Anti-Inflammatory Food in Asthma Prepared from Combination of Raphanus sativus L., Allium hookeri, Acanthopanax sessiliflorum, and Dendropanax morbiferus Extracts via Bioassay-Guided Selection

Asthma is a highly prevalent inflammatory disease of the respiratory airways and an increasing health risk worldwide. Hence, finding new strategies to control or attenuate this condition is necessary. This study suggests nutraceuticals that are a combination of herbal plant extracts prepared from Acanthopanax sessiliflorum (AS), Codonopsis lanceolate (CL), Dendropanax morbiferus (DM), Allium hookeri (AH), and Raphanus sativus L. (RS) that can improve immunomodulatory ability through the detoxification and diuresis of air pollutants. Herbal parts (AH whole plant, RS and CL roots, AS and DM stems, and DM leaves) were selected, and four types of mixtures using plant extracts were prepared. Among these mixtures, M2 and M4 exhibited antioxidant activities in potent 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 1,1-diphenyl-β-picrylhydrazine (DPPH) radical assays. Moreover, M4 exhibited a marked increase in glutathione S-transferase (GST) activity and significantly inhibited the inflammatory mediator, nitric oxide (NO) and proinflammatory cytokines, interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α generation. Furthermore, M4 exhibited the strongest antioxidant, hepatoprotective, and anti-inflammatory effects and was selected to prepare the product. Before manufacturing the product, we determined that the active mixture, M4, inhibited gene expression and generation of proinflammatory cytokines IL-1β, IL-6 and TNF-α in ovalbumin (OVA)-, lipopolysaccharide (LPS)-, and particulate matter (PM)-induced asthmatic rat models. The granular product (GP) was manufactured using M4 along with additives, i.e., lactose, oligosaccharide, stevioside extract, and nutmeg seed essential oils (flavor masking), in a ratio of 1:4 using a granulation machine, dried and ultimately packaged. The GP inhibited the generation of proinflammatory cytokines IL-1β, IL-6 and TNF-α in OVA-, LPS- and PM-induced asthmatic rat models. These results suggest that GP prepared from a combination of herbal plants (AS, CL, DM, AH and RS) is a potent functional food with anti-inflammatory activity that can be used to treat asthma caused by ambient air pollutants.


Introduction
Inflammation is a complicated physiological response of body tissues to detrimental stimuli and may result in inflammatory disorders such as allergies, asthma, autoimmune diseases, coeliac disease, hepatitis, inflammatory bowel disease, and reperfusion injury [1][2][3][4]. Asthma, a chronic condition of the respiratoty airway, causes airway disturbance and bronchial hyper-responsiveness, accompanied by symptoms involving coughing, wheezing, shortness of breath, chest tightness, and pain [5][6][7][8]. Allergic asthma is prolonged lung inflammation caused by the permeation of eosinophils, leukocytes and neutrophils

Product Preparation
The powder prepared from RS and CL roots, AH whole plant, AS and DM stems and DM leaves was added to 10 volumes of water, mixed, and extracted under reflux at 100 • C for 5 h. The crude extract was filtered using filter paper (Whatman, Merck, Darmstadt, Germany), evaporated in vacuo (N-1300EW, EYELA, Tokyo, Japan) and freeze-dried. The material was prepared by mixing freeze-dried powder according to the ratio M4, which has potent anti-inflammatory activity. Granular product (GP) was manufactured by mixing the material with additives, i.e., lactose, oligosaccharide, stevioside extract and nutmeg seed essential oil, in a ratio of 1:4 using a granulation machine. Finally, the granules were dried and packaged.

In Vitro Antioxidant Activity
Antioxidant activity of the samples was examined using 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 1,1-diphenyl-β-picrylhydrazine (DPPH) radical assays. The ABTS radical assay was performed in accordance with the ABTS cation decolorization assay, with slight modifications [30]. ABTS (7 mM; Sigma-Aldrich, St. Louis, MI, USA) was mixed with 2.45 mM potassium persulfate and reacted for 16 h at 4 • C. Typically, sample (50 µL) and ABTS solution (100 µL) were mixed and allowed to react at 23 • C for 20 min, and absorbance was measured at 732 nm using a microplate spectrophotometer (Epoch, Biotek Instruments, Inc., Winooski, VT, USA). The DPPH radical assay was performed in accordance with a previously reported, slightly modified method [31]. Briefly, an equal volume of DPPH solution (0.2 mM) and the samples were mixed in a 96 well plate for 5 s. The mixture was reacted for 30 min in the dark, and absorbance was measured at 517 nm. Garlic acid was used as the positive control of ABTS and DPPH radical assays.

Hepatoprotective Effects
The hepatoprotective effect of all extracts was investigated by measuring glutathione Stransferase (GST) activity using a spectrophotometric assay [32]. Hepa-lclc7 cells (1 × 10 4 cells/well) were treated in a serum-free medium for 48 h, lysed with 2% Triton X-100 and mixed for 10 min. The lysate was mixed with 100 µL of 2.5 mM GSH in 0.1 M potassium phosphate buffer and 1 mM 1-chloro-2,4-dinitrobenzene (DCDNB) for 1 min, and absorbance was measured at 405 nm for 5 min. GST activity was calculated as slope/min/mg protein and expressed as the ratio of GST activity of the cells treated with extracts to that of the control.

Anti-Inflammatory Effects
Nitric Oxide Production Assay RAW 264.7 cells (1 × 10 5 cells/well) were treated in a serum-free medium for 1 h and then stimulated with lipopolysaccharide (LPS; 1 µg/mL). After 16 h, the supernatant and equal volumes of Griess reagent A (0.1% (w/v) N-(1-naphthyl) ethylenediamine in DW) and B (1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid) were added and incubated at room temperature (RT) for 15 min, and the absorbance was measured at 540 nm. Nitric oxide (NO) production was measured using a serum-free culture medium as a control.

Cytokines Generation Assay
Cytokine generation in LPS-induced RAW 264.7 cells was measured using enzymelinked immunosorbent assay (ELISA) kits (BD OptEIATM, San Diego, CA, USA) according to the manufacturer's instructions.

Animals
Male Sprague-Dawley (SD) rats (160-190 g each) were used to determine the antiinflammatory effects of M4 and products prepared with M4. The animals were housed (3-5 per cage) in a laminar-air-flow cabinet maintained at 23 ± 1.5 • C, a humidity of 55 ± 15%, and lighting that followed a 12 h on/12 h off cycle. The rats were provided a laboratory diet and tap water ad libitum during the experimental period. All animals were approved by the Institutional Animal Care and Use Committee of Dongshin University (No. DSU2016-10-01).
were sensitized twice with an intraperitoneal (i.p.) injection of OVA (1 mg/kg), emulsified in aluminum potassium sulfate in phosphate-buffered saline (PBS), and LPS (5 mg/kg) every five days. Nebulized 2% PM (Standard Reference Matter, NIST, Gaithersburg, MD, USA), 3% OVA and 5% LPS in PBS were administered by inhalation for 15 min at 4 h intervals for a total of four times on days 6, 7, 9 and 11 after i.p. injection. The gene levels of OVA-, LPS-and PM-induced cytokine generation were assessed using real-time RT-PCR with specific primers (Table 1). Total RNA was extracted from OVA-, LPS-and PM-treated lung tissue using an extraction kit (TRIzol) according to the manufacturer's protocol. For complementary DNA (cDNA) synthesis, 5 μg of total RNA was reverse-transcribed using oligo dT, (2.5 μL) and RT premix (Bioneer, Daejeon, Korea). cDNA samples (50 μL) were amplified using RT-PCR with specific primers (Table 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. RT-PCR conditions were as follows: pre-denaturation at 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 55-60 °C for 30 s and extension at 72 °C for 1 min.

Cytokines Generation Assay
Proinflammatory cytokines, namely interleukin (IL)-1β, IL-6, and TNF-α produced by OVA-, LPS-and PM-stimulated rats were determined using an ELISA kit (BIOMATIK, Ontario, Canada) and Invitrogen (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Cytokine generation in LPS-induced RAW 264.7 cells was measured using ELISA kits (Invitrogen; Thermo Scientific) according to the manufacturer's instructions.

Statistical Analysis
Comparisons were made through one-way analysis of variance (ANOVA) and Duncan's multiple range test using IBM SPSS Statistics 26 (Chicago, IL, USA). Values are reported as the mean ± standard error (SE), and statistical significance was set at p < 0.05.

Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
The gene levels of OVA-, LPS-and PM-induced cytokine generation were assessed using real-time RT-PCR with specific primers (Table 1). Total RNA was extracted from OVA-, LPS-and PM-treated lung tissue using an extraction kit (TRIzol) according to the manufacturer's protocol. For complementary DNA (cDNA) synthesis, 5 µg of total RNA was reverse-transcribed using oligo dT, (2.5 µL) and RT premix (Bioneer, Daejeon, Korea). cDNA samples (50 µL) were amplified using RT-PCR with specific primers (Table 1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. RT-PCR conditions were as follows: pre-denaturation at 94 • C for 5 min, 30 cycles of denaturation at 94 • C for 30 s, annealing at 55-60 • C for 30 s and extension at 72 • C for 1 min. Table 1. Primer sequence used in this study.

Primer
Forward Reverse

Cytokines Generation Assay
Proinflammatory cytokines, namely interleukin (IL)-1β, IL-6, and TNF-α produced by OVA-, LPS-and PM-stimulated rats were determined using an ELISA kit (BIOMATIK, Ontario, Canada) and Invitrogen (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Cytokine generation in LPS-induced RAW 264.7 cells was measured using ELISA kits (Invitrogen; Thermo Scientific) according to the manufacturer's instructions.

Statistical Analysis
Comparisons were made through one-way analysis of variance (ANOVA) and Duncan's multiple range test using IBM SPSS Statistics 26 (Chicago, IL, USA). Values are reported as the mean ± standard error (SE), and statistical significance was set at p < 0.05.

In Vitro Antioxidant and Hepatoprotective Effects of Mixture
Herbal plants, namely RS, AH, AS, DM and CL, were selected for the preparation of functional foods to treat inflammatory disorders caused by asthma. As shown in Table 2, these plants were extracted with water and mixed in various ratios. Antioxidant activity has been reported to affect various bioactivities such as skin whitening and anti-inflammation [33]. ABTS and DPPH radical scavenging actions offer redoxfunctionalized proton ions for unstable free radicals and play a critical role in stabilizing detrimental free radicals; these assays were conducted to assess the antioxidant activity of each mixture [31,32]. The IC 50 values of M1, M2, M3 and M4 for ABTS radical scavenging activity were 214, 61, 150 and 57 (µg/mL), respectively ( Figure 2A). M2, M3 and M4 showed strong ABTS radical scavenging activity (Figure 2A). The IC 50 values of M1, M2, M3 and M4 for DPPH radical scavenging activity were 321, 171, 209 and 197 µg/mL, respectively ( Figure 2A). Among the mixture, M2 and M4 exhibited potent DPPH radical scavenging activity (Figure 2A). To examine the hepatoprotective effects, Hepa-lc1c7 cells were treated with a mixture of extracts at 50, 100 and 200 µg/mL, and GST activity was investigated. As shown in Figure 2B, the GST activity of M4 was 4.09 ± 0.58, which was 1.78 times higher than that of M1 (2.77 ± 0.44).

In Vitro Antioxidant and Hepatoprotective Effects of Mixture
Herbal plants, namely RS, AH, AS, DM and CL, were selected for the preparation of functional foods to treat inflammatory disorders caused by asthma. As shown in Table 2, these plants were extracted with water and mixed in various ratios. Antioxidant activity has been reported to affect various bioactivities such as skin whitening and anti-inflammation [33]. ABTS and DPPH radical scavenging actions offer redox-functionalized proton ions for unstable free radicals and play a critical role in stabilizing detrimental free radicals; these assays were conducted to assess the antioxidant activity of each mixture [31,32]. The IC50 values of M1, M2, M3 and M4 for ABTS radical scavenging activity were 214, 61, 150 and 57 (μg/mL), respectively ( Figure 2A). M2, M3 and M4 showed strong ABTS radical scavenging activity (Figure 2A). The IC50 values of M1, M2, M3 and M4 for DPPH radical scavenging activity were 321, 171, 209 and 197 μg/mL, respectively ( Figure  2A). Among the mixture, M2 and M4 exhibited potent DPPH radical scavenging activity (Figure 2A). To examine the hepatoprotective effects, Hepa-lc1c7 cells were treated with a mixture of extracts at 50, 100 and 200 μg/mL, and GST activity was investigated. As shown in Figure 2B, the GST activity of M4 was 4.09 ± 0.58, which was 1.78 times higher than that of M1 (2.77 ± 0.44).

Anti-inflammatory Effects of Mixture
To investigate anti-inflammatory effects, the levels of NO and proinflammatory cytokines were examined in LPS-induced murine macrophages. All mixtures inhibited LPSinduced NO production without causing cytotoxicity ( Figure 3A). Specifically, M4 exhibited the strongest inhibition of LPS-induced NO production in a dose-dependent manner ( Figure 3A). To confirm the anti-inflammatory effects of the mixture, the levels of proinflammatory cytokines were measured using an ELISA kit. Compared with the untreated

Anti-Inflammatory Effects of Mixture
To investigate anti-inflammatory effects, the levels of NO and proinflammatory cytokines were examined in LPS-induced murine macrophages. All mixtures inhibited LPS-induced NO production without causing cytotoxicity ( Figure 3A). Specifically, M4 exhibited the strongest inhibition of LPS-induced NO production in a dose-dependent manner ( Figure 3A). To confirm the anti-inflammatory effects of the mixture, the levels of proinflammatory cytokines were measured using an ELISA kit. Compared with the untreated control group, the levels of LPS-induced proinflammatory cytokines IL-1β, IL-6 and TNF-α increased. When macrophages were pretreated with the extracts, we observed extract-mediated downregulation of proinflammatory activation at 10, 25, 50 and 100 µg/mL ( Figure 3B). In particular, M4 exhibited the strongest inhibitory activity against proinflammatory cytokines compared with the other mixture groups, and hence was prepared based on bioassay-guided selection. Based on these results, M4 with the most potent anti-inflammatory and antioxidant effects was selected for the preparation of products to protect against inflammatory disorders. mediated downregulation of proinflammatory activation at 10, 25, 50 and 100 μg/mL (Figure 3B). In particular, M4 exhibited the strongest inhibitory activity against proinflammatory cytokines compared with the other mixture groups, and hence was prepared based on bioassay-guided selection. Based on these results, M4 with the most potent anti-inflammatory and antioxidant effects was selected for the preparation of products to protect against inflammatory disorders.

Antiasthmatic Effects of M4
The antiasthmatic effect of M4 was assessed by measuring the proinflammatory cytokine levels in OVA-, LPS-and PM-induced rat models. The gene expression of proinflammatory cytokines in the lungs of OVA-, LPS-and PM-treated rats was determined. RT-PCR analysis revealed that treatment with M4 modulated the gene expression of proinflammatory cytokines, such as IL-1β, IL-6 and TNF-α in a dose-dependent manner ( Figure 4A). Bronchoalveolar lavage fluid (BALF) was collected from the lungs to investigate the generation of inflammatory cytokines. As shown in Figure 4B, the levels of inflammatory cytokines (IL-1β, IL-6 and TNF-α) in the BAL of the OVA-, LPS-and PM-treated rats were higher than those in the PBS-treated group. However, the M4-treated group showed significantly reduced levels of these cytokines in a dose-dependent manner ( Figure 4B).

Antiasthmatic Effects of M4
The antiasthmatic effect of M4 was assessed by measuring the proinflammatory cytokine levels in OVA-, LPS-and PM-induced rat models. The gene expression of proinflammatory cytokines in the lungs of OVA-, LPS-and PM-treated rats was determined. RT-PCR analysis revealed that treatment with M4 modulated the gene expression of proinflammatory cytokines, such as IL-1β, IL-6 and TNF-α in a dose-dependent manner (Figure 4A). Bronchoalveolar lavage fluid (BALF) was collected from the lungs to investigate the generation of inflammatory cytokines. As shown in Figure 4B, the levels of inflammatory cytokines (IL-1β, IL-6 and TNF-α) in the BAL of the OVA-, LPS-and PM-treated rats were higher than those in the PBS-treated group. However, the M4-treated group showed significantly reduced levels of these cytokines in a dose-dependent manner ( Figure 4B).

Sulfur Flavor-Reduction Effects Using Essential Oil of Nutmeg Seed
To reduce sulfur flavor in the M4, essential oils of nutmeg and cinnamon were used. As shown in Figure 5A, the essential oil content of nutmeg seed and cinnamon bark was 11.5 mL/kg and 9.5 mL/kg, respectively. Nutmeg seeds contain many essential oils, and their components have been analyzed. The essential oils of nutmeg seed included 15 flavor components, with alpha-pinene, sabinene and beta-pinene as the major components ( Figure 5B). Therefore, the essential oils of nutmeg seeds were added to the product using M4 to remove the sulfur flavor.

Sulfur Flavor-Reduction Effects Using Essential Oil of Nutmeg Seed
To reduce sulfur flavor in the M4, essential oils of nutmeg and cinnamon were used. As shown in Figure 5A, the essential oil content of nutmeg seed and cinnamon bark was 11.5 mL/kg and 9.5 mL/kg, respectively. Nutmeg seeds contain many essential oils, and their components have been analyzed. The essential oils of nutmeg seed included 15 flavor components, with alpha-pinene, sabinene and beta-pinene as the major components ( Figure 5B). Therefore, the essential oils of nutmeg seeds were added to the product using M4 to remove the sulfur flavor.

Anti-Inflammatory Effects of Product
Based on these results, GP was manufactured using adjusted additives such as oligosaccharides, steviosides, and essential oils of nutmeg seeds as their main ingredients. The anti-inflammatory activity of the product was assessed in the BALF of the lungs from OVA-, LPS-, and PM-induced rat models. As shown in Figure 6, the levels of the proinflammatory cytokines IL-1β, IL-6 and TNF-α in the BALF of OVA-, LPS-and PM-stimulated rats were higher than those in the PBS-stimulated group. However, the GP-treated group showed reduced levels of IL-1β, IL-6 and TNF-α in a dose-dependent manner ( Figure 6).

Anti-Inflammatory Effects of Product
Based on these results, GP was manufactured using adjusted additives such as oligosaccharides, steviosides, and essential oils of nutmeg seeds as their main ingredients. The anti-inflammatory activity of the product was assessed in the BALF of the lungs from OVA-, LPS-, and PM-induced rat models. As shown in Figure 6, the levels of the proinflammatory cytokines IL-1β, IL-6 and TNF-α in the BALF of OVA-, LPS-and PM-stimulated rats were higher than those in the PBS-stimulated group. However, the GP-treated group showed reduced levels of IL-1β, IL-6 and TNF-α in a dose-dependent manner (Figure 6).

Discussion
Asthma is a chronic and complex airway disease that affects more than 350 million people worldwide and is heavily influenced by diverse lifestyles, genetics and environmental factors [6]. Allergic asthma is characterized by type 2 airway inflammation. Am- Figure 6. Anti-inflammatory effects of the GP. Generation of proinflammatory cytokines such as IL-1β, IL-6 and TNF-α in OVA-, LPS-and PM-induced asthmatic rat model. Data are expressed as the means ± SD (n = 5). A value of p-value < 0.05 was considered statistically significant.

Discussion
Asthma is a chronic and complex airway disease that affects more than 350 million people worldwide and is heavily influenced by diverse lifestyles, genetics and environmental factors [6]. Allergic asthma is characterized by type 2 airway inflammation. Ambient air pollution caused by many industries has raised great concern worldwide [8]. PM is an extrinsic factor that can trigger inflammation, a set of complicated immunovascular reactions designed to protect the host against external or internal stimuli [9,12]. Asthma medications include anti-inflammatory drugs and bronchodilators; however, they cause side effects such as depression, agitation and sleep disturbance [1]. Hence, there is an urgent need to develop nutraceuticals that can prevent adverse effects caused by drugs and PM-induced inflammatory diseases such as asthma that affect the human body. Natural products have attracted extensive attention owing to their usefulness in drug discovery and functional food production. The combination of herbal medicines is effective in the treatment and prevention of various diseases owing to synergistic effects [13,14]. In the present study, we assessed the optimal ratio of herbal plant extracts that produced antiinflammatory effects for asthma treatment using a bioassay-guided selection. The herbal plants RS, AH, AS, DM and CL were selected, and mixtures of various ratios were prepared ( Table 2). Among the mixtures, M2 and M4 exhibited strong scavenging activities of ABTS and DPPH radicals (Figure 2A). GST catalyzes the conjugation of the reduced form of GSH to xenobiotic substrates for detoxification [32]. To assess the hepatoprotective effects of the mixture, GST activity was examined in murine hepatocytes (hepa-1c1c7). Among the mixtures, M4 exhibited the strongest GST activity ( Figure 2B).
Inflammation is the response of the immune system to hazardous stimuli such as pathogens, damaged cells, toxic compounds, inflammatory cytokines or irradiation. It acts by eliminating injurious stimuli and initiating the healing process; therefore, it is a defense mechanism that is vital to health. Macrophages are major inflammatory and immune effector cells, and their activation is triggered by exposure to bacterial LPS or proinflammatory cytokines. Activated macrophages produce inflammatory mediators such as NO and proinflammatory cytokines such as IL-1β, IL-6 and TNF-α. The excessive production of these molecules can lead to chronic inflammatory diseases. Our results showed that M4 most strongly inhibited NO, IL-1β, IL-6 and TNF-α generation without causing cytotoxicity ( Figure 3A,B).
Allergic asthma, triggered by various environmental factors such as air pollutants, is a prolonged lung inflammatory disorder caused by the infiltration of eosinophils, neutrophils, and leukocytes into the bronchoalveolar space [1,8,9]. Furthermore, proinflammatory cytokines enhance inflammatory diseases, such as asthma and chronic obstructive pulmonary disease [34]. We examined the anti-inflammatory effects of M4 on asthma using OVA, -LPSand PM-induced rat models. Our results showed that M4 inhibited the gene expression and generation of the proinflammatory cytokines, IL-1β, IL-6 and TNF-α ( Figure 4A,B). AH, belonging to the Allium family, has a strong sulfur flavor; hence, essential oils of nutmeg seed, which contains a higher essential oil content than cinnamon bark, were added to reduce its flavor ( Figure 5A). Our results showed that the essential oil of nutmeg seed had 15 types of flavor components, with alpha-pinene, sabinene and beta-pinene as major components ( Figure 5B), which are also found to exhibit antiallergic effects when extracted from Lepechinia betonicifolia and Gynoxys miniphylla [35][36][37]. Major components were detected in essential oils from Flos magnoliae with inhibitory effects on allergic rhinitis [38,39]. We suggest that the addition of nutmeg seed essential oils effectively reduces the sulfur flavor. Based on these results, GP was prepared using M4 supplemented with the essential oils of nutmeg seeds according to the food manufacturing process. Cytokines play a significant role in asthma and promote the survival of various inflammatory cells [1,34]. Proinflammatory cytokines such as IL-1β, IL-6 and TNF-α are found in increased amounts in the sputum and BALF of patients with asthma [34]. Our results demonstrated that GP inhibited the generation of IL-1β, IL-6 and TNF-α in OVA-, LPS-and PM-induced asthmatic animal models ( Figure 6). Proinflammatory cytokines amplify inflammation through the activation of NF-κB, leading to the increased expression of inflammatory genes [1][2][3][4]34,40]. Overall, the results of our study indicate, for the first time, that the product prepared using an herbal mixture suppresses inflammatory responses in diseases such as asthma (Figure 7). Further studies on the regulatory action of products in inflammatory signaling are needed to ensure their potential therapeutic application in the treatment of inflammatory disease including asthma. Taken together, our results suggest that functional foods containing a mixture of herbal plants can be a potential candidate for the treatment of inflammatory diseases.
tively reduces the sulfur flavor. Based on these results, GP was prepared using M4 supplemented with the essential oils of nutmeg seeds according to the food manufacturing process. Cytokines play a significant role in asthma and promote the survival of various inflammatory cells [1,34]. Proinflammatory cytokines such as IL-1β, IL-6 and TNF-α are found in increased amounts in the sputum and BALF of patients with asthma [34]. Our results demonstrated that GP inhibited the generation of IL-1β, IL-6 and TNF-α in OVA-, LPS-and PM-induced asthmatic animal models ( Figure 6). Proinflammatory cytokines amplify inflammation through the activation of NF-κB, leading to the increased expression of inflammatory genes [1][2][3][4]34,40]. Overall, the results of our study indicate, for the first time, that the product prepared using an herbal mixture suppresses inflammatory responses in diseases such as asthma (Figure 7). Further studies on the regulatory action of products in inflammatory signaling are needed to ensure their potential therapeutic application in the treatment of inflammatory disease including asthma. Taken together, our results suggest that functional foods containing a mixture of herbal plants can be a potential candidate for the treatment of inflammatory diseases.

Conclusions
Four types of mixtures were prepared using five kinds of herbal plant extracts: RS and CM roots, AH whole plant, AS stems and DM leaves. M4 exhibited the strongest increase in GST activity and anti-inflammatory activity in asthma and thus was selected to prepare the product. GP was manufactured using M4 and additives including nutmeg seed essential oils. GP inhibited the generation of proinflammatory cytokines IL-1β, IL-6 and TNF-α in OVA-, LPS-and PM-induced asthmatic rat models. These results suggest that GP prepared with M4 is a potent functional food with anti-inflammatory activity that can be used to treat inflammatory diseases such as asthma caused by ambient air pollutants.

Conclusions
Four types of mixtures were prepared using five kinds of herbal plant extracts: RS and CM roots, AH whole plant, AS stems and DM leaves. M4 exhibited the strongest increase in GST activity and anti-inflammatory activity in asthma and thus was selected to prepare the product. GP was manufactured using M4 and additives including nutmeg seed essential oils. GP inhibited the generation of proinflammatory cytokines IL-1β, IL-6 and TNF-α in OVA-, LPS-and PM-induced asthmatic rat models. These results suggest that GP prepared with M4 is a potent functional food with anti-inflammatory activity that can be used to treat inflammatory diseases such as asthma caused by ambient air pollutants.

Institutional Review Board Statement:
The study on all animals were approved by the Institutional Animal Care and Use Committee of Dongshin University (No. DSU2016-10-01).

Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the corresponding author.