Traditional Herbal Medicine and Allergic AsthmaView this Special Issue
Research Article | Open Access
Ethanol Extract of Perilla frutescens Suppresses Allergen-Specific Th2 Responses and Alleviates Airway Inflammation and Hyperreactivity in Ovalbumin-Sensitized Murine Model of Asthma
This study was to investigate the effects of different fractions of Perilla frutescens (Pf) leaves extracted by water or ethanol on asthma. BALB/c mice sensitized intraperitoneally and challenged with ovalbumin (OVA) were divided into six groups. Each group of mice was tube-feeding with 0 (control), 80 μg (PfWL), or 320 μg (PfWH) water extracts or 80 μg (PfEL) or 320 μg (PfEH) ethanol extracts of perilla leaves daily for 3 weeks. A negative control group (PBS) was neither sensitized nor treated with Pf. The effects of perilla leave extracts on allergic immune response were evaluated. The results showed that OVA-specific IL-5 and IL-13 secretions from OVA-stimulated splenocytes were significantly suppressed in the ethanol extract groups PfEL and PfEH. Serum level of anti-OVA IgE tended to be lower in the PfEH group. The inflammatory mediators, such as eotaxin and histamine, and total cells, particularly eosinophils in bronchoalveolar lavage fluid (BALF), were also decreased in the PfEL and the PfEH groups. Therefore, the PfEL and the PfEH groups had significantly lower methacholine-induced hyperresponsiveness (AHR). In conclusion, ethanol extracts, rather than water extract, of perilla leaves could significantly suppress Th2 responses and airway inflammation in allergic murine model of asthma.
Allergic asthma is a chronic disease that clinically augments bronchial hyperresponsiveness and inflammation. The asthmatic inflammation is clearly associated with the high level of type 2 T cell (Th2) cytokines that induced immunoglobulin (Ig) E production and eosinophilic infiltration . The Th2 cytokines IL-4, IL-5, and IL-13 are the major cytokines for development of atopic diseases, such as asthma, rhinitis, and dermatitis . IL-4 promotes the immunoglobulin class switch from IgM to IgE . IL-5 induces eosinophilia activity and infiltration, which is the critical response in the allergic asthma . IL-13 acts to induce airway hyperresponsiveness (AHR) that contributes to atopic disease . The suppression of Th2 responses is a feasible attempt to attenuate the symptoms of allergic asthma. Therefore, biologic targeted therapies have been developed to target the specific molecular pathways to treat asthma, especially those targets at IL-4, IL-5, IL-13, and IgE . However, due to complex clinical symptoms and multiple mechanisms involved, the outcome of these trials has not been satisfied. Epidemiological studies indicate that patients often turn to complementary and alternative therapies, including dietary supplements . Studies also showed that dietary oil, adlay, and medicinal herbs, such as Ganoderma and Andrographis, decreased Th2 cytokines productions and thus alleviated allergic responses in a murine model of asthma [8–12], suggesting the potential application of traditional herbal medicine for immunomodulation.
Perilla frutescens (Pf) leaf is a kind of aromatic vegetables, which is also used as the traditional medicine in Asia. Perilla has been demonstrated to exert antiobesity, anti-dyslipidemia, antioxidant, and anti-infammation [13–19]. Recently, some researches focused on its antiallergic effects [20–23]. The studies suggested water extract of Pf inhibited histamine release from mast cells . Subcutaneous injection with perilla seed water/ethanol extract, as a traditional oriental therapeutic herbal acupuncture, seemed to reduce IgE, IL-4, IL-5, and IL-13 in BALF in OVA-induced asthma in mice .
However, they are either intraperitoneal injection , type II allergy evaluated by ear-passive cutaneous anaphylaxis or edema [20, 21], or lack of the important outcome of treatment in asthma, airway hyperresponsiveness (AHR) result . There are few studies investigating the effects of oral supplementation of perilla extracts on overall allergic responses of asthmatic murine model. In addition, the crude polysaccharide isolated from Pf by hot water extraction was shown to increase nitric oxide and TNFα and IL-6 production both in vitro and in vivo . Therefore, whether water or ethanol extracts of Perilla frutescens exert antiasthma effects via oral supplementation was investigated in this study using OVA-sensitized and challenged BALB/c mice.
2. Materials and Methods
2.1. Preparation of Extracts of Perilla frutescens
The crude extracts of Perilla frutescens (L.) Britt leaves were extracted as follows. Dried Perilla frutescens leaves were extracted with 5-fold (w/v) hot water for 60 min. The water extracts (PfW) were filtered through filter paper and concentrated using a rotary evaporator and then freeze-dried. The yield of PfW was 21.70% (w/w, dry basis). The ethanol extracts of dried Perilla frutescens leaves (PfE) were extracted with 15-fold (w/v) 95% ethanol for 24 hr. The PfE were filtered by filter paper and then evaporated in a rotary evaporator to remove the solvent. The yield of PfE was 9.22% (w/w, dry basis).
2.2. Animal Study, Sensitization, and Airway Challenge
Female BALB/c mice were purchased from the Animal Center at the National Taiwan University College of Medicine, Taipei, Taiwan, and maintained at the Department of Biochemical Science and Technology at the National Taiwan University. Animal care and handling conformed to accepted guideline .
The allergic asthma model, as well as the diet, was manipulated as described . As shown in Figure 1, 8-week-old female BALB/c mice were sensitized intraperitoneally (i.p.) with 10 μg ovalbumin (OVA; Sigma, St. Louis, Mo) in alum adjuvant (Imject Alum; Pierce, Rockford, IL). After three times OVA-sensitization, sensitized mice were grouped randomly into six groups for tube-feeding with 0 μg (Control), 80 μg (PfWL), or 320 μg (PfWH) water extract or 80 μg (PfEL) or 320 μg (PfEH) ethanol extract of perilla leaves daily for 3 weeks. During these 3 weeks, the OVA-sensitized mice were challenged twice with 5% aerosolized OVA in PBS buffer by inhalation. In addition, mice without Pf supplement, that received alum without OVA, and that inhaled aerosolized PBS are used as the negative control group (PBS). After 3 weeks of Pf supplement, mice that received a third challenge were euthanized by CO2 inhalation and the BALF, serum, and splenocytes of mice were collected for further analysis.
2.3. Determination of Airway Hyperresponsiveness (AHR)
Airway function was measured by the whole-body plethysmography as described previously . Twenty-four hours after the secondary challenge, AHR was measured when mice were stimulated with methacholine (Sigma) using whole-body plethysmography (Buxco, Wilmington, NC). Mice were placed in the main chamber of a whole-body plethysmography and challenged with aerosolized methacholine, increasing concentration from 12.5 to 50 mg/mL in PBS buffer. The AHR is expressed as the enhanced pause (Penh) values measured during each 3 min period.
2.4. Determination of Anti-OVA Antibodies
Serum anti-OVA IgE antibody titers were measured by ELISA method as previously described . Briefly, 96-well plates were coated with 10 μg OVA/mL NaHCO3 buffer. After overnight incubation at 4°C and being blocked with 1% bovine serum albumin (BSA)/PBS buffer for 2 hours at room temperature, the serum samples and positive control sera were appropriately diluted with blocking buffer and added to the 96-well plate. After 2 hours, the biotin-conjugated anti-mouse IgE (PharMingen, San Diego, CA) was added for 2 hours of incubation. Then, streptavidin-conjugated peroxidases and the enzyme substrate, 2,2′-azino-bis-3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), were added and incubated for 20 min at room temperature. The antibody levels of the samples were compared with the positive control sera. The positive control was a pool of serum collected from OVA-sensitized mice with strong response (optical density > 1). The results of the antibody titer were expressed in ELISA units (EU), .
2.5. Collection of BALF and Splenocytes
The BALF was collected with 5 instillations of 0.5 mL saline. Approximately 2.5 mL of fluid was recovered with each sample and the volume did not differ significantly among groups. The fluid was collected and kept at −80°C for eotaxin and histamine analysis. The cell pellet was resuspended in 250 μL saline containing 10% BSA. The total cells were counted with a hemocytometer using the trypan blue dye exclusion method. BALF cells with a concentration of 2 × 105 were cytocentrifuged and then stained with Liu’s stain for eosinophil counts.
The splenocytes were prepared by aseptically removing spleens from sensitized and challenged BALB/c mice . Splenocytes were counted with a hemocytometer using the trypan blue dye exclusion method. A concentration of 5 × 106 cells/mL was cultured in 48-well plates in RPMI-1640 medium supplemented with TCM medium in the absence or presence of antigen, 50 and 100 μg/mL OVA, for 48 hours. The supernatants in cell cultures were collected and stored at −80°C for cytokines analysis.
2.6. Cytokines Assay
The cytokine levels in splenocytes culture supernatants were measured by sandwich ELISA methods. Briefly, the anti-cytokine antibody was coated in the 96-well plates (Nunc, Roskilde, Denmark). After overnight incubation at 4°C and being blocked with 1% BSA/PBS buffer for 30 min, the samples and standards were added to the 96-well plates for 2 hours of incubation. The biotin-conjugated antibodies were added and incubated. After washing, the streptavidin-conjugated peroxidase was added for 1 hour. The substrate ABTS was added to each well for 20 min. The plates were read in a microplate autoreader (microplate autoreader; Bio-Tek Instrument, Inc. Winooski, VT) at 405 nm.
2.7. Eotaxin and Histamine Assay
The eotaxin concentration in BALF was determined by mouse eotaxin sandwich ELISA kit (R&D Systems, Minneapolis, MN). The eotaxin concentration was assayed according to the manufacturer’s instructions. After the color reagent was added, the plate was incubated at room temperature for 20 min for color development. The absorbance was measured at 630 nm in the microplate autoreader. The eotaxin level in BALF was determined using the standard curve.
The histamine level was determined by Histamine-ELISA kit (IBL Hamburg, Germany). The manipulation was according to manufacturer’s instructions for use. In brief, the culture supernatants and plasma standards were acylated with acylation reagent first. Then, aliquots of 50 μL acylated sample and acylated standards were loaded into the 96-microplate wells, respectively. Aliquots of 50 μL enzyme conjugate and 50 μL antiserum were pipetted into each well to react for 3 hours. The plate was washed four times and 200 μL tetramethylbenzidine (TMB) substrate solution was added for 30 min. Then the reaction was stopped by adding 100 μL of stop solution into each well. The optical density was detected by the microplate autoreader at 450 nm. The histamine concentration is determined using the standard curve.
2.8. Statistical Analysis
Data were expressed as mean ± SD. The significance of difference between the Pf and the control groups was analyzed statistically by Student’s -test of the SAS program system (Strategic Application Software; SAS windows version 8.2, SAS Institute Inc., Cary, NC) throughout the study.
3.1. Ethanol Extracts of Perilla frutescens Suppressed Th2 Responses of OVA-Sensitized Mice
After feeding of two doses of water extract of Pf (PfWL and PfWH) or ethanol extract of Pf (PfEL and PfEH) for 3 weeks, splenocytes were isolated from OVA-sensitized mice and stimulated with OVA to examine the effects of PfW and PfE on allergen-specific Th2 responses. The Th1 cytokines IL-2 and IFNγ were not significantly affected (data not shown). However, Th2 cytokines IL-5 and IL-13 productions of OVA-stimulated splenocytes in the PfEL and the PfEH groups were significantly lower than those of the control group, as shown in Figure 2. The OVA-stimulated IL-4 productions were low and not affected by either water or ethanol extracts. In addition, PHA-stimulated IL-13 productions by splenocytes were also suppressed by PfE (data not shown). These data suggested that ethanol extracts of Pf could inhibit allergen-specific stimulated Th2 cells activity.
The IgE antibody produced by Th2 cell-activated B cells tended to be lower in serum of OVA-sensitized mice supplemented with PfEH (Figure 3). The serum levels OVA-specific IgG1 and IgG2a were not significantly affected (data not shown). This data suggested that ethanol extracts of Perilla frutescens might have potential to suppress serum IgE levels in OVA-sensitized mice.
3.2. Ethanol Extracts of Perilla frutescens Decrease Cells Infiltration in Airway Allergic Inflammation of OVA-Sensitized Mice
The BALF was collected after OVA-inhalation challenge prior to sacrifice and the cell number in BALF was determined and shown in Figure 4. Few total cells and eosinophils were in the PBS negative control group. The infiltrated cells number increased after OVA-challenge as shown in the control group. The total cell number in BALF significantly decreased in asthmatic mice supplemented with PfEH (Figure 4(a)). Particularly, the number of eosinophils, the major cells contributing to airway inflammation of allergic asthma, was decreased by Pf supplement, significantly in both the PfEL and the PfEH groups (Figure 4(b)). These data suggested that Perilla frutescens inhibited the infiltration of inflammatory cells to BALF in asthmatic mice, especially the ethanol extracts.
The proinflammatory mediators such as histamine and eotaxin in BALF of OVA-sensitized mice were also suppressed by Pf, as shown in Figure 5. The histamine levels were significantly lower in both the PfEL and the PfEH groups. The eosinophil chemotactic protein eotaxin in BALF was also reduced in the PfEH group. Th2 cytokines in BALF of OVA-sensitized mice were also measured but were not found significantly lower by Pf in this experiment (data not shown). These results indicated that ethanol extracts of Perilla frutescens alleviated inflammatory cell infiltration and thus reduced the allergic inflammation in airway of OVA-sensitized and challenged mice.
3.3. Ethanol Extracts of Perilla frutescens Decrease Airway Hyperresponsiveness (AHR) of OVA-Sensitized Mice
OVA-sensitized mice supplemented with two doses of water or ethanol extracts, respectively, were challenged with 50 mg/mL aerosolized OVA to further induce AHR. The results shown in Figure 6 demonstrated that the control group had significantly higher AHR, measured as Penh value, than the PBS negative control group after methacholine challenge. Ethanol extract of Pf significantly reduced AHR as significantly lower Penh values were detected in both the PfEL and the PfEH groups. Water extract of Pf tended to have low AHR () at high dose (PfWH group) though the low dose PfWL group did not reach statistical significance. This data indicates that Perilla frutescens extracts possess inhibitory effects of airway hyperresponsiveness, possibly due to lower cell infiltration and inflammation in bronchiole and lung of allergic asthma.
Allergic asthma is a chronic inflammatory disease in airway, which was induced by Th2-prone responses. The present allergic asthma murine model demonstrated that Perilla frutescens extracts, especially the ethanol extracts, decrease Th2 cytokines production, serum IgE level, cells infiltration, allergic mediator secretions, and AHR.
Early study examined the effects of different extracts of Pf on TNFα levels in inflammatory mice and found that water extracts had stronger inhibition than n-hexane or ethyl acetate extracts . Water extract of Pf inhibited the histamine released from rat peritoneal mast cells in vitro . Glycoprotein derived from the hot water extract of Pf demonstrated the effective component of Perilla frutescens Britton to inhibit mast cells degranulation . These studies suggested the potential antiallergic effect of Pf. Further, our study showed that the ethanol extract of Pf had more effective inhibition than water extract on the histamine release in BALF of OVA-challenged mice. We suggested that this inhibition might be related to the suppression of Th2 activities.
It has been known that IL-4 plays a critical role in IgE production. IL-4 receptor was significantly associated with asthma risk [31, 32]. Allergen-stimulated IL-4 secretions by splenocytes from sensitized mice were low in this study and not suppressed by Pf, which might explain the reason why sera IgE levels were not significantly lowered by Pf. The tendency of lower serum IgE in the PfEH group might be partially due to lower Th2 activities such as allergen-stimulated IL-5 and IL-13 productions. IL-13 polymorphisms were consistently associated with asthma and serum IgE in asthma populations . Our study showed that the ethanol extracts of Pf significantly decreased IL-5 and IL-13 productions from splenocytes of OVA-sensitized mice. Blocking the binding of IgE to its receptors is the most effective therapy strategy for allergic disease . The association between IL-13 and IgE productions in asthma [35, 36] suggests that ethanol extracts of Pf suppressed IL-13 secretion and thus tended to reduce IgE production .
IL-13 induced the eotaxin release by airway epithelial cells in vitro . Our data also indicated that ethanol extracts of Pf decreased eotaxin production in BALF, consistent with its lower allergen-induced IL-13 secretion. The inflammatory cells, such as eosinophils, recruited into airway are the major clinical manifestations in allergic asthma. IL-5 is best characterized for eosinophilia that dominates airway inflammation on allergic asthma . When asthmatic patients were given anti-IL-5 (mepolizumab), bronchial mucosal eosinophils decreased . Furthermore, eosinophils not only act as terminal effector cells but also act to actively amplify allergic responses by promoting Th2 cell immunity , indicating the close relationship between eosinophils and airway hyperresponsiveness . Th2-cell-derived cytokine IL-5, together with eotaxin, plays the critical roles in the induction of airway hyperreactivity and the development of chronic airway wall remodeling . Present report indicated that high dose of ethanol extracts of Pf inhibited eosinophils and eotaxin levels in BALF and also decreased AHR. These data suggested that ethanol extracts of Pf exert attenuate airway hyperresponsiveness and inflammation in allergic asthma through inhibition of eosinophils and proallergic mediators.
However, the percentage of eosinophil in total cell in BALF was low in this study. It may be due to the OVA-challenge protocol. Continuous daily inhalation or intranasally challenge with allergen may drive more eosinophils in BALF. Our previous studies showed ~10% eosinophils in BALF of mice when challenged with OVA inhalation every three days , but only ~3% eosinophils were counted with challenge every seven days. The majority of cell populations in this study were neutrophils/basophils, which were 54% in the control group and 35–45% in the Pf groups.
Study showed that water extracts of perilla leaves improve atopic dermatitis  and identified the active constituent to be luteolin . Rosmarinic acid extracts in Pf strongly inhibited hexosaminidase release from RBL-2H3 mast cells  and were shown to decrease the neutrophils and eosinophils recruitment in nasal lavage fluid of seasonal allergic rhinoconjunctivitis patients . Recent report revealed that 80% ethanol extracts of purple perilla leaves contains 8.47% (w/w) of rosmarinic acid which is the major phenolic acid in perilla and commonly found in aromatic plants . Our data suggested that the water extracts of Pf attenuated AHR in OVA-sensitized mice, but they are less effective than ethanol extract. It may be due to the same doses of different extracts used in our study. Recent report indicated that ethanol extracts of Pf significantly decreased TNFα production in BALF from LPS-induced airway inflammation and suggested that phenylpropanoids may contribute to the inhibitory activity of ethanol extracts of Pf on the lung inflammatory response . As Perilla frutescent leaves are aromatic vegetables and can be consumed raw, cooked, or pickled , their application is feasible and expectable. In our study, the most effective dose for decreasing Th2 responses and AHR is 80~320 mg PfE daily for mouse. According to the dose translation from animal to human , it corresponds to 1.5~6 g (~5~20 pieces) of fresh Perilla frutescens leaves daily for a 60 kg adult and 0.7~3 g (~2.5~10 pieces) for 20 kg child, respectively.
In conclusion, ethanol extracts of Perilla frutescent leaves could downregulate Th2 activities to secrete less IL-5 and IL-13 and thus lower serum IgE level when counting allergen challenge. The cell infiltration, particularly eosinophils, and proinflammatory mediators such as histamine and eotaxin in BALF were significantly suppressed. As a result, AHR is alleviated by the extracts of Perilla frutescent leaves, suggesting that Perilla frutescent leaves are a potential herbal medicine for immunomodulation.
Conflict of Interests
The authors have declared that no conflict of interests exists.
- S. J. Galli, M. Tsai, and A. M. Piliponsky, “The development of allergic inflammation,” Nature, vol. 454, no. 7203, pp. 445–454, 2008.
- Y.-T. Lin, C.-T. Wang, P.-S. Chao et al., “Skin-homing CD4+ Foxp3+ T cells exert Th2-like function after staphylococcal superantigen stimulation in atopic dermatitis patients,” Clinical & Experimental Allergy, vol. 41, no. 4, pp. 516–525, 2011.
- S. M. Grunewald, M. Teufel, K. Erb et al., “Upon prolonged allergen exposure IL-4 and IL-4Rα knockout mice produce specific IgE leading to anaphylaxis,” International Archives of Allergy and Immunology, vol. 125, no. 4, pp. 322–328, 2001.
- A. N. Menzies-Gow, P. T. Flood-Page, D. S. Robinson, and A. B. Kay, “Effect of inhaled interleukin-5 on eosinophil progenitors in the bronchi and bone marrow of asthmatic and non-asthmatic volunteers,” Clinical and Experimental Allergy, vol. 37, no. 7, pp. 1023–1032, 2007.
- M. T. Kasaian and D. K. Miller, “IL-13 as a therapeutic target for respiratory disease,” Biochemical Pharmacology, vol. 76, no. 2, pp. 147–155, 2008.
- J. B. Bice, E. Leechawengwongs, and A. Montanaro, “Biologic targeted therapy in allergic asthma,” Annals of Allergy, Asthma and Immunology, vol. 112, no. 2, pp. 108–115, 2014.
- W. Jiratchariyakul and G. B. Mahady, “Overview of botanical status in EU, USA, and Thailand,” Evidence-Based Complementary and Alternative Medicine, vol. 2013, Article ID 480128, 13 pages, 2013.
- B.-F. Lin, C.-C. Lai, K.-W. Lin, and B.-L. Chiang, “Dietary oxidized oil influences the levels of type 2 T-helper cell-related antibody and inflammatory mediators in mice,” British Journal of Nutrition, vol. 84, no. 6, pp. 911–917, 2000.
- H.-Y. Hsu, B.-F. Lin, J.-Y. Lin, C.-C. Kuo, and W. Chiang, “Suppression of allergic reactions by dehulled adlay in association with the balance of Th1/Th2 cell responses,” Journal of Agricultural and Food Chemistry, vol. 51, no. 13, pp. 3763–3769, 2003.
- J.-Y. Lin, M.-L. Chen, B.-L. Chiang, and B.-F. Lin, “Ganoderma tsugae supplementation alleviates bronchoalveolar inflammation in an airway sensitization and challenge mouse model,” International Immunopharmacology, vol. 6, no. 2, pp. 241–251, 2006.
- M.-L. Chen and B.-F. Lin, “Effects of triterpenoid-rich extracts of Ganoderma tsugae on airway hyperreactivity and Th2 responses in vivo,” International Archives of Allergy and Immunology, vol. 143, no. 1, pp. 21–30, 2007.
- W.-W. Chao and B.-F. Lin, “Isolation and identification of bioactive compounds in Andrographis paniculata (Chuanxinlian),” Chinese Medicine, vol. 5, article 17, 2010.
- M.-J. Kim and H. K. Kim, “Perilla leaf extract ameliorates obesity and dyslipidemia induced by high-fat diet,” Phytotherapy Research, vol. 23, no. 12, pp. 1685–1690, 2009.
- Y. Izumi, A. Matsumura, S. Wakita et al., “Isolation, identification, and biological evaluation of Nrf2-ARE activator from the leaves of green perilla (Perilla frutescens var. crispa f. viridis),” Free Radical Biology and Medicine, vol. 53, no. 4, pp. 669–679, 2012.
- G. Zhao, C. Yao-Yue, G.-W. Qin, and L.-H. Guo, “Luteolin from Purple Perilla mitigates ROS insult particularly in primary neurons,” Neurobiology of Aging, vol. 33, no. 1, pp. 176–186, 2012.
- H. Ueda and M. Yamazaki, “Inhibition of tumor necrosis factor-α production by orally administering a perilla leaf extract,” Bioscience, Biotechnology and Biochemistry, vol. 61, no. 8, pp. 1292–1295, 1997.
- J.-H. Ryu, H. J. Son, S. H. Lee, and D. H. Sohn, “Two neolignans from Perilla frutescens and their inhibition of nitric oxide synthase and tumor necrosis factor-α expression in murine macrophage cell line RAW 264.7,” Bioorganic and Medicinal Chemistry Letters, vol. 12, no. 4, pp. 649–651, 2002.
- Y.-K. Yim, H. Lee, K.-E. Hong et al., “Anti-inflammatory and immune-regulatory effects of subcutaneous Perillae fructus extract injections on OVA-induced asthma in mice,” Evidence-Based Complementary and Alternative Medicine, vol. 7, no. 1, pp. 79–86, 2010.
- M. Igarashi and Y. Miyazaki, “A review on bioactivities of perilla: progress in research on the functions of perilla as medicine and food,” Evidence-Based Complementary and Alternative Medicine, vol. 2013, Article ID 925342, 7 pages, 2013.
- H. Ueda and M. Yamazaki, “Anti-inflammatory and anti-allergic actions by oral administration of a perilla leaf extract in mice,” Bioscience, Biotechnology and Biochemistry, vol. 65, no. 7, pp. 1673–1675, 2001.
- T. Makino, Y. Furuta, H. Fujii et al., “Effect of oral treatment of Perilla frutescens and its constituents on type-I allergy in mice,” Biological and Pharmaceutical Bulletin, vol. 24, no. 10, pp. 1206–1209, 2001.
- C. Sanbongi, H. Takano, N. Osakabe et al., “Rosmarinic acid in perilla extract inhibits allergic inflammation induced by mite allergen, in a mouse model,” Clinical and Experimental Allergy, vol. 34, no. 6, pp. 971–977, 2004.
- J.-C. Heo, D.-Y. Nam, M.-S. Seo, and S.-H. Lee, “Alleviation of atopic dermatitis-related symptoms by Perilla frutescens Britton,” International Journal of Molecular Medicine, vol. 28, no. 5, pp. 733–737, 2011.
- T.-Y. Shin, S.-H. Kim, Y.-K. Kim et al., “Inhibitory effect of mast cell-mediated immediate-type allergic reactions in rats by Perilla frutescens,” Immunopharmacology and Immunotoxicology, vol. 22, no. 3, pp. 489–500, 2000.
- K.-H. Kwon, K.-I. Kim, W.-J. Jun, D.-H. Shin, H.-Y. Cho, and B.-S. Hong, “In vitro and in vivo effects of macrophage-stimulatory polysaccharide from leaves of Perilla frutescens var. crispa,” Biological and Pharmaceutical Bulletin, vol. 25, no. 3, pp. 367–371, 2002.
- National Research Council, Guide for the Care and Use of Laboratory Animals, 1996.
- J.-Y. Lin, M.-L. Chen, and B.-F. Lin, “Ganoderma tsugae in vivo modulates Th1/Th2 and macrophage responses in an allergic murine model,” Food and Chemical Toxicology, vol. 44, no. 12, pp. 2025–2032, 2006.
- H. Ueda and M. Yamazaki, “Inhibition of tumor necrosis factor-α production by orally administering a perilla leaf extract,” Bioscience, Biotechnology and Biochemistry, vol. 61, no. 8, pp. 1292–1295, 1997.
- T.-Y. Shin, S.-H. Kim, S.-H. Kim et al., “Inhibitory effect of mast cell-mediated immediate-type allergic reactions in rats by Perilla frutescens,” Immunopharmacology and Immunotoxicology, vol. 22, no. 3, pp. 489–500, 2000.
- M. Asada, Y. Fukumori, M. Inoue et al., “Glycoprotein derived from the hot water extract of mint plant, Perilla frutescens Britton,” Journal of Agricultural and Food Chemistry, vol. 47, no. 2, pp. 468–472, 1999.
- W. Nie, Y. Zang, J. Chen, and Q. Xiu, “Association between interleukin-4 receptor α chain (IL4RA) I50V and Q551R polymorphisms and asthma risk: an update meta-analysis,” PLoS ONE, vol. 8, no. 7, Article ID e69120, 2013.
- S. Al-Muhsen, A. Vazquez-Tello, A. Alzaabi, M. S. Al-Hajjaj, H. H. Al-Jahdali, and R. Halwani, “IL-4 receptor alpha single-nucleotide polymorphisms rs1805010 and rs1801275 are associated with increased risk of asthma in a Saudi Arabian population,” Annals of Thoracic Medicine, vol. 9, no. 2, pp. 81–86, 2014.
- R. W. B. Bottema, I. M. Nolte, T. D. Howard et al., “Interleukin 13 and interleukin 4 receptor-α polymorphisms in rhinitis and asthma,” International Archives of Allergy and Immunology, vol. 153, no. 3, pp. 259–267, 2010.
- D. Y. M. Leung, H. A. Sampson, J. W. Yunginger et al., “Effect of anti-IgE therapy in patients with peanut allergy,” New England Journal of Medicine, vol. 348, no. 11, pp. 986–993, 2003.
- F.-M. Lu, C.-C. Chou, B.-L. Chiang, and K.-H. Hsieh, “Immunologic changes during immunotherapy in asthmatic children: increased IL-13 and allergen-specific IgG4 antibody levels,” Annals of Allergy, Asthma and Immunology, vol. 80, no. 5, pp. 419–423, 1998.
- X. Wang, C. Ma, Y. Zhang, L. Ning, H. Chen, and F. Zhou, “Clinical significance of the dynamic changes in serum eotaxin, interleukin 13 and total IgE in children with bronchial asthma,” Iranian Journal of Pediatrics, vol. 23, no. 5, pp. 525–530, 2013.
- S. Rayees, F. Malik, S. I. Bukhari, and G. Singh, “Linking GATA-3 and interleukin-13: implications in asthma,” Inflammation Research, vol. 63, no. 4, pp. 255–265, 2014.
- V. Provost, A. Langlois, F. Chouinard et al., “Leukotriene D4 and interleukin-13 cooperate to increase the release of eotaxin-3 by airway epithelial cells,” PLoS ONE, vol. 7, no. 8, Article ID e43544, 2012.
- T. Kouro and K. Takatsu, “IL-5- and eosinophil-mediated inflammation: from discovery to therapy,” International Immunology, vol. 21, no. 12, pp. 1303–1309, 2009.
- P. T. Flood-Page, A. N. Menzies-Gow, A. B. Kay, and D. S. Robinson, “Eosinophil's role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway,” American Journal of Respiratory and Critical Care Medicine, vol. 167, no. 2, pp. 199–204, 2003.
- J. R. MacKenzie, J. Mattes, L. A. Dent, and P. S. Foster, “Eosinophils promote allergic disease of the lung by regulating CD4+ Th2 lymphocyte function,” The Journal of Immunology, vol. 167, no. 6, pp. 3146–3155, 2001.
- A. A. Humbles, C. M. Lloyd, S. J. McMillan et al., “A critical role for eosinophils in allergic airways remodeling,” Science, vol. 305, no. 5691, pp. 1776–1779, 2004.
- K. L. Asquith, H. S. Ramshaw, P. M. Hansbro, K. W. Beagley, A. F. Lopez, and P. S. Foster, “The IL-3/IL-5/GM-CSF common β receptor plays a pivotal role in the regulation of Th2 immunity and allergic airway inflammation,” Journal of Immunology, vol. 180, no. 2, pp. 1199–1206, 2008.
- H. Ueda, C. Yamazaki, and M. Yamazaki, “Luteolin as an anti-inflammatory and anti-allergic constituent of Perilla frutescens,” Biological and Pharmaceutical Bulletin, vol. 25, no. 9, pp. 1197–1202, 2002.
- F. Zhu, T. Asada, A. Sato, Y. Koi, H. Nishiwaki, and H. Tamura, “Rosmarinic acid extract for antioxidant, antiallergic, and α-glucosidase inhibitory activities, isolated by supramolecular technique and solvent extraction from Perilla leaves,” Journal of Agricultural and Food Chemistry, vol. 62, no. 4, pp. 885–892, 2014.
- H. Takano, N. Osakabe, C. Sanbongi et al., “Extract of Perilla frutescens enriched for rosmarinic acid, a polyphenolic phytochemical, inhibits seasonal allergic rhinoconjunctivitis in humans,” Experimental Biology and Medicine, vol. 229, no. 3, pp. 247–254, 2004.
- H.-I. Jun, B.-T. Kim, G.-S. Song, and Y.-S. Kim, “Structural characterization of phenolic antioxidants from purple perilla (Perilla frutescens var. acuta) leaves,” Food Chemistry, vol. 148, pp. 367–372, 2014.
- H.-J. Lim, K.-W. Woo, K.-R. Lee, S.-K. Lee, and H.-P. Kim, “Inhibition of proinflammatory cytokine generation in lung inflammation by the leaves of Perilla frutescens and its constituents,” Biomolecules and Therapeutics, vol. 22, no. 1, pp. 62–67, 2014.
- Y. Li, J. Gong, C. Liu et al., “Phytochemicals, nutritional analysis and in vitro antioxidant activities of pickled Perilla frutescens ethanolic leaf extract,” European Journal of Medicinal Plants, vol. 4, no. 3, pp. 303–314, 2014.
- S. Reagan-Shaw, M. Nihal, and N. Ahmad, “Dose translation from animal to human studies revisited,” The FASEB Journal, vol. 22, no. 3, pp. 659–661, 2008.
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