Screening and Identification of an Anti-inflammatory and Anti-adipogenic Constituent from Ligularia taquetii Nakai

Abstract

Ligularia taquetii (H. Lev. & Vaniot) Nakai has traditionally been used to treat inflammation and skin swelling in the Jeju Island, Korea. The objective of this study was to investigate the anti-inflammatory and anti-adipogenic effects of Ligularia taquetii ethanoic extract (LTE), in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and 3T3-L1 adipocytes. Lipopolysaccharide-induced inflammation was reduced by LTE in a concentration-dependent manner, via the nuclear factor-κB signaling pathway. Ligularia taquetii ethanoic extract (100 µg/mL) inhibited the LPS-induced production of nitric oxide (NO) and inducible nitric oxide synthase (iNOS), by 60% and 100%, respectively. In comparison, 200 and 100 µg/mL LTE suppressed the LPS-stimulated production of prostaglandin-2 (PGE₂) and cyclooxygenase-2 by 50% and 80%, respectively. Ligularia taquetii ethanoic extract also inhibited the secretion of interleukin-1β and interleukin-6 at 300 and 100 μg/mL by 15% and 30%, respectively. High-performance liquid chromatography-photodiode array analysis, combined with mass analysis, revealed chlorogenic acid (CGA) as the anti-inflammatory constituent of LTE. Conversely, 25, 50, 100, and 200 μg/mL LTE lowered the lipid accumulation by 6%, 8%, 25%, and 60%, respectively, while simultaneously increasing cell viability by 7%, 14%, 34%, and 78%. The anti-adipogenic effect of LTE at 100 µg/mL was equivalent to that of CGA at 50 µg/mL. However, LTE treatment promoted cell proliferation by about 30% compared to its CGA-treated counterpart. These results suggest the potential of LTE as a new resource in the discovery of anti-inflammatory and anti-obesity drugs.

Inflammation is a common and diverse pathology, acting as the first line of defense against environmental and internal stressors. Oxygen radicals (such as nitric oxide or NO) and oxidative stress are both known to be involved in the initiation and perpetuation of inflammation. ¹ Macrophages play a central role in the immune response, allergy, and inflammation, while protecting our body from invading pathogens through phagocytosis. The production of inflammatory markers, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), NO, prostaglandinE2 (PGE₂), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), is markedly increased during inflammatory events. ^2,3 However, short-term or prolonged overproduction of NO causes septic shock and inflammation and can also lead to several severe pathological conditions through the formation of peroxynitrite (ONOO⁻) from NO and another free radicals, resulting in structural changes to lipids, proteins, and DNA within the body. ^4,5 These include aging, Alzheimer’s disease, cancer, atherosclerosis, and diabetes. The overabundance of macronutrients in the adipose tissues can also facilitate the release of pro-inflammatory mediators, such as IL-6 and TNF-α. ⁶

The Jeju Island of Korea is well known for its diverse geological formations and climates where unique plant species prosper. The plant Ligularia taquetii (H. Lev. & Vaniot) Nakai, a species from the Compositae family, is nationally protected and its aboveground parts were traditionally used for medicine and food. ^7,8 Ligularia fischeri, a close relative of L. taquetii, has been widely studied for its pharmaceutical properties, ^9,10 with the biological properties and constituent compounds of L. taquetii yet to be investigated.

The inhibitory effects of L. taquetii ethanoic extract (LTE) on the generation of pro-inflammatory markers, such as IL-1β, IL-6, NO, PGE₂, iNOS, and COX-2 in lipopolysachharide (LPS)-stimulated macrophages, were identified in the pilot study. We further examined the anti-inflammatory effects of LTE during the initiation and resolution of inflammation, via the regulation of the signaling mechanisms of nuclear factor-κB (NF-κB), extracellular signal-regulated kinases (ERK), and P38 mitogen-activated protein kinases (p38). Chlorogenic acid (CGA) was isolated and identified as a constituent compound of LTE, following the mechanistic investigation of the anti-inflammatory effect. The potent anti-adipogenic effect of LTE in 3T3-L1 adipocytes was also revealed. Chlorogenic acid, an esterification product of caffeic and quinic acid, has shown diverse therapeutic properties like having antioxidant, anti-obesity, antiviral, and anti-inflammatory effects. ¹¹ Further research is required to describe other biologically active compounds of L. taquetii.

Cytokines act as mediators in the activation, proliferation, and differentiation of immune cells. Typical pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, play active roles in the modulation of inflammatory responses, both in vitro and in vivo. ¹² No cytotoxicity was observed in the studied LTE concentration range of 50 to 200 μg/mL (Figure 1(a)). The suppressive effect of LTE on the production of 3 pro-inflammatory cytokines IL-1β, IL-6, and TNF-α was examined during LPS-triggered inflammation (Figure 1(b)). Interleukin-6 production decreased in a LTE concentration dependent manner and was inhibited by 30% at 200 µg/mL. Conversely, IL-1β production was decreased to 81% and 76% at 50 and 100 µg/mL LTE, respectively, but increased slightly at 200 µg/mL LTE (Figure 1(b)). Tumor necrosis factor-α production remained above 100% at all tested LTE concentrations (Figure 1(b)). The effect of LTE on the generation of NO and iNOS, 2 key mediators in the inflammatory response, was also examined (Figure 2(a)). Relative to the LPS-only treated group, iNOS protein expression and iNOS-mediated NO production were decreased exponentially in the LTE concentration range of 50 to 200 μg/mL. The half-maximal inhibitory concentrations (IC₅₀) of LTE against the generation of NO and iNOS were below 50 µg/mL (Figure 2(a)). The increased generation of COX-2 and COX-2-catalyzed PGE₂ is strongly associated with typical signs of inflammation such as redness, pain, swelling, and fever. ¹³ PGE₂ production was decreased during treatment with 50 to 200 μg/mL LTE. This reduction reached 50% at the peak concentration of 200 µg/mL (Figure 2(b)). Similarly, COX-2 proteins were downregulated by 10%, 30%, and 70% at 50, 100, and 200 µg/mL LTE, respectively (Figure 2(b)). Therefore, the simultaneous reduced expression of intracellular COX-2 proteins and suppression of extracellular PGE₂ production indicated a COX-2-dependent PGE₂ production and an inhibitory effect of LTE on COX-2 expression. The effect of LTE on the phosphorylation of mitogen-activated protein kinase (MAPK) proteins and the activation of NF-κB signaling during LPS-induced inflammation was subsequently investigated. Relative to an untreated control, NF-κB inhibitor-α (IκB-α) expression was downregulated, whereas phosphorylated NF-κB p65 (P-p65) was upregulated in the presence of LPS (Figure 2(c)). The expression level of P-p65 normalized to IκB-α reached the maximum level at 1 µg/mL LPS but dropped by almost 60% in the presence of LTE (50, 100, and 200 µg/mL) (Figure 2(c)). Relative to their respective untreated counterparts, the protein expression levels of both phospho-ERK (p-ERK) and phospho-p38 (P-p38) were upregulated with 1 µg/mL LPS and remained at 100% or above at LTE concentrations of 50 to 200 μg/mL (Figure 2(d)). These results demonstrate that the anti-inflammatory activity of LTE, in LPS-stimulated macrophages, was modulated via a NF-κB/IκB signaling pathway.

Figure 1

Cell viability assay of Ligularia taquetii ethanoic extract (a) and the suppressive effect of Ligularia taquetii ethanoic extract on the production of interleukin-6, interleukin-1β, and tumor necrosis factor-α (b). Cells were treated with 1 µg/mL alone or in combination with Ligularia taquetii ethanoic extract varying concentrations (50, 100, and 200 µg/mL) for 24 hours. All values were expressed as a percentage relative to the untreated control (a) or the lipopolysaccharide-only treated group (b), *P < 0.05, ^# P < 0.05, and ^& P < 0.05.

Figure 2

The inhibitory effect of Ligularia taquetii ethanoic extract on the productions of NO (a) and PGE₂ (b) via nuclear factor-κB (c) and mitogen-activated protein kinase (d) signaling pathways. Cells were treated with 1 µg/mL lipopolysachharide alone or in combination with Ligularia taquetii ethanoic extract varying concentrations (50, 100, and 200 µg/mL) for 24 hours. All values were expressed as a percentage of the lipopolysaccharide-only treated group, *P < 0.05 and ^# P < 0.05.

Screening and isolation of anti-inflammatory compounds from LTE ensued as described in the experimental section. Based on the typical mass fragmentations and high-performance liquid chromatography-photodiode array (HPLC-PDA) spectra patterns of CGA (Figure 3), CGA (Figure 3(a)) was suggested to be responsible for the anti-inflammatory effect of LTE. The tallest single peak was purified from LTE (Figure 3(c)). The LTE fraction (Figure 3(b)) and CGA (Figure 3(d)) results matched in their respective HPLC-PDA chromatograms and mass spectra. The chromatographic profiles and UV spectra of CGA and LTE were obtained as described in Figure 4. As expected, the standard curve of peak area vs concentration range of CGA of 10 to 250 μg/mL was linear. It was calculated that 8.5% of a LTE’s dry mass consists of CGA. The retention time and UV spectrum of the target peak in LTE were also found to be identical to the chromatographic profiles of CGA (Figures 3 and 4). Additionally, the HPLC peak spiked at a retention time of 4.6 minutes after the co-injection of equal volumes of LTE and CGA. These results therefore confirm the presence of CGA in LTE.

Figure 3

High-performance liquid chromatography-photodiode array chromatograms and high-resolution liquid chromatography-quadruple time-of-flight electrospray ionization mass spectrometry analyses. The structure of chlorogenic acid and its exact mass, 355.10 m /z [M+H]⁺ (a). UV spectra at 7.3 minutes retention time, high-performance liquid chromatography-photodiode array spectra at 326 nm, and mass spectra for a purified fraction of Ligularia taquetii ethanoic extract (b), Ligularia taquetii ethanoic extract (c), and chlorogenic acid (d). The solid arrows indicate the peaks and parent masses for chlorogenic acid. The 3 representative chlorogenic acid mass fragments of 355.10, 163.03, and 135 m /z [M+H]⁺ were detected in all 3 mass spectra (b-d). The 3 UV spectra (on the left of (b-d)) also shared a similar pattern.

Figure 4

Determination of the content of chlorogenic acid in Ligularia taquetii ethanoic extract by high-performance liquid chromatography-photodiode array. A standard curve (a) was constructed by plotting the mean peak area at the retention time of 4.6 minutes against the corresponding standard chlorogenic acid concentration. The equation and correlation coefficient (R ²) for the linear regression line are shown in an inset box within the graph (a). The high-performance liquid chromatography peak spiked at the retention time of 4.6 minutes after the co-injection of equal volumes of Ligularia taquetii ethanoic extract and chlorogenic acid (b).

The investigation of the anti-adipogenic properties of LTE was ensued. Our data showed that 25, 50, 100, and 200 µg/mL LTE lowered lipid accumulation by 6%, 8%, 25%, and 60%, but simultaneously increased cell viability by 7%, 14%, 34%, and 78% (Figure 5). Fat accumulation was reduced by 30% when treated with 50 µg/mL CGA, while its cell viability remained unchanged compared to a control. This is consistent with the previous findings by Jang et al. ¹⁴ It is noteworthy that the anti-adipogenic effect of LTE at 100 µg/mL was equivalent to 50 µg/mL CGA. However, LTE treatment enhanced cell viability by about 30% compared to its CGA-treated counterpart. These results showed that LTE not only contains CGA but also other potential anti-adipogenic compounds.

Figure 5

Anti-adipogenic effect of Ligularia taquetii ethanoic extract and its constituent compound chlorogenic acid on lipid accumulation. Confluent 3T3-L1 preadipocytes were differentiated into mature adipocytes in the presence of varying concentrations of Ligularia taquetii ethanoic extract (25-200 µg/mL) or 50 µg/mL chlorogenic acid for 10 days. Stained lipids for each treatment were quantified by measuring the absorbance at 500 nm. All values were expressed as a percentage relative to the untreated control, *P < 0.05 and ^# P < 0.05.

Chlorogenic acid was isolated and purified from LTE for the first time in this study. Lee et al ¹⁵ reported that 58 µM CGA exerted a 50% inhibition in the production of NO, but was unable to lower PGE₂ levels in LPS-stimulated RAW264 cells. Based on the standard curve (Figure 4), 100 µg/mL LTE was estimated to contain 20 µM CGA. However, the results of this study showed that 100 µg/mL LTE inhibited the production of NO, iNOS, COX-2, PGE₂, and IL-6 by 60%, 100%, 80%, 30%, and 30%, respectively. Ligularia taquetii ethanoic extract also suppressed the lipid accumulation of 3T3-L1 adipocytes in a concentration-dependent manner, without compromising cell viability. Our results confirmed the in vitro anti-inflammatory and anti-adipogenic potential of LTE.

Experimental

Preparation of Plant Extracts

The whole plants of L. taquetii (H. Lev. & Vaniot) Nakai including fruits, roots, stems, and flowers were obtained from Jeju Biodiversity Institute, Jeju-do, Korea and certified by Agricultural Corporation Company Biodiversity Resources, Jeju-do, Korea under the voucher specimen number 000000028. The plant parts were submerged in 70% v/v ethanol for 24 hours at room temperature to retrieve extracts. The whole extract was then filtered and concentrated using an evaporator and the dried extract was stored at −20°C before being dissolved in phosphate buffered saline (PBS) for later use.

Cell Culture

RAW264.7 mouse macrophage cells were kindly provided by Dr Chang-Gu Hyun (Department of Chemistry and Cosmetics, Jeju National University, Korea), and mouse embryo 3T3-L1 cell lines (Cat. no. CL-173) were obtained from the ATCC (American Type Culture Collection). Raw264.7 was cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Cat. no. LM001-05, Welgene, Gyeongsan-si, Korea) supplemented with 10% v/v fetal bovine serum (Cat. no. PK004-01, Welgene, Gyeongsan-si, Korea), 100 U/mL penicillin, and 100 µg/mL streptomycin (Cat. no. LS202-02, Welgene, Gyeongsan-si, Korea) at 37°C, 5% CO₂, and subcultured every 2 or 3 days. 3T3-L1 preadipocytes were grown in high glucose DMEM supplemented with 10% heat-inactivated (56°C for 30 minutes) newborn calf serum (HI-CSF) (Cat. no. 16010159, Gibco, Waltham, United States) at 37°C, 5% CO₂ and expanded before the cells became 85% confluent.

Cytotoxicity Assay

RAW264.7 cells were plated at 8 × 10⁴ cells/mL in a 24-well plate and incubated for 18 hours prior to the 24-hour treatment of LPS (1 µg/mL) and three different concentrations of LTE (50, 100, and 200 µg/mL) at 37°C, 5% CO₂. ¹⁶ Thiazolyl blue tetrazolium bromide (MTT) reagent (Cat. no. M2128, Sigma, St. Louis, United States) was then added at 0.5 mg/mL to each well and incubated for 4 hours. The dried formazan crystals were dissolved in dimethyl sulfoxide (DMSO) (Cat. no. D4540, Sigma, St. Louis, United States) and the absorbance at 540 nm was read using a microplate reader (Spectrophotometer, ThermoFischer, Waltham, United States). 3T3-L1 preadipocytes were placed in 24-well plates at a density of 5000 cells/cm² and fed every 2 to 3 days using 10% v/v HI-CSF-added DMEM. Once the cells were confluent, the cells were treated with four different concentrations of LTE (25, 50, 100, and 200 μg/mL) and incubated for 3 days. MTT assay was then conducted as described above.

Oil-Red O Staining

3T3-L1 preadipocytes were differentiated into mature adipocytes in the presence or absence of LTE. ¹⁷ Chlorogenic acid was used as a positive control for the anti-adipogenic effects of LTE. Briefly, 3T3-L1 preadipocytes were placed in 24-well plates at a density of 5000 cells/cm² and fed every 2 to 3 days using 10% v/v HI-CSF-added DMEM until the cells became growth arrested. To investigate the anti-adipogenic effect of LTE and CGA, the culture medium was then replaced with 3T3-L1 Differentiation Medium (Cat. no. DM-2-L1, ZenBio, Research Triangle Park, United States) containing LTE and a 0.1% v/v DMSO vehicle. After 72 hours of incubation, the culture medium was replaced with the same concentrations of LTE diluted in 3T3-L1 Adipocyte Maintenance Medium (Cat. no. AM-1-L1, ZenBio, Research Triangle Park, United States). This maintenance medium (along with LTE) was replenished every 2 to 3 days until adipocyte staining with Oil Red O (Cat. no. O0625, Sigma, St. Louis, United States). Following 10 days after LTE treatment, the cells were fixed in 4% v/v paraformaldehyde (pH 7.0) for 30 minutes and washed twice in PBS. The cells were then stained with a 0.2 µm syringe-filtered 0.2% w/v Oil Red O solution for 20 minutes. The cells were washed 4 times with distilled water and photographed. Stained oil droplets were eluted with 100% isopropanol and measured using a microplate reader at 500 nm.

Inhibitory Activity of LTE on NO Production

RAW264.7 cells were plated in triplicate at 8 × 10⁴ cells/mL in a 24-well plate and incubated for 18 hours prior to 24-hour treatment of 1 µg/mL LPS (Cat. no. L6529, Sigma, St. Louis, United States) and different doses of LTE extract (50, 100, and 200 µg /mL) at 37°C, 5% CO₂. Equal volumes of supernatant from each well and reconstituted Griess reagent (Cat. no. G4410, Sigma, St. Louis, United States) were mixed and incubated for 10 minutes, followed by taking the absorbance measurement at 540 nm, using a microplate reader.

Suppressive Activity of LTE on PGE₂, IL-1β, IL-6, and TNF-α Production

RAW264.7 cells were cultured as described above and treated with varying concentrations of LTE (50, 100, and 200 µg/mL) (then with or without LPS [1 µg/mL]) for 24 hours. The concentrations of pro-inflammatory cytokines (PGE₂, TNF-α, IL-1β, and IL-6) in culture supernatant were determined using enzyme-linkded immunosorbent assay (ELISA) kits (Prostaglandin E₂ ELISA Kit, R&D Systems, Minneapolis, United States; Mouse TNF alpha ELISA Kit, Invitrogen, United States; Mouse IL-6 ELISA Kit, BD, Franklin Lakes, United States; and Mouse IL-1β/IL-1F2, R&D Systems, Minneapolis, United States).

Western Blot

The protein expression of iNOS and COX-2 and the phosphorylation of MAPK proteins were assessed via Western blot analysis. ^18,19 As described above, RAW264.7 cells were cultured and treated with LPS (1 µg/mL) alone or in combination with varying concentrations of LTE. After incubation, the cells were collected and rinsed twice with cold PBS. The cells were then lysed in a lysis buffer (1× RIPA buffer [Cat. no. R2002, Biosesang, Seongnam-si, Korea], proteinase inhibitors [Cat. no. PPI 1015, Quartett, Berlin, Germany], 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na₃VO₄) and maintained on ice for 30 minutes. Aliquots of the lysate (10 µg/mL of protein) were loaded to a 8% sodium dodecyl sulfate (SDS)-polyacrylamide gel. The separated proteins were subsequently transferred to a polyvinylidene difluoride (PVDF) membrane using the Trans-Blot Turbo RTA Transfer Kit (Cat. no. 170-4272, Bio-Rad, Hercules, United States), according to the manufacturer’s instructions. The membrane was blocked and incubated with primary antibodies. The proteins were detected using Clarity Max Western ECL Substrate (Cat. no. 1705062, Bio-Rad, Hercules, United States) and photographed using Image Reader (LAS-400, Fujifilm, Tokyo, Japan). Western blot densitometry was performed using Image J (NIH, United States).

Fractionation of Crude Extract and Identification of Anti-inflammatory Compound

The crude LTE was dissolved in 80% v/v ethanol and subjected to HPLC-PDA (Shimadzu Spectro Monitor 3200 digital UV/Vis detector) connected with C18 column (Shim-pack GIS [4.6 × 250 nm, 5 µm]). ²⁰ Gradient elution with a mobile phase composed of 0.1% trifluroacetic acid-added water (A) and 100% acetonitrile (B) was applied at 40°C and a flow rate of 1 mL/min: 10% to 100% B (0-30 minutes), 100% B (30-32 minutes), 100% to 10% B (32-35 minutes), and 10% B (35-40 minutes). The exact mass of the component compounds of LTE were then analyzed by high-resolution liquid chromatography-quadruple time-of-flight electrospray ionization mass spectrometry (HR-LC-QTOF-ESI/MS) (ACQUITY [UPLC, Waters Corp., Milford, MA, United States]-SYNAPT GS-Si [Waters Corp.]) in positive ion mode. The potential fractions with anti-inflammatory properties, determined by LC-QTOF-ESI/MS and HPLC-PDA, were fractionated by a preparative HPLC-UV/Vis (Prominence HPLC, Shimadzu, Kyoto, Japan). For the purpose of fractionation, ²⁰ the linear gradient elution started 5% B at 0 minute and ended at 25% B over 30 minutes. The purified fractions were later compared to both chlorogenic acid and crude extract for their featuring peaks and exact masses by HPLC-PDA and LC-QTOF-ESI/MS, respectively.

Determination of Chlorogenic Acid Content in LTE

The aforementioned HPLC-PDA assay (sample duplicates) was employed to determine CGA content except gradient elution: 20% to 24% B (0-15 minutes), 24% B (15-17 minutes), 24% to 20% B (17-20 minutes), and 20% B (20-25 minutes). The standard curve was constructed by plotting the mean peak areas against the respective standard CGA concentrations (10, 50, and 250 µg/mL). The regression formula and correlation coefficient (R ²) were derived as y = 0.0005x − 0.6361 (where x is the peak area and y is the CGA concentration). In addition, equal volumes of CGA and LTE were co-injected into a column for a HPLC peak spike experiment.

Statistical Analysis

All experiments were performed in triplicate unless specified. Statistical analysis was performed using Microsoft Excel 2010. Data points are reported as mean ± standard deviation. Statistical differences between the means of 2 sample groups were resolved by Tukey-Kramer post hoc test after one-way analysis of variance. P values less than 0.05 were considered statistically significant.