Inflammation is a natural biological defense mechanism triggered by physical wound or infection caused by microorganisms. Inflammatory reactions remove pathogenic factors and restore damaged tissues to their normal structure and function ^(1). Allergic inflammatory reactions are steadily increasing worldwide, and mast cells are the main cells involved in various allergic reactions ⁽²⁾. Inhibition of mast cell activation and degranulation is important for treating allergic inflammatory reactions ⁽³⁾. Therefore, research is needed on the mechanism that regulates mast cell activity to treat allergic inflammation. In response to various stimuli, mast cells induce various mediators, including histamine, interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α ⁽⁴⁾. Increased levels of mediators may be of critical importance in the development of allergic inflammatory disorders ⁽⁵⁾. Attenuating histamine production by mast cells may help treat allergic inflammation ⁽⁶⁾. Thus, suppressing mast cell-derived inflammatory mediators could contribute to the development of a promising therapeutic strategy against allergic inflammatory diseases ⁽⁷⁾.

The nuclear factor-κB (NF-κB) is a key regulator of immune responses, cell death, and allergic inflammation ⁽⁸⁾. During inflammatory progress, the IκBkinase complex is phosphorylated and degraded, leading to NF-κB dissociation from IκB-α and its translocation to the nucleus, where it activates gene transcription of inflammatory ⁽⁹⁾. Increased NF-κB activity, associated with elevated IL-6 and IL-8 levels, plays a role in skin inflammation ⁽¹⁰⁾. Caspase-1, a member of the caspase family, is also involved in inflammatory reactions ⁽¹¹⁾. Its activation induces the inflammatory response by increasing the levels of inflammatory mediators and inflammatory cell recruitment. Previous studies revealed that caspase-1 is a major modulator of sepsis-induced inflammatory tissue injury. Additionally, it was reported that serum caspase-1 was elevated in patients with inflammatory disorders ⁽¹²⁾. Caspase-1 deficiency was reported to attenuate the allergic inflammation in mice ⁽¹³⁾. These findings suggest that targeting the NF-κB pathway/caspase-1 could be an effective approach for treating inflammatory diseases.

Natural products are valuable sources of drugs due to diverse biological properties. Angelica gigas Nakai (AG) has traditionally used to treat hepatic steatosis, hyperlipidemia, and hypercholesterolemia ^(14,15). Recent studies have reported that AG enhances immunity by modulating MAPK/NF-κB pathways and inhibits cellular injury by AMPK pathway ^(16,17). Additionally, AG suppressed the expressions of inflammatory mediators in RAW 264.7 cells ⁽¹⁸⁾. However, accurate mechanisms of action of AG on mast cell-mediated inflammatory responses is not well understood. To explore the anti-inflammatory mechanisms of AG, we evaluated the effect of AG on the production of inflammatory cytokines, histamine as well as the activation of NF-κB and caspase-1 in activated human mast cells-1 (HMC-1).

1. Reagents

Gallic acid, potassium persulfate, Folin-Denis, avidin peroxidase (AP), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazy (DPPH), and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma-Aldrich. The MTS assay kit was obtained from Promega. The enzyme-linked immunosorbent assay (ELISA) kits for human IL-6/IL-8/TNF-α were acquired from BD Biosciences. Caspase-1 assay kit was acquired from R&D System Inc. The bicinchoninic acid (BCA) kit and enhanced chemiluminescence (ECL) reagent were procured from Thermo Fisher Scientific Inc. NF-κB and IκB-α antibodies (Abs) were purchased from Santa Cruz Biotechnology.

2. Preparation of AG extract

AG was sourced from Daehak Oriental Drugstore and authenticated by Dr. Noh-Yil Myung (Wonkwang Digital University). Dried AG (100 g) was pulverized into a fine powder decocted in distilled water (1 L) for 3 hours, and concentrated under vacuum rotary evaporator. The extract was filtered, freeze dried (FDU-2000; Eyela) and stored at –20°C (yield, 7.67%). The extract was dissolved in distilled water and filtered via a syringe filter.

3. DPPH radical scavenging assay

The DPPH-radical scavenging ability of AG was measured according to by the method described by Naczk and Shahidi ⁽¹⁹⁾. 100 μL of the AG (0.01, 0.1, and 1 mg/mL) was mixed with 100 μL of DPPH solution in ethanol at concentration of 0.45 mM, and reacted in the dark for 30 minutes. Subsequently, the absorbance was assessed at 517 nm, and reduction was expressed as a percentage.

4. ABTS⁺ radical scavenging assay

The ABTS cation radical scavenging activity was measured following the method described by Re et al ⁽²⁰⁾. The test solution was prepared by adding 7 mM ABTS and 2.45 mM potassium persulfate to distilled water, and incubating at room temperature for 16 hours to generate ABTS cations. The solutions were diluted to obtain the absorbance value of 0.70 ± 0.03 at 734 nm. Next, 100 μL of AG (0.01, 0.1, and 1 mg/mL) was added to 100 μL of the ABTS⁺ and absorbance was measured after 6 minutes. The reduction in absorbance compared to the absorbance of the negative control group (2.45 mM potassium persulfate buffer) was expressed as a percentage.

5. Total polyphenol content

The amount of total polyphenol was evaluated using a Folin-Denis reagent. Briefly, 1 mL of the AG extract was added to each tube, followed by Folin-Denis reagent (1 mL) was added and allowed to react for 5 minutes. Subsequently, 5 mL of 10% Na2CO3 was added, mixed and reacted in the dark for 30 minutes. Absorbance was evaluated, and the total polyphenol content was calculated using a standard calibration curve prepared using garlic acid as the standard.

6. Cell culture

HMC-1 were cultured in Dulbecco’s Modified Eagle’s medium containing 1% penicillin-streptomycin and 10% fetal bovine serum at 37°C in 5% CO₂ atmosphere. HMC-1 was activated with 50 nM of PMA plus 1 mg/mL of calcium Ionophore A23187 (PMACI).

7. MTS assay

To assess cell viability at various AG concentrations, the MTS assay was performed using a kit (Promega). Cells were incubated with AG (0.01, 0.1, and 1 mg/mL) for 24 hours, followed by the addition of MTS. After 2 hours of incubation, the absorbance was assessed at 490 nm using a microplate reader.

8. Cytokine assay

The levels of human IL-6, IL-8, and TNF-α was assessed using modified ELISA. Briefly, plates were coated with monoclonal Abs of anti-human IL-6, IL-8, and TNF-α at 4°C for 24 hours. After washing, AG samples or standard recombinant proteins of IL-6, IL-8, and TNF-α were added to each well and reacted at 37°C for 2 hours. After washes, the plates were incubated with biotinylated anti-human IL-6, IL-8, and TNF-α at 37°C for 1 hour. After further washing, AP and substrates were sequentially mixed and the reaction stopped. The amount of IL-6, IL-8, and TNF-α at 405 nm were analyzed using an ELISA reader.

9. Histamine assay

The amount of histamine released from mast cells was determined using a histamine assay kits (Neogen), following the manufacturer’s protocol.

10. Western blot analysis

Cells were washed, and cytosol/nuclear lysates were prepared using nuclear extraction reagent kit (Pierce Thermo Scientific) according to the manufacturer’s instructions. After protein quantification using BCA assay kit, samples were added with sample buffer, separated by gel electrophoresis, and transferred onto membranes. Membrane was blocked by 5% skimmed milk for 1 hour and subsequently reacted with IκB-α and NF-κB (p65) primary Abs at 4°C for 24 hours. After washing, membranes were reacted with secondary Abs for 2 hours. After further washing, proteins were visualized using an ECL detection kit.

11. Caspase-1 activity assay

Caspase-1 ability was assessed using colorimetric assay kit, following the manufacturer’s protocols. After protein quantification, the sample was reacted with 50 μL reaction buffer and 5 μL caspase-1 substrate at 37°C for 2 hours. The absorbance was evaluated at 405 nm using a microplate reader.

12. Statistical analysis

Results are shown as the mean ± standard deviation of three experiments. Statistical analyses were conducted using an independent t-tests and one-way ANOVA with a Tukey post hoc test. The significance levels between experimental groups were set at P < 0.05.

1. Anti-oxidant activity of AG

The DPPH and ABTS radical scavenging assay are widely used to evaluate the antioxidant activity. To evaluate the antioxidant activity of AG, both DPPH radical scavenging activity and ABTS⁺ radical scavenging activity were assessed. The DPPH and ABTS radical scavenging abilities of AG at various concentrations are shown in Fig. 1A, 1B. AG showed dose-dependent DPPH and ABTS⁺ radical scavenging activities. Specifically, DPPH radical scavenging capacities of AG were approximately 25.8% (0.01 mg/mL), 46.2% (0.1 mg/mL), and 77.6% (1 mg/mL), respectively. In the evaluation of ABTS⁺ radical scavenging ability, AG demonstrated efficacies of 20.5% (0.01 mg/mL), 36.3% (0.1 mg/mL), and 69.8% (1 mg/mL) respectively (Fig. 1B).

Fig. 1. Antioxidant effects of Angelica gigas Nakai (AG). Antioxidant activity was evaluated by (A) 2,2-diphenyl-1-picrylhydrazy (DPPH) free radical scavenging assay, (B) 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)⁺ cation radical scavenging assay, (C) Total polyphenol amounts. The antioxidant properties of AG were prepared as described in the MATERIALS AND METHODS section. Results are shown the mean ± standard deviation of experiments. ^#P < 0.05, significantly different from control (vehicle).

Phenolic compounds are secondary metabolites widely distributed in plants ⁽²¹⁾. They have various physiologically active function, such as antioxidant, antibacterial, and anti-inflammatory actions, and are used as functional ingredient ⁽²²⁾. The anti-oxidant activity of natural product is correlated with polyphenol content. In this study, to determine the total polyphenol content of AG, we applied the Folin-Denis method. As a result, the total polyphenol content of AG (1 mg/mL) was 69.2 mg GAE/g (Fig. 1C). Thus, we verified that AG extract has antioxidant properties.

2. Effect of AG on cell viability and histamine secretion in activated-HMC-1 cells

To explore the effect of AG on cell viability, we used an MTS assay. Cells were incubated with or without AG (0.01, 0.1, and 1 mg/mL) for 24 hours, and the cytotoxic effects of AG was evaluated. The results indicated that cell viability was consistent across various concentrations of AG, suggesting that AG was non-cytotoxic at these experimental levels (Fig. 2A).

Fig. 2. Effects of Angelica gigas Nakai (AG) on cell viability and histamine release in activated human mast cells-1. (A) Cells (3 × 10⁵ cells/well) were treated with various concentration of AG (0.01, 0.1, and 1 mg/mL) for 24 hours and cell viability was measured using the MTS assay. (B) Cells were treated with various concentration of AG (0.01, 0.1, and 1 mg/mL) for 1 hour before activation with PMA plus 1 mg/mL of calcium Ionophore A23187 (PMACI) for 2 hours. Dexamethasone (Dex, 100 nM) was used as positive control. The histamine concentration was assessed using histamine assay kits. Results are shown the mean ± standard deviation of experiments. ^#P < 0.05, significantly different from control (vehicle); *P < 0.05, significantly different from PMACI-treatments.

Mast cell-derived histamine initiates allergic inflammation and its appropriate regulation may help treat allergic inflammation ⁽⁶⁾. Thus, we determined that AG inhibited effects of AG on PMACI-enhanced the histamine release from mast cells. The cells were incubated with AG (0.01, 0.1, and 1 mg/mL) and activated with PMACI for 3 hours. Histamine amount derived from mast cell was measured using histamine assay kit. The results showed that PMACI increase histamine release compared to the normal group, but AG suppressed the PMACI-enhanced the histamine levels in a concentration-dependent manner (Fig. 2B). The maximal inhibition of histamine release by AG (1 mg/mL) was approximately 36.4% (P < 0.05). In this study, dexamethasone was used as positive control.

3. Effect of AG on inflammatory cytokines levels in activated-HMC-1 cells

Attenuation of inflammatory cytokine level is the therapeutic strategies against inflammatory disease ⁽¹⁾. Therefore, we investigated the inhibitory effect of AG on secretion of IL-6, IL-8, and TNF-α in activated HMC-1 cells. Cells were incubated without AG (0.01, 0.1, and 1 mg/mL) and activated by PMACI for 8 hours. The results showed that PMACI significantly increased the IL-6, IL-8, and TNF-α levels compared to the normal group. However, AG attenuated the PMACI-enhanced the IL-6, IL-8, and TNF-α secretion in a concentrations-dependent manner in activated-HMC-1 cells (Fig. 3). The maximal inhibition of IL-6, IL-8, and TNF-α amount by AG (1 mg/mL) was approximately 28.6% (P < 0.05), 32.5% (P < 0.05), and 41.4% (P < 0.05), respectively.

Fig. 3. Effects of Angelica gigas Nakai (AG) on interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α levels in activated human mast cells-1. Cells were incubated with various concentration of AG (0.01, 0.1, and 1 mg/mL) for 1 hour before activation with PMA plus 1 mg/mL of calcium Ionophore A23187 (PMACI) for 8 hours. (A-C) The IL-6, IL-8, and TNF-α amounts were measured by ELISA kits. Dexamethasone (Dex, 100 nM) was used as positive control. Results are shown the mean ± standard deviation of experiments. ^#P < 0.05, significantly different from control; *P < 0.05, significantly different from PMACI-treatments.

4. Effect of AG on NF-κB pathway in activated-HMC-1 cells

Since down-regulation of NF-κB has been related to anti-inflammatory reaction ⁽¹⁰⁾, we predicted that NF-κB pathway regulation might be the molecular mechanism of AG. Cells were incubated with or without AG and subsequently activated with PMACI for 2 hours. We investigated the effect of AG on IκB-α degradation and NF-κB translocation using a western blot analysis. As shown in Fig. 4, it was observed that the movement of NF-κB from the cytoplasm into the nucleus was significantly inhibited by AG treatment, and at the same time, the degradation of IκB-α in the cytoplasm was also significantly recovered in activated-HMC-1 cells.

Fig. 4. Effect of Angelica gigas Nakai (AG) on (A) IκB‐α degradation and (B) nuclear factor-κB (NF-κB) activation in activated human mast cells-1. Cells (7 × 106 cells/well) were incubated with various concentration of AG (0.1 and 1 mg/mL) for 1 hour and then stimulated by PMA plus 1 mg/mL of calcium Ionophore A23187 (PMACI) for 2 hours. Cells were harvest and cytosol and nuclear proteins were isolated. Cytosolic extracts for IκB‐α expression and nuclear extracts for NF-κB were evaluated by Western blot analysis. Dexamethasone (Dex, 100 nM) was used as positive control. Results are shown the mean ± standard deviation of experiments. ^#P < 0.05, significantly different from control; *P < 0.05, significantly different from PMACI-treatment.

5. Effect of AG on caspase-1 activation in activated-HMC-1 cells

Caspase-1 activation is associated with an inflammatory response through increase in the expression of inflammatory genes in mast cell ⁽¹¹⁾. To determine the molecular mechanism underlying the AG’s involvement in mast cell-mediated inflammatory responses, we investigated the effects of AG on caspase-1 activity. We found that the PMACI–enhanced caspase-1 activity was significantly reduced by AG in a dose-dependent manner (Fig. 5). The maximal inhibition of caspase-1 activity by AG (1 mg/mL) was approximately 38.9% (P < 0.05).

Fig. 5. Effect of Angelica gigas Nakai (AG) on caspase-1 activity in activated human mast cells-1 (HMC-1). Cells (5 × 106 cells/well) were pretreated with AG (0.01, 0.1, and 1 mg/mL) for 1 hour before activation by PMA plus 1 mg/mL of calcium Ionophore A23187 (PMACI) for 3 hours. The caspase-1 enzymatic activity was assessed using a colorimetric assay kit. Dex, dexamethasone. The values are represented as the mean ± standard deviation of independent experiments. ^#P < 0.05, significantly different from control; *P < 0.05, significantly different from PMACI-treatment.

Herb medicines are used globally to promote health, though precise mechanisms of action remain to be fully elucidated. Continued research is essential to identify novel therapeutic with immunomodulatory property. AG has traditionally been used to treat for various disease. AG has been reported to ameliorate diabetes, inhibit platelet aggregation, improve liver disease, and exert anticancer effects. This study demonstrated the antioxidant activity and anti-inflammatory mechanism of AG, validating its potential as a functional material. Our findings indicate that AG has robust free-radical scavenging properties. AG inhibited the PMACI-enhanced histamine as well as IL-6, IL-8, and TNF-α production in activated HMC-1cells. Additionally, AG exhibited inhibitory mechanism on mast cell-mediated inflammation could be resulting through the suppression of NF-κB/caspase-1 activation.

Inflammatory reactions aim to removes pathogenic factors and restores damaged tissues to their normal structure and function. Oxidative stress and inflammation are closely associated, making, substances with antioxidant or anti-inflammatory effects play an important role in the prevention of various chronic inflammatory diseases ⁽²²⁾. Oxidative stress can lead to cell damage, accelerating skin aging and inflammation ⁽²³⁾. Antioxidants play a crucial role in human body by reducing oxidative processes and the harmful effects of free radicals ⁽²⁴⁾. Natural antioxidants therefore, have potential as therapeutic agents against many diseases. To investigate the anti-oxidative activity of AG, we assessed its effects of different concentrations of AG on DPPH and ABTS⁺ radical scavenging activities. AG exhibited strong scavenging effects on both DPPH and ABTS⁺ free radicals. The total polyphenol content of AG (1 mg/mL) was 69.2 mg GAE/mg (Fig. 1C). Therefore, we concluded that AG possesses potent antioxidant properties.

Mast cell activation is critical in allergic inflammation, including psoriasis, and atopic dermatitis ⁽²⁵⁾. In chronic inflammatory response, mast cell generates a variety of mediators, including histamine, TNF-α, tryptase, IL-8, leading to tissue damage ^(4,5). Increased in these factors may be critical for the development of allergic inflammatory disorders ⁽²⁶⁾. Histamine plays a critical role in the pathogenesis of allergic inflammatory diseases by inducing leukotrienes, cytokines, and chemokines ⁽²⁷⁾. Mast cell-derived IL-8 is a chemotactic factor that activates inflammatory response. Moreover TNF-α secreted from mast cells, accumulates white blood cells, resulting in allergic inflammation ⁽²⁸⁾. Therefore, inhibiting excess inflammatory mediators is a valuable therapeutic strategy against allergic inflammatory disease. In this study, AG shown that to decrease the secretion of histamine, IL-6, IL-8, and TNF-α in a concentration-dependent manner in activated-HMC-1 cells. Especially, inhibitory rates of IL-6, IL-8, and TNF-α by AG (1 mg/mL) were approximately 28.6% (P < 0.05), 32.5% (P < 0.05), and 41.4% (P < 0.05), respectively (Fig. 3). This suggests that AG exerts its anti-inflammatory effect by suppressing the production of mast cell-derived inflammatory mediators.

Emerging evidence has shown that increases of inflammation are associated with NF-κB pathway ⁽²⁹⁾. Increased NF-κB activity, associated with elevated IL-6 and IL-8 levels, plays a role in skin inflammation ⁽¹⁰⁾. Caspase-1 exerts its biological effects by activating inflammatory pathways. Caspase-1 deficiency inhibit inflammatory reaction in an allergic rhinitis experiment model ⁽¹¹⁾. To identify the anti-inflammatory mechanisms of AG, we investigated whether AG could attenuate PMACI-induced NF-κB/caspase-1 activation in HMC-1 cells. Our study found that the movement of NF-κB from the cytoplasm into the nucleus was significantly inhibited by AG treatment, and at the same time, the degradation of IκB-α in the cytoplasm was also significantly recovered in activated-HMC-1 Additionally, AG ameliorated the PMACI-induced caspase-1 activation in a dose-dependent manner. Taken together, our results indicated that the regulatory mechanism of AG on mast cell-mediated inflammation likely results from the suppression of NF-κB/caspase-1 activation. AG is comprised of several bioactive components such as decursin, decursinol, and nodakenin ⁽³⁰⁾. Decursin, major active component, has anti-oxidative activity by regulation of AMPK ⁽¹⁷⁾. Additionally, decursin attenuates inflammatory mediators through blocking NF-κB activation in macrophages ⁽³¹⁾. Although AG attenuated NF-κB/caspase-1 activation in mast cell-mediated inflammation, the effect of decursin on NF-κB/caspase-1 activation was not elucidated in this study. Therefore, further study is necessary to clarify the role of active component in mast cell-mediated allergic inflammation.

In conclusion, AG exhibits anti-oxidative and the anti-inflammatory activities, attributable to the scavenging of DPPH and ABTS⁺ free radicals and amelioration of mast cell-mediated inflammatory mediators in activated HMC-1 cells. Its anti-inflammatory mechanisms are attributed to down-regulation the NF-κB pathway and caspase-1 activation. Our novel results indicated evidence that AG is a potential as a therapeutic agent against allergic inflammation.

Following are results of a study on the Leaders in industry-university Cooperation 3.0 Project, supported by the Ministry of Education and National Research Foundation of Korea and was supported by Basic Science Research Program (NRF-2021R1F1A1047814).

No potential conflict of interest relevant to this article was reported.

Following are results of a study on the Leaders in industry-university Cooperation 3.0 Project, supported by the Ministry of Education and National Research Foundation of Korea and was supported by Basic Science Research Program (NRF-2021R1F1A1047814).