Pyranocoumarins from Root Extracts of Peucedanum praeruptorum Dunn with Multidrug Resistance Reversal and Anti-Inflammatory Activities

In the search for novel herbal-based anticancer agents, we isolated a new angular-type pyranocoumarin, (+)-cis-(3′S,4′S)-3′-angeloyl-4′-tigloylkhellactone (1) along with 12 pyranocoumarins (2–13), two furanocoumarins (14, 15), and a polyacetylene (16) were isolated from the roots of Peucedanum praeruptorum using chromatographic separation methods. The structures of the compounds were determined using spectroscopic analysis with nuclear magnetic resonance (NMR) and high-resolution-electrospray ionization-mass spectrometry (HR-ESI-MS). The multidrug-resistance (MDR) reversal and anti-inflammatory effects of all the isolated compounds were evaluated in human sarcoma MES-SA/Dx5 and lipopolysaccharide (LPS)-induced RAW 264.7 cells. Among the 16 tested compounds, two (2 and 16) downregulated nitric oxide (NO) production and five (1, 7, 8, 11, and 13) inhibited the efflux of drugs by MDR protein, indicating the reversal of MDR. Therefore, these compounds may be potential candidates for the development of effective agents against MDR forms of cancer.

P. praeruptorum roots and the constituents have been reported to have modulatory effects on tumor cells such as chemopreventive [14], anti-inflammatory [10][11][12], and multidrug resistance reversal [41,42]. Therefore, we examined the biological activities of the compounds isolated from the root extracts of P. praeruptorum using several in vitro assays to evaluate various aspects of their potential anticancer properties. First, the cytotoxic effects of the isolated compounds were investigated using A549 human non-small cell lung cancer cells, which were treated with varying concentrations of the test compounds at up to 100 μM for 48 h. Then, the cell viability was measured using a water-soluble tetrazolium salt (WST) assay (Ez-Cytox, Daeil Lab Service, Seoul, Korea). As expected, none of the compounds showed significant cytotoxicity or growth arrest in A549 lung cancer cells (data not shown). Next, we examined the effects of the isolated compounds on nitric oxide (NO) production in Raw 264.7 mouse macrophages stimulated with LPS. NO is mainly produced from L-arginine by the inducible nitric oxide synthase (iNOS) and is known to play a role in the host defense system against bacterial or viral infections or both by inducing inflammatory condition [43]. However, prolonged or hyper-stimulated NO production not only has the propensity to damage host cells but also contributes to cancer development by regulating the expression of genes involved in tumorigenesis [44][45][46]. Based on these scientific observations, NOS inhibitors and compounds that From the spectral data obtained, compound 1 was found to be a new angular-type pyranocoumarin, 3 1 -angeloyl-4 1 -tigloylkhellactone, which was similar to (+)-praeruptorin B (3), (+)-cis-(3 1 S,4 1 S)-3 1 ,4 1 -diangeloylkhellactone, except for the tigloyl group. The cis configuration between the two chiral centers, C-3 1 /C4 1 was determined based on its large coupling constant J 3 1 4 1 as 4.8 Hz and large differences in chemical shifts (∆ = 2.6 ppm) between two germinal methyl signals in the 13 C-NMR spectrum [6,18]. The absolute configuration was determined by comparing the optical rotation value (rαs 25 D + 9.5, CHCl 3 ) with those of some known analogues [19]. Therefore, compound 1 was established as (+)-cis-(3 1 S,4 1 S)-3 1 -angeloyl-4 1 -tigloylkhellactone.
P. praeruptorum roots and the constituents have been reported to have modulatory effects on tumor cells such as chemopreventive [14], anti-inflammatory [10][11][12], and multidrug resistance reversal [41,42]. Therefore, we examined the biological activities of the compounds isolated from the root extracts of P. praeruptorum using several in vitro assays to evaluate various aspects of their potential anticancer properties. First, the cytotoxic effects of the isolated compounds were investigated using A549 human non-small cell lung cancer cells, which were treated with varying concentrations of the test compounds at up to 100 µM for 48 h. Then, the cell viability was measured using a water-soluble tetrazolium salt (WST) assay (Ez-Cytox, Daeil Lab Service, Seoul, Korea). As expected, none of the compounds showed significant cytotoxicity or growth arrest in A549 lung cancer cells (data not shown). Next, we examined the effects of the isolated compounds on nitric oxide (NO) production in Raw 264.7 mouse macrophages stimulated with LPS. NO is mainly produced from L-arginine by the inducible nitric oxide synthase (iNOS) and is known to play a role in the host defense system against bacterial or viral infections or both by inducing inflammatory condition [43]. However, prolonged or hyper-stimulated NO production not only has the propensity to damage host cells but also contributes to cancer development by regulating the expression of genes involved in tumorigenesis [44][45][46]. Based on these scientific observations, NOS inhibitors and compounds that reduce the upregulation of NO production are considered as possible cancer chemotherapeutic candidate agents [43]. Of the compounds (1-16) isolated from the roots of P. praeruptorum, two of them (2 and 16) reduced the production of NO dose-dependently and by more than 70% at 100 µM in Raw 264.7 cells stimulated with 1 µg/mL LPS than the vehicle control did (Figure 3a,b). In addition to NO, pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 are secreted from macrophages during inflammatory response and recognized as pivotal markers of inflammation [47,48]. Hence we further confirmed the anti-inflammatory effect of compound 2 and 16 on the secretion of these cytokines from the LPS-stimulated Raw 264.7 cells. Stimulation of cells with LPS markedly induced the release of IL-1β (Figure 4a), IL-6 ( Figure 4b), and TNF-α (Figure 4c), which were suppressed by both compound 2 and compound 16 in a dose dependent manner, indicating that these compounds isolated from the roots of P. praeruptorum inhibit the early phase of LPS-stimuated inflammatory response.
Molecules 2015, 20, page-page 4 reduce the upregulation of NO production are considered as possible cancer chemotherapeutic candidate agents [43]. Of the compounds (1-16) isolated from the roots of P. praeruptorum, two of them (2 and 16) reduced the production of NO dose-dependently and by more than 70% at 100 μM in Raw 264.7 cells stimulated with 1 μg/mL LPS than the vehicle control did (Figure 3a,b). In addition to NO, pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 are secreted from macrophages during inflammatory response and recognized as pivotal markers of inflammation [47,48]. Hence we further confirmed the anti-inflammatory effect of compound 2 and 16 on the secretion of these cytokines from the LPS-stimulated Raw264.7 cells. Stimulation of cells with LPS markedly induced the release of IL-1β (Figure 4a), IL-6 ( Figure 4b), and TNF-α (Figure 4c), which were suppressed by both compound 2 and compound 16 in a dose dependent manner, indicating that these compounds isolated from the roots of P. praeruptorum inhibit the early phase of LPS-stimuated inflammatory response.
(a) (b) Figure 3. Effects of isolated compounds on nitric oxide production. Raw 264.7 cells were stimulated with 1 μg/mL LPS and co-treated with (a) 100 μM of each compound or vehicle (0.1% DMSO in PBS) as a control or (b) indicated compounds. Nitrite concentration in media was quantified using nitrite standard reference curve supplied in commercial Griess Reagent System. Data are means ± SD of one duplicated representative experiment. Differences between each treatment group against LPS control group were analyzed and statistical significances are denoted as * p < 0.05 or ** p < 0.01. LPS, lipopolysaccharide; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; SD, standard deviation.   It is a well-known fact that many drug-resistant tumor cells overexpress P-glycoprotein (Pgp), multidrug resistance-associated proteins (MRPs), or both, which decrease the cellular concentration of anticancer drugs and lead to MDR [41]. Furthermore, it has been reported that pyrocoumarins isolated from P. praeruptorum Dunn such as (±)-3′-angeloyl-4′-acetoxy-cis-khellactone (Pd-la), can suppress Pgp expression, reversing the MDR it induces, and consequently sensitize drug-resistant cancer cells to common anticancer agents [42]. In the present study, we used calcein-AM, a cell-permeable MDR protein substrate to test the MDR reversing activities of the compounds isolated from the roots of P. praeruptorum in the multidrug-resistant MES-SA/Dx5 cancer cell line. As shown in Figure 4, a few compounds showed enhanced calcein-AM fluorescence intensities, indicating a reduction in the drug-eliminating activities of MDR proteins. In particular, five compounds (1, 7, 8, 11, and 13) showed considerably significant activities compared to those of known MDR inhibitors (verapamil and cyclosporine A, Figure 5). Previous phytochemical investigations revealed that compounds 2 and 4 inhibited LPS-induced NO production in macrophages [12] and compounds 2-4 showed MDR reversal activities in cancer cells [42,46]. However, our study appears to be the first report of the anti-inflammatory potential of compound 16 and the potential MDR reversal activity of compounds 1, 7, 8, 11, and 13 in tumor cells. It is a well-known fact that many drug-resistant tumor cells overexpress P-glycoprotein (Pgp), multidrug resistance-associated proteins (MRPs), or both, which decrease the cellular concentration of anticancer drugs and lead to MDR [41]. Furthermore, it has been reported that pyrocoumarins isolated from P. praeruptorum Dunn such as (˘)-3 1 -angeloyl-4 1 -acetoxy-cis-khellactone (Pd-la), can suppress Pgp expression, reversing the MDR it induces, and consequently sensitize drug-resistant cancer cells to common anticancer agents [42]. In the present study, we used calcein-AM, a cell-permeable MDR protein substrate to test the MDR reversing activities of the compounds isolated from the roots of P. praeruptorum in the multidrug-resistant MES-SA/Dx5 cancer cell line. As shown in Figure 4, a few compounds showed enhanced calcein-AM fluorescence intensities, indicating a reduction in the drug-eliminating activities of MDR proteins. In particular, five compounds (1, 7, 8, 11, and 13) showed considerably significant activities compared to those of known MDR inhibitors (verapamil and cyclosporine A, Figure 5). Previous phytochemical investigations revealed that compounds 2 and 4 inhibited LPS-induced NO production in macrophages [12] and compounds 2-4 showed MDR reversal activities in cancer cells [42,46]. However, our study appears to be the first report of the anti-inflammatory potential of compound 16 and the potential MDR reversal activity of compounds 1, 7, 8, 11, and 13 in tumor cells. , while verapamil or cyclosporine A were controls. This was followed by addition of cell-based Calcein AM/Hoechst dye staining solution, and its uptake was analyzed using a plate reader and normalized to cell densities measured using fluorescence intensity of Hoechst dye staining. Data are means ± SD of one duplicated representative experiment. Differences between each treatment group against LPS control group were analyzed and statistical significances are denoted as ** p < 0.01. DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; SD, standard deviation.

Figure 5.
Inhibitory effects of isolated compounds against multidrug-resistant (MDR) protein-mediated drug efflux. MES-SA/Dx5 cells were treated with 10 µM of each compound and vehicle (0.1% DMSO in PBS), while verapamil or cyclosporine A were controls. This was followed by addition of cell-based Calcein AM/Hoechst dye staining solution, and its uptake was analyzed using a plate reader and normalized to cell densities measured using fluorescence intensity of Hoechst dye staining. Data are means˘SD of one duplicated representative experiment. Differences between each treatment group against LPS control group were analyzed and statistical significances are denoted as ** p < 0.01. DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; SD, standard deviation.

Plant Material
The dried roots of P. praeruptorum were purchased from Kwangmyungdang Medicinal Herbs Co., (Ulsan, Korea) and identified by Dr. Go Ya Choi, K-herb Research Center, Korea Institute of Oriental Medicine, Korea. A voucher specimen (KIOM-CRC-50) was deposited at the KM Convergence Research Division, Korea Institute of Oriental Medicine, Korea.

Extraction and Isolation of Compounds
The plant material (10 kg) was ground and extracted thrice with 70% EtOH (40 L for 48 h each time) by maceration at room temperature. The extracts were filtered (Whatman filter paper, No. 2, Whatman International, Maidstone, UK), concentrated (EYELA rotary evaporation system, 20 L scale, 40˝C, Tokyo Rikakikai, Tokyo, Japan), and dried (WiseVen vacuum oven, WOW-70, Daihan Scientific, Seoul, Korea) to obtain the EtOH extract (2.0 kg). Then, 1.0 kg of the EtOH extract was suspended in distilled water and subsequently partitioned with organic solvents to obtain the n-hexane-, EtOAc-, n-BuOH-, and water-soluble extracts with yields of 118.7, 15.8, 77.3, and 786.2 g, respectively.

Cell Culture and Cell Viability
The MDR human uterine sarcoma MES-SA/Dx5 and RAW 264.7 mouse macrophage cell lines were cultured in McCoy's 5A and Dulbecco's modified Eagle's medium (DMEM), respectively, each supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA), and maintained at 37˝C in a humidified incubator with 5% (v/v) CO 2 atmosphere. All the cell lines used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cell viability was quantified using the Ez-Cytox cell viability assay kit (Daeil Lab Service, Seoul, Korea) as previously described [49].

NO Assay
Raw 264.7 cells were inoculated at a density of 5ˆ10 5 cells/well in 48-well cell culture plates, cultured overnight, and then treated with 1 µg/mL LPS (Sigma-Aldrich) in the presence or absence of varying concentrations of the test compounds. After 20 h, the concentration of nitrite, a stable metabolite of NO, in the culture medium was measured using the Griess Reagent System (Promega, Madison WI, USA) following the manufacturer's instructions.

Measurement of IL-1β, IL-6, and TNF-α
Raw 264.7 cells were inoculated at a density of 5ˆ10 5 cells/well in 48-well cell culture plates, cultured overnight, and then treated with 1 µg/mL LPS in the presence or absence of varying concentrations of the test compounds. After 24 h, culture supernatants were collected after centrifugation at 14,000 rpm for 10 min. Levels of IL-1β, IL-6, and TNF-α in the culture media from each group were determined by enzyme-linked immunosorbent assay (ELISA; R & D Systems, Minneapolis, MN, USA) per manufacturer's instructions.

MDR Assay
MES-SA/Dx5 cells were seeded at a density of 5ˆ10 4 cells/well in 96-well plates containing 100 µL culture medium and grown overnight. Then, the cells were treated with 10 µM of the test compounds or vehicle control, as well as cyclosporine A or verapamil as the positive controls for 30 min at 37˝C in a humidified incubator with a 5% (v/v) CO 2 atmosphere. Following the treatments, the MDR protein modulatory activities of the test compounds were measured using Calcein AM/Hoechst dye staining solution (Cayman Chemical, Ann Arbor, MI, USA) as described in the manufacturer's instructions.