Novel Antifungal Dimers from the Roots of Taiwania cryptomerioides

Five new dimer compounds, namely Taiwaniacryptodimers A–E (1–5), were isolated from the methanol extract of the roots of Taiwania cryptomerioides. Their structures were established by mean of spectroscopic analysis and comparison of NMR data with those of known analogues. Their antifungal activities were also evaluated. Our results indicated that metabolites 1, 2, 4, and 5 displayed moderate antifungal activities against Aspergillus niger, Penicillium italicum, Candida albicans, and Saccharomyces cerevisiae.


Introduction
Taiwan cedar (Taiwania cryptomerioides Hayata), also known as Asia cedar, is an essential economic tree species native to Taiwan. It belongs to the same Mesozoic and Cenozoic Tertiary relics with Ginkgo biloba, Sequoiadendron giganteum, and Metasequoia glyptostroboides. It is a rare and precious tree species [1]. It belongs to the gymnosperm phylum, Coniferae, Coniferales, Taxodiaceae, Taiwania, one genus and one species. In 1904, the Japanese Prof. S. Konisi was first discovered in Nantou County, Taiwan. It was named by the botanist Prof. Hayata Bunzo in 1906 and named after Taiwan as its genus, "Taiwania". The meaning of the species name "cryptomerioides" is "like Cryptomeria". It can be seen that the morphological characteristics of Taiwan fir is similar to Cryptomeria japonica (L. f) D. Don. As far as the nature of the wood is concerned, Taiwan cedar is straight, delicate, and beautiful, with a light and soft texture, easy to process, and its heartwood has a beautiful yellow-red texture, which is quite distinctive. Weather resistance, decay resistance, and ant resistance are similar to those of cypress. It is a material with superior properties and is often used for construction, furniture, door panels, and thin panels. The fresh inner skin of Taiwan fir is mashed to apply it to poisonous snake bites. The leaves also have the effects of mashing juice and involving poison and are used for anti-inflammatory, diuretic, and gonorrhea treatment.
Taiwania cryptomerioides (Taxodiaceae) is taxonomically included in one genus and one species of endemic plants in Taiwan. It contains more than 6% of essential oil in its heartwood [1]. T. cryptomerioides is a vital building material with high value in Taiwan. Earlier phytochemical investigations of this plant mainly focused on its heartwood [2][3][4] and bark constituents [5][6][7][8][9] have also been found. In the past, interesting compounds isolated from the heartwood and bark of T. cryptomerioides and those with biological activity prompted us to study the chemical composition of their roots. Several kinds of sesquiterpenes with unique and novel structures have also been obtained from its roots [10][11][12][13][14]. However, compared with the composition of different parts of T. cryptomerioides in the past literature, it is found that there is very little research on the roots, and it is worth continuing to explore its composition. In this paper, we would like to report the five new dimers, namely, Taiwaniacryptodimers A-E (1)(2)(3)(4)(5), isolated from the roots of the T. cryptomerioides. The MeOH extract of the roots of T. cryptomerioides was suspended in H 2 O and partitioned between H 2 O and EtOAc. The EtOAc-soluble portion was subjected to repeated silica gel column chromatography and semipreparative normal phase-HPLC to afford compounds 1-5. Herein, the isolation and structure elucidation of five new compounds and their antifungal activity is described. ] + , as well as forty carbon signals found in the 13 C NMR spectrum hinted that 1 was a dimeric diterpenoid ( Figure 1). The UV maxima at 229, 284, and 314 nm suggested the presence of a benzoyl functionality. The absorption bands for hydroxyl (3582 cm −1 ), γ-lactone (1791 cm −1 ), and aromatic (1616, 1499 cm −1 ) groups were observed in its IR spectrum. The 1 H and 13 C NMR data of 1 (Tables 1 and 2 -20]. Those data proved that the structure of constituent upper monomer-1 was related to demethylsalvicanol [15]. The 1 H and 13 C NMR data of 1 (Table 1) also exhibited another set of signals for constituent lower monomer-2 (B): two methylenedioxy groups (δ H 5.94 (2H, s, C4 ,5 -OCH 2 O-), 5.98/6.00 (each 1H, J =1.6 Hz, C4 ,5 -OCH 2 O-) at two benzene moieties, respectively, and CH 2 protons in a γ-position of a γ-lactone ring (δ H 3.94 (1H, dd, J = 13.2, 2.8 Hz, H-9 ), 4.81 (1H, dd, J = 13.2, 2.8 Hz, H-9 ). Additionally, two ABX systems (6 H) of aromatic protons were observed (δ H 6.91 (1H, d, J = 8.0 Hz, H-2 ), 6.94 (1H, s, H-6 ), 6.80 (1H, d, J = 8.0 Hz, H-3 ), and δ H 6.42 (1H, s, H-6 ), 6.44 (1H, d, J = 8.0, H-2 ), 6.73 (1H, d, J = 8.0 Hz, H-3 )), besides two benzylic protons (δ H 5.06 (1H, s, H-7 ), 6.47 (1H, s, H-7 )). The 13 C-NMR spectrum and DEPT experiment showed 20 signals including a γ-lactone carbonyl C-atom (δ C 171.7 (C-9 )), twelve aromatic C-atoms, and three CH 2 (δ C 101.4 (C4 ,5 -OCH 2 O-), 101.4 (C4 ,5 -OCH 2 O-), 69.6 (C-9 )) and two CH groups (δ C 77.4 (C-7 ), 128.7 (C-7 )). The above NMR spectroscopic data of constituent lower monomer-2 are very similar to that of Taiwanin A [16], suggesting constituent lower monomer-2 was a lignanoid-derivative. Thus, the gross structure of 1 is composed of demethylsalvicanol and Taiwanin A. After calculating the degree of unsaturation, subtracting the above-inferred part, there is still one degree of unsaturation. The C-7 /C-12 and C-8 and C-11 linkage of the two monomers in 1 was through a O-linkage functionality; judged by calculating the degree of unsaturation, subtracting the above inferred parts A and B, there is still one degree of unsaturation. Its molecular formula contains nine oxygen atoms and the downfield shift of H-7 (δ H 5.06) in constituent monomer-1, compared to that of the Taiwanin A analogues.

Results and Discussion
8′ and C-11 linkage of the two monomers in 1 was through a O-linkage functionality; judged by calculating the degree of unsaturation, subtracting the above inferred parts A and B, there is still one degree of unsaturation. Its molecular formula contains nine oxygen atoms and the downfield shift of H-7′ (δH 5.06) in constituent monomer-1, compared to that of the Taiwanin A analogues.     ] + , indicated that 2 was also a dimeric diterpenoid. The IR spectrum indicated the presence of hydroxyl (3576 cm −1 ), ester carbonyl (1791 cm −1 ), and aromatic (1616, 1499, 1447 cm −1 ) groups. The UV maxima at 229, 284, and 314 nm indicated a benzoyl functionality. Comparison of 1 H and 13 C NMR data of 1 and 2 (Tables 1 and 2) showed that the signals of constituent upper monomer-1 of 2 were almost the same as those of 1; thus, the structure of constituents upper monomer-1 and lower monomer-2 were related to demethylsalvicanol and Taiwanin A, respectively. Compounds 1 and 2 are speculated to be 9(10→20)abeoabietane type diterpenoids (demethylsalvicanol) combined with 8,8 -lignans (Taiwanin A). After calculating the degree of unsaturation, subtracting the above inferred part, there is still 1 degree of unsaturation. Therefore, the combination of the two monomer compounds should be adjacent to the oxygen atom and cyclized. The Z-configuration of the extra-cyclic olefinic proton at C-7 was confirmed by the observation of the 1 H-1 H NOESY correlation between H-7 and H-9 in compounds 1-3 (Table 3).  . Although there are other chiral centers, the absolute configurations of Parts A and B have been confirmed according to the literature. Therefore, it can only be based on the large difference between the positive and negative values of the optical rotation value that the relative stereo configuration of the C-7 position should be the opposite. Compounds 1 and 2 were a pair of C-7 stereoisomerism. Therefore, we temporarily decided that the relative configuration at C-7 of 1 is β-orientation. According to the reverse optical rotation value, the relative configuration at C-7 of 2 is proposed as α-form. However, the absolute configurations of Compounds 1 and 2 in C-7 and C-8 are uncertain. These data, supported by the 13 C NMR (Table 2), DEPT, COSY (Table 3), HSQC, and HMBC (Table 3) spectra, were in agreement with a dimer system bearing two partial structures A and B. The structures of 1 and 2 were established as taiwaniacryptodimers A and B (rel-(3S,12aS,Z)-3-(benzo[d] [1,3]dioxol-5-yl)-4 -(benzo[d] [1,3]dioxol-5-ylmethylene)-12a-hydroxy-5-isopropyl-9,9-dimethyl-4 ,5 ,8,8a,9,10,11,12,12a, 13-decahydro-2 H,3H,7Hspiro [benzo [5 ,6 ] 9 ) in its HR-EI-MS. UV absorptions (λ max bands at 227, 284, and 308 nm) confirmed the presence of a benzenoid nucleus. The IR spectrum revealed the presence of ester lactone (1791 cm −1 ), and aromatic rings (1618, 1498, and 1447 cm −1 ), respectively. Twenty-one degrees of insaturation were determined from the molecular formula, 13 C-NMR (Table 1), and DEPT spectra. Because part of the all spectra data of 3 were similar to 1 and 2, it is inferred from the molecular formula and mass fragments that Compound 3 is also a dimer compound. In conjunction with the 13 C-NMR spectrum, there is a lactone signal at δ C 171.4 (C-9 ). From the above characteristics, we know that Compound 3 is similar to 1 and 2. The main difference is that the part A C-7 of 3 changes from a secondary carbon to an oxygenated tertiary carbon, which is a 9(10→20)abeoabietane diterpenoids combined with 8,8 -lignan. It can also be known from the IR spectrum that there is no absorption of hydroxyl groups. The C-7 and C-10 of the Part A form epoxide through the connection of oxygen atoms, which is also consistent with the calculated degree of unsaturation. Part A can be confirmed by comparison with the known compound brussonol (5,6-dihydroxysalviasperanol) [17,18].
The structure was further confirmed by 13 C NMR (Table 1), DEPT, COSY (Table 3), and NOESY (Table 3) experiments. Thus, the structure of 3 was determined to be (3 S*,7 S*,8a S*, 12a S*,Z)-3 -(benzo[d] [1,3] Compound 4 was isolated as a yellowish gum; its molecular formula C 35 H 50 O 3 was established by 13 C-NMR and HR-EI-MS data, and the eleven degrees of insaturation were determined from the molecular formula, the 13 C-NMR spectrum, and the DEPT experiment ( Table 1). Analysis of its IR spectrum suggested that 4 contains an OH group (3423 cm −1 ) and an aromatic moiety (1615, 1464 cm −1 ). The 13 C-and 1 H-NMR spectrum (Table 1) data together with the UV absorption bands at λ max 214 and 234 nm suggested that Compound 4 is very similar to hinokione [19].
The 1 H NMR spectrum shows that there are two isopropyl groups [δ H 1.14 (d, J = 6. . The 13 C NMR spectrum shows that 10 olefinic carbons are forming a benzene ring, a terminal double bond, a four-substituted double bond, and three oxygenated carbons at δ C 78.7 (C-3 ), 73.5 (C-5), 71.2 (C-4). From the above signals, it is inferred that it is a dimer formed by combining two monomers (A and B). Part A is a cadinane and Part B is an abietane skeleton.

Biological Studies
The antifungal activities of the roots of Taiwania cryptomerioides were tested against the following fungi: Aspergillus niger (BCRC-31512), Penicillium italicum (BCRC-30567), Candida albicans (BCRC-21538), and Saccharomyces cerevisiae (BCRC-20822). The antifungal data are shown in Table 4 and clinically used antifungal agent, ketoconazole, was used as positive control. Our results indicated that Metabolites 1, 2, 4, and 5 present moderate antifungal activities compared with ketoconazole, for which Compound 3 was weak. From the results of the antifungal tests, the following conclusions can be drawn regarding these isolates: Compounds 1, 2, 4, and 5 were further tested for their inhibitory activity against A. niger, P. italicum, C. albicans, and S. cerevisiae by using a method as described in the experimental section (Table 5). Compound 1 was found to show inhibitory activity against A. niger strain with MIC value 54.87 µg/mL. Compounds 2 and 4 were found to show inhibitory activity against S. cerevisiae or P. italicum strain with MIC values 58.92 and 42.78 µg/mL. Compound 5 was also revealed to show inhibitory activity against A. niger and S. cerevisiae with MIC values of 62.86 and 56.32 µg/mL, respectively. Their bioactivity was weaker than reference compound ketoconazole (with MIC values of 3.25, 6.72, 11.79, and 3.16 µg/mL for A. niger, P. italicum, C. albicans, and S. cerevisiae, respectively). No antifungal activity (MIC > 100) was observed for Compound 3 at concentrations below 100 µg/mL in this bioassay. Among 9(10→20)abeoabietane type diterpenoids combined with 8,8 -lignans series analogues 1-3, taiwaniacryptodimers C (3) (possess the C-7 and C-10 of the part A form epoxide through the connection of oxygen atoms exhibited less effective inhibition than their analogues, taiwaniacryptodimers A (1) and B (2). Compounds 1 and 2 are a pair of diastereomers, with little difference in activity. Compounds 4 and 5 are dimers that belong to the combination of abietane and cadinane skeleton compounds. It has been reported in the literature that the compounds in the heartwood of T. cryptomerioides have good anti-fungal and anti-wood-destroying fungus effects, which shows that the extracted components of T. cryptomerioides have the potential to be used as food additives or health-care medical supplies, etc. As for how to develop and utilize, further research and exploration are needed.

Plant Material
The roots of T. cryptomerioides were collected from Taichung, Taiwan, in August 1996. The plant was identified by Dr. Shang-Tzen Chang, Professor of the Department of Forestry, National Taiwan University. A voucher specimen (no. 013542) has been deposited in the Herbarium of the Department of Botany of the National Taiwan University, Taipei, Taiwan.

Isolation and Characterization of Secondary Metabolites
Air-dried roots of T. cryptomerioides

By Disc Diffusion Assay
The antifungal susceptibility test of the isolated compounds was performed with the following strains: Aspergillus niger, Penicillium italicum, Candida albicans, and Saccharomyces cerevisiae by the disc diffusion method and applied following the CLSI M44-A, M44-S2 (for yeasts) [20,21], and M-51P (for filamentous fungi) guideline [22]. A standard disk of ketoconazole was used as a positive control, while the disk imbued with 50 µL of pure DMSO was used as a negative control. The diameters of the inhibition zones were measured in millimeters and means of a slide caliper. Each test was performed in triplicate and repeated three times, and results were analyzed for statistical significance [20][21][22].

By Broth Dilution Assay
The MIC determination for the antifungal assay was performed according to the CLSI (Clinical and Laboratory Standard Institute) using the broth dilution assay methods [23][24][25]. Extract stock solutions and partitions were prepared in 5% DMSO, and twofold serial dilutions were prepared in RPMI in 96-well microtiter plates (Corning Incorporated, Corning, NY, USA). The final concentrations ranged from 0.98 to 2.000 g mL −1 . Test organisms (100 µL) were added to each well in the microtiter plates. The growth control contained medium and inoculum. Blank controls contained medium only. The microtiter plates were then incubated at 35 • C and the endpoints were read after 48 h. The lowest concentration for each test compound at which color change occurred was recorded as its primary MIC value. The average of primary values from three individual tests were calculated, and that was taken as the final MIC value for each of the test compounds.

Conclusions
The research object of this research material Taiwania cryptomerioides (Taxodiaceae) is taxonomically included in one genus and one species of endemic plants in Taiwan. It contains more than 6% of essential oil in its heartwood. T. cryptomerioides is an important building material with high value in Taiwan. Previously, we investigated the chemical components of the heartwood and bark of this plant because of its antifungal and decayresistant characteristics as well as of its beautiful yellowish-red color with distinct purplish-pink streaks. The interesting compounds and those conferring biological activities isolated from the heartwood and bark of T. cryptomerioides prompted us to study the chemical components of its roots. In this study, we explored one novel constituent from the roots that had not been published in the past and evaluated and screened the metabolites for antifungal activity. It is found that the new dimer skeleton components 1 and 2 have moderate antifungal activity, one epoxide dimer 3 has weak activity, and two diterpenes 4 and 5 also possess moderate antifungal activities. The unique structure and antifungal activity of those have activities that make it an interesting material for further development.