Effects of Shiranuhi flower extracts and fractions on lipopolysaccharide-induced inflammatory responses in murine RAW 264.7 cells

Preparation of Shiranuhi flower extracts

Shiranuhi flowers were collected from Citrus Research Institute (RDA, Jeju Island) and were identified by Dr. Young Hun Choi. A voucher specimen was prepared and deposited at the Cosmetic Science Center at Jeju National University (Korea).

The dried flowers (1.39 kg) were soaked in 5 L of 80% ethanol at room temperature for 24 h, after which the solvent was evaporated under vacuum. The residue obtained was freeze-dried and stored in a desiccator at 4°C before use. The weight of the dried sample was 52.48 g; therefore, the yield of the extraction was 3.78%. The dried extract (20 g) was then sequentially suspended in water (1 L) and extracted with ethyl acetate (1 L). The recoveries of the extract from the ethyl acetate and water fractions were 12.5% (2.49 g) and 70.5% (14.1 g), respectively.

For the water extract, the dried flowers (700 g) were ground into powder and extracted with 3 L of water in a water bath at 80°C for 5 h. Next, the mixture was filtered and the filtrate was concentrated to about 250 mL. The water extract of the Shiranuhi flower was thereafter obtained by lyophilizing the concentrate. The yield was 25.4 g (3.6%). In this study, each fraction and extracts were dissolved in DMSO and filtered through syringe filters (0.45 μm pore size).

Reagents and cell culture

Murine RAW 264.7 macrophages were purchased from American Type Culture Collection (Manassas, VA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). LPS, antibiotics (penicillin and streptomycin), and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Lactate dehydrogenase (LDH) cytotoxicity detection kit and enzyme-linked immunosorbent assay (ELISA) antibody sets were purchased from Promega (Madison, WI, USA) and R&D Systems Inc. (Minneapolis, MN, USA), respectively. Primary antibodies for iNOS, COX-2, phospho-ERK, ERK, phosphor-p38, p38, phospho-JNK, JNK, and horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Nitrocellulose membrane was obtained from Invitrogen Inc. (Seoul, Korea). The RAW 264.7 cells were maintained at subconfluence at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The medium used for routine subculture was DMEM containing 10% FBS, penicillin (100 units/mL), and streptomycin (100 μg/mL).

Cell viability assay

Cell viability was analyzed by using the LDH cytotoxicity detection kit according to the manufacturer’s instructions. The RAW 264.7 macrophages (1.8×10⁵ cells/mL) were plated in 24-well plates and incubated for 18 h, after which they were treated with the extract samples for 2 h. The macrophages were then challenged with LPS (1 μg/mL) for an additional 24 h. The release of LDH from the macrophages was used to assess the cytotoxicity of each sample. This method determines LDH activity from the production of NADH during the conversion of lactate to pyruvate. Absorbance was measured at 450 nm using an ELISA multiplate reader (Sunrise, Tecan, Switzerland). Percentage cytotoxicity was determined relative to the viability of the control cells. All experiments were performed in triplicate.

Measurement of NO levels

The RAW 264.7 cells were plated at 1.8×10⁵ cells/mL in 24-well plates and incubated for 18 h, and pretreated with the various samples for 2 h, after which they were stimulated with LPS (1 μg/mL) for 24 h. The nitrite levels in the culture media were determined using Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in distilled water) and incubated at room temperature for 5 min. Absorbance was then measured at 540 nm using the ELISA multiplate reader. The quantity of nitrite in the samples was calculated using sodium nitrite as the standard.

Determination of cytokine levels

The concentrations of PGE₂, TNF-α, and IL-6 in the supernatant were assayed using a commercial mouse ELISA kit according to the manufacturer’s instructions. The RAW 264.7 cells were plated at 1.8×10⁵ cells/mL in 24-well plates and incubated for 18 h, and pretreated in absence or presence the ethyl acetate fraction (HEF; 25, 50, or 100 μg/mL) for 2 h, after which they were stimulated with LPS (1 μg/mL) for 24 h. Supernatant samples were obtained 24 h later and frozen until analysis by ELISA. Cytokine concentrations were measured from standard curves that were constructed using known concentrations of recombinant TNF-α, IL-6, and IL-1β.

Western blot analysis

Western blot analysis was performed as previously described with some modifications [25]. First, for iNOS and COX-2 protein expression analysis, RAW 264.7 macrophages were pre incubated for 18 h in a 6-well culture plate at a density of 1.0×10⁶ cells/well. And then, the cells were pretreated stimulated with LPS (1 μg/mL) in the absence or presence of HEF (25, 50, or 100 μg/mL) for 24 h, after which they were stimulated with LPS (1 μg/mL) for 24 h. Next, for MAPKs protein expression analysis, RAW 264.7 macrophages were pre incubated for 18 h in a 6-well culture plate at a density of 1.0×10⁶ cells/well. And then, the cells were pretreated in the absence or presence of HEF (25, 50, or 100 μg/mL) for 2 h, after which they were stimulated with LPS (1 μg/mL) for 10 min. After incubation, the cells were collected and washed twice with cold phosphate-buffered saline. The macrophages were lysed in a lysis buffer (radioimmunoprecipitation assay buffer, 1% Nonidet P-40, and 1% protease inhibitor cocktail) for 1 h, collected into microtubes, and then centrifuged at 15,000 rpm for 15 min at 4°C. The protein contents of the cell lysates were then determined by the Bradford Assay (Bio-Rad, Richmond, CA, USA). BSA was used as the standard for the analysis. Equal amounts of the lysates (30–50 μg of protein) were separated on a 4–12% Bis-Tris mini gel. After electrophoresis, the proteins were transferred onto a nitrocellulose membrane (Invitrogen Inc.). The membrane was washed in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TTBS) and blocked in TTBS containing 5% BSA solution for 2 h. The membrane was then incubated overnight at 4°C with primary antibodies (iNOS, COX-2, p-JNK, JNK, p-ERK, ERK, p-p38, and p38) diluted in TTBS (1:1000) and washed 4 times in TTBS. Each membrane was incubated for 1 h with secondary peroxidase-conjugated goat immunoglobulin G (IgG, 1:5000) and washed again with TTBS. The target proteins were detected using enhanced chemiluminescence solution. The immunoreactive bands were detected and then exposed to X-ray film. Protein levels were quantified by scanning the immunoblots.

2,2-diphenyl-1-picrylhydrazyl (DPPH assay)

The modified method of Kim et al. [26] was used. Twenty microliter of sample solution was mixed with 180 μL of 0.2 mM DPPH solution in a microtiter plate. After incubation for 15 min in the dark, the absorbance was measured at 517 nm. The % DPPH scavenging activity of samples are calculated as:

Inhibition (%)=[1−(Abssample−Abssample blank)/(Abscontrol−Abscontrol blank)]×100

2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS assay)

The modified method of Yang et al. [27] was used. To make ABTS+solution, 7 mM ABTS and 2.45 mM potassium persulfate were mixed and left in the dark for 16 h. one hundred and eighty microliter of ABTS⁺ solution was added to 20 μL of sample solution in a microtiter plate. After incubating for 15 min in the dark, the absorbance was measured at 700 nm. The % ABTS scavenging activity of samples are calculated as:

Inhibition (%)=[1−(Abssample−Abssample blank)/(Abscontrol−Abscontrol blank)]×100

HPLC fingerprint

Chromatographic analysis of the Shiranuhi flower performed using a HPLC system (Waters, Milford, MA, USA) with a YMC Pro-Triart C18 RS column (250×4.6 mm, S-5 μm, 8 nm; YMC Co., Kyoto, Japan) and a Waters 2489 UV visible detector. The mobile phases for the HPLC system were acetonitrile (A) and water containing 20 mM phosphoric acid (2:8) (B) at a flow rate of 2 mL/min. The samples were filtered through 0.22 μm polyvinylidene difluoride membrane filter. The following flavonoid compounds were analyzed in the citrus sample: rutin, naringin, hesperidin, neohesperidin, and narirutin. UV detection was performed at 280 nm.

Statistical analysis

The results have been expressed as mean±SD. All the quantitative data are representative of at least three independent experiments. Student’s t-test was used to determine statistically significant differences between each treated group and the negative control (LPS group). Statistical significance was considered at p<0.05 (*) or p<0.01 (**).

Effect of Shiranuhi flower on NO synthesis in activated macrophages

In murine RAW 264.7 macrophages, bacterial LPS alone can induce iNOS transcription, protein synthesis, and subsequent NO production. Therefore, the RAW 264.7 cell line is a common model for evaluating anti-inflammatory agents by inhibiting pathways that trigger iNOS and NO productions [1, 2, 10]. To investigate the effect of Shiranuhi flower extract on NO production, we measured the levels of nitrite, a stable oxidized product of NO, in culture media containing RAW 264.7 cells that had been stimulated with LPS in the presence or absence of the Shiranuhi flower extract.

It was observed that the nitrite level in the medium containing the LPS-stimulated cells increased significantly and was higher than that in the medium containing the control cells. To evaluate whether Shiranuhi flower can modulate NO production by activated macrophages, we examined the effects of the hot water and ethanolic extracts, as well as those of the ethyl acetate and water fractions on NO production in the murine RAW 264.7 macrophages. As well, the effects of 2-amino-4-methylpyridine (10 μM) and dexamethasone (20 μM) on the cells were investigated. As shown in Figure 1A, among the four Shiranuhi flower preparations, HEF (100 μg/mL) markedly inhibited LPS-induced NO production in the RAW 264.7 cells by 85%. Furthermore, 2-amino-4-methylpyridine, which was used as an NO inhibitor and the positive control, also inhibited LPS-induced NO production by 91%. As shown in Figure 1B, the numbers of viable activated macrophages were not altered by HEF, 2-amino-4-methylpyridine, or dexamethasone, as were observed from the LDH assays. This was an indication that the inhibition of NO production by HEF was not simply due to cytotoxic effects.

Figure 1:

Inhibitory effects of Shiranuhi flower extracts and fractions on cytotoxicity and nitric oxide (NO) production in murine RAW 264.7 cells.

RAW 264.7 cells were pretreated with the Shiranuhi flower extracts and fractions (each at concentrations of 25, 50, and 100 μg/mL), 2-amino-4-methylpyridine (A, 10 μM), dexamethasone (B, 20 μM), and vehicle (DMSO, the solvent in which the dried extracts and fractions are dissolved) for 2 h, and then treated with lipopolysaccharide (1 μg/mL) for 24 h. (A) The nitrite levels in the culture media were determined using Griess reagent. (B) Cell viability was analyzed by using the LDH cytotoxicity detection kit according to the manufacturer’s instructions. The data are expressed as mean±SD (n=3). *indicates p<0.05 and **indicates p<0.01.

Effect of HEF on PGE₂ production in LPS-induced RAW 264.7 cells

Prostaglandins are small pro-inflammatory molecules derived from arachidonic acid that play roles in a multitude of biological processes including inflammation. COX-2 expression, which is induced by cytokines and endotoxins such as LPS, results in the release of large amounts of PGE₂ at sites of inflammation [1, 2, 10]. Therefore, we examined the effects of HEF on PGE₂ production in the LPS-stimulated RAW 264.7 macrophages. As shown in Figure 2, LPS increased PGE₂ production by approximately 12-fold, whereas pretreatment with HEF (25, 50, or 100 μg/mL) markedly reduced LPS-induced PGE₂ production in the cells in a dose-dependent manner. Furthermore, dexamethasone, which is a COX-2 selective inhibitor, had a significant inhibitory effect on PGE₂ production.

Figure 2:

Inhibitory effect of the ethyl acetate fraction of Shiranuhi flower extract (HEF) on prostaglandin (PG)E₂ production in murine RAW 264.7 cells.

HEF was prepared by suspending the residue obtained after drying the ethanolic extract of Shiranuhi flower in water, followed by extraction with ethyl acetate. The cells were stimulated with of LPS alone or with a combination of LPS and various concentrations (25, 50, or 100 μg/mL) of HEF for 24 h. PGE2 produced and released into the culture media were determined using ELISA Kit according to the manufacturer’s instructions. The data are expressed as mean±SD (n=3). *indicates p<0.05 and ** indicates p<0.01.

Effects of HEF on LPS-induced expression of iNOS and COX-2 proteins

Western blot analyses were performed to determine whether the inhibitory effects of HEF on NO and PGE₂ productions were related to modulations of iNOS and COX-2 expressions, respectively. As shown in Figure 3, we did not detect iNOS and COX-2 protein expressions in the control cells. However, iNOS and COX-2 levels were markedly upregulated by the LPS treatment. On the other hand, HEF did not affect the expression of β-actin, which is a housekeeping gene. In general, these results suggest that the inhibitory effects of HEF on LPS-induced NO and PGE₂ productions are the results of the suppression of iNOS and COX-2 expression levels, respectively.

Figure 3:

Inhibitory effects of the ethyl acetate fraction of Shiranuhi flower extract (HEF) on the expression levels of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in murine RAW 264.7 cells.

The RAW 264.7 cells were plated at 1.0×10⁶ cells/mL in 24-well plates and incubated for 18 h, and pretreated with HEF (25, 50, or 100 μg/mL), 2-amino-4-methylpyridine (A, 10 μM), or dexamethasone (B, 20 μM) for 2 h, after which they were stimulated with LPS (1 μg/mL) for 24 h. COX-2 and iNOS protein level was determined by immunoblotting.

Effect of HEF on the production of pro-inflammatory cytokines

Because TNF-α and IL-6 are markers of early-phase inflammation, elevated levels of these molecules can be detected during several acute and chronic inflammatory diseases. Therefore, we evaluated the effects of HEF on the levels of these cytokines in the LPS-treated cells by enzyme immunoassay. As expected, the LPS-treated RAW 264.7 cells displayed notable increases in the levels of TNF-α and IL-6. Moreover, the induction of pro-inflammatory cytokines was significantly decreased by HEF in a dose-dependent manner (p<0.05). As shown in Figure 4, the productions of TNF-α and IL-6 were decreased at the IC₅₀ values of HEF (41 and 35 μg/mL, respectively). These indicate that HEF efficiently suppressed LPS-induced TNF-α and IL-6 productions, which suggests that HEF inhibited the initial phase of the LPS-induced inflammatory response.

Figure 4:

Inhibitory effects of the ethyl acetate fraction of Shiranuhi flower extract (HEF) on the productions of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in murine RAW 264.7 cells.

The cells were stimulated with of LPS alone or with a combination of LPS and various concentrations (25, 50, or 100 μg/mL) of HEF for 24 h. TNF-α and IL-6 produced and released into the culture media were determined using ELISA Kit according to the manufacturer’s instructions. The data are expressed as mean±SD (n=3). *indicates p<0.05 and **indicates p<0.01.

Effect of HEF on the phosphorylation of MAPKs in LPS-stimulated RAW 264.7 cells

MAPKs play critical roles in the regulation of cell growth and differentiation, as well as in the control of cellular responses to cytokines and stressors. One of the most extensively investigated transduction pathways involved in inflammation is the MAPK pathway. Previous studies have shown that MAPKs play significant roles in regulating the levels of COX-2 and iNOS and the production of pro-inflammatory cytokines by modulating nuclear factor κB (NF-κB) and activator protein 1 (AP-1) signals in LPS-stimulated macrophages [3, 8, 13]. Therefore, Western blot analyses were performed to determine whether the inhibitory effects of HEF on the phosphorylations of ERK1/2, p38, and JNK MAPKs were related to the modulations of NF-κB and AP-1 signals. As shown in Figure 5, the LPS treatment significantly promoted the phosphorylations of ERK1/2, JNK, and p38 MAPKs in the RAW 264.7 cells. At all the concentrations studied (25, 50, and 100 μg/mL), HEF dramatically reduced the phosphorylations of ERK1/2, p38, and JNK MAPKs, each in a dose-dependent manner. However, the expression levels of unphosphorylated ERK1/2, p38, and JNK MAPKs were unaffected in cells that were treated with either LPS alone or both LPS and HEF. These results suggest that the phosphorylation of MAPKs may be involved in the mechanisms underlying the inhibitory effects of HEF on LPS-induced inflammation in murine RAW 264.7 cells.

Figure 5:

Effect of the ethyl acetate fraction of Shiranuhi flower extract (HEF) on lipopolysaccharide (LPS)-induced protein expression of p-JNK (phosphorylated-JNK)/JNK, p-ERK (phosphorylated-ERK)/ERK, and p-p-38 (phosphorylated-p-38)/p-38 in murine RAW 264.7 macrophages.

RAW 264.7 cells (1.0×10⁶ cells/mL) were pre-incubated for 18 h, incubated for 2 h with HEF, and then stimulated for 10 min with LPS (1 μg/mL). Immunoblotting was used to determine the levels of p-JNK (phosphorylated-JNK)/JNK, p-ERK (phosphorylated-ERK)/ERK, and p-p-38 (phosphorylated-p-38)/p-38.

Investigation of the relationship between the anti-inflammatory and antioxidant activities

It has been shown that reactive oxygen species (ROS) trigger the phosphorylation of MAPKs and the production of pro-inflammatory cytokines [28], [29]. Therefore, 70% ethanol extracts and HEF of Shiranuhi flower was used to investigate the relationship between the data sets of anti-inflammatory and the anti-oxidant activities. In this study, 70% ethanol extracts and HEF were screened free radical-scavenging effects such as DPPH and ABTS. Two extracts, HEF was showed good anti-oxidative effects with IC₅₀ of 149.44 μg/mL for DPPH and 54.57 μg/mL for ABTS (Figure 6). The anti-oxidative evaluation demonstrates that the same fraction was the best to inhibit the pro-inflammatory cytokines and MAPK mechanism.

Figure 6:

Antioxidant activities of 70% ethanol extracts and HEF.

DPPH and ABTS scavenging activities of 70% ethanol extracts (A and C) and HEF (B and D) at different concentrations. Each value represents mean±SEM of three replicates.

HPLC fingerprint of 70% ethanol extracts and HEF

Finally, in other to apply 70% ethanol extracts and HEF for anti-inflammatory agents in cosmetic industry, standard and/or functional materials have to be investigated. Therefore, the flavonoid constituents of the 70% ethanol extracts and HEF were also investigated using HPLC analysis in the present study. As shown in Figure 7, a total of 2 citrus flavonoids were identified on the basis of the compared to those in our HPLC library. The HEF is a predominantly composed of narirutin (3.13±0.43 g) and hesperidin (6.03±0.59 g) in 100 g of HEF. On the other hands, the 70% ethanol extract is composed of narirutin (0.69±0.07 g) and hesperidin (1.75±0.98 g).

Figure 7:

HPLC fingerprinting analysis of the HEF(A) and 70% ethanol (B) extracts.

The lower side (C) represents standard flavonoids. The wavelength of flavonoids is 280 nm.