New Terpenoids from Chamaecyparis formosensis (Cupressaceae) Leaves with Modulatory Activity on Matrix Metalloproteases 2 and 9

Chamaecyparis formosensis is Taiwan’s most representative tree, and has high economic value. To date, only a few active chemical constituents have been reported for C. formosensis. In this study, 37 secondary metabolites, including three new compounds (1–3), were extracted from the leaves of C. formosensis. The compounds isolated from the ethyl acetate layer were used at different concentrations to treat HT-1080 human fibrosarcoma cells and to evaluate their effects on matrix metalloprotease 2 (MMP-2) and 9 (MMP-9) expression. Based on extensive analysis of data from high-resolution mass spectrometry (HR-MS) as well as nuclear magnetic resonance (NMR), infrared (IR), and ultraviolet (UV) spectroscopy, the new compounds were identified as 11,12-dihydroxyisodaucenoic acid (1), 12-hydroxyisodaucenoic acid (2), and 1-oxo-2α,3β-dihydroxytotarol (3). Known compounds 4–37 were identified by comparing their spectroscopic data with data reported in the literature. Biological activity tests by gelatin zymographic analysis revealed that seven compounds, including new compound 2, have no cytotoxic effect on HT-1080 cells and were found to increase MMP-2 or MMP-9 expression by 1.25- to 1.59-fold at lower concentrations of 10–50 µM. These naturally derived regulatory compounds could potentially serve as a novel pharmaceutical basis for medical purposes.


Introduction
The genus Chamaecyparis is the main genus of the family Cupressaceae, with five species, C. formosensis, C. lawsoniana, C. obtusa, C. pisifera, and C. thyoides, and one variety, C. obtusa var. formosana, spread throughout the world [1]. The genus is native to eastern Asia (Japan and Taiwan), as well as the western and eastern regions of the United States. Besides C. formosensis and C. obtusa var.

Evaluation of Modulatory Effects of Compounds on MMP-2 and MMP-9 Expression in HT-1080 Cells
To obtain the modulatory compounds for MMP-2 and MMP-9 expression, we analyzed the compound-rich EA layer by HPLC. The concentrated extract was subjected to silica gel chromatography. Six fractions were isolated and screened using the bioassay-guided method for their respective MMP activity. Compounds in three fractions with effects of elevating and decreasing MMP-2 expression were further isolated and purified through semi-HPLC techniques.
Thirty-seven purified compounds were classified into eight types of compounds: sesquiterpenoid, diterpenoid, lignan, flavonoid, biflavonoid, aromatic, aliphatic, and steroid compounds. All compounds were individually applied to human fibrosarcoma cell line HT-1080 cells to assess cytotoxicity and MMP-2 and MMP-9 expression. Five out of the eight types, a total of seven compounds, were identified to have modulatory effects on MMP-2 and MMP-9 levels ( Table 2). Table 2. Compounds that modulate matrix metalloprotease 2 (MMP-2) and MMP-9 expression to various degrees. One of the new sesquiterpenoid compounds had distinct effects on MMP-2 and MMP-9 expression when compared with PMA (a positive control that is known to improve MMP-2 and MMP-9 expression) or EGCG (a negative control that represses MMP-2 and MMP-9 expression). As shown in Table 2, compound 2 elevated MMP-2 expression 1.26-fold (50 µM) and 1.32 fold (10 µM). By observing the structure, we found that the presence of a double bond on C11 of 2 ( Figure 1) seemed relevant to the regulation of MMP expression. Little is known about the biological activity of carotene-structured sesquiterpenoids. However, research has been done on Schisanwilsonenes A, a compound structurally similar to 2 (except the carboxyl group on C4 is substituted with a methene alcohol, and a different substituent is attached to C10) with antiviral function [46]. The novel finding of the MMP modulatory effect of carotene type sesquiterpenoid compound 2 can potentially provide insight into the cellular regulatory mechanism. As for the other newly discovered diterpenoid 3, no changes were detected in MMP-2 or MMP-9 levels.
When analyses were performed on lignan compounds, 18 and 19 had individual effects on MMP-2 and MMP-9 expression ( Table 2). Results showed that the hydroxyl groups on C8 or C8 of lignans could be responsible for regulating the expression of MMP-2 and MMP-9. Flavonoids such as 23 and 24 differ in the stereo position of the hydroxyl group on C3, which resulted in various levels of MMP-2 expression. A related compound with physical significance is epicatechin gallate, which is gallic acid derived from C3 of epicatechin. It is considered to prevent lung cancer metastasis by repressing MMP-2 expression [47,48]. Furthermore, we speculate that the expression of MMP-9 on 26 and 27 is modulated by the number of methyl groups and binding on the C3 -C6" or C4 -O-C6" position of a biflavonoid compound. Lee et al. [49] concluded that biflavonoid affects MMP activity and cell viability via binding variation on C2 -C8" positions, such as C2 -C8" on 2 ,8"-biapigenin, C3 -C8" on sumaflavone, and C3 -C3" on taiwaniaflavone, corroborating our speculation. All compounds with regulatory effects on MMP expression in this study possess more than 80% viability through HT-1080 MTT cell viability assay. In summary, to investigate the functional groups regulating MMP-2 and MMP-9 expression, we compared the structures of some biologically significant compounds with those of the MMP modulatory compounds in our study. Some distinctive findings show promise to serve as the basis for advanced biochemical examination.
Because of its rarity, Chamaecyparis formosensis is understudied and little is known about the species or its composition. However, we discovered several new compounds from leaf extract of C. formosensis through bioassay-guided fractionation. These newly identified compounds are highly unique to the species, and we also studied their individual regulatory effect on MMP-2 and MMP-9 levels of HT-1080 human fibrosarcoma cells. Elevated MMP-2 and MMP-9 is thought to induce angiogenesis, inflammation, and cancer metastasis [17], and may also be related to progression of diseases such as hypertension, hyperlipidemia, and diabetes mellitus [50]. On the other hand, MMP-2 and MMP-9 could also be involved in the process of wound healing [20,21]. In this paper, we isolated seven compounds with the ability to upregulate MMP-2 and MMP-9 expression. These naturally derived compounds could serve as candidates to investigate the mechanisms of pathogenesis and wound healing.

General Methods
Optical rotations were measured on a JASCO P-1020 polarimeter. IR spectra were recorded on a JASCO 4100 FT-IR spectrometer. UV spectra were measured on a Helios spectrophotometer (Thermo Scientific, Waltham, MA, USA). NMR data were recorded on a Bruker DRX-500 SB 500 MHz instrument (Bruker, Rheinstetten, Germany) with CDCl 3 and acetone-d6 as solvent and TMS as internal standard. HRESIMS data were acquired using an LTQ-Orbitrap XL mass spectrometer (Thermo Scientific, Bremen, Germany). Semipreparative HPLC was performed on a Hitachi L-7110 HPLC with a refractive index detector (Thermo Separation Products, Sunnyvale, CA, USA). A Phenomenex Luna silica column (5 µm, 10 × 250 mm, Torrance, CA, USA) and Hibar ® Fertigsäule silica column (5 µm, 10 × 250 mm, Merck, Darmstadt, Germany) were used for normal-phase separations. Silica gel (Geduran ® Si 60 0.063-0.2 mm, Merck, Darmstadt, Germany) was used for column chromatography. All solvents were either ACS or HPLC grade and were obtained from JT Baker (Phillipsburg, NJ, USA).

Plant Material
Leaves of Chamaecyparis formosensis were collected at Cile Hill in Nantou County, Taiwan, in the summer of 2009 and kindly provided to our laboratory by the Taiwan Forestry Research Institute. The sample was authenticated by Dr. Sheng-you Lu (Taiwan Forestry Research Institute), and a voucher specimen (CFL2009-1) was deposited at the School of Pharmacy, Taipei Medical University, Taipei, Taiwan.

Extraction and Isolation
Dried leaves (5.95 kg) of C. formosensis were smashed and extracted 3 times with 50 L of methanol, which was then filtered and rotary evaporated to give a brown-black residue (510 g). This residue was then suspended in 3 L of water and partitioned with an equal volume of ethyl acetate 3 times. The ethyl acetate layer was evaporated to dryness under vacuum (200 g). Subsequently, the dried ethyl acetate layer was mixed with 300 g of silica gel (70-230 mesh, Merck), and loaded onto a conditioned open column packed with 2500 g of silica gel and eluted via a stepwise gradient method using mixtures of n-hexane, ethyl acetate, and methanol. Five hundred milliliters were collected for each fraction and analyzed using thin-layer chromatography (TLC). TLC was performed on silica gel 60 F254 plates (Merck) using mixtures of n-hexane-ethyl acetate for development, and the spots were detected after heating by spraying with vanillin-sulfuric acid. Then, 216 fractions were combined into 6 portions (I-VI) according to the results of the TLC analyses; they were then redissolved in a minimum volume of n-hexane/ethyl acetate mixtures for subsequent HPLC analysis.

Cell Culture
HT1080 human fibrosarcoma cells were purchased from American Type Culture Collection (ATCC: CCL-121) and cultured in RPMI-1640 medium (Gibco), which was supplemented with 10% heat-inactivated fetal bovine serum, 100 U mL −1 penicillin, and 100 mg mL −1 streptomycin. The cell cultures were maintained in a humidified incubator at 37 • C in 5% CO 2 /95% air.

Gelatin Zymography
Gelatin zymography was used to determine expression and activities of MMP-2 and MMP-9 [16]. HT1080 cells were seeded in 24-well plates using serum-free medium for 24 h cell adhesion and growth. After giving the cells some time for cell adhesion and growth, they were treated with various concentrations (10 µM, 50 µM, and 100 µM) of compounds 1-37 for 48 h before the MMP-2 and MMP-9 activities were analyzed. The medium was collected for data analysis.

Statistical Analysis
The experimental results are expressed as mean ± SEM. Data were analyzed using one-way ANOVA and subsequently Student-Newman-Keuls test with p < 0.05, p < 0.01, and p < 0.001 to examine statistical significance.