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A flavonoid component and a phenyl compound are selected as nine propolis components were used in the experiment. The selected components are the most closely related components to the propolis functions. Nine selected components were as follows: Pinocembrin, quercetin, chrysin, naringin, gallic acid, p-coumaric acid, cinnamic acid, caffeic acid and caffeic acid penethyl ester (CAPE). The nine propolis components were purchased from Sigma Aldrich (U.S.A.).

The raw264.7 mouse macrophages were purchased from the Korea Cell Line Bank (KCLB) and maintained with high glucose Dulbecco’s modified eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 units/mL penicillin-streptomycin as antibiotics. DMEM, FBS and penicillin-streptomycin solution for subculture of Raw264.7 macrophages were purchased from Gibco (U.S.A.). FBS was used after inactivation in 56℃ for 50 min. Subculture of the cells are aided with the use of cell scraper without a trypsin cell detachment solution and maintained in an incubator set at 37℃ and 5% CO₂.

To investigate how each propolis components affect the cell survival, cytotoxicity assay was carried out. Cytotoxicity assay used were EZ-Cytox MTT assay kit was purchased from Dainbio (Korea). Raw264.7 macrophage was inoculated into 96-well plate on 2×10⁴ cells/well and incubated for 24 hr. Components of propolis were dissolved to absolute EtOH at 1 μg/μL. Each propolis component was filtered by 0.2 μm syringe filter for treatment. Raw264.7 macrophage was treated with each propolis component on dose-dependent (1, 2.5, 5, 10, 20, 25 and 50 μg/mL) exposure for 24 hr. As a control in this experiment, we compared only media treated cells. As a solvent control, only EtOH treatment was compared. After 24 hr exposure, EZ-Cytox solution was added to media at 1/10 volume and the cell was incubated for another 2 hr. The optical density of the media was measured at a wavelength of 450 nm.

Raw264.7 macrophage inoculated into 24-well plate was 2×10⁵ cells / well incubated for 24 hr. LPS used as positive reagent for inflammation reaction was purchased from Sigma Aldrich. The components of propolis were treated into the cells was 25 μg/mL for 24 hr except CAPE. Treated CAPE concentration is at 1 μg/mL. To compare the NO production, only the media treated cells and EtOH treated cells was considered as a negative control. LPS was treated into the cells at 1 μg/mL was made as the positive control. Cell supernatants were subjected to centrifugation at 3,000 rpm for 10 min after 24 hr. The supernatants without cell debris were collected to be used for NO assay. Generated NO was measured with NO assay reagents kit supplied by iNtRON (Korea). NO assay was carried out following the manufacture’s protocol. The optical density of produced NO was determined at a wavelength of 560 nm.

To investigate the effect of propolis components in LPS-mediated inflammation progression three groups of experimental set up for NO generation was used. First, pre-incubation of LPS for 24 hr on Raw264.7 cells and then added with the components of propolis for 24 hr. Second, pre-incubation of the components of propolis for 24 hr on Raw264.7 cells and then added with LPS for 24 hr. Lastly, LPS and the components of propolis simultaneously treated on Raw264.7 cells for 48 hr. The experiment procedure was progressed in the same protocol as described above. However, three propolis components such as quercetin, chrysin and CAPE were selected based on the result of the NO assay. Treated LPS concentration was 1 μg/mL, and quercetin and chrysin were treated on three dose-dependent levels (1, 5 and 25 μg/mL). The CAPE concentration used was 0.01, 0.1 and 1 μg/mL.

The proteins for Western blotting were extracted from Raw264.7 cells. Cells were washed twice with 1× phosphate-buffered saline (PBS) and lysed with 200 μL of nonidet P (NP)-40 lysis buffer (Dainbio). The lysate supernatants were cleared by centrifugation for 15 min at 13,000 rpm. The concentrations of cleared lysates were determined by bicinchronic acid (BCA) assay. Cell lysated at 20 μg were used in Western blotting. The lysates were added with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. Non-specific binding was blocked by incubation of membranes in 2% skim milk at 1X Tris-buffered Tween 20 (1× TBST) for 1 hr at room temperature. The membranes were probed with primary antibodies such as anti-iNOS, anti-IL-1β, anti-NF-κB and anti-GAPDH antibodies. iNOS and IL-1β antibody were purchased from Cell Signaling (U.S.A.) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and glyceraldehyde 3-phosphate dehydrogtenase (GAPDH) antibody were purchased from Santacruz biotechnology (U.S.A.). Except for anti-GAPDH, all antibodies were incubated for 16 hr. Anti-GAPDH were incubated for only 2 hr. Protein expression were detected by incubation with secondary horseradish peroxide (HRP)-conjugated anti-goat antibody and was visualized with an enhanced chemiluminescence (ECL) pico detection system.

The statistical significance of the results obtained from the experiment was used by the R (3.4.1 version, NewZealnd) statistics program, and the mean significance of the experiment was verified by the ANOVA test at p<0.05.

To investigate the cell viability of propolis components, nine propolis components were treated as described previously to Raw264.7 macrophage. The cells were incubated with each propolis component at different dose-dependent levels (0, 1, 2.5, 5, 10, 20, 50 and 100 μg/mL) for 24 hr. The components: naringin, p-coumaric acid, cinnamic acid and caffeic acid have not affected cell viability. In this result, four propolis components did not show cytotoxicity. However, pinocembrin, quercetin, chrysin and gallic acid decreased the cell viability in dosage of more than 50 μg/mL. As shown in Fig. 1, CAPE showed the most potent cytotoxicity effect of more than 2.5 μg/mL dosage. The component can be grouped into compounds that have cytotoxicity and no cytotoxicity effects. Based on the results, the concentration of propolis components for immune-regulation can be determined. The concentration of another component for immune-regulation was set up at 25 μg/mL maximum except CAPE which was determined at 1 μg/mL.

Fig. 1. 
Propolis components influencing cell viability. Raw264.7 macrophage cells placed in to 96-well plates on a 2×10⁴ cells/well. After 24 hr, each propolis component was treated into the cells at dose-dependent. Cell viability was measured by EZ-Cytox MTT assay after 24 hr incubation.

NO molecule as representative marker in inflammation mediator is produced from L-arginine by nitric oxide synthase (NOS). L-arginine is converted to L-citrulline and NO, and iNOS are known as key molecule in this process. When cells are stimulated by external factor such as inflammation factor, bacteria and virus, it was expressed as iNOS. Generated NO molecule through this reaction were severely promoting inflammation responses such as cytotoxicity and tissue injury. To confirm the propolis components related to NO production, LPS was treated as inflammation inducing factor and each propolis component was prepared at 1 μg/mL for 24 hr. As a control, LPS treatment group was not only set up but also each propolis component treatment group. EtOH treatment group is a solvent control for propolis diluent.

As shown in Fig. 2A, inflammation response significantly increased in LPS-treated group, but not in propolis component-treated group. However, LPS and propolis components-treated group has different NO generation. Although all propolis components have decreased NO production, the component such as quercetin, chrysin and CAPE has significantly reduced NO production by more than half. The results indicate that the specific component of propolis has directly participated in NO reduction. It has been confirmed that inhibition of three components specially quercetin, chrysin and CAPE, whether the dose-dependent have been the most effective in NO reduction. Among the most effective concentrations for quercetin and chyrsin are 1, 5 and 25 μg/mL, and for CAPE is 0.01, 0.1 and 1 μg/mL. As shown in Fig. 2B, three components of propolis reduced the generated NO by LPS depending on dose-dependent levels. The results indicate that three components of propolis play essential role in reducing NO generation by inflammation response.

Fig. 2. 
Propolis component, quercetin, chrysin and CAPE inhibit NO generation. Raw264.7 macrophage cells placed into 24-well plates on a 2×10⁵ cells/well. All propolis components treatments was at 25 μg/mL except CAPE (M: Media, E: EtOH, L: LPS, P: Pinocembrin, Q: Quercetin, C: Chrysin, N: Naringin, Ga: Gallic acid, Co: p-coumaric acid, Ci: Cinnamic acid, Ca: Caffeic acid, CP: CAPE) (A). Three components of propolis as quercetin, chrysin and CAPE was measured generated NO on dose-dependent (B). The supernatants were cleared with centrifugation at 3,000 rpm for 10 min. Generated NO was measured following manufacture’s protocol (Quer: Quercetin, Chry: Chrysin).

The synergistic effects of quercetin, chrysin and CAPE in generated NO reduction by LPS-mediated inflammation response was investigated. The concentration of each propolis component was determined at 1 μg/mL and combined the components as follows. (1) Quercetin and chrysin; (2) Quercetin and CAPE; (3) Chrysin and CAPE; (4) Quercetin, chrysin and CAPE combination. Media and EtOH treatment is the control group for LPS and propolis components. LPS-treated group is a positive control for inflammation response.

As shown in Fig. 3, generated NO reduction is better in the combined components than single component treatment only. Especially, when quercetin and chrysin are combined reduced NO generation is about 50% compared with each quercetin and chrysin only. CAPE has shown the most reduction of generated NO. The results mean that CAPE plays essential role in reduction of NO production related to inflammation, while the combination of quercetin and chrysin provides synergistic effects on inflammation reduction.

Fig. 3. 
The synergistic effects on generated NO inhibition. Raw264.7 macrophage cells placed into 24-well plates on a 2×10⁵ cells/well. The treatment of quercetin and chrysin were 25 μg/mL, and CAPE treatment was 1 μg/mL. LPS and each component combined mixture were incubated for 24 hr, and generated NO was measured with NO assay kit (Quer: Quercetin, Chry: Chrysin).

For inflammation prevention, three experimental groups are determined whether each propolis component exhibits therapeutic or prevention effects. The first group is induced inflammation response by LPS treatment for 24 hr, and was treated with the components of propolis. The second group has pre-treated propolis components for 24 hr, and was LPS-treated for 24 hr. The final group, propolis components and LPS were simultaneously used as treatment. The generated NO was measured on each experimental group. The media only was used as the negative control, EtOH treatment as the solvent control and LPS treatment group as the positive control. Propolis components that were selected are quercetin, chrysin and CAPE which had the most effect on the inhibition of NO production. All components concentration is set at 1 μg/mL.

As shown in Fig. 4A, when the component of propolis is pre-treated and LPS is treated, generated NO was decreased. However, when LPS is pre-treated then propolis component is treated, generated NO did not decrease. In simultaneously treatment group, CAPE has shown the most reduction of NO. Quercetin and chrysin have the same results as the previously held experiment. In other words, three components are related in terms of inflammation response reduction, with CAPE as the most efficacy on inflammation. Three components of propolis have the better effect when they are pre-treated to cells. Therefore, all three components are expected to have more prevention effects than therapeutic effects on inflammation.

Fig. 4. 
Propolis components prevention effects on inflammation response. Raw264.7 cells were placed into a 24-well plate on a 2×10⁵ cells/well. The quercetin and chrysin treatments were set at 25 μg/mL while CAPE treatment was at 1 μg/mL for 24 hr. The treatment of LPS was 1 μg/mL for 24 hr (Quer: Quercetin, Chry: Chrysin) (A). Each propolis component mixture and LPS were treated for 24 hr. Each reagent concentration is same with previously conducted experiment (Q: Quercetin, C: Chrysin; CP: CAPE) (B).

Although three components of propolis showed NO reduction, confirmation of synergistic effects between three components were determined. As shown in Fig. 4B, the combination of the components showed more NO reduction effects than single component treatment. The results suggest that three components which showed NO reduction improved its effect by combination. Therefore, propolis is effective in inflammation prevention, especially quercetin, chrysin and CAPE in relation to NO reduction.

In relation to NO inhibition caused by each component of propolis we carried out Western blotting whether each component regulate expression of protein in the cells. The regulation of expression of protein cell was determined using iNOS which involved NO production process, IL-1β which representative cytokine and NF-κB expression in inflammation signaling cascade.

As shown in Fig. 5A, the single treatment of quercetin, chrysin and CAPE did not have related protein expression in the cells. The protein expression level, however, was present when LPS and propolis components was treated into the cells as shown in Fig. 5B. LPS-mediated iNOS protein level was decreased by crude propolis treatment. Quercetin directly regulated iNOS expression. On the other hand, chrysin and CAPE did not affect to iNOS expression. In the combined components, iNOS expression decreased when quercetin was combined, while chrysin and CAPE mixing did not show inhibition of iNOS expression.

Fig. 5. 
Propolis components influenced protein expression level in the cells. All lysates were used at 20 μg for Western blotting. Each antibody diluted at 1 : 1,000 in 2% skim milk incubated overnight except for GAPDH. GAPDH was made to be housekeeping control for Western blotting and its dilution is 1 : 5,000 (Qu: Quercetin, Ch: Chrysin, CP: CAPE).

In the NF-κB protein expression, which is important in cell immune signaling, CAPE treatment group has the lowest expression level. The component quercetin and chrysin have shown similar protein expression. For IL-1β which is known representative marker in inflammation response has shown the most reduction of protein expression in quercetin treatment group. The chrysin treatment group also showed reduced protein expression. CAPE, on the other hand, does not reduce the IL-1β protein expression level. The results of the combined components showed an inhibition in IL-1β expression only when quercetin was mixed, but chrysin and CAPE had no significant effect on IL-1β expression. GAPDH is a loading control in this protein expression experiment.