**Efficacy of Flavonoids for Patients with Japanese Cedar Pollinosis**

Toshio Tanaka

102 Current Insights in Pollen Allergens

*Biochemistry*, Vol. 121, pp. 811-817.

Yoneda, Y. (1997). How proteins are transported from cytoplasm to the nucleus. *Journal of* 

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/45882

#### **1. Introduction**

The worldwide prevalence of allergic diseases such as asthma, atopic dermatitis and allergic rhinitis has increased during the last two decades (Holgate, 1999; Eder et al., 2006). Allergic rhinitis now affects 400-500 million people worldwide (Greiner et al., 2011; Ozdoganoglu & Songu, 2012) and adversely affects social life, school performance, and work productivity (Bousquet et al., 2001). The first case of Japanese cedar pollinosis in Japan was reported in the mid-1960s (Horiguchi & Saito, 1964), but now half of the Japanese population have become sensitized to Japanese cedar pollens and 24-29% of the population is suffering from the disease (Kaneko et al., 2005), so that Japanese cedar pollinosis is now rated as one of the most common diseases in Japan (Okamoto et al., 2009). The complicated interaction between genetic and environmental factors is thought to cause the development of allergic diseases. Many genetic loci related to atopy, a genetic tendency to produce immunoglobulin E (IgE) in response to environmental allergens, have been identified through genome-wide association studies (Grammatikos, 2008). However, changes in the environment have made a more significant contribution than genetic factors to the recent increase in the prevalence of allergic diseases (Nolte et al., 2001; Ho, 2010), since it seems unlikely that genes would have changed during the last two decades. Dietary change has been proposed as one of the environmental factors responsible for the increasing prevalence or the worsening symptoms of allergic diseases (Devereux & Seaton, 2005; Devereux, 2006; Kozyrskyj et al., 2011; Nurmatov et al., 2011; Allan & Devereux, 2011). Indeed foods include both allergypromoting and anti-allergic nutrients (McKeever & Britton, 2004), and flavonoids, which are plant secondary metabolites, can have powerful antioxidant, anti-allergic and immunemodulating effects (Hollman & Katan, 1999; Middleton et al., 2000; Manach et al., 2004). This review article introduces the anti-allergic properties and efficacy of flavonoids for patients with Japanese cedar pollinosis and discusses the possibility that an appropriate intake of

© 2012 Tanaka, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Tanaka, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

flavonoids may constitute an effective complementary and alternative medicine as well as a preventative strategy for allergic diseases.

Efficacy of Flavonoids for Patients with Japanese Cedar Pollinosis 105

Flavonols constitute a separate class of flavonoids that possess the 3-hydroxyflavone backbone. Typical flavonoids such as quercetin, kaempferol, fisetin and myricetin belong to flavonols while luteolin and apigenin are classified as flavones. Flavonoids have been found to exert several biological activities including antioxidant, anti-bacterial and anti-viral activities, and to have anti-inflammatory, anti-angionic, analgestic, hepatoprotective, cytostatic, apoptotic, estrogenic or anti-estrogenic and immune-modulating effects as well as anti-allergic properties (Harborne & Williams, 2000; Williams & Grayer, 2004; Chirumbolo, 2010; Visioli et al., 2011; Calderon-Montano et al., 2011; Russo et al., 2012). As a result, considerable interest has been paid to the role of flavonoids in the prevention of chronic diseases, including cardiovascular diseases, cancers, type 2 diabetes, neurodegenerative

Mast cells and basophils expressing the high-affinity IgE receptor (FcεRI) play an important role in allergic inflammation by releasing chemical mediators such as histamine and cyteinyl leukotrienes, cytokines and chemokines (Stone et al., 2010). As for the anti-allergic activities of flavonoids, Fewtress and Gomperts first identified the inhibition by flavones of transport ATPase in histamine secretion from rat mast cells (Fewtrell & Gomperts, 1997). Fisetin, quercetin, myricetin and kaempferol were found to inhibit histamine release while morin and rutin showed little effect. Subsequently, quercetin was reported to inhibit histamine release by allergen-stimulated human basophils (Middleton et al., 1981; Middleton & Kandaswami, 1992). Flavonoids such as apigenin, luteolin, 3,6-dihydroxy flavones, fisetin, kaempferol, quercetin, and myricetin, all with IC50 values of less than 10 μM, were found to inhibit hexosaminidase release from rat mast cells (Cheong et al., 1998). In addition, flavonoids have also been shown to suppress cysteinyl leukotriene synthesis through inhibition of phospholipase A2 and 5-lipoxygenase (Lee et al., 1982; Yoshimoto et al., 1983). As for the suppressive effect of flavonoids on cytokine expression, Kimata et al were the first to report that luteolin, quercetin and baicalein inhibited the secretion of granulocyte macrophage-colony stimulating factor by human cultured mast cells in response to crosslinkage of FcεRI (Kimata et al., 2000a) and subsequently showed that these compounds also inhibited IgE-mediated tumor necrosis factor (TNF)-α and interleukin (IL)-6 production by bone marrow-derived cultured murine mast cells (Kimata et al., 2000b). These findings thus indicate that flavonoids are inhibitors of chemical mediator release and cytokine production by mast cells and basophils. One of the characteristic features of allergic diseases is overproduction of IgE in response to environmental allergens. The differentiation of B cells into IgE-producing cells requires both the interaction of the CD40 ligand with CD40 and the action of IL-4 or IL-13 on B cells (Rosenwasser, 2011), which are provided with these signals by Th2 cells, basophils and mast cells (Gauchat et al., 1993). Basophils were then used to examine the effects of flavonoids on IL-4, IL-13 and CD40 ligand expression. It was found that fisetin suppressed in a dose-dependent fashion both IL-4 and IL-13 synthesis by allergen- or anti-IgE antibody-stimulated peripheral blood basophils and that the IC50 value of fisetin for inhibition of IL-4 synthesis was 5.8 μM (Higa et al., 2003; Hirano et al., 2004). Fisetin also inhibited IL-4, IL-5 and IL-13 production by KU812 cells, a basophilic cell line, in response to the calcium ionophore, A23187 plus phorbol myristate acetate (PMA), but the suppressive effect of fisetin on IL-6, IL-8 and IL-1β synthesis was relatively weak (Higa et

diseases, osteoporosis and allergic diseases (Sealbert et al., 2005).

#### **2. Flavonoids possess anti-allergic activity**

In the mid-1990s we evaluated the clinical efficacy of one kind of traditional vegetarian diet on adult patients with severely to moderately active atopic dermatitis. After a two-month treatment period, the severity of dermatitis had decreased from 49.9 to 27.4 based on the SCORAD index, a score of atopic dermatitis severity, in association with a reduction in the number of peripheral blood eosinophils and the amount of urinary secretion of 8-hydroxy-2'-deoxyguanosine, a marker of oxidative DNA damage (Kouda et al., 2000; Tanaka et al., 2001). What factor(s) led to this amelioration of dermatitis remained unknown but subsequently it was found that one of the characteristics of the remedy was a high daily intake of flavonoids.

**Figure 1.** Structures of basic flavonoid skeletons

Flavonoids are comprised of a large group of low-molecular-weight polyphenolic secondary plant metabolites that are found in fruit, vegetables, cereals and beverages, and thus are common substances in the daily diet (Hollman & Katan, 1999; Middleton et al., 2000). Based on their skeleton, flavonoids are classified into eight groups: flavans, flavanones, isoflavanones, flavones, isoflavones, anthocyanidins, chalcones and flavonolignans (Fig. 1). Flavonols constitute a separate class of flavonoids that possess the 3-hydroxyflavone backbone. Typical flavonoids such as quercetin, kaempferol, fisetin and myricetin belong to flavonols while luteolin and apigenin are classified as flavones. Flavonoids have been found to exert several biological activities including antioxidant, anti-bacterial and anti-viral activities, and to have anti-inflammatory, anti-angionic, analgestic, hepatoprotective, cytostatic, apoptotic, estrogenic or anti-estrogenic and immune-modulating effects as well as anti-allergic properties (Harborne & Williams, 2000; Williams & Grayer, 2004; Chirumbolo, 2010; Visioli et al., 2011; Calderon-Montano et al., 2011; Russo et al., 2012). As a result, considerable interest has been paid to the role of flavonoids in the prevention of chronic diseases, including cardiovascular diseases, cancers, type 2 diabetes, neurodegenerative diseases, osteoporosis and allergic diseases (Sealbert et al., 2005).

104 Current Insights in Pollen Allergens

intake of flavonoids.

preventative strategy for allergic diseases.

**Figure 1.** Structures of basic flavonoid skeletons

**2. Flavonoids possess anti-allergic activity** 

flavonoids may constitute an effective complementary and alternative medicine as well as a

In the mid-1990s we evaluated the clinical efficacy of one kind of traditional vegetarian diet on adult patients with severely to moderately active atopic dermatitis. After a two-month treatment period, the severity of dermatitis had decreased from 49.9 to 27.4 based on the SCORAD index, a score of atopic dermatitis severity, in association with a reduction in the number of peripheral blood eosinophils and the amount of urinary secretion of 8-hydroxy-2'-deoxyguanosine, a marker of oxidative DNA damage (Kouda et al., 2000; Tanaka et al., 2001). What factor(s) led to this amelioration of dermatitis remained unknown but subsequently it was found that one of the characteristics of the remedy was a high daily

Flavonoids are comprised of a large group of low-molecular-weight polyphenolic secondary plant metabolites that are found in fruit, vegetables, cereals and beverages, and thus are common substances in the daily diet (Hollman & Katan, 1999; Middleton et al., 2000). Based on their skeleton, flavonoids are classified into eight groups: flavans, flavanones, isoflavanones, flavones, isoflavones, anthocyanidins, chalcones and flavonolignans (Fig. 1). Mast cells and basophils expressing the high-affinity IgE receptor (FcεRI) play an important role in allergic inflammation by releasing chemical mediators such as histamine and cyteinyl leukotrienes, cytokines and chemokines (Stone et al., 2010). As for the anti-allergic activities of flavonoids, Fewtress and Gomperts first identified the inhibition by flavones of transport ATPase in histamine secretion from rat mast cells (Fewtrell & Gomperts, 1997). Fisetin, quercetin, myricetin and kaempferol were found to inhibit histamine release while morin and rutin showed little effect. Subsequently, quercetin was reported to inhibit histamine release by allergen-stimulated human basophils (Middleton et al., 1981; Middleton & Kandaswami, 1992). Flavonoids such as apigenin, luteolin, 3,6-dihydroxy flavones, fisetin, kaempferol, quercetin, and myricetin, all with IC50 values of less than 10 μM, were found to inhibit hexosaminidase release from rat mast cells (Cheong et al., 1998). In addition, flavonoids have also been shown to suppress cysteinyl leukotriene synthesis through inhibition of phospholipase A2 and 5-lipoxygenase (Lee et al., 1982; Yoshimoto et al., 1983). As for the suppressive effect of flavonoids on cytokine expression, Kimata et al were the first to report that luteolin, quercetin and baicalein inhibited the secretion of granulocyte macrophage-colony stimulating factor by human cultured mast cells in response to crosslinkage of FcεRI (Kimata et al., 2000a) and subsequently showed that these compounds also inhibited IgE-mediated tumor necrosis factor (TNF)-α and interleukin (IL)-6 production by bone marrow-derived cultured murine mast cells (Kimata et al., 2000b). These findings thus indicate that flavonoids are inhibitors of chemical mediator release and cytokine production by mast cells and basophils. One of the characteristic features of allergic diseases is overproduction of IgE in response to environmental allergens. The differentiation of B cells into IgE-producing cells requires both the interaction of the CD40 ligand with CD40 and the action of IL-4 or IL-13 on B cells (Rosenwasser, 2011), which are provided with these signals by Th2 cells, basophils and mast cells (Gauchat et al., 1993). Basophils were then used to examine the effects of flavonoids on IL-4, IL-13 and CD40 ligand expression. It was found that fisetin suppressed in a dose-dependent fashion both IL-4 and IL-13 synthesis by allergen- or anti-IgE antibody-stimulated peripheral blood basophils and that the IC50 value of fisetin for inhibition of IL-4 synthesis was 5.8 μM (Higa et al., 2003; Hirano et al., 2004). Fisetin also inhibited IL-4, IL-5 and IL-13 production by KU812 cells, a basophilic cell line, in response to the calcium ionophore, A23187 plus phorbol myristate acetate (PMA), but the suppressive effect of fisetin on IL-6, IL-8 and IL-1β synthesis was relatively weak (Higa et al., 2003). In order to determine the basic structure of the flavonoids that accounts for their inhibition of IL-4 production and to identify more active compounds, 45 kinds of flavones, flavonols and their related compounds were screened (Hirano et al., 2004; Kawai et al., 2007).

Efficacy of Flavonoids for Patients with Japanese Cedar Pollinosis 107

shown that AhR is a regulator of differentiation of naïve CD4 positive T cells into effector T cell subsets (Marshall & Kerkvliet, 2010), suggesting that flavonoids modulate immune

As mentioned previously, flavonoids are contained in vegetables, fruit, cereals and beverages. Epidemiological studies have reported that a high intake of fresh fruit and vegetables may provide protection against asthma (La Vecchia et al., 1998; Butland et al., 1999). The Mediterranean diet, which has high antioxidant content because of the preponderance of fruit, vegetables, legumes, nuts and wholegrain cereals, has been associated with a reduced likelihood of asthma, wheezing and allergic rhinitis in crosssectional studies of children (Chatzi et al., 2007; Garcia-Marcos et al., 2007; Tamay et al., 2007; De Batlle et al., 2008; Castro-Rodriguez et al., 2008). Shaheen et al reported that the results of a population-based case-control study of 607 cases and 864 controls in South London indicated that apple consumption and red wine intake were negatively associated with, respectively, asthma prevalence and severity, perhaps due to the protective effect of flavonoids (Shaheen et al., 2001), while the follow-up study made it clear that dietary intake of catechins, flavonols and flavones was not significantly associated with asthma prevalence and severity (Garcia et al., 2005). A cohort epidemiological study of 10,054 adults in Finland regarding the association between flavonoid intake and risk of several chronic diseases found that asthma incidence was lower for higher quercetin, naringenin, and hesperetin

A study of 1,253 five-year-old children reported that maternal apple intake during their mothers' pregnancy was associated with beneficial results for ever wheeze, ever asthma and doctor-confirmed asthma (Willers et al., 2007). The Irish Lifeways Cross-Generation Cohort Study determined an association between high maternal fruit and vegetable intake during pregnancy and reduced likelihood of asthma in 632 three-year-old children (Fitzsimon et al., 2007). A third cohort study also demonstrated that wheeze and atopic sensitization in 460 children aged 6-7 years was less frequent if their mothers had followed a Mediterranean diet during pregnancy (Chatzi et al., 2008). Although there have been few reports of case-control or longitudinal studies examining direct associations between flavonoid intake and the prevalence or incidence of allergic diseases, the findings of the epidemiological studies mentioned here suggest that higher flavonoid intake is beneficial for protection against

The anti-allergic characteristics of flavonoids observed *in vitro* led to a study using NC/Nga mice to test whether intake of flavonoids might be effective for the prevention or the amelioration of allergic symptoms. NC/Nga mice spontaneously develop severe eczema, scratching behaviour and serum IgE elevation with aging under nonspecific pathogen-free

**3. The relationship between flavonoid intake and the prevalence,** 

functions through their binding to AhR.

intakes (Knekt et al., 2002).

allergic diseases.

**4. Efficacy of flavonoids in allergic models** 

**incidence or severity of allergic diseases** 

**Figure 2.** Basic structure of flavonoids for inhibitory activity of IL-4 synthesis by basophils

Luteolin, apigenin and fisetin were found to be the strongest inhibitors with an IC50 value of 2.7-5.8 μM (Fig. 2). Quercetin and kaempferol are representative of flavonoids associated with a substantial daily intake and had an intermediate inhibitory effect on IL-4 synthesis with an IC50 value of 15.7-18.8 μM, but myricetin showed no such effect. These analyses of structure-activity relationships revealed the fundamental structure required for the action. For maximal effect, hydroxylation in positions 7 and 4' is essential while the presence of OH in either position 3 or 5 is also required. In addition, luteolin, apigenin and fisetin were found to suppress CD40 ligand expression by activated basophils and KU812 cells in a dosedependent manner, whereas myricetin even at 30 μM did not have such an effect (Hirano et al., 2006). These inhibitory properties indicate that flavonoids such as luteolin, apigenin and fisetin are natural IgE inhibitors.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor that mediates the toxic and biological actions of many aromatic environmental pollutants such as dioxins (Connor & Aylward, 2006). An AhR-based in vitro bioassay for the dioxin [2,3,7,8 tetrachlorodibenzo-*p*-dioxin (TCDD)] revealed that the flavonoids, apigenin, luteolin, baicalein, quercetin, kaempferol and myricetin had noticeable inhibitory effects on AhR activation with an EC70 value (equal to 70% of the maximal response to TCDD) of 1.9-5.1 μM, while marked AhR activation was displayed by daidzein, resveratrol, naringenin and baicalein at higher concentrations (Amakura et al., 2008). Moreover, it has recently been shown that AhR is a regulator of differentiation of naïve CD4 positive T cells into effector T cell subsets (Marshall & Kerkvliet, 2010), suggesting that flavonoids modulate immune functions through their binding to AhR.
