Coreolanceolins A–E, New Flavanones from the Flowers of Coreopsis lanceolate, and Their Antioxidant and Anti-Inflammatory Effects

(1) Background: Many flavonoids derived from natural sources have been reported to exhibit antioxidant and anti-inflammatory effects. Our preliminary study suggested that Coreopsis lanceolata flowers (CLFs) include high flavonoid content; (2) Methods: CLFs were extracted in 80% (v/v) aqueous methanol and fractionated into ethyl acetate, n-butanol, and water fractions. Repeated column chromatographies for the organic fractions led to the isolation of seven flavanones. Quantitative analysis of the flavanones was carried out using reversed-phase high-performance liquid chromatography. All flavanones were evaluated for their antioxidant and pro-inflammatory inhibition effects; (3) Results: Spectroscopic analyses revealed the chemical structure of five new flavanones, coreolanceolins A–E, and two known ones. The content of the seven flavanones in extracts were determined from 0.8 ± 0.1 to 38.8 ± 0.3 mg/g. All flavanones showed radical scavenging activities (respectively 104.3 ± 1.9 to 20.5 ± 0.3 mg vitamin C equivalents (VCE)/100 mg and 1278.6 ± 26.8 to 325.6 ± 0.2 mg VCE/100 mg) in the DPPH and 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays and recovery activities in Caco-2 (59.7 to 41.1%), RAW264.7 (87.8 to 56.0%), and PC-12 (100.5 to 69.9%) cells against reactive oxygen species. Furthermore, all flavanones suppressed nitric oxide production (99.5% to 37.3%) and reduced iNOS and COX-2 expression in lipopolysaccharide-treated RAW 264.7 cells; (4) Conclusions: Five new and two known flavanones were isolated from CLF, and most of them showed high antioxidant and pro-inflammatory inhibition effects.


Introduction
The Coreopsis genus is the phanerogam in the Asteraceae family. Coreopsis is commonly called "Calliopsis" or "tickseed." The flowers are usually yellow or yellow-and-red bicolor with a toothed tip. The Coreopsis genus is widely distributed in North and South America, as well as Eastern Asia [1]. Coreopsis lanceolata, C. drummondii, and C. tinctoria are universal Coreopsis plants distributed all over Biotechnology (Santa Cruz, CA, USA) and Cell Signaling Technology (Beverly, MA, USA). The PC-12 and RAW 264.7 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). The Caco-2 cell line was purchased from the Korean Cell Line Bank (Seoul, Republic of Korea).

Quantitative Analysis of Compound 1-7 Using Reversed-Phase HPLC
Quantitative analysis of the flavonoids was performed using reversed-phase high-performance liquid chromatography (HPLC) (Alliance e2690; Waters Corp., Milford, MA, USA) with a C18 column (Poroshell 120 SB-C 18 ; 120 Å, 2.7 µm, 4.6 × 150 mm; Agilent Technologies, Santa Clara, CA, USA). The column oven temperature was 40 • C, the sample injection volume was 5 µL, and the detection wavelength was set to 360 nm. Solvent A (H 2 O: formic acid = 99.9: 0.1, v/v) and solvent B (acetonitrile) were used in the mobile phase, and the flow rate was set to 0.8 mL/min. The solvent elution was graded as follows: 95% A/5% B at 0 min, 95% A/5% B at 1 min, 80% A/20% B at 3 min, 80% A/20% B at 8 min, 77% A/23% B at 10 min, 77% A/23% B at 13 min, 72% A/28% B at 15 min, 72% A/28% B at 20 min, 20% A/80% B at 22 min, 95% A/5% B at 24 min, and 95% A/5% B at 26 min. For the quantitative analysis of compound 1-7 isolated from the CLF extract, 1 mg of each flavanone was accurately weighed and dissolved in MeOH to obtain stock solutions with a concentration of 1.0 mg/mL. Calibration curves were developed for each standard with six different concentrations (100, 50, 25, 12.5, 6.25, and 3.125 µg/mL). A volume of 1 milligram obtained from the CLF extract was also accurately weighed and dissolved in 80% (v/v) aqueous MeOH to create stock solution with a concentration of 5.0 mg/mL. The quantitative analysis was repeated three times.

Free Radical Scavenging Activity
Antioxidant capacities were determined using the 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays [16,17]. Briefly, the ABTS radical solution was adjusted to an absorbance of 0.650 ± 0.020 at 734 nm. Reactions between the ABTS radical solution and the diluted compound 1-7 (satisfying the standard curve range) were allowed to proceed at 37 • C for 10 min, and decreases in the absorbance of the resulting solution were measured using a spectrophotometer (SPECTRONIC 200; Thermo Fisher Scientific Inc., Waltham, MA, USA). For the DPPH assay, the absorbance of DPPH radicals in 80% (v/v) aqueous methanol was set to 0.650 ± 0.020 at 517 nm. Reactions between the DPPH radical solution and the diluted compound 1-7 (satisfying the standard curve range) were allowed to proceed at ambient temperature for 30 min. Decreases in the absorbance of the resulting solution were monitored at 517 nm using a spectrophotometer (SPECTRONIC 200). Antioxidant capacities were expressed as mg vitamin C equivalent (VCE)/100 mg.

Cell Culture and Cytotoxicity
For the cell culture, a complete medium (with the addition of 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL of streptomycin) was used. RAW 264.7 and Caco-2 cells were cultured in complete Dulbecco's modified Eagle's medium (DMEM), and PC-12 cells were cultured in complete Roswell Park Memorial Institute (RPMI) 1640 medium. All cell lines were sub-cultured when approximately 90% of the distribution was in the culture dish.
To determine the non-toxic maximal concentration of compound 1-7, cytotoxicity was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction assay [18]. PC-12, Caco-2, and RAW 264.7 cells were seeded at a density of 2 × 10 4 cells/well in a 96-well plate in RPMI 1640 or DMEM medium containing FBS and left for 24 h. After removing the medium, cells were treated with a serum-free medium containing compound 1-7 at various concentrations. Following a 24 h incubation period, the medium was removed from each well. Then, MTT reagent was added, the plate was incubated for 3 h, and 50 µL of DMSO was added. The absorbance was measured using a microplate reader (Infinite M200; Tecan Austria GmbH, Grödig, Austria) at 570 nm (test wavelength) and 630 nm (reference wavelength). The cytotoxic effect was expressed as the percentage (%) of metabolically active cells relative to control cells cultured without test samples.

Measurement of Intracellular OS
Intracellular OS levels were evaluated using 2 ,7 -dichlorofluorescein diacetate (DCFH-DA) following previously reported methods [19]. Briefly, Caco-2, RAW 264.7 and PC-12 cells were seeded (2 × 10 4 cells/well in a 96-well plate) in their respective complete media and incubated for 3 h in a humidified incubator with 5% CO 2 at 37 • C. The cells were then treated with non-toxic concentrations (0.625-10 µg/mL) of compound 1-7. After removing the supernatant, 50 µM DCFH-DA in phosphate-buffered saline (PBS) was added and incubated for 30 min, and then the cells were treated with 200 µM H 2 O 2 for 30 min. Fluorescence was measured using a microplate reader (Infinite M200) with excitation at 485 nm and emission at 530 nm.

Determination of NO Production
NO produced by RAW 264.7 cells was determined using a method reported in the literature [3]. In brief, RAW 264.7 cells at a density of 4 × 10 5 cells/well in a 96-well plate were pre-cultured for 24 h. The cells were then stimulated with 1 µg/mL of lipopolysaccharide (LPS) in the presence of samples for 24 h. A supernatant was obtained to evaluate the nitrite level using the Griess reagent system. The nitrite level was determined by measuring the absorbance at 540 nm with a microplate reader (Infinite M200). The nitrite concentration was extrapolated from the standard curve of sodium nitrite.

Western Blot Analysis for Protein Expression
The intracellular content of pro-inflammatory enzymes was measured by Western blotting. RAW 264.7 cells at a density of 2 × 10 6 cells/well were pre-cultured in a 6-well plate for 24 h and then stimulated with 1 µg/mL LPS and 1 ng/mL β-actin in the presence of samples for 24 h. Total cell extracts were obtained by a lysis buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1mM EDTA; 20 mM NaF; 0.5% NP-40; 1% Triton X-100) containing a protease inhibitor cocktail (GenDEPOT, Barker, TX, USA). The protein concentration was quantified using the Bradford assay. Cell extracts were loaded onto an 12% sodium dodecyl sulfate polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were incubated with iNOS (1:500) and COX-2 (1:500) overnight at 4 • C. Subsequently, horseradish-peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody was used for 1 h at ambient temperature. Blots were detected using EzWestLumi plus (ATTO, Tokyo, Japan) and analyzed using an EZ-Capture MG (ATTO). The band density was quantified using Image J software (Bogdan, 2001, Stuttgart, Baden-Wurttemberg, Germany).

Statistical Analysis
Results (mean ± standard deviation; n = 3) were assessed using one-way analysis of variance and the Tukey-Kramer honestly significant difference (HSD) test with p < 0.05 considered to represent statistical significance. All statistical analyses were performed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA).

Quantitative Analysis of Compound 1-7 in the Extracts from CLF
Quantitative analysis of compound 1-7 in the extract was conducted. Gradient elution using water and acetonitrile was performed, using a Poroshell 120 SB-C18 column. Most peaks were eluted within 26 min and detected at 360 nm. The peaks that appeared at 8.78, 12.99, 13.58, 17.99, 18.67, 18.67, and 24.20 min were identified as flavanomarein (3), coreolanceolin B (4), coreolanceolin C (5), 8methoxybutin (1), coreolanceolin D (6), coreolanceolin E (7), and coreolanceolin A (2), respectively, by comparing their retention times with those of standard compounds ( Figure 3). This method was reliable since the r 2 values of the regression curves were all bigger than 0.99. The contents of compound 1-7 in CLF are presented in Table 3.

Quantitative Analysis of Compound 1-7 in the Extracts from CLF
Quantitative analysis of compound 1-7 in the extract was conducted. Gradient elution using water and acetonitrile was performed, using a Poroshell 120 SB-C 18 (3), coreolanceolin B (4), coreolanceolin C (5), 8-methoxybutin (1), coreolanceolin D (6), coreolanceolin E (7), and coreolanceolin A (2), respectively, by comparing their retention times with those of standard compounds ( Figure 3). This method was reliable since the r 2 values of the regression curves were all bigger than 0.99. The contents of compound 1-7 in CLF are presented in Table 3.

Radical Scavenging Activities of Compound 1-7 Using DPPH and ABTS Assays
The radical scavenging activities of compound 1-7 in the DPPH and ABTS radical assays were presented in Table 4. In the DPPH radical scavenging assay, compound 1 displayed the highest scavenging potential and compounds 2, 3, and 4 showed significant activity, whereas compounds 5-7 showed low antioxidant activity. In the ABTS assay, compounds 1 and 2 exhibited slightly higher antioxidant activity than that of vitamin C. These results indicate unambiguous structure-activity relationships. The aglycone compounds 1 and 2 showed higher activity than the monoglucoside compounds 3-7. The sugar moiety in flavanones reduces antioxidant activity because the free 7-hydroxy group in the A-ring is the key structure enabling antioxidant activity, which was clearly proven for naringenin and naringenin 7-rutinoside [21,22]. The DPPH scavenging activity of compound 1 was higher than that of compound 2, and the activities of compounds 3 and 4 were higher than those of compounds 5-7. Notably, compound 1, 3, and 4 contain a 3,4-dihydroxyphenyl group, a catechol structure. The presence of −OH groups in the aromatic ring of flavonoids is considered essential for radical scavenging activity, and a catechol moiety in the B-ring confers high stability to the phenoxyl radical via electron delocalization after hydrogen dissociation [23,24],. The DPPH scavenging action of compound 3 was higher than that of compound 4, and the action of compound 5 was higher than of compounds 6 and 7 because the 8-hydroxy or/and 4 -hydroxy groups of compounds 6 and 7 were protected. The influence of flavanone glycosides on antioxidant activity was reported as follows; 8-methoxyl or/and 4 -methoxyl groups were consistently less active than corresponding compounds with a free hydroxyl group [22]. Therefore, the catechol group at the B-ring, the free hydroxyl group of C-8 or C-4 , and the absence of an attached sugar are the key factors that contribute to the potency of the radical scavenging capacity. ROS are reducing metabolites of oxygen which are generated by metabolic processes or external elements in normal cells in the body, and most of them have an unstable state that allows them to lose or obtain electrons and a stable state. These properties are known to cause OS in DNA and cell membranes in vivo, causing damage and various diseases, inflammation, and aging [25,26]. DCFH-DA is a representative material for measuring ROS in cells. It can freely pass through the cell membrane and is deacetylated with non-fluorescent DCFH when the acetate group is removed by intracellular esterase. Deacetylated DCFH is oxidized by ROS, such as H 2 O 2 , and thus becomes a strong fluorescent DCF. The isolated compounds from CLF inhibited intracellular ROS in Caco-2 colon epithelial, PC-12 neuronal, and RAW 264.7 macrophage cells (Figure 4). ROS levels in all three cell lines were increased by OS (200 µM H 2 O 2 ) compared to the levels in control cells (Caco-2: 265.7%, RAW 264.7: 188.0%, and PC-12: 136.6%). After treating the cells with isolated flavanones (10 µM), we confirmed that all the flavanones significantly lowered the ROS-induced stress. However, the reduction rates varied by cell line. In Caco-2 cells, flavanones 2 and 7 lowered ROS stress levels; flavanones 1−5 were efficacious in RAW 264.7 cells, and all flavanones lowered ROS stress levels in PC-12 cells. These results also exhibited unambiguous structure-activity relationships. In the Caco-2 cells, flavanones 2 and 7 were the most efficacious, indicating that the catechol structure in the B-ring hindered ROS effects. Flavanone 4, which has an additional glucose compared with flavanone 1, showed a little more efficacy than flavanone 1. In the RAW 264.7 cells, flavanones 2 and 7, which are aglycones, were more effective than the glycosides. Among flavanones 3, 4 and 6, the more methoxy groups in the flavanones, the lower recovery effects. Therefore, flavanones 7 and 8 showed the lowest recovery effects because they have attached sugars and two methoxy groups. In PC-12 cells, all the compounds restored intracellular OS to the control level, and no deviations caused by differences in structure, such as −OH, −glucose, and −OCH 3 , were observed. Flavonoids have different absorption and transmission rates in cells depending on their structure [27,28]. Therefore, compounds with different functional groups are thought to have different degrees of access and absorption in different types of cells. In addition, the flavonoids have different antioxidant properties depending on the number or position of their OH and various other structural characteristics, such as double bonds, protection of OH, and number of sugars [29]. For this reason, even though the aglycone compounds were the strongest antioxidants, flavanones 3-7 had a better ability to reduce OS in cells than flavanones 1 and 2. Therefore, the ability to reduce the OS in a particular cell depends on a combination of absorption, permeability, and molecular antioxidant capacity.
Antioxidants 2020, 9,539 12 of 17 and transmission rates in cells depending on their structure [27,28]. Therefore, compounds with different functional groups are thought to have different degrees of access and absorption in different types of cells. In addition, the flavonoids have different antioxidant properties depending on the number or position of their OH and various other structural characteristics, such as double bonds, protection of OH, and number of sugars [29]. For this reason, even though the aglycone compounds were the strongest antioxidants, flavanones 3-7 had a better ability to reduce OS in cells than flavanones 1 and 2. Therefore, the ability to reduce the OS in a particular cell depends on a combination of absorption, permeability, and molecular antioxidant capacity.

Inhibitory effects of flavanones 1-7 on NO production in RAW 264.7 cells
NO, a signaling molecule that plays a key role in the pathogenesis of inflammation, is produced by mouse macrophages in response to LPS [30]. NO is thought to cause several chronic inflammatory diseases such as inflammatory bowel disease and arthritis as well as certain autoimmune disorders [31][32][33]. Therefore, we evaluated the effects of flavanones 1-7 on NO production in RAW 264.7 cells at dose of none cytotoxic effects. All compounds showed no cytotoxicity lower than 100 μM. ( Figure  5)

Inhibitory Effects of Flavanones 1-7 on NO Production in RAW 264.7 Cells
NO, a signaling molecule that plays a key role in the pathogenesis of inflammation, is produced by mouse macrophages in response to LPS [30]. NO is thought to cause several chronic inflammatory diseases such as inflammatory bowel disease and arthritis as well as certain autoimmune disorders [31][32][33]. Therefore, we evaluated the effects of flavanones 1-7 on NO production in RAW 264.7 cells at dose of none cytotoxic effects. All compounds showed no cytotoxicity lower than 100 µM. ( Figure 5). NO, a signaling molecule that plays a key role in the pathogenesis of inflammation, is produced by mouse macrophages in response to LPS [30]. NO is thought to cause several chronic inflammatory diseases such as inflammatory bowel disease and arthritis as well as certain autoimmune disorders [31][32][33]. Therefore, we evaluated the effects of flavanones 1-7 on NO production in RAW 264.7 cells at dose of none cytotoxic effects. All compounds showed no cytotoxicity lower than 100 μM.  Figure 6 showed that LPS treatment induced an increase in NO production. NO is synthesized by endothelial nitric oxide synthase. All seven flavanones significantly reduced NO production at 100 μM. In particular, flavanone 1 inhibited NO production to the control level ( Figure 6C), similarly to 10 μM AMT, which is commonly used as a positive control (83% inhibited to control) [34]. Flavanones 3, 4, and 7 inhibited NO production more than flavanones 2 and 5. Therefore, flavanones 1, 3, 4, and 7 have potential as pro-inflammatory inhibition materials. A distinct structure-activity relationship was also observed in this case. Flavanones 1, 3, and 4 were notably effective in inhibiting NO  Figure 6 showed that LPS treatment induced an increase in NO production. NO is synthesized by endothelial nitric oxide synthase. All seven flavanones significantly reduced NO production at 100 µM. In particular, flavanone 1 inhibited NO production to the control level ( Figure 6C), similarly to 10 µM AMT, which is commonly used as a positive control (83% inhibited to control) [34]. Flavanones 3, 4, and 7 inhibited NO production more than flavanones 2 and 5. Therefore, flavanones 1, 3, 4, and 7 have potential as pro-inflammatory inhibition materials. A distinct structure-activity relationship was also observed in this case. Flavanones 1, 3, and 4 were notably effective in inhibiting NO production because of the catechol moieties in their B-rings. The catechol groups inhibit NO production through the inhibition of LPS signaling and direct scavenging of NO [35].
Antioxidants 2020, 9,539 13 of 17 production because of the catechol moieties in their B-rings. The catechol groups inhibit NO production through the inhibition of LPS signaling and direct scavenging of NO [35].

Inhibitory effects of flavanone 1-7 on expression of iNOS and COX-2 in RAW 264.7 cells
When inflammation occurs in response to activating signals such as LPS and/or cytokines, iNOS produces NO in macrophages [13,33,36]. In addition, the level of COX-2, a pro-inflammatory enzyme, can also indicate the extent of inflammation [13,36,37]. The effect of the flavanones on iNOS and COX-2 expression was evaluated using western blot experiments. β-Actin, a housekeeping gene that is expressed at a stable level in varying cellular conditions, was used as an internal loading control for normalization. Flavanones 1, 3, 4, 6, and 7 inhibited iNOS expression in RAW 264.7 cells relatively strongly. (Figure 7) This result showed the same tendency as their ability to inhibit NO production. A previous study also reported that NO and iNOS share generation mechanisms [38]. In addition, flavanones 1, 3, 4, 6, and 7 are thought to not only inhibit NO production, but also affect the gene promoter that produces iNOS. However, we did not determine the detailed for that effect in this study. The inflammatory enzymes iNOS and COX-2 are regulated by the same factors and are often expressed together in inflammatory reactions. COX-2 is undetectable in most tissues but is expressed during inflammatory responses [39]. All seven flavanones markedly inhibited COX-2 expression in RAW 264.7 cells . Flavanones 1 and 3 showed the highest inhibitory effects on the expression of iNOS and COX-2 because of the presence of catechol groups in the B-ring [35].

Inhibitory Effects of Flavanone 1-7 on Expression of iNOS and COX-2 in RAW 264.7 Cells
When inflammation occurs in response to activating signals such as LPS and/or cytokines, iNOS produces NO in macrophages [13,33,36]. In addition, the level of COX-2, a pro-inflammatory enzyme, can also indicate the extent of inflammation [13,36,37]. The effect of the flavanones on iNOS and COX-2 expression was evaluated using western blot experiments. β-Actin, a housekeeping gene that is expressed at a stable level in varying cellular conditions, was used as an internal loading control for normalization . Flavanones 1, 3, 4, 6, and 7 inhibited iNOS expression in RAW 264.7 cells relatively strongly. (Figure 7) This result showed the same tendency as their ability to inhibit NO production. A previous study also reported that NO and iNOS share generation mechanisms [38]. In addition, flavanones 1, 3, 4, 6, and 7 are thought to not only inhibit NO production, but also affect the gene promoter that produces iNOS. However, we did not determine the detailed for that effect in this study. The inflammatory enzymes iNOS and COX-2 are regulated by the same factors and are often expressed together in inflammatory reactions. COX-2 is undetectable in most tissues but is expressed during inflammatory responses [39]. All seven flavanones markedly inhibited COX-2 expression in RAW 264.7 cells . Flavanones 1 and 3 showed the highest inhibitory effects on the expression of iNOS and COX-2 because of the presence of catechol groups in the B-ring [35]. relatively strongly. (Figure 7) This result showed the same tendency as their ability to inhibit NO production. A previous study also reported that NO and iNOS share generation mechanisms [38]. In addition, flavanones 1, 3, 4, 6, and 7 are thought to not only inhibit NO production, but also affect the gene promoter that produces iNOS. However, we did not determine the detailed for that effect in this study. The inflammatory enzymes iNOS and COX-2 are regulated by the same factors and are often expressed together in inflammatory reactions. COX-2 is undetectable in most tissues but is expressed during inflammatory responses [39]. All seven flavanones markedly inhibited COX-2 expression in RAW 264.7 cells . Flavanones 1 and 3 showed the highest inhibitory effects on the expression of iNOS and COX-2 because of the presence of catechol groups in the B-ring [35].

Conclusions
New anti-inflammatory flavanones from CLF were identified and quantified. Five new flavanones, coreolanceolins A-E, along with two previously known flavanones, were isolated through repeated CC using silica gel, octadecyl silica gel, and Sephadex LH-20 resins, and their chemical structures were determined without ambiguity based on the intensive analysis of 1D-, 2D-NMR, UV, IR, MS, and CD data. Most flavanones showed significant radical scavenging activities, with flavanones 1 and 2 showing the highest radical scavenging activities. All seven flavanones showed potential to be powerful antioxidants by reducing OS in Caco-2 intestinal epithelial cells, RAW 264.7 macrophage cells, and PC-12 neuron cells. Moreover, these antioxidants suppressed pro-inflammatory enzymes iNOS and COX-2 in RAW 264.7 macrophages. We have also provided a reasonable explanation for the structure-activity relationship in our results. The catechol group at the B-ring, a free hydroxyl moiety at C-8 or C-4 , and the absence of an attached sugar are key factors that contribute to radical scavenging capacity, the suppression of ROS production, inhibition of NO production, and inhibition of iNOS and COX-2 expression. Although the exact mechanisms, such as the expression of specific proteins at the molecular level, remain to be determined, these results indicate that flavanones obtained from CLF could be used as potential antioxidant and anti-inflammatory agents.