Chemical Constituents of the Leaves of Butterbur (Petasites japonicus) and Their Anti-Inflammatory Effects

Two new aryltetralin lactone lignans, petasitesins A and B were isolated from the hot water extract of the leaves of butterbur (Petasites japonicus) along with six known compounds. The chemical structures of lignans 1 and 2 were elucidated on the basis of 1D and 2D nuclear magnetic resonance (NMR) spectroscopic data, electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectra. Petasitesin A and cimicifugic acid D showed significant inhibitory effects on the production of both prostaglandin E2 (PGE2) and NO in RAW264.7 macrophages. The expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were inhibited by compound 1 in RAW264.7 cells. Furthermore, compounds 1 and 3 exhibited strong affinities with both iNOS and COX-2 enzymes in molecular docking studies.


Introduction
Petasites japonicus Maxim (Asteraceae), known as butterbur, Japanese butterbur, and giant butterbur, is used as a botanical dietary supplement in the USA. The aerial parts of P. japonicus have been used in traditional Japanese folk medicine as an antipyretic, antitussive, or wound healing agent [1]. The constituents of P. japonicus have been reported and include flavonoids [2], sesquiterpenes [3][4][5], triterpenes [6], and various types of phenolic compounds [7]. Moreover, the leaves or stalks of P. japonicus are commonly consumed as vegetables in Korea and Japan. In the course of searching for active compounds from higher plants [8,9], the leaves of P. japonicus were selected for a detailed study since a hot water extract of the leaves of P. japonicus have shown inhibitory activity against nitric oxide (NO) production in RAW 264.7 cells [half maximal inhibitory concentration (IC 50 ) value:

General Experimental Procedures
General experimental procedures are described in the Supplementary Materials.

Plant Material
The leaves of Petasites japonicus (Asteraceae) were obtained from Nature Bio Co. (Seoul, Republic of Korea), in October 2016. The plant material was identified by one of the authors (D.S.J.) and the plant specimen (PEJA-2016) has been deposited in the Laboratory of Natural Product Medicine, College of Pharmacy, Kyung Hee University.

General Experimental Procedures
General experimental procedures are described in the Supplementary Materials.

Plant Material
The leaves of Petasites japonicus (Asteraceae) were obtained from Nature Bio Co. (Seoul, Republic of Korea), in October 2016. The plant material was identified by one of the authors (D.S.J.) and the plant specimen (PEJA-2016) has been deposited in the Laboratory of Natural Product Medicine, College of Pharmacy, Kyung Hee University.

Computational Methods
ECD and VCD calculations of compounds 1 and 2 were conducted as described previously [10,11]. In brief, their 3D models were built from Chem3D modeling. Conformational analysis was performed by the MMFF force field as implemented in Spartan'14 software (Wavefunction, Inc., Irvine, CA, USA; 2014). Geometrical optimization of the selected conformers was performed at the B3LYP/6-31 + G (d,p) level by Gaussian 09 software (Revision E.01; Gaussian, Inc., Wallingford, CT, USA; 2009). The theoretical ECD and VCD spectra were calculated at the CAM-B3LYP/SVP level with a CPCM solvent model (acetonitrile) and at the DFT [B3LYP/6-31 + G(d,p)] basis set level by the Gaussian 09 software, respectively.

Measurement of PGE 2
The RAW 264.7 macrophage cell lines were pretreated with various concentrations of the extract and isolates 1-8 for 1 h and then stimulated with or without LPS (1 µg/mL) for 24 h. A selective COX-2 inhibitor, NS-398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide; Sigma Aldrich, St. Louis, MO, USA) was used as a positive control for blocking PGE 2 production. PGE 2 levels in cell culture mediums were measured using the same methods as described in the previous paper [12].

Measurement of iNOS and COX-2 Expression
Quantitative polymerase chain reaction (qPCR) using Thermal Cycler Dice Real Time PCR System (Takara Bio Inc., Shiga, Japan) was used to determine the steady-state mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) as reported previously [12].

Molecular Docking Studies
The software AutoDock Vina with AutoDock Tools (The Scripps Research Institute, La Jolla, CA, USA: ADT 1.5.6) using the hybrid Lamarckian Genetic Algorithm (LGA) was used for performing molecular docking simulations as reported in the literature [13,14]. In short, the 3D crystal structures (resolution: 2.5 Å) of iNOS (PDB code: 3E6T) and COX-2 (PDB code: 1PXX) were obtained from the RCSB Protein Data Bank. The configurations of compounds 1 and 3 were determined by their nuclear overhauser effect spectroscopy (NOESY) spectra and time-dependent density functional theory (TDDFT) ECD calculations. Chem3D Pro 14.0 software (CambridgeSoft, Waltham, MA, USA) was used for construction of the standard 3D structures (PDB format) of compounds 1 and 3.
The remaining two quaternary carbons (δ C 160.9 and 128.2) were derived from a double bond.  The absolute configuration at C-7′ of compound 1 was established by comparing its experimental ECD spectrum with those calculated spectra of (7′R) and (7′S) models using the timedependent density functional theory (TDDFT) method. The experimental ECD spectrum of compound 1 exhibited a positive Cotton effect (CE) at 239 nm (Δε +2.1) and negative CEs at 214 nm (Δε −10.4) and 254 nm (Δε −5.3). The experimental data ( Figure 3) was in agreement with the calculated ECD spectrum of the (7′R) model, suggesting the absolute configuration of compound 1 as (7′R). Thus, the structure of 1 was elucidated as (R)-9-(3, 4-dihydroxyphenyl)-6,7-dihydroxy-4,9dihydronaphtho[2¨C-c]furan-1(3H)-one, and was named as petasitesin A. The compound 2 was isolated as a dark brownish powder. Its molecular formula was established as C18H16O7 by HRESIMS (m/z 343.0810 [M−H] − ; calculated for C18H15O7, 343.0818). The 1 H and 13 C NMR data of 2 were similar to those of 1 (Table 1), although the NMR solvents were different from each other due to the different solubility of the compounds. Comparison of the 13 C NMR data and molecular weights from 1 and 2 suggested that the carbons C-8 and C-8′ of 1 with a double bonded linkage (δC 160.9 and 128.2) were replaced by an oxygenated quaternary (δC 78.0) and methine (δC 56.2) carbon atoms. The correlation spectroscopy (COSY) correlation between H-7′ (δH 4.18) and H-8′ (δH 3.24), and the HMBC experiment revealed aryltetralin lactone type lignan (Figure 4). Considering a The absolute configuration at C-7′ of compound 1 was established by comparing its experimental ECD spectrum with those calculated spectra of (7′R) and (7′S) models using the time-  The compound 2 was isolated as a dark brownish powder. Its molecular formula was established as C18H16O7 by HRESIMS (m/z 343.0810 [M−H] − ; calculated for C18H15O7, 343.0818). The 1 H and 13 C NMR data of 2 were similar to those of 1 (Table 1), although the NMR solvents were different from each other due to the different solubility of the compounds. Comparison of the 13 C NMR data and molecular weights from 1 and 2 suggested that the carbons C-8 and C-8′ of 1 with a double bonded linkage (δC 160.9 and 128.2) were replaced by an oxygenated quaternary (δC 78.0) and methine (δC 56.2) carbon atoms. The correlation spectroscopy (COSY) correlation between H-7′ (δH 4.18) and H-8′ (δH 3.24), and the HMBC experiment revealed aryltetralin lactone type lignan (Figure 4). Considering a ) and heteronuclear multiple bond correlation spectroscopy (HMBC, →) (in acetone-d 6 and methanol-d 4 ).
The absolute configuration at C-7 of compound 1 was established by comparing its experimental ECD spectrum with those calculated spectra of (7 R) and (7 S The absolute configuration at C-7′ of compound 1 was established by comparing its experimental ECD spectrum with those calculated spectra of (7′R) and (7′S) models using the time-  The compound 2 was isolated as a dark brownish powder. Its molecular formula was established as C18H16O7 by HRESIMS (m/z 343.0810 [M−H] − ; calculated for C18H15O7, 343.0818). The 1 H and 13 C NMR data of 2 were similar to those of 1 (Table 1), although the NMR solvents were different from each other due to the different solubility of the compounds. Comparison of the 13 C NMR data and molecular weights from 1 and 2 suggested that the carbons C-8 and C-8′ of 1 with a double bonded linkage (δC 160.9 and 128.2) were replaced by an oxygenated quaternary (δC 78.0) and methine (δC 56.2) carbon atoms. The correlation spectroscopy (COSY) correlation between H-7′ (δH 4.18) and H-8′ (δH 3.24), and the HMBC experiment revealed aryltetralin lactone type lignan (Figure 4). Considering a The compound 2 was isolated as a dark brownish powder. Its molecular formula was established as C 18 (Table 1), although the NMR solvents were different from each other due to the different solubility of the compounds. Comparison of the 13 C NMR data and molecular weights from 1 and 2 suggested that the carbons C-8 and C-8 of 1 with a double bonded linkage (δ C 160.9 and 128.2) were replaced by an oxygenated quaternary (δ C 78.0) and methine (δ C 56.2) carbon atoms. The correlation spectroscopy (COSY) correlation between H-7 (δ H 4.18) and H-8 (δ H 3.24), and the HMBC experiment revealed aryltetralin lactone type lignan (Figure 4). Considering a biogenetic relationship with 1, the absolute configuration at C-7 of 2 was suggested to be (R)-configuration [15].
The coupling constant of 3.0 Hz between H-7 and H-8 suggested the cis-geometry of C-7 and C-8 . It was further confirmed by the NOESY interaction of H-7 and H-8 [16].

3.2.Anti-inflammatory Effects of the Isolates
As shown in Table 2, cimicifugic acid D (3) and the new compound 1 (petasitesin A) exhibited significant inhibitory activities against NO production with IC50 values of 12 ± 1.1 and 15 ± 1.4 µM, respectively, without affecting the cell viability ( Figure S15). 4,5-Dicaffeoylquinic acid showed mild activity with an observed IC50 value of 38.9 ± 0.72 µM. On the other hand, compound 1 showed the most potent inhibitory effect on PGE2 production with an IC50 value of 17 ± 3.2 µM (Table 2) in a dosedependent manner ( Figure 5). These results suggest that compound 1 might have an antiinflammatory effect due to inhibition of the production of NO and PGE2 which are the key inflammatory mediators of macrophages. It is worth noting that compound 1 significantly suppressed the expression of NO and PGE2 synthesis enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively (Figure 6), in a concentration-dependent manner. The data indicate that the inhibitory effect of compound 1 on NO and PGE2 production in macrophages is related to the regulation of iNOS and COX-2 expression. To determine the absolute configuration C-8 of 2, experimental ECD spectrum of 2 was compared with the calculated spectra of (8R,7 R,8 R) and (8S,7 R,8 R) models using the TDDFT method. The experimental ECD spectrum of 2 showed positive CEs at 203 nm (∆ε +4.0) and 229 nm (∆ε +1.9), and negative CEs at 210 nm (∆ε −5.4) and 222 nm (∆ε −4.4). The experimental spectrum (Figure 3) was also in agreement with the calculated ECD spectrum of (8S,7 R,8 R) model. Moreover, the VCD spectrum of 2 was measured additionally to establish the configuration at C-8. The conformity of the experimental IR and VCD spectra and theoretical spectra of 2 suggested the absolute configuration of 2 as (8S,7 R,8 R) (Figure 4). Therefore, the structure of 2 was proposed as (9R,3aS,9aR)-9-(3,4-dihydroxyphenyl)-6,7,3a-trihydroxy-4,9,3a,9a-tetrahydronaphtho[2,3-c]furan-1(3H)-one, and was named as petasitesin B.

Anti-inflammatory Effects of the Isolates
As shown in Table 2, cimicifugic acid D (3) and the new compound 1 (petasitesin A) exhibited significant inhibitory activities against NO production with IC 50 values of 12 ± 1.1 and 15 ± 1.4 µM, respectively, without affecting the cell viability ( Figure S15). 4,5-Dicaffeoylquinic acid showed mild activity with an observed IC 50 value of 38.9 ± 0.72 µM. On the other hand, compound 1 showed the most potent inhibitory effect on PGE 2 production with an IC 50 value of 17 ± 3.2 µM (Table 2) in a dose-dependent manner ( Figure 5). These results suggest that compound 1 might have an anti-inflammatory effect due to inhibition of the production of NO and PGE 2 which are the key inflammatory mediators of macrophages. It is worth noting that compound 1 significantly suppressed the expression of NO and PGE 2 synthesis enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively (Figure 6), in a concentration-dependent manner. The data indicate that the inhibitory effect of compound 1 on NO and PGE 2 production in macrophages is related to the regulation of iNOS and COX-2 expression.   a The values represent the means of the results from three independent experiments with similar patterns. l-N 6 -(1-Iminoethyl)lysine (l-NIL) and N-[2-(cyclohexyloxy)-4nitrophenyl]methanesulfonamide (NS-398) were used as a positive control substance for NO [half maximal inhibitory concentration IC50) value = 1.62 ± 0.08 µM] and prostaglandin E2 (PGE2) productions (IC50 value = 3.3 ± 0.15 µM), respectively. Three known compounds, fukinolic acid, 3,4-dicaffeoylquinic acid, and 3,5-dicaffeoylquinic acid were inactive (IC50 value > 50 µM) in this assay system.        To better understand the molecular mechanism of inhibitory activities against NO and PGE 2 production, the most active compounds 1 and 3 were subjected to molecular docking studies. The results showed that 1 and 3 had strong affinities with both NO and PGE 2 synthesis enzymes, iNOS and COX-2 ( Figure 7, Table 3). The binding residues and logarithms of free binding energy are given in Table 3. These results implicated that 1 and 3 may directly interact with the cavity residues of Biomolecules 2019, 9, 806 8 of 10 iNOS and COX-2, leading to the activity reduction of free iNOS and COX-2 enzymes. Taken together, these results indicate petasitesin A (1), a novel lignan isolated from butterbur leaves extract, exhibits anti-inflammatory properties by suppressing NO and PGE 2 production via inhibiting the expression of iNOS and COX-2 and binding to the free iNOS and COX-2 enzymes.
Biomolecules 2019, 9, 806 8 of 10 To better understand the molecular mechanism of inhibitory activities against NO and PGE2 production, the most active compounds 1 and 3 were subjected to molecular docking studies. The results showed that 1 and 3 had strong affinities with both NO and PGE2 synthesis enzymes, iNOS and COX-2 ( Figure 7, Table 3). The binding residues and logarithms of free binding energy are given in Table 3. These results implicated that 1 and 3 may directly interact with the cavity residues of iNOS and COX-2, leading to the activity reduction of free iNOS and COX-2 enzymes. Taken together, these results indicate petasitesin A (1), a novel lignan isolated from butterbur leaves extract, exhibits antiinflammatory properties by suppressing NO and PGE2 production via inhibiting the expression of iNOS and COX-2 and binding to the free iNOS and COX-2 enzymes.

Discussion
In the present study, we isolated two new aryltetralin lactone lignans, petasitesin A and B (1 and 2) from the leaves of P. japonicus. To the best of our knowledge, this is the first report on the isolation of the aryltetralin lactone type lignans from the leaves of P. japonicas. Although cimicifugic acid D (3) has been isolated from Cimicifuga spp. including black cohosh (Cimicifuga racemosa) and possesses

Discussion
In the present study, we isolated two new aryltetralin lactone lignans, petasitesin A and B (1 and 2) from the leaves of P. japonicus. To the best of our knowledge, this is the first report on the isolation of the aryltetralin lactone type lignans from the leaves of P. japonicas. Although cimicifugic acid D (3) has been isolated from Cimicifuga spp. including black cohosh (Cimicifuga racemosa) and possesses vasoactive effect and hyaluronidase inhibitory activity [20,21], this is the first finding that it presents in P. japonicus and inhibits pro-inflammatory mediators, NO and PGE 2 .
A new lignan petasitesin A (1) showed a potent inhibitory effect on the production of both NO and PGE 2 in LPS-stimulated macrophages (IC 50 values < 20 µM). Our molecular docking studies reveal that petasitesin A (1) can interact with the cavity residues of both iNOS and COX-2. Interestingly, petasitesin A (1) also inhibited the mRNA expression of iNOS and COX-2 induced by LPS in macrophages. However, the molecular mechanism of action underlying the gene expression regulation by petasitesin A remains to be investigated. Considering that LPS binds to toll-like receptor 4 (TLR4), the TLR4-mediated NF-κB pathway is likely associated with the inhibition of iNOS and COX-2 expression by petasitesin A. In fact, NF-κB is a key transcriptional factor to regulate the iNOS and COX-2 gene in macrophages under the inflammatory condition. In this regard, the effect of petasitesin A on the NF-κB pathway can be further elucidated.

Conclusions
New lignans (compounds 1 and 2) and six known compounds were isolated and identified from the leaves of P. japonicus. Petasitesin A (1) and cimicifugic acid D (3) inhibit production of inflammatory mediators NO and PGE 2 . PetasitesinA (1) inhibits iNOS and COX-2 expression, and petasitesin A (1) and cimicifugic acid D (3) have strong affinities with both iNOS and COX-2 enzymes in molecular docking studies. Thus, petasitesin A (1) and cimicifugic acid D (3) are worthy of further pharmacological evaluation for their potential as anti-inflammatory drugs.