Secondary Metabolites with Antimicrobial Activities from Chamaecyparis obtusa var. formosana

Seven new compounds, including one dimer novel skeleton, chamaecyformosanin A (1); three diterpenes, chamaecyformosanins B–D (2–4); one sesquiterpene, chamaecyformosanin E (5); and two monoterpenes, chamaecyformosanins F and G (6 and 7) were isolated from the methanol extract of the bark of Chamaecyparis obtusa var. formosana. Their structures were established by the mean of spectroscopic analysis and the comparison of NMR data with those of known analogues. Their structures were elucidated on the basis of physicochemical evidence, in-depth NMR spectroscopic analysis, and high-resolution mass spectrometry. Furthermore, the isolated compounds were subjected to an evaluation of their antimicrobial activity. Metabolites 1, 3, and 4 present antibacterial activities. It is worth mentioning that the chemical composition of the bark of C. obtusa var. formosana has never been studied in the past. This is the first time the barks from C. obtusa var. formosana were studied and two new skeleton compounds, 1 and 7, were obtained.


Introduction
The global distribution of plants of the genus Chamaecyparis includes six species and one variety, and they are only distributed in Taiwan [1], Japan, and the United States, except for one species (C. formosensis) and one variety in Taiwan (C. obtusa var. formosana); there are two Japanese species (C. obtusa and C. pisifera) [2,3] and three American species (C. lawsoniana, C. nootkatensis, and C. thyoides) [4]. The Taiwan cypress was collected by Kawakami Takiya and Mori Uzumaki in June 1906 from the Yushan mountain, and was handed over to Hayada Bunzo for publication in 1908. There are many studies in the literature on the composition of plants of the genus Hinoki, and the composition of this genus mainly contains essential oils and lignans [5][6][7][8].
The research object of this paper is Taiwan's unique cypress family Hinoki (Chamaecyparis obtusa var. formosana), one of the five largest trees in Taiwan. Taiwan cypress (Chamaecyparis obtusa var. formosana), also known as yellow juniper or thick crust, is a specialty of Taiwan in the cloud and fog belt at an altitude of 1500 to 2500 m, and most of it is distributed in the area north of the central part, namely MaKau Ecological National Park. It is the most concentrated place in Taiwan. The wood is pungent, aromatic, and highly resistant to decay and insects [5][6][7][8]. The wood is also light, soft, flexible, easy to dry, and less warped. The shrinkage is very small and easy to split. It is easy to plan and process. It has a good nailing performance, good coating, and gluing properties with very high economic value. More importantly, it has high corrosion resistance and ant resistance, so it is regarded as the finest wood.
Nature contains abundant elements of pharmaceutical raw materials. Today, when the pharmaceutical industry is so developed, most drugs are still found in nature or artificially modified products that imitate nature. Although this subject has been developed for a long time, the continuous advancement of analytical instruments and technology today allows us to continuously make new discoveries in the analysis of natural substances [9][10][11]. It also allows us to organize natural objects more accurately and systematically.
We have previously investigated the chemical components of the heartwood of this plant and found various monoterpenes, sesquiterpenes, diterpenes, and lignans [1][2][3][4]. The chemical constituents and biological properties from the stem barks of this plant have never been demonstrated. Recently, about 1500 species of Formosan plants have been screened for antimicrobial activity and the bark of C. obtusa var. formosana was shown to be one of the active species [12]. The MeOH extract of the bark of C. obtusa var. formosana 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 seven compounds, namely, chamaecyformosanins A-G (1-7) ( Figure 1). The isolation and structural elucidation of these compounds and an assessment of their in vitro antimicrobial activities are described herein.

Structure Elucidation of Compounds
Compound 1 was isolated as yellowish oil with a positive optical rotation [α] 30 D = + 200. 8 (c 0.48,CHCl3) and UV λmax at 218, 246, 294, and 339 nm, revealing the presence of the conjugated system. The EI-MS of 1 (see in Supplementary Materials)showed a molecular ion peak at m/z 596 [M] + , and the molecular formula C40H52O4 of 1 was resolved using HR-EI-MS. The IR (KBr) spectrum of 1 showed absorption bands at 3401, 1621, and 1504 cm −1 ascribable to hydroxyl and aromatic groups. From the 13 C-NMR and EI-MS spectrometry analysis, it was found that compound 1 has forty carbons, and its unsaturation is 15; the 1 H-NMR signals at δH 2.87 (1H, sept, J = 6.9 Hz, H-15), 0.84 (3H,d,J = 6.9 Hz,and 0.67 (3H,d,J = 6.9 Hz, show an isopropyl attached to the benzene ring, and δH 6.48 (1H, s, H-14) and 6.91 (3H, s, H-11) show the para-position on the benzene ring. After examining the 13 C-NMR spectrum appearing in δC 151.5 (C-12), 147.0 (C-9), 131.2 (C-13), 127.6 (C-8), 125.7 (C-14), and 109.3 (C-11), the above signals can be determined as benzene moiety. In addition, based on the signals of the three methyl groups of δH 1.28 (3H, s, CH3-20), 1.26 (3H,s,, and 1.14 (3H, s, CH3-19), the above characteristics are inferred to be of an abietane-type skeleton [5]. Comparing the 13 C-NMR of the compound 1 with 4αhydroxyferrugiol [13], it was found that the signal is quite close. The difference between them is only that C-3 is connected to a hydroxyl group in 1, which results in a large difference between C-2, C-3, and C-5, and the 13 C-NMR signal of C-6 and C-7 is quite different from the literature value. Therefore, it can be inferred that it is possible to use this position
From the NOESY spectrum, H-20 is correlated to H-6 and H-19. Therefore, it can be confirmed that C-20 and C-19 are in the axial position, and the 1 H-NMR signal of H-18 has a lower magnetic field, so it was inferred that an oxygen atom attached to C-6 should be located in the equatorial position. Therefore, H-18 is affected by the lone pair electron on the oxygen atom and it is displaced to the lower magnetic field, that is, H-6 is located in the axial position. H-18 is correlated to H-3 and H-5 but not related to H-20. It can be determined that the CH 3 -19 on C-4 is located on the axial, and the hydroxyl (-OH) on C-3 occupies the equatorial position ( Figure 3).  The presence of NOE cross-peaks between H-6 and H-7 clearly established the βequatorial orientation of the H-7. The C-C linkage bond between C-7 and C-2 was assigned the α-axial orientation, since the naphthalene ring and diterpenoid exhibited vertical orientations, causing the chemical shift of H-14, H-15, H-16, and H-17 with a higher magnetic field than usual. Then, other 2D-NMR spectra were used to determine the compound 1 as chamaecyformosanin A.
The full assignment of 1 H and 13 C NMR resonances was supported by 1 H-1 H COSY ( Figure 2), DEPT, HSQC, NOESY, and HMBC ( Figure 2) spectral analyses. Thus, the structure of 5 was established as shown in Figure 1, and named chamaecyformosanin E.

Plant Material
The barks of C. obtusa var. formosana 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.  has been deposited in the Herbarium of the Department of Botany of the National Taiwan University, Taipei, Taiwan.

Isolation and Characterization of Secondary Metabolites
The air-dried bark of C. obtusa var. formosana (12.1 kg) was extracted two times with acetone (100 L) at room temperature (every 7 days). The acetone extract was concentrated, and the black residue was suspended in H 2 O (7 L) and then extracted with EtOAc. The EtOAc fraction (709 g) was subjected to CC (silica gel, hexane/EtOAc of increasing polarity (H
The disc diffusion method according to the NCCLS [19] was employed for the determination of the antimicrobial activities of the compounds. Briefly, a suspension of the tested microorganisms (0.1 mL of 10 8 cells per mL) was spread on the solid media plates. The following nutritive media were used: Antibiotic Medium 1 (Difco Laboratories, Detroit, Michigan, USA) for growing Gram-positive and Gram-negative bacteria and Tryptone soy agar (TSA; Torlak, Belgrade) for Aspergillus niger, Penicillium italicum, Candida albicans, and Saccharomyces cerevisiae. Nutritive media were prepared according to the instructions of the manufacturer. All agar plates were prepared in 90 mm Petri dishes with 22 mL of agar, giving a final depth of 4 mm. Sterile filter paper disks (8 mm in diameter; Advantec, Tokyo, Japan) were impregnated with 50 µL of the sample solution in dimethyl sulfoxide (DMSO), 1 mg/1 mL of DMSO (all solutions were filter-sterilized using a 0.45 mm membrane filter), and placed on inoculated plates. These plates, after standing at 4 • C for 2 h, were incubated at 37 • C for 24 h for bacteria and at 30 • C for 48 h for the fungi. Standard disks of tetracycline (for bactericidal purposes) and ketoconazole (for fungicidal purposes) were used as positive controls, 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 by means of a slide caliper. Each test was performed in triplicate and repeated three times.

Conclusions
C. obtusa var. formosana (also called Taiwan cypress, Taiwan yellow cedar, and Formosan hinoki) is a variant plant endemic to Taiwan. It grows in the middle and upper part of a hillside or the flat part of a ridge at a high altitude of about 1300-2700 m in the Central Mountain Range. It is also occasionally seen in depressions and highlands. The wood is extremely good for various uses: the wood material is dense and tough, without cracking, and is mainly used for construction, furniture, sleepers, bridges, shipbuilding, sculptures, and decorations. In addition, it has been reported in medicinal practice that essential oils containing hinokitiol and rhodinic acid have an antibiotic effect, and rhodinic acid can treat tuberculosis. This study explored seven new components in the bark of C. obtusa var. formosana that have not been published previously. Compounds 1 and 7 are new skeletons. We also screened sufficient compounds for antimicrobial properties (anti-fungi and bacteria assays), and the results were found that the new skeleton (1) has good antibacterial activity, one sesquiterpene (2) is inactive, and two diterpenoids (3 and 4) have moderate antibacterial activity. The results of this screening show the weak antifungal activity compared to that of ketoconazole. The active substances with a unique structure and antibacterial activity make it an interesting material to be further developed.