Inflammation is an immune response for defending the body from the damage caused by external stimuli. It is typically accompanied by localized swelling, redness, and pain. The inflammatory response is a critical system for alleviating tissue damage and maintaining homeostasis in the body [1]. However, if inflammation persists excessively, it can lead to chronic inflammation, which is the underlying cause of various diseases, such as arthritis, cardiovascular disorders, and degenerative diseases [2]. Inflammatory responses are regulated by the activation of macrophages, which are immune cells that respond to external substances or injuries. This regulation is achieved through the control of various inflammatory mediators, including nitric oxide (NO), prostaglandin E2, interferons, and cytokines [3].

However, the abnormal activation of macrophages due to prolonged external stimulation promotes the production of NO, which is synthesized by inducible nitric oxide synthase (iNOS) from L-arginine, resulting in chronic inflammation. Abnormally activated macrophages also exacerbate inflammation by increasing the expression of pro-inflammatory factors, such as prostaglandin E2 generated by cyclooxygenase-2 from arachidonic acid, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. This cascade of events can ultimately lead to tissue damage, genetic mutations, and nerve damage [1].

Excessive activation of macrophages can occur due to the continuous stimulation by substances such as lipopolysaccharide (LPS), a component of the cell membrane of gram-negative bacteria or reactive oxygen species (ROS) that act as endotoxins. The response to LPS is regulated through the Toll-like receptor 4 (TLR-4) signaling pathway, which activates the nuclear factor-kappa B (NF-κB). NF-κB forms a complex with IkappaB kinase (IκB) in the cytoplasm and is activated when IκB is phosphorylated by the activated IκB kinase in response to LPS stimulation. Separation of the NF-κB:IκB complex allows NF-κB to migrate to the nucleus, where it regulates the production of NO and the biosynthesis of inflammatory mediators, while the separated IκB is degraded. This series of processes is known to be closely associated with various signaling pathways [4].

Sipyungtang is a prescription that combines Soshihotang and Pyungwisan, also referred to as Pyeonghoeumja in traditional medicine, and is documented in Donguibogam as a remedy for various types of fevers [5]. Previous studies on Sipyungtang have investigated its efficacy in treating pediatric fever [6] and allergies [7]. However, few studies report its anti-inflammatory and antioxidant effects. Therefore, in this study we aimed to determine the anti-inflammatory and antioxidant effects of Sipyungtang.

1. Materials

1) Herbs

The herbs used in this study were purchased from Omniherb.

2) Prescription composition

The composition of the prescription was based on the formulas found in Donguibogam [5].

3) Cells

The RAW 264.7 cells used in this study were purchased from the Korean Cell Line Bank, cultivated in a Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and cultured at 37℃ in an incubator with 90% relative humidity and 5% CO₂.

4) Experimental materials

The following materials were used in this study: DMEM (Invitrogen); FBS 10% (Invitrogen); penicillin/streptomycin (Gibco) 1%; 3-(4,5-dimethyl thiazol-2-yl)-2,5 diphenyl-2H-tetrazolium bromide (MTT; Bio Basic); Griess reagent (Sigma-Aldrich); 2,2-diphenyl-1-picrylhydrazyl (DPPH; Sigma-Aldrich); ethanol (Sigma-Aldrich); 96-well plates (Falcon); and vitamin C (ascorbic acid; Sigma-Aldrich).

5) Experimental equipment

The following devices were used in this study: CO₂ Incubator (SANYO), Clean Bench (The Baker), Microplate Reader-SpectraMax 190 (Molecular Device), Microscope (EVOS), Plate Shaker (Titeck), Mastercycler EP Realplex (Eppendorf), Mastercycler Pro (Eppendorf), and Micro & Nano Spectrophotometer (Eppendorf).

2. Methods

1) Preparation of the Sipyungtang water extract

A total of 210 g of Sipyungtang (Table 1) was placed in a reflux extractor with 3,000 mL of distilled water. The mixture was heated for 4 hours from the point at which the solution began to boil. The extract was then vacuum filtered using a filter paper (Advantec No. 2; ADVANTEC) and the filtrate was concentrated using R-100 rotary evaporator (Bunch). The concentrated solution was dried using a freeze dryer, resulting in a powder sample. Altogether, 40.95 g of the freeze-dried extract was obtained, with a yield of 19.5%.

Table 1 . Components of Sipyungtang.

Pharmaceutical name	Scientific name	Amount (g)
Bupleuri Radix	Bupleurum chinense De Candole	8
Atractylodis Rhizoma	Atractylodes japonica	8
Scutelariae Radix	Scutellaria baicalensis Georgi	4
Citri Pericarpium	Citrus unshiu	4
Magnoliae Cortex	Magnolia officinalis	4
Pineliae Rhizoma	Pine lia pedatisecta Schott	4
Ginseng Radix	Panax ginseng C. A. Mey	2
Glycyrrhizae Radix	Glycyrrhiza uralensis Fischer	2
Zingiberis Rhizoma ecens	Zingiber officinale ROSC.	2
Jujubae Fructus	Zizyphus jujuba Mil. Var. inermis	2
Mume Fructus	Prunus mume siebold et zuccarrini	2
Total		42

2) Cell culture

The RAW 264.7 cells were cultured in a cell incubator at 37℃ and 5% CO₂ using a DMEM supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL). After sufficient proliferation of the RAW 264.7 cells in a 100-mm dish, the culture surface was washed with phosphate-buffered saline (PBS) solution every 3 days. Subsequently, 1 mL of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution was added per 100-mm dish and the cells were incubated at room temperature for 1 minute. The trypsin solution was discarded and the cells were incubated at 37℃ for 5 minutes to detach them for subculturing. The detached cells were mixed with 10 mL of DMEM culture containing 10% FBS and then transferred to a new 100-mm dish for cultivation at a ratio of 1:2.

3) Cytotoxicity

The RAW 264.7 cells were cultured in a 96-well plate at a density of 5 × 10⁴ cells/well for 24 hours. The cells were then treated with Sipyungtang at various concentrations and cultured for an additional 24 hours. The MTT solution (5 mg/mL; 10 μL per well) was added and the cells were incubated for 4 hours. The supernatant was removed and 100 µL of dimethyl sulfoxide was added to each well, followed by measuring the absorbance at 570 nm. Cell viability was calculated using the following formula: cell viability (%) = (absorbance of sample-added group / absorbance of sample-free group) × 100.

4) Inhibition of nitric oxide production

RAW 264.7 cells were seeded in a 96-well plate at a density of 5 × 10⁴ cells/well and cultured for 24 hours in DMEM containing 10% FBS. After removing the medium, LPS was administered at a concentration of 1 µg/mL along with the diluted samples of Sipyungtang in serum-free DMEM and cells were incubated for an additional 24 hours. After adding the Griess reagent to 100 µL of the culture supernatant, the mixture was incubated for 10 minutes and absorbance was measured at 540 nm.

NO inhibition (%) = (absorbance of sample-added group / absorbance of sample-free group) × 100.

5) Measurement of cytokine secretion

The effects of Sipyungtang samples on cytokine secretion were investigated. Cells were cultured on 96-well plates (100 μL per well) at a density of 1 × 10⁵ cells/mL in an incubator for 24 hours. After discarding the medium, cells were washed with 1× PBS solution. Then, cells were treated with LPS at a concentration of 1 µg/mL alone or in combination with various concentrations of Sipyungtang samples and incubated for an additional 24 hours. Then, the cell culture supernatant was collected and antibody-conjugated capture beads that had been pre-prepared in filter plates (96-well) were added to it. Each well of the filter plates containing the captured beads was washed with 150 µL wash buffer. After washing, the detection antibody was added to each well and incubated for 30 minutes. Following this, wells were washed thrice with wash buffer and streptavidin phycoerythrin was added to each well, followed by shaking at room temperature at 300–500 rpm for 30 minutes. After incubation, the wells were washed thrice with wash buffer, and 120 µL of reading buffer was added to each well, followed by shaking for 5 minutes under the same conditions (300–500 rpm at room temperature). Cytokine L-1β, IL-6, and TNF-α levels were compared using a Bio-Plex array reader (Bio-Plex 200).

6) Antioxidant (2,2-diphenyl-1-picrylhydrazyl) assay

The free radical scavenging activity of Sipyungtang samples was measured using the stable radical DPPH, dissolved in ethanol. The samples were dissolved in distilled water and diluted to various concentrations, and 100 µL were used for testing, to which 100 µL, 250-µM DPPH solution was added and the reaction mixture was allowed to stand for 30 minutes. Absorbance was measured at 515 nm after the reaction. As a positive control, 200-µM vitamin C (ascorbic acid) was used.

DPPH radical scavenging activity (%) = (1 − absorbance of sample-added group / absorbance of sample-free group) × 100.

7) Statistical analysis

All experimental results were measured under the same conditions and significance testing was conducted using an independent sample Student’s t-test. Differences with p-values of < 0.05 were considered statistically significant. The results of the DPPH experiments were determined under the same conditions and significance was confirmed by repeating each experiment three times.

1. Cell viability

The viability of RAW 264.7 cells treated with Sipyungtang was assessed. At a concentration of 7.50 mg/mL, the cell viability was 83.98%, as compared with that of the control group without sample treatment. No cytotoxicity was observed at concentrations of < 7.50 mg/mL (Fig. 1).

Figure 1. Cell viability according to the concentration of Sipyungtang in RAW 264.7 cells.

2. Inhibition of nitric oxide production

To evaluate the ability of Sipyungtang to inhibit NO production, cells were treated with the inflammatory mediator LPS, and the decrease in NO production was measured. Results revealed a significant increase in NO production when the RAW 264.7 cells were treated with LPS. When the NO production in the LPS-treated group was designated as 100, the NO levels observed upon the Sipyungtang treatment at various concentrations were as follows: 0.47 mg/mL resulted in 74.10 ± 9.90; 0.94 mg/mL resulted in 46.55 ± 2.32; 1.88 mg/mL resulted in 24.19 ± 3.25; and 3.75 mg/mL resulted in 18.95 ± 1.05. Overall, Sipyungtang treatment resulted in a concentration-dependent decrease in NO production at concentrations ranging from 0.47 mg/mL to 3.75 mg/mL (Fig. 2).

Figure 2. The NO production rate according to the concentration of Sipyungtang in LPS-treated RAW 264.7 cells. The mean of three independent experiments ± standard deviation. NO, nitric oxide; LPS, lipopolysaccharide. *p < 0.05.

3. Effect on interleukin-1β production increase

The RAW 264.7 cells were treated with Sipyungtang along with LPS (1 µg/mL) for 24 hours. When the IL-1β production in the LPS-treated group was designated as 100, the IL-1β production levels obtained with Sipyungtang treatment at various concentrations were as follows: 0.47 mg/mL resulted in 93.24 ± 11.33; 0.94 mg/mL resulted in 78.45 ± 9.90; 1.88 mg/mL resulted in 71.93 ± 5.94; and 3.75 mg/mL resulted in 51.85 ± 5.79. Sipyungtang significantly inhibited IL-1β production at concentrations of > 0.94 mg/mL (Fig. 3).

Figure 3. IL-1β production rate according to the concentration of Sipyungtang in LPS-treated RAW 264.7 cells. The mean of three independent experiments ± standard deviation. IL, interleukin; LPS, lipopolysaccharide. *p < 0.05.

4. Effect on interleukin-6 production increase

The RAW 264.7 cells were treated with Sipyungtang along with LPS (1 µg/mL) for 24 hours. When IL-6 production in the LPS-treated group was designated as 100, the IL-6 production levels upon Sipyungtang treatment at various concentrations were as follows: 0.47 mg/mL resulted in 86.87 ± 5.54; 0.94 mg/mL resulted in 96.53 ± 3.84; 1.88 mg/mL resulted in 68.66 ± 4.66; and 3.75 mg/mL resulted in 57.45 ± 15.56. Sipyungtang significantly inhibited the IL-6 production at concentrations of > 1.88 mg/mL (Fig. 4).

Figure 4. The IL-6 production according to the concentration of Sipyungtang in the LPS-treated RAW 264.7 cells. The mean of three independent experiments ± standard deviation. IL, interleukin; LPS, lipopolysaccharide. *p < 0.05.

5. Effect on tumor necrosis factor-α production increase

RAW 264.7 cells were treated with Sipyungtang along with LPS (1 µg/mL) for 24 hours. When the TNF-α production in the LPS-treated group was designated as 100, the TNF-α production levels upon Sipyungtang treatment at various concentrations were as follows: 0.47 mg/mL resulted in 96.78 ± 5.05; 0.94 mg/mL resulted in 77.70 ± 7.00; 1.88 mg/mL resulted in 65.00 ± 9.67; and 3.75 mg/mL resulted in 59.18 ± 8.65. Sipyungtang markedly inhibited the TNF-α production at concentrations of > 0.94 mg/mL (Fig. 5).

Figure 5. TNF-α production rate according to the concentration of Sipyungtang in the LPS-treated RAW 264.7 cells. The mean of three independent experiments ± standard deviation. TNF, tumor necrosis factor; LPS, lipopolysaccharide. *p < 0.05.

6. Antioxidant properties of Sipyungtang

Sipyungtang exhibited antioxidant effects ranging from 21.29% ± 1.72% to 72.20% ± 1.00% at concentrations from 0.16 to 2.50 mg/mL (Fig. 6). The antioxidant effects observed with Sipyungtang treatments at different concentrations were as follows: 0.16 mg/mL resulted in 21.29% ± 1.72%; 0.31 mg/mL resulted in 34.89% ± 4.61%; 0.62 mg/mL resulted in 56.42% ± 1.92%; 1.25 mg/mL resulted in 72.48% ± 3.46%; 2.5 mg/mL resulted in 72.20% ± 1.00%; and 5 mg/mL resulted in 67.58% ± 2.48% (Fig. 6).

Figure 6. Antioxidant rate according to the concentration of Sipyungtang. DW, distilled water; AA, ascorbic acid; DPPH, 2,2-diphenyl-1-picrylhydrazyl.

Inflammation is an immune response that protects against damage through the secretion of inflammatory mediators such as NO and IL-1β from activated macrophages in response to external stimuli including infections. These mediators interact through various intracellular signaling pathways. However, inflammation can also be a major cause of various diseases. Specifically, macrophages that are abnormally activated due to sustained stimulation may excessively secrete inflammatory mediators, leading to cancer or neurodegenerative diseases [8]. In degenerative diseases, levels of inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, may significantly increase, and the expression of iNOS can be higher than physiological levels. Excessive NO production can disrupt the blood-brain barrier and cause oxidative damage, exacerbating cerebral infarction. Accordingly, the substances that specifically inhibit NO production may help manage inflammatory diseases [9]. Controlling the excessive expression of inflammatory mediators is considered crucial in the prevention and treatment of various inflammation-related diseases. Biological resources with anti-inflammatory effects, particularly plant species, have been studied extensively. Recently, studies have also attempted to utilize insect resources along with plant-based materials.

Sipyungtang inhibits NO production. Previous studies have shown that Magnoliae cortex [10], Ginseng radix [11], Glycyrrhizae radix [12], Mume fructus [13], and Scutellariae radix [14-16] have anti-inflammatory effects. It is an important component of Sipyungtang. It is speculated that Sipyungtang exhibits anti-inflammatory effects by inhibiting the inflammatory cytokines, such as IL-1β and IL-6. IL-1β is an important inflammatory mediator known to regulate various immune responses, including assisting in the phagocytic activity of macrophages. It is produced by macrophages, neutrophils, as well as epithelial and endothelial cells and is induced by bacterial products, such as LPS or other cytokines such as TNF-α. Overproduction or prolonged secretion can lead to systemic invasive effects, causing fever, inflammation, tissue destruction, and shock [4]. In the present study, the Sipyungtang extract significantly inhibited the production of IL-1β at concentrations of 0.94, 1.88, and 3.75 mg/mL.

IL-6 is activated by the antigens IL-4 and IL-5, playing a crucial role in the final stage of differentiation of proliferating B lymphocytes to produce large quantities of secretory antibodies. It also induces the proliferation and differentiation of T lymphocytes, promoting protein synthesis and differentiation of neurocytes by proliferating blood liver and plasma cells, thereby exhibiting various biological activities [8]. In the present study, we treated RAW 264.7 cells with Sipyungtang for 24 hours, and found that this treatment markedly inhibited the increase in IL-6 production induced by LPS at concentrations of 1.88 and 3.75 mg/mL.

TNF-α is a cytokine secreted by various cells, including macrophages, monocytes, mast cells, neutrophils, astrocytes, and fibroblasts, which are activated by LPS. TNF-α is involved in cellular immune responses, cellular growth, and cell differentiation. It plays a crucial role in the initiation and maintenance of inflammation in many autoimmune diseases. In tumor cells, TNF-α exerts cytotoxic effects, whereas, in inflammatory cells, it has an inflammatory activity similar to IL-1, which is to regulate cell proliferation and differentiation. Additionally, the concentration of TNF-α is elevated in the serum and brain tissue of patients with stroke, as well as Parkinson’s, and Alzheimer’s diseases. This increase in TNF-α levels may play an important role in the pathogenesis of neuronal cell death [4]. In this study, the water extract of Sipyungtang significantly inhibited the production of TNF-α when applied at concentrations of 0.94, 1.88, and 3.75 mg/mL.

ROS cause oxidative stress in the body and react with lipids, proteins, and other molecules. ROS are unstable and possess strong oxidative power, thereby damaging biological tissues. They are also associated with aging and diseases. The antioxidant capacity refers to the ability to gain electrons, which reduces the oxidation state [17]. To measure the sample’s antioxidant capacity, a DPPH assay was conducted, revealing that Sipyungtang exhibited antioxidant effects of > 20% at concentrations ranging from 0.15 mg/mL to 2.50 mg/mL. The antioxidant efficacy showed a tendency to decrease at concentrations of > 5 mg/mL, and decreased as the concentration increased, exhibiting negative results at 40 mg/mL. This is presumed to be due to the bias caused by the color of the sample affecting the absorbance.

In the present study, the Sipyungtang extract markedly inhibited the increased production of various inflammatory mediators induced by LPS without exhibiting cytotoxicity. These results suggest that Sipyungtang could be utilized to alleviate musculoskeletal and neurological disorders caused by inflammatory mediators and that its antioxidant properties may also be beneficial owing to its anti-aging effects and in skincare. Further studies should be conducted to expand the clinical applications of Sipyungtang. Sipyungtang can be prepared through pharmacupuncture. Pharmacupuncture has a wide range of indications, has a rapidtherapeutic effect, and can be used for patients who have difficulty taking medication [18]. If Sipyungtang is utilized in pharmacupuncture, it can be used for symptoms such as high fever, chills, vomiting, cold sweat, and muscle pain, per previous reports. Our results can be used to support the reported efficacy of Sipyungtang Thus, further studies on Sipyungtang are necessary.

The present study experimentally confirmed the anti-inflammatory and antioxidant effects of Sipyungtang, serving as a foundational study for its potential applications in treating inflammatory diseases. To this end, its impact on cell viability, NO production, and levels of inflammatory-related cytokines, such as IL-1β, IL-6, and TNF-α, as well as its antioxidant effects, were examined.

As a result, > 80% of the cells survived even after treatment with Sipyungtang at a concentration of 7.5 mg/mL. Additionally, at concentrations of > 0.94 mg/mL, the NO production was significantly inhibited. The levels of inflammatory-related cytokines were mostly reduced in a concentration-dependent manner by Sipyungtang, with IL-1β showing a significant reduction in expression at concentrations of < 0.94 mg/mL, IL-6 at concentrations of < 1.88 mg/mL, and TNF-α at concentrations of < 0.94 mg/mL. Sipyungtang demonstrated an efficacy of >20% as an antioxidant at concentrations of > 0.15 mg/mL.

These findings suggest that Sipyungtang may have potential applications in the treatment of various pathologies, including musculoskeletal disorders related to inflammation and neurological diseases associated with excessive inflammatory responses, such as arthritis and pneumonia [19]. Particularly, if Sipyungtang is utilized in pharmacopuncture, the range of its clinical application is expected to further expand and its efficacy will further improve, the present study provides fundamental evidence of the efficacy of Sipyungtang. However, further studies are necessary to expand the clinical applications of Sipyungtang.

Conceptualization: NYJ. Data curation: NYJ. Formal analysis: NYJ. Funding acquisition: NYJ. Investigation: NYJ. Supervision: NYJ, CKL. Validation: EYL, JDR, CKL. Writing – original draft: NYJ. Writing – review & editing: All authors.

The authors have no conflicts of interest to declare.

This study was supported by the Semyung University Research Grant of 2022.

The present work was an experimental study and did not involve any human or animal experiments.