N-acetyl-L-cysteine (NAC, ROS scavenger), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), SB203580 (p38 inhibitor), PD98059 (ERK1/2 inhibitor), SP600125 (JNK inhibitor), BAY 11-7082 (IKK inhibitor), TAK-242 (Toll-like receptor 4 [TLR4] inhibitor), lipopolysaccharide (LPS), and neutral red were purchased from Sigma-Aldrich (St. Louis, MO, USA). C29 (TLR2 inhibitor) was purchased from BioVision (Milpitas, CA, USA). Primary antibodies against p-JNK, total-JNK, and β-actin and secondary antibody against anti-rabbit were purchased from Cell Signaling Technology (Beverly, MA, USA).

Cells were cultured according to a previous method with some modifications (Hong et al., 2017). Briefly, RAW264.7 mouse macrophage cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured using Dulbecco's Modified Eagle’s medium (DMEM)/F-12 1 : 1 modified medium (Lonza, Walkersville, MD, USA) mixed with 10% fetal bovine serum (Hyclone Laboratories Inc., Logan, UT, USA), 100 U/㎖ penicillin, and 100 ㎍/㎖ streptomycin (Gibco BRL, Grand Island, NY, USA) at 37℃ under a humidified atmosphere of 5% CO₂.

S. bumalda leaves (FMCSbHC1908 1 - FMCSbHC1908 3) were provided by Forest Medicinal Resources Research Center, Korea. The leaves were washed 2 - 3 times with distilled water, dried with a freeze dryer, crushed using a grinder, and stored at -20℃.

After immersing 10 g of S. bumalda leaves in 200 ㎖ of distilled water, the mixture was extracted with stirring at room temperature for 3 days and then centrifuged at 12,000 rpm for 10 min, and the supernatant was collected and lypophilized. The water extracts (SBL) were kept at -80℃ until use. Before the experiment, SBL was dissolved in sterile water and administered to cells. The absence of endotoxin in SBL was verified using a Plant Endotoxin ELISA Kit (San Diego, CA, USA).

The total phenolic compound contents were measured using the Folin-Ciocalteu assay (Park et al., 2008).

Briefly, 0.5 ㎖ of SBL (25 ㎎/㎖) was mixed with 0.5 ㎖ of 2 N Folin-Ciocalteu reagent for 5 min; then, 2 ㎖ of 7% (w/v) sodium carbonate was added. The mixtures were incubated for 90 min at room temperature. Then, the absorbance was measured at 750 ㎚ using a UV-Vis spectrophotometer (Human Co., Xma-3000PC, Seoul, Korea).

The total phenolic compound contents were measured according to a previous method with some modifications (Park et al., 2008).

Briefly, 20 ㎕ of SBL (25 ㎎/㎖), 80 ㎕ of distilled water, and 6 ㎕ of 5% NaNO₂ were mixed for 5 min at room temperature. Then, 12 ㎕ of 10% AlCl₃·6H₂O was added, and the mixtures were incubated for 6 min at room temperature. Next, 40 ㎕ of 1 N NaOH was added, and then the mixtures were incubated for 11 min at room temperature. The absorbance was measured at 510 ㎚ using a UV-Vis spectrophotometer (Human Co., Xma-3000PC, Seoul, Korea).

Tannic acid from SBL was analyzed using high-performance liquid chromatography (HPLC). For HPLC analysis, the Waters 1525 system with a Waters 2487-dual λ absorbance detector was used.

The column was equipped with the SUNFIRE C18 column (250 ㎜ × 4.6 ㎜). The binary mobile phase consisted of water containing 1% acetic acid (solvent A) and acetonitrile (solvent B). The flow rate was kept constant at 1.0 ㎖/min for a total run time of 33 min. The mobile phase was programmed consecutively in a linear gradient as follows: 0–10 min (90% A); 10–21 min (90% → 78% A); 21 min - 23 min (78% → 50% A); 23 min - 25 min (50% → 10% A); 25 min - 30 min (10% →90% A), and 30 min - 33 min (90% A). The injection volume of the extract was 10 ㎕. The elution was monitored at 280 ㎚. Tannic acid in SBL was identified by the chromatogram of the analytical tannic acid standard.

The MTT assay was performed according to a previous method with some modifications (Geng et al., 2018) to evaluate the cytotoxicity of SBL against RAW264.7 cells.

Briefly, after culturing RAW264.7 cells (1 × 10⁴ cells/well) in a 96-well plate for 24 h, cells were treated with various SBL concentrations and cultured for an additional 24 h. After 24 h, 50 ㎕ of MTT solution (1 ㎎/㎖) was added to each well and incubated for an additional 4 h. The cell culture medium was removed, and 100 ㎕ of dimethyl sulfoxide was added to elute the resulting crystals from the wells. The absorbance was measured at 570 ㎚ using a UV-Vis spectrophotometer (Human Co., Xma-3000PC, Seoul, Korea).

The neutral red uptake assay has been widely used to measure phagocytosis by macrophages (Wang et al., 2018).

Thus, this assay was performed to evaluate the effect of SBL on phagocytosis in macrophage cells. Briefly, after culturing cells (2 × 10⁵ cells/well) in a 12-well plate for 24 h, cells were treated with various SBL concentrations and cultured for an additional 24 h. After 24 h, each well was washed 3 times with 1× phosphate-buffered saline (PBS), and 1 ㎖ of a 0.01% neutral red solution was added to each well and incubated for an additional 2 h. Subsequently, each well was washed 3 times 1× PBS, and then 1 ㎖ of cell lysis buffer (ethanol acid : acetic acid = 1 : 1) was added to elute the neutral red absorbed by the cells.

The absorbance was measured at 540 ㎚ using a UV-Vis spectrophotometer (Human Co., Xma-3000PC, Seoul, Korea).

The Griess assay was performed according to a previous method with some modifications (Hong et al., 2017) to evaluate the effect of SBL on NO production in the cells.

Briefly, after culturing cells (2 × 10⁵ cells/well) in a 12-well plate for 24 h, cells were treated with various SBL concentrations and cultured for an additional 24 h. Next, 100 ㎕ of the cell culture medium was collected and reacted with the same amount of Griess reagent (Sigma-Aldrich Co., St. Louis, MO, USA) at room temperature for 15 min. The absorbance was measured at 540 ㎚ using a UV-Vis spectrophotometer (Human Co., Xma-3000PC, Seoul, Korea).

After culturing RAW264.7 cells (2 × 10⁵ cells/well) in a 12-well plate for 24 h, cells were treated with various SBL concentrations and cultured for an additional 24 h.

The levels of IL-1β, IL-6, and TNF-α were measured using ELISA kits for mouse IL-1β (Invitrogen, Carlsbad, CA, USA), mouse IL-6 and mouse TNF-α Cayman Chemical, Ann Arbor, MI, USA), respectively, according to the manufacturers’ protocols.

Western blot analysis was performed according to a previous method with some modifications (Hong et al., 2017).

Briefly, after all treatments were completed, the cells were washed 3 times with cold 1× PBS, recovered with a radioimmunoprecipitation assay buffer (Boston Bio Products, Ashland, MA, USA) containing protease inhibitor and phosphatase inhibitor (Sigma-Aldrich Co., St. Louis, MO, USA) to extract proteins, and incubated at 4℃ for 30 min. After centrifugation at 15,000 rpm at 4℃ for 10 min, the supernatant was collected, and the protein was quantitatively analyzed using the BCA protein assay (Thermo Scientific Inc., Waltham, MA USA).

Subsequently, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transfered to a polyvinylidene fluoride membrane, which was blocked for 1 h by stirring at room temperature with blocking buffer [Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and 5% non-fat dry milk]. After washing the membrane with TBS-T, it was treated with specific primary antibodies in 0.05% TBS-T containing 5.00% bovine serum albumin and incubated for 16 h with stirring at 4℃.

Subsequently, the membrane was washed with TBS-T and continuously treated with secondary antibodies in 0.05% TBS-T containing 5.00% non-fat dry milk by stirring at room temperature. After washing the membrane with TBS-T, chemiluminescence was detected with ECL Western Blotting Substrate (Amersham Biosciences Co., Piscataway, NJ, USA) and visualized using the LI-COR C-DiGit Blot Scanner (Li-COR Biosciences Inc., Lincoln, NE, USA). Western blot band densities were calculated using the UN-SCAN-IT gel version 5.1 software (Silk Scientific Inc., Orem, UT, USA).

RT-PCR analysis was performed according to a previous method with some modifications (Geng et al., 2018).

Briefly, after all treatments were completed, total RNA was separated from cells using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and was quantitatively analyzed. Then, cDNA was synthesized from 1 ㎍ of total RNA using a Verso cDNA Kit (Thermo Scientific Inc., Pittsburgh, PA, USA). PCR was performed using a PCR Master Mix Kit (Promega, Madison, WI, USA) and the primer presented in Table 1.

The PCR results were visualized using agarose gel electrophoresis. The density of mRNA bands was calculated using UN-SCAN-IT gel version 5.1 (Silk Scientific Inc., Orem, UT, USA).

All experiments were performed in triplicate. Data are presented as the mean ± standard error of the mean. Data analysis was carried out using SPSS software (version 19.0, IBM Co., Armonk, NY, USA).

One-way analysis of variance was considered as indicating a significant difference followed by Tukey's honestly significant difference test was used to compare differences among groups (p < 0.05).

To evaluate the effect of SBL on the production of immunomodulators in macrophages, the levels of NO, iNOS, IL-1β, IL-6, TNF-α, and MCP-1 were investigated after treating cells with SBL for 24 h.

As shown in Fig. 1A, SBL increased NO production in a concentration-dependent manner. Since an excessive NO level results in macrophage death (Taylor et al., 2003), the cytotoxicity of SBL toward macrophages was evaluated. As shown in Fig. 1B, SBL showed no cytotoxicity.

Fig. 1.  Effect of SBL on the activation of RAW264.7 cells.

RAW264.7 cells were treated with the indicated concentrations of SBL for 24 h. (A): NO level was measured using Griess assay. (B): Cell viability was measured using MTT assay. (C): mRNA level was measured using RT-PCR analysis. (D): Cytokine production was measured using ELISA kit. (E): phagocytic activity was measured using Neutral Red assay. ^*p < 0.05 compared to the cells without the treatment.

In addition, SBL increased the expression of major immunomodulators, such as iNOS, IL-1β, IL-6, TNF-α and MCP-1, in macrophages (Fig. 1C and 1D). It is known that these macrophage-secreted immunomodulators increase phagocytosis by macrophages, which is used as an indicator of macrophage activation (We et al., 2006).

Thus, we investigated the effect of SBL on macrophage phagocytosis through the neutral red assay. As shown in Fig. 1E, SBL was found to significantly increase macrophage phagocytosis. These results indicate that SBL activates macrophages (Fig. 1).

Since TLR2/4 are known to be major receptors associated with macrophage activation (Li and Cherayil, 2003; Kakutani et al., 2012), the effect of TLR2/4 SBL-induced immunomodulator production was evaluated.

First, after suppressing TLR2 or TLR4 with C29 (TLR2 inhibitor) or TAK-242 (TLR4 inhibitor), cells were treated with SBL for 24 h. As shown in Fig. 2A-2D, TLR2 and TLR4 inhibition by C29 and TAK-242 dramatically attenuated NO production and the expression of iNOS, IL-1β, IL-6, and MCP-1 induced by SBL. In addition, we observed that SBL increased TLR2 and TLR4 expression (Fig. 2E and 2F).

Fig. 2.  Effect of TLR2 and TLR4 on SBL-mediated production of immunomodulators in RAW264.7 cells.

(A, B, C, D) RAW264.7 cells were pretreated with C29 (TLR2 inhibitor) or TAK-242 (TLR4 inhibitor) for 2 h and then co-treated with SBL for 24 h. The level of NO or mRNA was measured using Griess assay and RT-PCR analysis, respectively. (E, F) RAW264.7 cells were treated with SBL for 24 h. mRNA level was measured using RT-PCR analysis. ^*p < 0.05 compared to the cells without the treatment.

The results shown in Fig. 2 indicate that SBL increases the production of immunomodulators by inducing TLR2/4 expression in the cells.

Since TLR2/4 are known to activate various signaling pathways, such as mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB), to induce the production of immunomodulators in macrophages (Shen et al., 2017; Zhang et al., 2017; Bai et al., 2019; Yang et al., 2019), we analyzed the signaling pathway related to SBL-mediated immunomodulator production in RAW264.7 cells.

RAW264.7 cells was pretreated with PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), or BAY 11-7082 (NF-κB inhibitor) to inhibit each signaling pathway, and then treated with SBL for 24 h. As shown in Fig. 3A, SBL induced NO production regardless of whether ERK1/2 and p38 were inhibited. However, JNK inhibition reduced the level of NO produced by SBL. In addition, the inhibition of NF-κB resulted in slight inhibition of SBL-mediated NO production (Fig. 3B). The results shown in Fig. 3A and 3B suggest that JNK is the major signaling pathway associated with SBL-mediated immunomodulator production in macrophages.

Fig. 3.  Effect of MAPKs and NF-κB signaling pathways on SBL-mediated production of immunomodulators in RAW264.7 cells.

RAW264.7 cells were pretreated with PD98059 (10 μM, ERK1/2 inhibitor), SB203580 (10 μM, p38 inhibitor), SP600125 (10 μM, JNK inhibitor) or BAY 11-7082 (10 μM, NF-κB inhibitor) for 2 h and then co-treated with SBL. (A, B) NO level was measured using Griess assay. (C) mRNA level was measured using RT-PCR analysis. (D) RAW264.7 cells were pretreated with PD98059 (10 μM, ERK1/2 inhibitor), SB203580 (10 μM, p38 inhibitor), SP600125 (10 μM, JNK inhibitor) or BAY 11-7082 (10 μM, NF-κB inhibitor) for 2 h and then co-treated with SBL. mRNA level was measured using RT-PCR analysis. ^*p < 0.05 compared to the cells without the treatment. ^#p < 0.05 compared to the cells with SBL treatment alone.

Thus, we investigated the effect of JNK on the expression of other immunomodulators induced by SBL. As shown in Fig. 3C, JNK inhibition reduced the expression of iNOS, IL-1β, IL-6, and MCP-1. Although ERK1/2, p38, and NF-κB signaling had no effect on SBL-induced NO production, we investigated the effects of these pathways on the production of other immunomodulators, such as iNOS, IL-1β, IL-6, and MCP-1.

As shown in Fig. 4D, ERK1/2 inhibition by PD98059 blocked IL-6 production by SBL, while SB230580-induced p38 inhibition attenuated SBL-mediated production of iNOS, IL-1β, and IL-6. However, NF-κB inhibition by BAY 11-7082 did not affect immunomodulator production. Therefore, the results depicted in Fig. 3 demonstrate that JNK is a major upstream kinase related to SBL-mediated immunomodulator production.

Fig. 4.  Effect of TLR2 and TLR4 on SBL-mediated JNK activation in RAW264.7 cells.

(A): RAW264.7 cells were treated with SBL for the indicated times. JNK phosphorylation was measured using Western blot analysis. (B and C): RAW264.7 cells were pretreated with C29 (TLR2 inhibitor) or TAK-242 (TLR4 inhibitor) for 2 h and then co-treated with SBL for 3 h. JNK phosphorylation was measured using Western blot analysis. (D): RAW264.7 cells were treated with SBL for the indicated times. ERK1/2 and p38 phosphorylation were measured using Western blot analysis. (E): RAW264.7 cells were pretreated with C29 (TLR2 inhibitor) or TAK-242 (TLR4 inhibitor) for 2 h and then co-treated with SBL for 3 h. ERK1/2 and p38 phosphorylation were measured using Western blot analysis. ^*p < 0.05 compared to the cells without the treatment. ^#p < 0.05 compared to the cells with SBL treatment alone.

We confirmed that SBL activated TLR2/4 and that JNK is a major upstream kinase involved in immunomodulator production in macrophages (Fig. 2 and 3).

Thus, we next analyzed whether SBL induces JNK activation. After treating cells with SBL for the indicated times, the phosphorylation level of JNK was investigated by western blot analysis. The results revealed that SBL phosphorylated JNK beginning at 1 h after the treatment (Fig. 4A).

Additionally, the effect of TLR2/4 on SBL-induced JNK phosphorylation was investigated by western blot analysis. As shown in Fig. 4B and 4C, inhibition of TLR2 by C29 or of TLR4 by TAK-242 significantly attenuated SBL-induced JNK phosphorylation. Because ERK1/2 and p38 signaling also partially influenced SBL-induced immunomodulator production, we investigated whether SBL activates ERK1/2 and p38 signaling.

As shown in Fig. 4D, SBL phosphorylated ERK1/2 and p38. In addition, TLR2 inhibition by C29 blocked SBL-induced phosphorylation of p38 but not that of ERK1/2, whereas TAK-242-mediated TLR4 inhibition attenuated SBL-induced phosphorylation of both p38 and ERK1/2. The results shown in Fig. 4 indicate that SBL may induce the activation of ERK1/2, p38, and JNK through TLR2/4.

As a well-known immune-enhancing agent, γ-PGA activates macrophages by increasing the production of immunomodulators (Jeon et al., 2015).

Thus, we compared the immunomodulator-inducing activity of SBL with that of γ-PGA to determine the degree of SBL’s immune-enhancing activity. As shown in Fig. 5A and 5B, the production of NO, iNOS, and IL-1β induced by SBL was found to be slightly higher than that induced by γ-PGA; conversely, SBL-mediated IL-6 and MCP-1 production was slightly lower than that mediated by γ-PGA. However, given the results overall, the immune-enhancing activity of SBL appears to be similar to that of γ-PGA.

Fig. 5.  Comparison of immune-enhancing activity of SBL and γ-PGA in RAW264.7 cells.

RAW264.7 cells were treated with SBL and γ-PGA for 24 h. RAW264.7 cells were treated with SBL and γ-PGA for 24 h. (A): NO level was measured using Griess assay. (B): mRNA level was measured using RT-PCR analysis. ^*p < 0.05 compared to the cells without the treatment.

If immunomodulator secretion by macrophages is excessive or abnormal, an inflammatory response occurs, with continual inflammation leading to chronic inflammation-related diseases.

Thus, we evaluated the effect of SBL on LPS-induced immunomodulator production in macrophages. As shown in Fig. 6A and 6B, SBL suppressed excessive production of NO, iNOS, and IL-1β. Since it is known that excessive immunomodulator production induced by LPS is also dependent on the activation of TLR2 or TLR4, we investigated the effect of SBL on LPS-induced expression of TLR2 and TLR4.

Fig. 6.  Anti-inflammatory activity of SBL in LPS-stimulated RAW264.7 cells.

RAW264.7 cells were pre-treated with SBL for 4 h and then co-treated with PBS or LPS for 20 h. (A): NO level was measured using Griess assay. (B and C): mRNA level was measured using RT-PCR analysis. ^*p < 0.05 compared to the cells without the treatment.

As shown in Fig. 6C, TLR2 and TLR4 expression was induced in cells treated with LPS alone, but SBL inhibited the expression of TLR2 and TLR4 induced by LPS. The results shown in Fig. 6 indicate that SBL suppresses excessive production of immunomodulators secreted by macrophages during excessive immune responses.

Since polyphenols, flavonoids, and tannins in natural products are known to enhance the body’s immune response to external pathogens (Kaneko et al., 2017; Ding et al., 2018; Reyes et al., 2018), we analyzed the contents of phenols, flavonoids, and tannins in SBL. As shown in Fig. 7A, the total phenolic and flavonoid contents were confirmed to be 40.1 ± 0.7 and 67.5 ± 0.7 ㎍/㎎ extract, respectively. Moreover, tannic acid was also analyzed in SBL, the contents of which were approximately 14.58 ± 1.27 ㎍/㎎ (RSD = 1.04) extracts.

Fig. 7.  Analysis of total phenolic compounds, total flavonoid compounds, and tannin contents of SBL.

(A): Contents of total phenolic compounds and total flavonoid compounds. (B): HPLC Chromatogram of a tannic acid standard and SBL.