The Fungicidal Terpenoids and Essential Oil from Litsea cubeba in Tibet

A new C9 monoterpenoid acid (litseacubebic acid, 1) and a known monoterpene lactone (6R)-3,7-dimethyl-7-hydroxy-2-octen-6-olide (2), along with three known compounds – vanillic acid (3), trans-3,4,5-trimethoxylcinnamyl alcohol (4), and oxonantenine (5) – were isolated with bioassay-guided purification from the fruit extract of Litsea cubeba collected in Tibet. The structure of 1 was elucidated by MS, 1H-NMR, 13C-NMR, COSY, HSQC, HMBC, NOE spectral data as 2,6-dimethyl-6-hydroxy-2E,4E-hepta-2,4-diene acid. Additionally 33 compounds were identified from the essential oil of L. cubeba. The preliminary bioassay results showed that 1 and 2 have good fungicidal activities against Sclerotinia sclerotiorum, Thanatephorus cucumeris, Pseudocer-cospora musae and Colletotrichum gloeosporioides at the concentration of 588 and 272 μM, and the essential oil has good fungicidal activities against T. cucumeris and S. sclerotiorum, with IC50 values of 115.58 and 151.25 μg/mL, repectively.


Introduction
Litsea cubeba (Lour.)Pers. is a plant of Lauraceae family. The essential oil from the fruit contains 75% citral, but the essential oil from the leaves contains more 1,8-cineole than citral [1]. In fact the compositions were different due to their location and collection times [2]. The essential oil is widely used as a flavor enhancer in foods, cosmetics and cigarettes; as raw material for the manufacture of citral, vitamins A, E and K, ionone, methylionone, and perfumes; and as an antimicrobial and insecticidal agent [3], and the complex and molecular microcapsules of L. cubeba oil with βcyclodextrin and derivatives were reported recently [4]. The oral and dermal LD 50 of the oils in mice were reported near 4,000 and 5,000 mg/kg of body weight, respectively. In addition lots of aporphine alkaloids [5][6][7][8][9], lactone [10], and flavone derivatives were isolated from the root, stem, barks of L. cubeba, and their structure-activity relationships against AchE for the aporphine alkaloids and their derivatives have been discussed in detail [11]. The crude extract of L. cubeba was screened in our laboratory and showed fungicidal activities. However, the ingredients of the essential oil, and the extracts from the fruit of L. cubeba produced in Tibet, and their fungicidal activities were unknown. In order to seek new lead fungicidal compounds, the components of essential oil, and extracts from the fruit of L. cubeba collected in Tibet, and their fungicidal activities are reported in this paper.
Bioassay-guided fractionation, macroporous resin column chromatography, silica gel chromatograph, and preparative TLC isolation of the EtOH extracts of the fruit of L. cubeba yielded five compounds. Four of them were characterized as (6R)-3,7-dimethyl-7-hydroxy-2-octen-6-olide (2) [12,13], vanillic acid (3) [14], trans-3,4,5-trimethoxyl-cinnamyl alcohol (4) [15], and aporphine alkaloid oxonantenine (5) [16]   7.27 and 6.58, 6.58 and 6.20 in the COSY spectrum of 1, and the coupling constants of 11.0 and 15.5 Hz, showed they are the adjacent protons connected on the conjugated olefin. The long range allylic coupling of 1.0 Hz between the olefin protons at δ 7.27 and the methyl group at δ 1.96 was confirmed due to the correlation between them in the COSY spectrum of 1. The carbon signals were assigned on the basis of HSQC and DEPT spectra of 1. The positions of substituent groups were determined depending on the correlations between the proton at δ 1.96 and the carbon at δ 172.18, 126.59, and 139.45, the proton at δ 7.27 and the carbon at δ 149.27 and 12.50, the proton at δ 6.58 and the carbon at δ 71.13, the proton at δ 6.20 and the carbon at δ 71.13 and 29.69, the proton at δ 1.38 and the carbon at δ 149.27. The E-configuration of double bonds at C 2 and C 4 positions were determined due to the NOE enhancement of the proton at δ 6.58 when irridiating the proton at δ 1.96 in the NOE difference spectrum, and the 15.5 Hz coupling constant of the protons at C 4 and C 5 . Thus the structure of 1 was elucidated to be 2,6-dimethyl-6-hydroxy-2E,4E-hepta-2,4-diene acid. To the best of our knowledge, it is found for the first time in Nature. As we know, 2,6-dimethyl-6-hydroxy-2E,4E-hepta-2,4-dienal (6) was found to be present in Labdanum oil [17], and the tradional Chinese medicine Alpinia oxyphilla Miq. [18]. Both 1 and 6 are C 9 monoterpenes, which including the other C 9 monoterpenoids such as dimetol originate from biosynthesis pathway of terpenoids, and 2 are relating structurally with citral and geranic acid existing in the essential oil of L. cubeba.  (2), and for the essential oil 300 μg/mL. The data in Table 1 indicate that compound 1 showed 56.7%∼86.7% inhibitory ratio against seven fungi, compound 2 showed 53.5%∼81.8% inhibitory ratio against 13 fungi, while the essential oil showed excellent fungicidal activities against S. sclerotiorum, T. cucumeris and A. mali with 100%, 100%, and 83.2% inhibitory rate, respectively. However, the data in Table 1 also indicated that 1, 2, and the essential oil showed no significant inhibition against the other fungi. These results suggested that the activities of 2 were relatively broad spectrum, which indicated the lactone ring in 2 is a very important moiety for the fungicidal activities of this kind of terpenoid compounds. The further precise determination of the toxicities for the essential oil indicated that the IC 50 values against S. sclerotiorum and T. cucumeris were 151.25 and 115.58 μg/mL, repectively.

General
Melting points were measured on a Yanagimoto apparatus and are uncorrected. Optical rotations were measured on a Perkin-Elmer 341 polarimeter. 1 H-NMR, 13 C-NMR, COSY, HMQC, HMBC, and NOESY spectra were recorded on Brüker DRX 500 and DPX 300 NMR Spectrometers with CDCl 3 as solvent and TMS as internal standard. ESI-MS data were analyzed with an Agilent LCQ LC-MSD iontrap mass spectrometer. HR-FAB-MS were obtained on Brüker Apex II mass spectrometer using nitrobenzoyl alcohol and sodium chloride as matrix. The solvents were analytical grade and without treatment before usage. Agilent 6890GC 5973I MSD GC-MS spectrometer with HP-5MS column (0.25 mm × 30 m × 0.25 μm) was used. The standard mixture of alkanes was provided friendly by Dr. Zhou Ligang, China Agricultural University. The NIST Library (2002 Ed.) for mass spectra search and RI values were used for peak identification of the essential oil analysis.

Plant Material
The fruit of Litsea cubeba was collected in Yigong workshop, Bomi County, Tibet, China on Oct., 2008, and authenticated by Prof. Wang Li of the Department of Plant Sciences, College of Biology, China Agricultural University. The voucher specimens were deposited at the Department of Plant Sciences, China Agricultural University.

Extraction and Isolation
The 10.93 g essential oil was obtained from 400 g of L. cubeba fruit by steam distillation for 12 h and dried over anhydrous MgSO 4 . The dried and pulverized fruit (4 Kg) of L. cubeba was extracted three times with 95% C 2 H 5 OH one week each time. The extracted material was dispersed into water, and re-extracted three times with petroleum ether and EtOAc, respectively. The EtOAc extract was evaporated to afford a residue (17 g). The water phase was absorbed on a macroporous resin column, washed glucose with water, and eluted with 95% C 2 H 5 OH, evaporated the eluents under reduced pressure to give a residue (10 g).

Bioassay of Fungicidal Activities
Fungicidal activities of compounds 1, 2 and the essential oil against B. cinerea, A.mali, A. alternate, F. oxysporurn, F. moniliforme, F. graminearum, P. infestans, S. sclerotiorum, C. lindemuthianum, V. dahliae, T. cucumeris, B. berengeriana, P. musae, and C. gloeosporioides were evaluated using the mycelium growth rate test [19]. The culture media with known concentration of the test compounds were obtained by mixing the solution of 1, 2, and the essential oil in acetone with potato dextrose agar (PDA), on which fungus cakes were placed. The blank test was made using acetone and carbendazim (7) was used as positive control. The culture was incubated at 25 ± 0.5 °C. Three replicates were performed. After the mycelium in the blank grew completely, the diameter of the mycelium was measured and the inhibitory ratio calculated according to the following formula. In which I is the inhibitory ratio, P 0 is the average diameter of the mycelium in the blank, and P 1 is the average diameter of the mycelium in the presence of the test samples. Mean measurements were calculated from the three replicates. The IC 50 values were calculated using linear relation between the inhibitory probability and concentration logarithm according to the method outlined by Finney [20].