforsch. 59c, 791Ð796 (2004); received July 22/August 8, 2004 is the source of the carotane sesquiterpenes carotol, daucol and -caryo- phyllene. These sesquiterpenic allelochemicals were evaluated against alternata isolated from the surface of carrot Perfekcja, a variety widely distributed in horticultural practise in Poland. Alternaria alternata is one of the most popular phytotoxic fungi infesting the carrot . The strongest antifungal activity was observed for the main constituent of carrot seed oil, carotol, which inhibited the radial growth of fungi by 65% at the following concentration. Key words: Carrot Seeds Oil, Carotol, Daucol, Antifungal Activity

Introduction storage, period of dormancy or at the very begin- ning of seedling development and emergency still Nowadays there is a clear tendency towards the remains unanswered. The fungal infections might utilisation of natural products, especially allelo- be considered as the main factor influencing the chemicals, as alternative compounds for pest and health of at these early stages of their plant disease control, safe for humans and envi- growth and development. Therefore, it is not sur- ronment. Therefore, the search of new natural prising that many of the sesquiterpenoid com- products including plant extracts, which might sub- pounds are known from their antifungal activity stitute synthetic agrochemicals or contribute to the against plant pathogenic fungi, including the most development of new agents for pest control, seems popular Alternaria sp. (Alvarez-Castellano et al., to be important. It is well recognised that among 2001; Skaltsa et al., 2000). For example the sesqui- other plant products essential oils, rich in terpe- terpene fulvoferruginin shows strong activity noids and non-terpenoid compounds, possess vari- against Gram-positive bacteria and significant an- ous and interesting allelopathic properties. Their tifungal activity towards Paecilomyces varioti. An- insecticidal action against specific pests and fungi- other carotane sesquiterpene, rugosal A, isolated cidal action towards some important plant patho- from Rosa rugosa, which is accumulated in the gens have been recently reviewed (Isman, 2000). trichomes, shows antifungal activity against Cla- Over the past decade a large volume of data dosporium herbarum (Ghisalberti, 1994). documenting the defence abilities of various seeds Large varieties of compounds synthesised in with a long period of dormancy were accumulated carrot tissues are known also for their allelopathic (Halloin, 1983; Harman, 1983; Kremer et al., 1984; activity. These include asarones (Guerin and Städ- Ceballos et al., 1998; Özer et al., 1999). Among ler, 1984; Jasicka-Misiak and Lipok, 2000), chlo- them antimicrobial activity of chemicals exuded rogenic acid (Cole et al., 1988) and trans-2-nonenal from seeds and acting on soil-rhizosphere inter- (Guerin and Ryan, 1980). In previous reports we face was reported (Helsper et al., 1994). Quite sur- described phytotoxic activity of carrot seed oil and prisingly, there is little information about the role its main terpenoic components (Jasicka-Misiak and importance of allelochemicals, which are pri- et al., 2002). mary constituents of seeds. The question whether To the best of our knowledge, the chemical com- these compounds might play any role during the position and in vivo antimicrobial activities of

0939Ð5075/2004/1100Ð0791 $ 06.00 ” 2004 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D 792 I. Jasicka-Misiak et al. · Antifungal Activity of Sesquiterpenes carrot seed oil were investigated several times fractions 9Ð20 having one spot of Rf 0.86, charac- (Guerin and Reveillere, 1985; Dwivedi et al., 1991; teristic for carotol, were combined. The fractions Kilibarda et al., 1996;, Friedman et al., 2002). Al- were evaporated to dryness (3.35 g) yielding a pale though, the information about allelopathic activity oil, which was identified by means of GC- of terpenes at all is massively accumulated in the MS and 1H NMR spectroscopy. literature, the biological role of carrot seed sesqui- Daucol. It was obtained by a series of silica gel terpenes is still poorly defined, especially in the column chromatographic steps. The first step was context of the low sensitivity of carrot seeds to the same as during carotol isolation. The fractions fungal infections. were controlled by TLC, and fractions 46Ð55, The structures and chemical properties of two showing a similar spotting patterns zone (Rf be- most specific carrot seed sesquiterpenes, carotol tween 0 and 0.32), were combined. After evapora- and daucol, were described (Sykora et al., 1961; tion, the residue (0.19 g) was characterised by GC- Hashidoko et al., 1992; Platzer et al., 1987; Bülow MS, showing the presence of 10 compounds. This and König, 2000), but nothing could be traced in mixture was applied onto a silica gel column the literature about their antifungal activity. The (Merck Kieselgel 60; 0.063Ð0.2 mm particle size; limited knowledge concerning the biological activ- 300 ¥ 15 mm ID). The column was eluted with ity of daucol and carotol is surprising if con- CH2Cl2/EtOAc (15:1 v/v). Eluates were collected sidering the fact that they are present in carrot as 26 ¥ 20 ml subfractions, analysed by TLC, and seed oil in high amounts. Moreover, the oil is rela- those containing daucol were combined (fractions tively cheap and commercially available therefore 10Ð17). The obtained compound was recrystal- it can be treated as a valuable source of antifun- lised from hexane at 4 ∞C for 48 h and analysed by gal substances. GC-MS and 1H NMR spectroscopy. In this work the novel, improved procedure of isolation of carotol and daucol is reported and the Structural studies antifungal activity of these compounds against Al- The analysis of the was performed ternaria alternata, the most popular phytotoxic using a Hewlett Packard 6890 gas chromatograph fungus, was examined. equipped with a FID detector. The diethyl ether solution (1 µl) was injected into a HP-1 capillary Materials and Methods column (30 m ¥ 0.32 mm bonded-phase fused sil- Starting material ica). The initial oven temperature was maintained at 60 ∞C for 2 min and then raised at 10 ∞C minÐ1 Carrot seed oil was purchased from Augustus to 280 ∞C. Helium was used as carrier gas. MS Oils Ltd., London. -Caryophyllene was pur- analyses were performed on a quadrupole Hewlett chased from Aldrich, Poland. The commercially Packard 6897 instrument with ionisation at 70 eV. available fungicide Funaben T containing thiuram The structure of the active compound was found (45%) and carbendazim (20%) was produced by using a peak matching library search to published Chemical Corporation Organika-Azot s.a., Poland. standard mass spectra and by comparison with lit- Seeds of carota L. var. Perfekcja, collected erature data. in 2001, were purchased from Torseed Co., The NMR experiments were carried out using Torun, Poland. a Bruker DRX 300 MHz spectrometer. Chemical shifts were referred to TMS (tetramethylsilane). Isolation of carotol and daucol The proton and carbon assignments were per- Carotol. A portion (10 g) of the carrot seed oil formed by means of COSY, TOCSY, HMQC, was subjected to silica gel (Merck Kieselgel 60; DEPT-135 and HMQC-TOCSY experiments 0.063Ð0.2 mm particle size; 650 ¥ 25 mm ID) col- using a spin lock time in the range 80Ð120 ms. The umn chromatography. The column was eluted with TOCSY spectra were acquired with total spin- CH2Cl2, followed by 1% MeOH in CH2Cl2 and locking time of 80 ms using the MLEV-17 mixing eluates were collected as 62 ¥ 20 ml fractions. sequence. In order to assign the carbon signal of Each fraction was analysed by TLC (plate 5553, the daucol molecule the HMBC spectra were ad- Merck; solvent system benzene/EtOAc, 19:1 v/v, ditionally performed. developed by spraying with 0.5% aldehyde in MeOH, heating at 105 ∞C for visualisation), and I. Jasicka-Misiak et al. · Antifungal Activity of Sesquiterpenes 793

Antifungal activity Table I. Levels (peak area percent) of major compo- nents of carrot seed oil purchased from Augustus Oils Fungal bioassays in 9-cm Petri dishes were de- Ltd. signed to evaluate the influence of tested sub- stances on mycelial growth of Alternaria alternata. Component Carrot seed oil The mixtures of: carotol, caryophyllene and daucol RTa RAb (%) 8:2:1 w/w/w (at the same weight ratio as it was found in carrot seed oil and in carrot seeds); caro- α-Thujene 3.06 1.90 α tol and daucol 8:1 w/w; carotol and caryophyllene -Pinene 3.20 3.94 Camphene 3.43 0.92 4:1 w/w; caryophyllene and daucol 2:1 w/w as well -Pinene 3.46 1.90 as carrot seed oil were tested for antifungal activ- -Myrcene 3.65 1.44 ity. Appropriate amounts of above terpenoids α-Terpinene 3.97 1.43 were mixed (before sterilization) with Czapek me- o-Cymene 4.03 1.34 Limonene 4.12 1.75 dium to obtain the final concentration of 150 mg/l. γ-Terpinene 4.49 1.43 The commercially available fungicide Funaben T Terpinolene 5.54 0.63 was used as a control. The agar was allowed to Linalool 6.08 0.51 solidify and experiments were initiated by placing Pinen-4-ol 7.32 0.42 6-mm fungal plugs taken from the growing mar- Terpinen-4-ol 8.16 0.22 3-Carene 8.60 0.83 gins of 9-day-old cultures, mycelial side down, on Neryl acetate 8.69 1.06 Czapek medium. Plates were incubated at 24Ð Calarene 8.80 3.23 25 ∞C. Radial growth of the strain was recorded Zingibren 8.99 2.13 α daily, by taking the mean diameter of colonies -Farnesene 9.10 3.35 -Caryophyllene 9.18 10.66 from each plate. The same experiments were car- α-Cedrene 9.35 2.74 ried out for individual compounds: carotol, daucol α-Himachalene 9.44 0.55 and caryophyllene. All the experiments were per- -Cubebene 9.57 0.53 α formed four times, with four replications with four -Longipinene 9.73 0.76 Aromadendrene 9.93 1.92 pure Czapek medium controls. -Farnesene 10.08 4.03 Levomenol 10.18 0.34 A aldehyde 10.33 0.66 Data analysis Isolimonene 10.56 3.24 Caryophyllene oxide 10.97 4.34 The data were subjected to analysis of variance Carotol 11.22 38.85 to test the significance of all factors examined in -Cadinene 11.49 0.25 each experiment. F test was done to determine the Daucol 11.57 2.00 homogeneity of error variances among runs. Treat- Total 99.30 ment means were separated using Tukey’s HSD a Retention time on HP-1 column in minutes. test at a 5% significance level (Statgraphics“ b Relative area (peak area relative to total peak area). Plus 5, 2000). The main components of the oil were carotol Results and Discussion (38.8%) and -caryophyllene (10.7%), accompa- nied by caryophyllene oxide (4.3%) and a second Carrot seed oil composition daucane sesquiterpene alcohol namely daucol The chemical composition of the carrot seed oil which is present in significant amounts (2.0%). was determined by GC-MS analysis. The results The other identified volatile components have are presented in Table I as percent of the total MS been reported previously as constituents of an- ion current. The compounds are listed according other organs of carrot (Seifert and Buttery, 1978; to their elution order. Monoterpenes and sesqui- Buttery et al., 1979; Kjeldsen et al., 2001). All of terpenes represent the major components of car- them, except the sesquiterpenic alcohols carotol rot seed oil. Out of 40 compounds detected in the and daucol are well-known compounds isolated chromatogram of the carrot seed oil 33 were iden- from many other plant sources. Especially, -ca- tified. In our studies, only those components which ryophyllene is a widespread sesquiterpene. Caro- were present in the oil in amounts higher than tol, daucol and -caryophyllene comprised 51.5% 0.1% have been taken into consideration. of the oil. 794 I. Jasicka-Misiak et al. · Antifungal Activity of Sesquiterpenes

11 11 OH Table III. 1H and 13C chemical shifts of daucol in chloro- 2 2 form (CDCl3). 3 12 3 12 10 1 10 1 9 9 O 1 a113 8 4 8 4 Number of H H C 5 5 7 6 6 carbon with 13 OH its proton 14 14 a b 1C 45.15 15 15 2CH2 1.28/1.68 1.28/1.68 40.09 Fig. 1. Structures of carotol (a) and daucol (b). 3CH 3.72 3.74 71.70 4C 85.22 5CH2 1.36/1.86 1.37/1.86 29.50 6CH2 1.55/2.15 1.58/2.15 41.15 Isolation of carotol and daucol 7C 91.63 The previously described methods for isolation 8CH 1.50 1.50 52.40 9CH2 1.70 1.71 26.20 of carotol and daucol are based on fractional dis- 10CH 1.25/1.30 1.24/1.28 32.90 tillation (Sykora et al., 1961; Hashidoko et al., 2 11CH3 1.06 1.06 22.42 1992). In our opinion column chromatography is a 12CH3 1.36 1.36 23.45 faster and simpler method since carotol was ob- 13CH 1.77 1.78 31.51 tained from carrot seed oil by only one step by 14CH3 0.81 0.82 21.79 15CH 1.06 1.06 22.93 means of silica gel column chromatography with 3 high efficiency (99%). Fractions with daucol were a Platzer et al., 1987. provided by the same column, however, they re- quired further purification by means of additional silica gel column chromatography. These three cule are in good agreement with those reported in chromatographic steps resulted in the isolation of the literature (Bülow and König, 2000; Platzer daucol of 99% purity. et al., 1987; Hashidoko et al., 1992). The proton 1 13 H NMR and C NMR spectra of carotol and chemical shifts of daucol are also in good consis- daucol (Fig. 1) assigned using the set of NMR ex- tence with only one up to now published data periments (see Materials and Methods) are sum- (Bülow and König, 2000), however, additional as- marized in Tables II and III.( The chemical shifts signments for carbon atoms were performed (Ta- of proton and carbon peaks for the carotol mole- ble III).

Table II. 1H and 13C chemical shifts of carotol in chloro- Antifungal activity form (CDCl3). The antifungal activity of carrot seed terpenoids Number of 1Ha13Ca1H 13C was tested on strains isolated from non-disinfected carbon and untreated seeds. Seven strains of fungi belong- with ing to the Alternaria family and one strain of its proton Acremonium were isolated from the surface of 1C 49.09 49.08 carrot seeds. Thus, Alternaria predominated 2CH2 1.70/2.26 38.62 1.70/2.26 38.62 among all the identified genera of fungi. Because 3CH 5.32 122.13 5.32 122.13 the phytopathogenic activity of Alternaria towards 4C 138.60 138.59 carrot plants is well documented they were chosen 5CH 2.08 29.45 2.08 29.45 2 for further experiments. 6CH2 1.94 34.45 1.63/1.94 34.41 7C 84.55 84.54 At the start, we tested the effect of crude carrot 8CH 1.80 52.54 1.79 52.53 seed oil and appropriate mixtures of carotol, 9CH2 24.39 1.52/1.68 24.39 caryophyllene and daucol in different weight ra- 10CH2 1.3 39.45 1.29/1.57 39.45 tios (see Materials and Methods) on the growth of 11CH3 0.95 21.47 0.95 21.47 12CH3 1.67 25.23 1.67 25.24 Alternaria alternata. The composition of the mix- 13CH 1.80 27.58 1.81 27.59 ture of the three terpenoids was set at the same 14CH3 1.00 24.04 1.00 24.05 ratio as it was found in crude oil. For comparison 15CH3 0.95 21.38 0.94 21.38 the additional tests with the commercially avail- 7C-OH 1.14 1.14 able fungicide Funaben T were also performed. a Bülow and König, 2000. The obtained results are shown in Fig. 2a. Carrot I. Jasicka-Misiak et al. · Antifungal Activity of Sesquiterpenes 795

11 10 control a carotol;caryophyllene (2:1 w/w) 9 carotol;caryophyllene (4:1 w/w) 8 carotol;daucol (8:1 w/w) 7 carotol;caryophyllene;daucol (8:2:1 w/w/w) Funaben T 6 5 4 3 2 1 0 12345678910 11

Colony diameter [cm] 10 b control 9 seed oil 8 daucol 7 carotol Funaben T 6

5

4

3

2

1

0 12345678910 Fig. 2. The effect of tested mixtures (a) and poor substances (b) at 150 mg/l on mycelial growth of Alternaria alternata Cultivation time [d] on solid media. seed oil exhibited moderate inhibitory effects on pounds started to influence the fungal growth after mycelium radial growth of Alternaria alternata the third day of incubation (see the standard devi- (20% of inhibition). Mixtures containing only ca- ation bars) and the differences in their action had rotol exhibited strong inhibitory activity. This ac- significantly grown with time. Carotol significantly tivity increased with an increasing content of caro- inhibited the growth of the fungi and reduced the tol in individual mixtures. The highest inhibition colony radial size by 65% at the 9th day of the of the growth of pathogenic fungi was determined experiment. Quite different effects were observed for a carotol and daucol mixture (66%) in which for daucol (second specific sesquiterpene alcohol the content of carotol was the highest (90%). of the oil) where slight stimulation of the develop- The experiments with individual substances, ment of Alternaria alternata was observed. Wide- namely carotol, caryophyllene and daucol were spread in various plants the sesquiterpene -ca- carried out to find out, whether the observed ac- ryophyllene failed to have any effect. Therefore, it tivity derives from the action of carotol only or can be concluded that carotol is the main agent from a synergetic nature. The kinetics of inhibition attributed for antifungal activity of carrot seeds. of Alternaria alternata by these terpenoids used at The activity of carotol is nearly as strong as of the the concentration 150 mgl/l in Czapek medium is commercially abailable fungicide Funaben T shown in Fig. 2b. It is clearly seen that these com- (85%). 796 I. Jasicka-Misiak et al. · Antifungal Activity of Sesquiterpenes

Acknowledgements This research was supported by Polish State Committee for Scientific Research (Komitet Ba- dan´ Naukowych) grants PBZ KBN Ð 060/T09/ 2001/37. Piotr Młynarz wishes to thank The Foun- dations for Polish Science for scholarship.

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