Asthma in the 21st Century — Unexpected Applications of Ancient Treatments

Asthma comes from the Greek word for “panting” and has been described as a pathological condition for centuries. It is a chronic inflammatory disorder of the airways in which many immunological cells play a role, including mast cells and eosinophils. In susceptible individ‐ uals, this inflammation causes symptoms which are usually associated with widespread variable airflow obstruction that is often reversible, either spontaneously or with treatment, and causes an associated airway hyperresponsiveness (AHR) to a variety of stimuli. The clinical features of asthma include dyspnea, wheezing and coughing.


Introduction
Asthma comes from the Greek word for "panting" and has been described as a pathological condition for centuries. It is a chronic inflammatory disorder of the airways in which many immunological cells play a role, including mast cells and eosinophils. In susceptible individuals, this inflammation causes symptoms which are usually associated with widespread variable airflow obstruction that is often reversible, either spontaneously or with treatment, and causes an associated airway hyperresponsiveness (AHR) to a variety of stimuli. The clinical features of asthma include dyspnea, wheezing and coughing.
During the last forty years there has been an increased understanding of the wide spectrum of this disease and as a result a number of effective treatments have been developed. Despite these advances, however, the mortality continues to increase and approximately 500 Canadians and 3500 Americans die each year from asthma. It remains a major cause of morbidity, as the leading cause of school absenteeism and the third leading cause of work absenteeism. The prevalence of asthma in North America has been on a constant rise over the last 25 years and it is estimated that currently over 3 million Canadians and 25 million Americans suffer from asthma. Worldwide the prevalence rates of asthma are rising on average by 50% each decade and developing a better understanding of the risk factors associated with this trend is critical. These may be broadly classified as either host genetic factors or environmental factors ( Table  1).
One of the marked risk factors of asthma associated with the westernized lifestyle is our changing diet and/or nutritional status. It has been hypothesized that the significant change in our diet plays a dominant role in the etiology of asthma. Seaton et al. have proposed that asthma prevalence has increased in UK because of an alteration in diet associated with industrialization [1]. This has lead to a substantial decline in the consumption of fresh fruits, green vegetables, fish and red meat, and as a result decrease in pulmonary antioxidant defences and an increase in susceptibility to inhaled irritants and allergens [1]. These foods are the main sources of antioxidants, substances that protect cells against the effects of free radicals generated during oxidative stress.
Oxidative stress is important in the pathophysiology of asthma [2] and development of AHR [3]. A large number of epidemiologic studies have reported the protective effects of dietary antioxidants such as micronutrients vitamin A, C, and E, polyphenol, and carotenoids against the development of asthma and decline of lung function. In a study on American children higher levels of antioxidants beta-carotene and Vitamin C, along with antioxidant trace mineral selenium is associated with a lower risk of asthma [4]. Dietary vitamin C intake is positively associated with 1 Second Forced Expiratory Volume (FEV 1 ) in children and adults [5][6][7][8] but less frequently with asthma or wheeze in children and adults [4,[9][10][11]. Dietary vitamin E intake is positively associated with ventilatory function [5,6,12] but negatively associated with asthma and wheeze in children [13], adult-onset wheeze [11] and the likelihood of atopic sensitization in adults [14]. Fresh fruits intake is inversely associated with wheeze [15] and chronic lung disease onset [16] and is positively associated with FEV 1 [17]. Total fruit and vegetable intake is inversely related to asthma prevalence [18] but not to FEV 1 [19] or airway obstruction [20]. Vegetables may protect against chronic bronchitis, asthma [21], and wheeze [22]. Moreover, dietary polyphenols intake are associated with lower disease risk with beneficial clinical outcomes attributed to both the antioxidants and anti-inflammatory properties of polyphenols [23]. Polyphenols consist of a large group of natural antioxidants extracted from plants and flavonoids comprise the most studied group.
In addition to antioxidants, intake of fats, particularly the changing composition of polyunsaturated fatty acids (PUFA) in westernized diets, has been implicated in the etiology of asthma. There has been a reduced intake of saturated fat accompanied by an increase in n-6 PUFA consumption, particularly linoleic acid and arachidonic acid. In addition, there has been a decrease in consumption of n-3 PUFA such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Thus, it has been postulated that the increased ratio of n-6:n-3 PUFA in diets of industrialized countries may also contributed to the increased asthma incidence.
Inspite of the advances made in the field of asthma treatments, some patients remain less responsive to conventional therapies than others. Current treatment strategy includes the combinations of bronchodilators, particularly short or long acting β 2 -adrenergic agonists (SABA, LABA), and inhaled and oral corticosteroids. The current approach to the management of asthma includes the addition of drugs in a stepwise fashion based on the severity of symptoms, however the stronger drugs include more severe side effects. The treatment aims to reverse airflow obstruction and reduces asthma exacerbations thus improving quality of life. However, long-term use of high dose inhaled corticosteroids therapy may lead to detrimental effects, such as cataracts [46], osteoporosis in elderly patients [47], and stunting of growth in children [48]. Moreover, the combination therapy may not modify the disease progression and are not curative.
The limited efficacy and side effects associated with conventional treatments has lead to the introduction of nutraceuticals as a "safer"alternative therapy and for those whom symptoms are not improved with current therapies. Nutraceuticals is a very general term which encompasses many classifications of food products and derivatives that have the potential to either prevent or treat pathological conditions in humans or animals. For example, micronutrients such as vitamins and minerals and non-nutritive components of plant products such as polyphenols have some anti-inflammatory activity and have been used to supplement some foods to improve their health benefits. Table 3 summarizes some of the major nutraceuticals used to treat allergy and asthma currently.
The following sections discuss the current knowledge on the effects of nutraceuticals on inflammation associated with asthma with a focus on the cellular and molecular mechanism involved.

Anti-mediator agents
Anti-mediator agents are a group of drugs that antagonize the release of granule-associated preformed mediators, lipid mediators, cytokines, chemokines, and growth factors released by allergen-activated inflammatory cells. Several important groups of specific inhibitors against many of these inhibitors have been developed. The allergen enters tissues through disrupted epithelium. It is then captured by antigen-presenting cells (APCs), usually dendritic cells (DCs), but also alveolar macrophages and B cells [27]. Allergen-loaded DCs migrate to regional lymph nodes where they present peptides to naïve T cells [28]. Presentation of processed allergen in the form of peptide fragments on MHC class II molecules to naïve T cells signals activation and clonal expansion of T cells [27]. In the presence of interleukin (IL)-4, naïve T cells acquire the characteristic of T helper 2 (Th2) cells [29]. Activated Th2 cells secrete greater amounts of IL-4 and IL-13 which triggers the isotype switch to immunoglobulin (Ig)E synthesis [29,30]. IgE produced by B cells diffuses locally, enters the blood and is then distributed systematically. Allergen-specific or non-specific IgE binds to the highaffinity receptor for IgE (FcεRI) on the surface of tissue-resident mast cells and peripheral blood basophils, thereby sensitizing them to future allergen exposures [30].
Using Old Solutions to New Problems -Natural Drug Discovery in the 21st Century

Lipid mediator blockers
Montelukast is a current FDA approved drug used in asthma treatment and serves a prototypical drug for Lipid Mediator Blocking class of drugs. Its mechanism of actions works through the blocking of the CysLT receptor for leukotriene D 4 which reduces bronchoconstriction and inflammation. Zileuton, a related drug in the same class, is a 5-lipoxygenase inhibitor which blocks the synthesis of cysLTs and leukotriene B 4 . These drugs while not natural products serve as a models in the search for nutraceuticals whom may share same or related mechanism of action and therefore may prove useful in asthma management.  The EAR occurs within 30 min of allergen exposure and is principally initiated by mast cell and basophil activation [26]. Mast cells are widely distributed throughout the human respiratory tract and are found in large numbers in the walls of the alveoli and airways [31]. Asthmatics have allergen-specific IgE bound to the FcεRI receptors on mast cell surface. Upon cross-linking of adjacent IgE molecules by allergen, aggregation of FcεRI triggers a complex intracellular signaling process [32]. Activated mast cells release a diverse array of biologically active mediators: preformed granule-associated mediators, lipid-derived mediators, and de novo synthesized cytokines, chemokines, growth factors and other biologically active molecules ( Table 2) [33]. The release of mast cell-derived mediators contributes to acute signs and symptoms associated with EAR that may range from mild rhinitis to anaphylactic shock. These mediators induce vasodilation, contraction of the bronchial smooth muscle (producing airflow obstruction and wheezing) and increased mucus secretion (exacerbating airflow obstruction in the lower airways) [26].

Cytokines blockers
Cytokines exhibit pleiotropy and have overlapping functions in the pathogenesis of asthma, making them a major target for new asthma therapies. Allergic inflammation is driven by an imbalance between Th1 and Th2 cytokines, favoring the Th2 arm of the immune response and inhibition of Th2 cytokines IL-4, IL-5 and IL-13 prevents asthma progression in animal models. Anti-IL-4 administration in mice prevents development of acute and chronic allergic inflammation [53], therefore, natural products that specifically target cytokines or their receptors have the potential to be effective asthma treatments.
Our current pharmacological approach include the use humanized monoclonal antibodies against specific cytokine or receptor targets. This class of drugs, known as the biologics, has been approved for use in treatment of cancer, autoimmune and inflammatory diseases. Omalizumab is a drug currently approved for the management of asthma and is antibody targeting IgE. While not specifically a cytokine blocker it functions through the same mechanism of action. These drugs while very effective carry the risk of unforeseen side effects and under current production treatment costs remain very high ranging upwards from $15,000 to 60,000 per annum. Others examples including humanized IL-4-specific antibodies that block IL-4 receptor α that are under clinical trial [54]. Neutralizing antibodies against IL-5 (Mepolizumab and Reslizumab) and IL-5 receptor α (MEDI-563) remarkably inhibits IL-5 related pathways resulting in reduction of asthma exacerbations [55]. Tralokinumab, an anti-IL-13 monoclonal antibody, prevents the development of asthmatic phenotype, both in murine model as well asthmatic patients [56]. Suplatast tosilate inhibits IL-4 and IL-5 production from T cells and reduces AHR in asthmatic patients.
Some of the phytochemicals and potential treatments indicated against cytokines function are listed in Table 5. These products if proved to be effective, could be cost effective alternatives and being natural products have the potential to have less side effects.

Ginkgetin
Inhibits TNF expression in activated macrophages. Using Old Solutions to New Problems -Natural Drug Discovery in the 21st Century 290
Inhibits TNF and IL-6 production in HMC-1 cells (100 μM). Attenuates production of TNF in lungs of mice with LPS-induced acute lung injury (10 mg/kg body wt. IP).
EPA (120 μM) suppress TNF and IL-1β expression and production in LPS-stimulated alveolar macrophages from asthmatic patients Ref? Table 5. Phytochemical inhibitors of proinflammatory cytokines

Chemokines and chemokine receptors blockers
Chemokines (CC) and their receptors (CCR) play a crucial role in the recruitment of inflammatory cells into the airways and development of asthma. CC-chemokine receptor 3 CCR3, CCR4, and CRTH2 antagonists are being targets currently being evaluated for the treatment of asthma. A study found that treatment of asthmatic mice with an anti-CCR3 monoclonal antibody inhibits allergen-induced eosinophilia and CD34 + progenitor cell infiltration into the lung, which is accompanied by reduced AHR [114,115]. RS-1748, a CCR4 antagonist, inhibits OVA-induced airway inflammation in guinea pigs [116]. The number of CCR4-expressing Th2 cells is increased in the airways of asthmatic patients which can be blocked by a selective CCR4 antagonist [117], and therefore could be an effective therapy for asthma. Ramatroban and closely related TM30089 are antagonists for CRTH2, a chemokine receptor expressed on Th2 cells. They have been shown to attenuate allergen-induced EAR and LAR in animal models of asthma [118,119]. Some of the phytochemicals indicated against chemokines function are listed in Table 6. Inhibits eotaxin production in human dermal fibroblasts (10 μg/ mL). [59,122] Baicalin Reduces IL-8 levels in plasma and BALF in cigarette smoke-induced COPD rat model. Inhibits eotaxin production in human dermal fibroblasts (10 μg/mL). [60,122] Using Old Solutions to New Problems -Natural Drug Discovery in the 21st Century 292

Miscellaneous: IgE, histamine, enzymes
The activation of mast cells involves the cross-linking of IgE bound to the F C εRI surface receptor. Activation is measured in the laboratory by the release of the Beta-hexosaminidase enzyme (β-hex) from cytosolic granules into the interstitial fluid. The following compounds (Table 7) have been found to be inhibitors of β-Hex release in vitro and have to potential to be inhibitor of IgE-antigen activation.

Inhibitors of intracellular signaling pathways
The previous section focused on compounds that affected the function of extracellular, cell surface/receptor and cell to cell interactions. The following group of compounds affect the intercellular functions of the cells particular the components of cell signalling pathways.

Protein kinase inhibitor
Protein kinases have a key role in the expression and activation of inflammatory mediators implicated in airway inflammation. Enhanced activation of p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), spleen tyrosine kinase (Syk), and phosphoinositol 3-kinase (PI3K) signaling pathways have all been proposed to have a role in the pathogenesis of asthma.
p38 MAPK is involved in the airway inflammation and remodeling. A selective synthetic p38 MAPK inhibitor SB2439063 reduces synthesis of Th2 cytokines [54] and thus has a potential application in asthma treatment. Inhaled p38 MAPK antisense oligonucleotide attenuates asthma in OVA-sensitized and -challenged mice [165]. The natural product limonene inhibits eosinophil migration in p38 MAPK dependent manner and was investigation in an in vitro bronchial asthma model [166].
JNK activity is increased in corticosteroid-resistant asthma and SP600125, a JNK inhibitor, reduces cytokines expression and inflammatory cells accumulation in BALF of asthmatic animal models. Celastrol, a natural compound, modulates the expression of JNK in asthma [167]. It supresses allergen-induced mouse asthma by decreasing expression of MAP kinases, ERK and JNK [168].
Syk is a protein kinase involved in signal transduction in many inflammatory cells, and its aberrant regulation is associated with asthma, thus is considered an interesting target for asthma therapies. BAY 61-3606, a synthetic Syk inhibitor, inhibits inflammatory mediator release from mast cells, basophils, eosinophils, and monocytes, and reduces allergic asthma in rats [169]. Eupatilin, a biological extract, inhibits Syk and blocks downstream signaling pathways in mast cell from guinea pig lung tissues, leading to inhibition of mediator release [170]. Thus, Syk inhibitors may have use clinically as a treatment for asthma.
The PI3K pathway plays a major role in the pathogenesis of asthma by promoting eosinophil and neutrophil recruitment and degranulation [171]. Sorbus commixta water extract, an antiinflammatory medicinal plant, remarkably blocks PI3K activity in antigen-activated macrophages, suggesting the usefulness of PI3K inhibitors in asthma [172].

Transcription factor inhibitors
The increased expression of various inflammatory proteins seen in asthma is the result of enhanced gene transcription, since many of the genes are not expressed in normal cells but are selectively induced during inflammation. Changes in gene transcription are under the control of transcription factors which therefore play a key role in the pathogenesis of asthma.
Transcription factors such as nuclear factor-κB (NF-κB), GATA-3, signal transducers and activators of transcription protein (STAT)s, nuclear factor of activated T cells (NFAT), and peroxisome proliferator-activated receptors (PPAR) have been implicated in asthma and therefore represent therapeutic targets.
NF-κB is induced by many factors involved in asthmatic inflammation and is implicated in glucocorticoid-resistant asthma. Inhibition of IκB (inhibitor of NF-κB) by small molecule inhibitors suppresses inflammatory responses in mast cell [173], OVA-induced rat model of airway inflammation [174], and macrophages from BALF of asthmatic patients. A number of herbal preparations (i.e. andrographolide and narigenin) have been demonstrated to inhibit airway inflammation and AHR by inhibiting NF-κB activity in OVA-induced murine asthma [78,175]. Many inhibitors of NF-κB have been identified belonging to the Flavonoids, Polyphenols and Terpenoids classes of compounds as well as n-3 PUFA (see Table 8).  Table 8. Naturally occurring NF-κB inhibitors GATA-3 is a critical transcription factor that is specifically expressed by Th2 cells and is involved in their differentiation [176]. GATA-3 has been indicated in the development of airway eosinophilia and expression is increased in atopic asthma [177] and therefore is an obvious target for inhibition. Direct inhibition by using a specific antisense oligonucleotide or interference RNA promises a novel approach for asthma treatment [178]. Polyphenols, such as apigenin and quercetin, ameliorate asthma symptoms, and suppress the translocation of GATA-3 in the cytosol of lung tissue of OVA-sensitized and -challenged mice [179].
NFAT transcription factor is mostly involved in the production of Th2 cytokines through its interaction with GATA-3 and activator protein-1 (AP-1) [180]. Immunosupressive drugs cyclosporin A and FK506 block NFAT activation [181]. The use of peptides known as inhibitors of NFAT-calcineurin association (INCA) represents an alternative asthma treatment strategy [182].
PPARs are transcription factors belonging to the nuclear receptor superfamily activated by PUFA derivatives, oxidized fatty acids and phospholipids. PPARγ activation might exhibit anti-inflammatory properties in different inflammatory processes. In a murine model of asthma, treatment with PPARγ ligand ciglitazone significantly reduces AHR and lung inflammation [183]. PPARα and PPARγ ligands also decreases allergen-induced AHR, lung inflammation as well as serum IgE levels in different asthma models [184]. Popular antiasthmatic Traditional Chinese Medicine San-ao Decoction (SAD), comprising Herba Ephedrae, Radix et Rhizoma Glycyrrhizae and Seneb Armeniacae Amarum, has a significant effect on PPARγ activation [185].

Inhibitors of oxidative stress
Oxidative stress plays a critical role in the development of asthmatic conditions. Oxidative stress and its by-products drive a Th2-dependent immune response. A number of antioxidants have been explored for their anti-inflammatory and anti-asthmatic properties, and a number of natural products have emerged as promising candidates. Resveratrol, a component of red wine, possesses anti-inflammatory and antioxidant properties. It inhibits inflammatory cytokines release in patients with chronic obstructive pulmonary diseases (COPD) [186] and may be beneficial in asthma. Several other biological compounds such as Sanguisorba offcinalis [187], aqueous extract from the root of Platycodi Radix [188], stem and bark of Ulmus davidiana [189], and Alpinia katsumadai seed extracts [190] attenuate oxidative stress and asthmatic activity in OVA-induced murine asthma. The flavonoids and polyphenols are the main groups of compounds that display anti-oxidative properties as listed in table 9.

Experimental models of asthma
Understanding respiratory sensitization mechanisms is the first step to designing therapeutic agents that may relieve patients of their asthma symptoms. A number of in vitro and in vivo experimental models are able to reproduce one or more features of allergic response and have been studied for a few decades. Animal models of asthma are the best characterized in terms of the inflammatory and remodeling processes. The use of gene knockout and transgenic animals and the therapeutic administration of antibodies or pharmacological antagonists/ inhibitors have helped to identify a range of pre-clinical targets for subsequent evaluation in humans. Small animal models of asthma, using mice, rats and guinea pigs, are most commonly used. Most of these models are based on active sensitization to an allergen such as OVA via the airways. In vitro model systems using inflammatory cells and airway-related cell types are widely used in studies on immuno-biological mechanisms of asthma. A more detailed description of the most commonly used models of asthma can be found in Table 10. Release of proinflammatory mediators such as cytokines and chemokines.

Antioxidants
The airways are continuously exposed to oxidants, either generated endogenously by various metabolic reactions (e.g. from mitochondrial respiration or released from phagocytes) or derived from exogenous sources (e.g. air pollutants and cigarette smoke). Allergen-activated inflammatory cells from asthmatic patients produce more ROS than from healthy individuals. In addition, several inflammatory mediators including histamine, lipid mediators, cytokines, chemokines, ECP, and EPO are potential stimuli for ROS production in the airways, leading to asthma exacerbation.
Deficiency of endogenous antioxidant defenses has been reported in asthma [195]. Since a diet rich in vitamin A or carotenoids, vitamin C vitamin E, and flavonoids, has been associated with a decreased prevalence of asthma, understanding the relationship between dietary antioxidants and asthma-associated inflammatory responses has been a recent focus.

Vitamin A and carotenoids
A systemic review and meta-analysis by Allen et al. has shown that dietary vitamin A intake is significantly lower in asthmatic patients than in healthy subjects [196]. Asthmatic children have a lower serum vitamin A concentration than healthy controls [197]. Supplementation of the diet with lycopene, a carotene found in tomatoes and carrots, has a protective effect against asthma development in a murine model [198].
All-trans retinoic acid (ARTA), a derivative of vitamin A, inhibits airway inflammation in asthmatic rats. ARTA inhibits total cell counts and the proportion of inflammatory cells in BALF, suppresses the expression of NF-κB and intercellular adhering molecule-1 (ICAM-1), and increases the expression of iκB [199]. Retinoid acid also downregulates the expression of Th1 and Th2 chemokines in monocytes, including macrophage-derived chemokine and IP-10, which are all important in the inflammatory process [200]. Airway smooth muscle cell migration, which contributes to the airway remodeling in chronic asthma is also inhibited by ARTA [201]. However, excessive intake of vitamin A exacerbates pulmonary hyperresponsiveness in murine asthma model, suggesting that excessive vitamin A may increase the risk and severity of asthma [202].
Mechanistically, vitamin A may regulate bronchial hyperreactivity by altering the function and abundance of the muscarinic M(2) receptors in bronchial tissue [203]. Moreover, carotenoids may regulate activation of a variety of transcription factors. Treatment of cells exposed to oxidative stress with β-carotene suppresses oxidative stress-induced activation of NF-κB and production of IL-6, TNF, and inflammatory cytokines. Carotenoids may influence the process of apoptosis in healthy cells. While the pro-apoptotic protein Bax is downregulated after induction of external stimuli, β-carotene is able to increase expression of the antiapoptotic protein Bcl-2 in normal cells. In addition, β-carotene exhibits a pro-apoptotic effect in colon and leukemic cancer cells, and this effect occurs by a redox-dependent mechanism linked with NF-κB activity. These dual roles of vitamin A, including carotenoids, on apoptosis provide the capability of carotenoids as an effective anti-inflammatory agent in various diseases.

Vitamin C
Many observational studies have reported associations between reduced dietary/blood vitamin C levels and reduced lung functions. Asthmatic children undergoing an exacerbation have significant lower serum levels of vitamin C [204]. There is a positive correlation between serum vitamin C levels and asthma development in children (OR=0.72 per mg/dl, 95% CI=0.55, 0.95) [10]. Furthermore, asthma patients have significantly lower vitamin C level in both the cellular and fluid-phase fraction in induced sputum [205]. Higher maternal intake of citrus fruits rich in vitamin C during pregnancy is significantly associated with a reduced risk of allergic inflammation in the offspring [206]. Administration of vitamin C in OVA-challenged mice decreases AHR, influx of inflammatory cells in BALF and attenuates lung inflammation [207]. Similarly, high dose vitamin C supplementation significantly reduces eosinophilic infiltration in BALF and increases the Th1/Th2 cytokine secretion ratio; thus, skewing the Th1/ Th2 balance toward non-allergic Th1 immune response in asthmatic mice [208].
A randomized, placebo controlled, double-blinded crossover trial has shown that vitamin C supplementation (1500 mg/day) attenuates asthma symptoms. Moreover, exhaled nitric oxide, urinary leukotriene C 4 , D 4 , E 4 and 9α, 1β-prostaglandin F 2 after exercise are downregulated [209]. On the contrary, there are also studies showing no significant effect of vitamin C supplementation on asthma symptoms. For example, in a randomized, placebo-controlled, double-blind parallel group trial three hundred asthma patients provided with 1 g/day vitamin C or placebo for 6 weeks do not show any improvements of asthma symptoms [210], therefore, there is insufficient evidence from randomised-controlled trials to support the use of vitamin C for asthma treatment [211].
As its mechanism of action, vitamin C may regulate factors that can influence gene expression, apoptosis, and other cellular functions indicated in inflammation. In fact, vitamin C protects Using Old Solutions to New Problems -Natural Drug Discovery in the 21st Century against cell death triggered by various stimuli, and major proportion of this protection is associated with its antioxidant ability [212]. Vitamin C inhibits the AP-1 activation by regulating MAPK-ERK pathway [213]. Treatment of cells exposed to UV-B irradiation with vitamin C results in a 50% decrease in JNK phosphorylation, which activates AP-1, therewith inhibiting the JNK/AP-1 signaling pathways [214]. At present, however, evidence from randomized controlled trials is insufficient to recommend a specific role for vitamin C in the treatment of asthma due to variable study design and generally poor reporting system.

Vitamin E
The body of evidence from multiple studies suggests that a positive association between asthma outcomes and vitamin E intake or serum vitamin E levels. Asthmatic children have significantly lower serum levels of vitamin E than non-asthmatic children [204,215]. A longitudinal birth cohort study has explored association between maternal plasma vitamin E, fetus and fetal lungs growth, and childhood asthma. The findings have shown that maternal vitamin E status has a positive effect on the growth of fetus and fetal lungs during early pregnancy and better asthma outcomes during childhood [216]. Moreover, high maternal vitamin E intake during pregnancy also reduces the risk of infantile wheeze [206]. Vitamin E intake is higher in control subjects than in asthma patients [217]. However there is no relationship found between serum vitamin E level and asthma [4,218]. On the other hand, administration of vitamin E for 6 weeks does not have an effect on asthma features and serum immunoglobulin levels in adults [219].
Role of Vitamin E has been investigated in animal models of allergic asthma. Administration of Vitamin E to allergen-challenged mice reduces mitochondrial dysfunction, Th2 cytokines production, allergen-specific IgE, and expression of lipid mediators in lung leading to alleviation of asthmatic features [220]. Expression of IL-5 mRNA and protein in lung, and plasma IgE level are reduced after OVA sensitization and challenge compared to wild type mice in vitamin E transfer protein knockout mice [221]. Moreover, dietary supplementation with vitamin E affords variable degree of protection against ozone-induced enhanced airway response in allergen-sensitized guinea pigs [222]. However, oral α-tocopherol has no protective effect on lung response in rat model of allergic asthma. There is no improvement in OVAinduced AHR, the inflammatory cell infiltrate and histological changes [223]. The observed opposite effects of vitamin E could be associated with the study design in an animal model of asthma. The effect of vitamin E deserves further evaluation.
Vitamin E may induce immunological effects via modulation of the functional activity of T cells and enhancing the phagocytic activity of peripheral granulocytes [224]. A derivative γtocopherol appears to be a more potent anti-inflammatory agent than α-tocopherol. It decreases systemic oxidative stress, cytokine release from monocytes in asthmatic patients, and inhibits monocyte response to LPS and LPS-induced degradation of IκB and JNK activation [225]. There is a contradictory study demonstrating that γ-tocopherol elevates inflammation and ablates the anti-inflammatory benefit of the α-tocopherol by regulation of endothelial cell signals during leukocyte recruitment in experimental asthma [226]. Dietary tocopherols are taken up from the intestine and transported via the lymph to the blood and then to the liver.
In the liver, α-tocopherol is transferred to plasma lipoproteins, resulting in retention of γtocopherol in tissues at 10% that of α-tocopherol. On interpreting these two contradictory results, one should consider their serum levels with caution since low plasma level of γtocopherol (1.2-7.0 μM) may act as prooxidant, while higher level of γ-tocopherol (19.5 μM at 8 days) exerts antioxidative and anti-inflammatory effects.

Vitamin D
Over the past several years, the role of vitamin D in immunomodulation has been studied and shown to have a significant impact on innate and adaptive immunity to infections, including the pathophysiology of allergic asthma. It has been proposed that the increase in allergy and asthma is a consequence of widespread vitamin D insufficiency which appears to be frequent in industrialized countries, reflecting the insufficient intake of diet-sourced vitamin D.
The serum vitamin D level is associated with asthma in children as well as adults. A randomized, placebo controlled clinical study with 1024 children suffering from mild-to-moderate persistent asthma has shown that Vitamin D deficiency ias associated with a higher rate of severe asthma [227]. There is a significant positive correlation between forced vital capacity percent predicted and serum vitamin D level children with asthma. Moreover, 91.6% of these asthmatic children are not sufficient in serum vitamin D level [228]. Low level of vitamin D in serum are also associated with increased hyperresponsiveness and reduced glucocorticoid response in adults with asthma [229]. These studies have indicated that the low serum vitamin D level is related to reduced lung function and higher risk of asthma. Reduced the risk of asthma exacerbation triggered by acute respiratory tract infection is observed in a vitamin D supplementation [230]. Higher consumption of vitamin D during pregnancy may reduce the risk of childhood wheeze and asthma.
One possible mechanism of vitamin D's protective effect against asthma can be that it inhibits the maturation process of dendritic cells by suppressing the expression of costimulatory molecules HLA-DR, CD86, CD80, the maturation marker CD83, and IL-12 which are important for the recruitment of Th1 cells [231]. Vitamin D also upregulates the expression of IL-10 receptor in dendritic cells, which is an anti-inflammatory cytokine. In addition, it can promote the production of FoxP3 positive and IL-10 positive regulatory T cells, and induce the release of IL-10, TGF-β and CTLA-4 [232]. Furthermore, it may reverse steroid-resistance in asthmatic patients through induction of IL-10 secreting T-regulatory cells [233], and vitamin D has been shown to regulate expression of many genes in ASM cells, including genes previously implicated in asthma predisposition and pathogenesis [234].

Flavonoids
Flavonoids interfere with oxidation of lipids and other molecules and this strong antioxidative property makes them protective against airway diseases linked to oxidative stress. In fact, several epidemiologic studies suggest the beneficial effects of flavonoids on asthma. A population-based case-control study has shown that apple consumption and red wine intake are inversely associated with asthma prevalence or severity, perhaps due to a protective effect Using Old Solutions to New Problems -Natural Drug Discovery in the 21st Century of flavonoids [18]. Moreover, a 30-year longitudinal epidemiological study has reported that the incidence of asthma is lower in populations with higher intake of flavonoids [235]. Beyond antioxidative effects, flavonoids inhibit the release of histamine and other preformed granule associated mediators by inhibiting the activation of basophils and mast cells [66]. Flavonoids inhibit synthesis of IL-4, IL-13, and CD40 ligand but initiate generation of new phospholipid-derived mediators. One of the well-characterized flavonoids, quercetin, inhibits eosinophilic secretion of Charcot-Leyden crystal protein and ECP in a concentration-dependent manner. Very recently, Li et al. demonstrated that apigenin exhibits an anti-inflammatory activity in a murine asthma model and can switch the immune response to allergens toward the Th1 profile. These findings suggest that flavonoids are anti-allergenic and anti-inflammatory agents effective in treating/preventing asthma.
Vascular changes are one of the major components of asthmatic pathogenesis. These changes include an increase in vascular permeability, vascular dilation/engorgement, and vasculogenesis/angiogenesis. Flavonoids and their related compounds have been shown to modulate expression of HIF-1, VEGF, matrix metalloproteinases (MMPs), and epidermal growth factor receptor but also inhibit NF-κB, PI3K/Akt, and ERK1/2 signaling pathways [236]. These observations suggest that flavonoids as well as their related compounds inhibit certain steps of angiogenesis including cell migration, microcapillary tube formation, and MMP expression.
Many flavonoids have been tested for their anti-asthma effect. Quercetin decreases the eosinophil recruitment, reduces IL-5 and IL-4 levels, and inhibits NF-κB activation in BALF in OVA-induced mouse model [237]. It also regulates Th1/Th2 balance by enhancing IFN-γ and decreasing IL-4 levels in mouse asthma model [179]. Naringenin alleviates airway inflamma-tion and reactivity by decreasing serum total IgE level and IL-4, IL-13 level in BALF and inhibiting NF-κB activity [78]. Licorice is a Traditional Chinese Medicine, which contain many flavonoids. Flavonoids extracted from licorice attenuates LPS-induced acute pulmonary inflammation by inhibiting inflammatory cells infiltration and inflammatory mediator release [238]. Neutrophils, macrophages and lymphocytes accumulation in BALF, lung TNF and IL-1β mRNA expression and lung myeloperoxidase activity are reduced; whereas BALF superoxide dismutase activity is increased [238]. Flavonoids from red algae decrease eosinophil infiltration, levels of TNF, IL-4 and IL-5 in BALF, airway luminal narrowing, AHR and level of allergen-specific IgE in the serum [239]. A complex mixture of bioflavonoids derived from purple passion fruit peel extract supplemented to asthma patients in a randomized, placebo-controlled, double-blinded trial alleviates asthma clinical symptoms, including FVC and FEV1 [240].
Recent studies have reported that resveratrol activates sirtuin1 (SIRT1) which is modulates apoptosis and has been shown to increase longevity in some experimental systems [242]. SIRT1 modulates poly (ADP-ribose) polymerase-1 (PARP-1) activity upon DNA damage. Activation of SIRT1 by resveratrol leads to a decrease in PARP-1 activity and promotes cell survival, which can attenuate the inflammatory reaction. We investigated the effects of resveratrol on human mast cell activation in comparison to the anti-allergic drug tranilast. The results show that resveratrol inhibits mast cell degranulation, cytokine, chemokine and leukotriene release, and is more efficacious than tranilast [316].
Resveratrol is able to modulate innate immune response by inhibiting expression of costimulatory molecules (CD80 and CD86) and major histocompatibility complex classes I and II in bone marrow-derived dendritic cells and inhibit angiogenesis pathway that is mediated through expression of MMPs, VEGF, cathepsin D, ICAM-1, and E-selectin [236]. These findings suggest that resveratrol can be a very attractive compound for preventing/treating asthma since this compound displays multiple therapeutic effects, showing antioxidative, antiinflammatory, immune modulating, and vascular protective property.
Resveratrol has been shown to inhibit the airway inflammation and hyperresponsiveness in OVA-induced mouse asthma by reducing eosinophil/neutrophils infiltration, the levels of IL-4 and IL-5 in plasma and BALF [81]. It can modulate Th1/Th2 balance, polarization of naive CD4 + T cells to the Th2 phenotype, and the expression of Th2 regulatory transcription factor, GATA-3 [81]. It also inhibits cytokine release in vitro by alveolar macrophages from patients with COPD, including IL-8 and GM-CSF [186].

Selenium
Selenium is an important molecule in both innate and adaptive immune responses. It stabilizes activated platelets by inhibiting platelet aggregation and secretion of adenine nucleotide, thus possibly blocking the release of acrachidonic acid from platelet membrane [243]. In asthma, platelets participate by acting as inflammatory cells, by releasing mediators, spasmogens and/ or by interacting with other inflammatory cell types [168]. Selenium affects the expression of endothelial cell adhesion molecules, E-selectin, P-selectin, ICAM-1, VCAM-1, and ELAM-1, which are crucial in the inflammatory process for recruitment of inflammatory cells into the target tissue [244].
Some studies have reported that asthma patients have lower selenium level in platelets and serum compared to healthy controls [245,246]. While others studies have found no relationship between serum selenium level and asthma in Japanese and Europe populations [247,248]. Selenium supplementation studies in mouse OVA-induced asthma models have shown that selenium has some protective effects on asthma-associated inflammation. Mice with decreased and increased levels of selenium intake show lower cytokine levels, airway inflammatory cell infiltration, serum anti-OVA IgE, airway hyperreactivity, and phosphorylated STAT-6 levels in the lung compared to medium selenium intake [249]. Selenium supplementation does not show any clinical benefit in adult asthma patients [250].
Despite the data showing positive effects of selenium on some of the pathologies associated with asthma, there are still some conflicting findings of selenium supplementation in animal and human studies. Thus the issue regarding selenium is not conclusive.

Avenanthramides
Avenanthramides (Avns) are extracted from oats and those synthetically prepared exhibit potent antioxidant properties in vitro and in vivo. The antioxidant activity of Avns is 10-30 times greater than that of oats' other phenolic antioxidants such as vanillin and caffeic acid. Avn-C, one of the three major Avns of oats, often comprises about one-third of the total concentration of Avns in oat grain (although the relative proportion of Avns is highly variable), it has the highest antioxidant activity in vitro. By far, these Avns constitute the major phenolic antioxidants present in the oat kernel. The antioxidant activity of Avn-enriched extract of oats has been investigated in laboratory animals. Supplementing the diet of rats at 100 mg/kg diet (providing about 20 mg Avns/kg body weight) has been reported to increase superoxide dismutase (SOD) activity in skeletal muscle, liver, and kidneys, and to enhance glutathione peroxidase activity in heart and skeletal muscles [251]. Supplementation at 200 mg/kg diet, which provides about 40 mg Avns/kg body weight in rats, attenuated the exercise-induced production of ROS [251].
In addition to demonstrating antioxidant activity, Avn compounds may also interact with cellular components, through their interactions with the molecular and signaling pathways that govern cellular responses during inflammation. Using the human aortic endothelial cell (HAEC) culture system, the potentially beneficial health effects of oat Avns was found to be mediated via modulation of the cellular and molecular processes that are known to play an important role in the inflammation of arteries and the development of atherosclerosis [251]. They have been shown to inhibit vascular endothelial cell expression of adhesion molecules, including ICAM-1, VCAM-1, and E-selectin. Suppression of these adhesion molecules resulted in inhibition of monocyte adhesion to HAEC monolayers and reduced production of several inflammatory cytokines and chemokines, including IL-6, IL-8, and MCP-1, the inflammatory components involved in fatty streak formation in arteries. The production of proinflammatory cytokines, chemokines, and adhesion molecules by endothelial cells has been shown to be regulated by redox-sensitive signal transduction involving nuclear transcription factor NF-κB. The above-observed effects of Avns on HAEC and other cells are reported to be mediated through inhibition of NF-κB. More recently, dihydroavenanthramide (DHAv), a synthetic analog of Avn, has been shown to protect pancreatic β-cells from damage via inhibition of NF-κB. In a series of experiments, Guo et al. determined that suppression of the expression of NF-κB activity by Avns is mediated via inhibition of the phosphorylation of IKK and iκB, and by suppression of proteasome activity in endothelial cells [252]. A study by Sur et al. demonstrated anti-inflammatory activity of Avns in skin, inhibiting the degradation of IκB-α in human keratinocytes which correlates with decreased activation of NF-κB and subsequent reduction in IL-8 release [253]. Topical application mitigates skin inflammation in murine model of contact hypersensitivity and neurogenic inflammation and reduces pruritogen-induced scratching in murine itch model [253]. Taken together these observations suggest that Avns are potent anti-inflammatory agents with a potential application in asthma treatment.

Herbal preparations
Herbs have been used to treat airway diseases including asthma for thousands years in many nations, especially in Asian and African countries. In recent decades, some Chinese, Japanese, Indian, and African herbs have been tested for their anti-asthmatic effects.

Boswellia serrata
Boswellia serrata, Indian frankincense, is commonly found in many regions of the world, such as South Asia, Northern Africa, and Middle East. Traditional medicine using extract made from sap, has long been used to treat inflammatory diseases [204]. These extracts contain resin, amino acids, phenols, terpenes, polysaccharides [205] and β-boswellic acid the major active anti-inflammatory component [206].
Extract of Boswellia Serrata or β-boswellic acid has been reported to inhibit hypersensitivity reactions by regulating both the humoral and cellular immune systems They decrease primary antibody synthesis, inhibit polymorphonuclear leukocyte proliferation and infiltration, enhance the phagocytotic function of macrophages, and suppress the classical and alternate complement pathways [254,255] and suppress the inflammation process, one of the critical pathological features in asthma. It has been shown that β-boswellic acid inhibits the production of proinflammatory cytokines, including TNF, IL-1, IL-2, IL-6, IL-12 and IFN-γ by suppressing the activation of NF-κB [256]. It also inhibits histamine release from mast cells challenged with G protein stimulator c48/80 in a dose-dependent manner [257]. β-boswellic acid can downregulate the synthesis of prostaglandins by inhibiting COX-1 in intact human platelets [258].
The synthesis of 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B 4 from rat peritoneal polymorphonuclear leukocytes, which contribute to bronchoconstriction, and increased vascular permeability, are reduced by Boswellia Serrata extract as a result of 5-LO inhibition [259]. These results suggest that Boswellia Serrata might be effective in controlling the inflammation process and contraction of airway smooth muscle in asthmatic condition by inhibiting enzymes required for production of proinflammatory mediators and bronchoconstrictor.

Boswellia serrata
Preliminary clinical investigation has shown Boswellia Serrata's potential therapeutic effect on asthma. In a double-blind, placebo-controlled clinical study [260], 40 patients took 300 mg of extract daily for six weeks, while a control group received a lactose placebo for the same period of time. Lung and immune functions were recorded, including dyspnoea, rhonchi, frequency of attacks, FEV1, FVC, peak expiratory flow rate (PEFR), eosinophil count and erythrocyte sedimentation rate. In the treatment group 70% of patients and 27% in the control group showed improvement in terms of recorded physical symptoms and signs. These results suggest that Boswellia Serrata extract has potential benefit for asthma patients, although the age for control and treatment group was not perfectly matched. However, there is not enough evidence to draw a conclusion on the potential use of Boswellia Serrata for treating asthma in human.

Bromelain
Bromelain is an extract from the pineapple stem, Ananas comosus, containing a mixture of cysteine proteases, peroxidase, acid phosphatase, protease inhibitors, and calcium, with cysteine proteases being the main functional components [214].
Bromelain modulates immune responses both in vitro and in vivo. In vitro, it downregulates mRNA expression of IL-2, IL-4, and IFN-γ in T cells induced by phorbol myristate acetate (PMA), with the mechanism thought to be the inhibitionof p21ras and subsequent ERK-2 [261,262]. In a study using peripheral blood mononuclear cells (PBMC), Bromelain decreases the expression of migration/activation related cell surface markers on leukocyte by proteolysis, including CD14, CD16, CD21, CD25, CD44, CD45RA, CD62L [263]. In addition, it can dosedependently reduce CD25 expression in anti-CD3 antibody-stimulated CD4 + T cells, which is upregulated when T cells are activated in inflammation, autoimmunity and allergy [264].
These results indicate that Bromelain may regulate inflammatory process by interfering the migration and activation of immune cells, primarily T cells. In vivo, Bromelain may inhibit IgG production and decrease IL-2 gene transcription in spleen, and significantly reduce blood CD4 + T cell count [262]. In addition, it downregulates IFN-γ mRNA expression in spleen [265].
These results indicate that Bromelain has regulatory effects on the adaptive immunity, principally by targeting T cell responses.
Bromelain administration via intraperitoneal injection alleviates some of the features of airway inflammation in the OVA-induced murine asthma model. It reduces the total numbers of leukocytes, eosinophils, CD4 + and CD8 + T cells in BALF, and decreases IL-13 concentration, which is a critical mediator for AHR in asthma [266]. In separate study, oral supplementation has been shown to suppress airway methacholine sensitivity, decrease IL-13 level, and eosinophils, CD19 + B cells and CD8 + T cells counts in BAL [267]. These results suggest that Bromelain modulates airway reactivity by altering the presence of leukocytes in airway, which is consistent with the in vitro results mentioned above. However, there is no clinical report available on the use of Bromelain against asthma so far.

Butterbur (Petasites hybridus)
Butterbur is a member of the perennial sunflower family found in Europe and northern Asia. The ancient Greeks used butterbur roots to treat airway diseases and alleviate bronchial spasms [268].

Petasin
Extract from the flower bud, leaves and root have been shown to inhibit β-hexosaminidase release, leukotriene C4/D4/E4 synthesis, and TNF production from IgE-sensitized RBL-2H3 cell [223]. A group of Japanese researchers reported that Japanese butterbur contains multiple active compounds including two eremophilane-type sesquiterpenes, six polyphenolic compounds, and two triterpene glycosides [223], and based on its inhibitory activity on mast cell degranulation, fukinolic acid is believed to be the most active component [269]. Another active component petasin, can reduce leukotriene and ECP production from eosinophils activated Using Old Solutions to New Problems -Natural Drug Discovery in the 21st Century by platelet-activating factor (PAF) or C5a via suppression of cytosolic phospholipase A2 (cPLA2) activity, decreasing intracellular calcium concentration and inhibiting 5-LO translocation from the cytosol to nuclear membrane [270]. Pepsin inhibits leukotriene production from macrophages [271] and suppresses bronchial constriction induced by histamine, carbachol, KCl and leukotriene D 4 in isolated guinea pig trachea [272]. In the OVA murine model, butterbur extract given intranasally together with antigen challenge has been shown to inhibit airway inflammation induced by OVA and hyperresponsiveness to aerosolized methacholine, reduce eosinophil count and decrease Th2 cytokine production including IL-4, IL-5 and RANTES in BALF [227]. These results suggest that Butterbur may have inhibitory effects on proinflammatory mediator release from a broad range of immune cells.
In 2003, a prescription-based Butterbur extract was approved in Switzerland for the treatment of seasonal allergic rhinitis and in response some researchers have tested Butterbur extract for the treatment of asthma. Ziolo et al conducted an open clinical study on its effects on bronchial reactivity in asthma patients. Provided orally in a single dose for three time periods patients show significant improvement on FEV1, especially subjects those in longer treatment group [273]. In another randomized, double-blind, placebo-controlled clinical study, results have shown that the signs of asthma are significantly suppressed by Butterbur treatment including FEV1, exhaled NO, serum ECP and peripheral blood eosinophil count, suggesting Butterbur reduces some of the inflammatory markers associated with allergic respiratory inflammation [274], However, some long term adverse side effects have ben reported including abdominal pain, flatulence, and sneezing in pediatric patients and hair loss, cough, dyspnea, and severe depression for adult patients [230]. More studies with larger sample size are needed for the evaluation of Butterbur's clinical use on asthma.

Curcumin
Curcumin is a yellow polyphenol compound, extracted from the rhizomes of Curcuma longa [231]. In ancient time, curcumin containing turmeric plants were widely used to treat swelling and wounds in Southern Asia [275]. Many pharmacological effects of curcumin have been reported, including antioxidative, antiinflammatory and antimicrobial activities [276]. In terms of its antioxidative effects, curcumin is thought to be more potent than vitamin E [277] with the mechanism including downregulation of NO production, scavenging free radicals, and inducing heme oxygenase-1 to repair the oxidative damage caused by free radicals [278][279][280]. Curcumin can inhibit the production of proinflammatory cytokines such as IL-1β and IL-8, suppress inducible iNOS and NO production, and modulate steroid activity. It's effect on steroid activity may be the result of inhibition of NF-κB through blocking IKK activity [281][282][283][284].
During allergic inflammation, curcumin may modulate both early and late phase responses by altering Th2 responses. In a murine latex-induced allergy model, characterized by an increased serum total IgE and latex specific IgG 1 , elevated peripheral blood eosinophils count, and enhanced lung tissue IL-4, IL-5 and IL-13, intragastric curcumin administration reduces lung inflammation. Protein expression of costimulatory molecules CD80, CD86, and OX40-Ligand, and RNA expression of MMP-9, ornithine aminotransferase (OAT), and thymic stromal lymphopoietin (TSLP) in antigen-presenting cells are all decreased. These results suggest that curcumin may disrupt antigen presentation, so that has potential therapeutic value on allergen triggered airway inflammation [285].
Curcumin has been shown to have anti-asthmatic effects in both in vivo and in vitro studies. In OVA-induced asthma model in guinea pigs, curcumin treatment during OVA sensitization or following antigen challenge shows significant protective effects through attenuation of bronchial constriction and hyperreactivity [286]. This indicates curcumin has both preventive and therapeutic effects on asthma. In another study in an OVA-induced murine asthma model, curcumin's anti-asthmatic function is attributed to the suppression of iNOS and subsequent NO production, inhibition of inflammatory cytokine synthesis and downregulation of eosinophil recruitment to airway [91]. In vitro, curcumin supplementation inhibits IgE/antigen activation of mast cells through the principal activation pathway mediated by FcεRI directly inhibiting Syk kinase phosphorylation, which is critical for the propagation of signaling cascade. Subsequently, the phosphorylation of MAP kinases including p38, ERK 1/2 and JNK are supressed, which are crucial for gene transcription and production of proinflammatory cytokines [287]. In addition, curcumin inhibits HDM-induced lymphocyte proliferation and production of IL-2, IL-4, IL-5, and GM-CSF by lymphocytes from asthma patients [246]. These results indicate that curcumin may attenuate asthma symptom by inhibiting production of cytokines related to eosinophil function and IgE synthesis, and suppressing IgE-mediated reactions and hyperreactivity.

Licorice root (Glycyrrhiza glabra)
Licorice root has been widely used around the world to treat cough since ancient time [247]. It contains the active compounds including glycyrrhizin, glycyrrhetinic acid, flavonoids, isoflavonoids, and chalcones [248]. Glycyrrhizin and glycyrrhetinic acid are considered to be the main active components [249] and are potent inhibitors of cortisol metabolism, due to their steroid like structures inhibiting the key steroid metabolic enzymes, delta 4-5-reductase, 11 beta-hydroxysteroid dehydrogenase and 20-hydroxysteroid dehydrogenase [250,251]. Therefore, the benefits and side effects of steroid are both expected to be enhanced in the presence of glycyrrhetinic acid and glycyrrhizin. The anti-inflammatory effect of glycyrrhizin during virus infection has been well documented [288][289][290] and may alleviate allergic inflammation as well. In a contact skin hypersensitivity mouse model, glycyrrhizin and its metabolite 18 β-glycyrrhetinic acid-3-O-β-D-glucuronide show protective effects in terms of reduced passive cutaneous anaphylaxis and inflammation, with glycyrrhizin being more potent than 18 β-glycyrrhetinic acid-3-O-β-D-glucuronide [291].
In an OVA-induced murine asthma model, glycyrrhizin provided orally alleviates airway constriction and hyperreactivity, pulmonary inflammation. In BAL, IFNγ level is increased, while IL-4, IL-5 levels and eosinophil count are decreased. It also reduces OVA-specific IgE levels and upregulates total IgG 2a in serum as well [292]. These results indicate that glycyrrhizin interfere the production of IgE by decreasing the IgE-stimulating cytokines. not liquiritigenin reduces the level of IgE in serum [293]. Flavonoids extracted from licorice root quench LPS-induced pulmonary inflammation by inhibiting the recruitment of neutrophils, macrophages and lymphocytes in BALF, and suppressing the mRNA expression of TNF and IL-1β in LPS-challenged lung tissue in mice [238]. The reported side effects of licorice root includes headache, hypertension, hypokalemia, premature birth, muscle weakness, and increase body weight, which were attributed to its function on inhibiting the steroid metabolism [294].

Modified Mai-Men-Dong-Tang
Mai-Men-Dong-Tang is an old Chinese herb formula commonly used for treating lung diseases, which contains Ophiopogon, Ginseng, Pinellia, Licorice, Jujube, and Oryza [260]. It is reported to increase the cough threshold to inhaled capsaicin in asthmatic patients. Also, the eosinophil count in peripheral blood, sputum eosinophil ratio, and serum eosinophil cationic protein level are significantly decreased, especially in patients with severe airway inflammation [261], which suggests that Mai-Men-Dong-Tang may alleviate asthma-related cough by inhibiting eosinophil function.
Modified Mai-Men-Dong-Tang (mMMDT) contains five herbs, Ophiopogon, American ginseng, Pinellia, Licorice root, and Lantern tridax [262]. The efficacy and safety of this formula to persistent, mild to moderate asthma has been evaluated in a double-blind, randomized clinical study of 100 patients with mild to moderate asthma. After 4 months, improvements in FEV1 and symptom scores has been reported in mMMDT treatment groups with decreased serum IgE and no drug-related adverse effects seen in terms of blood test, and liver, kidney functions [295]. Modified Mai-Men-Dong-Tang is a potential effective herb formula in treatment of childhood asthma for long time use. However, recommendation cannot be made because of small sample size used in the study.

Ding-Chuan-Tang
Ding-Chuan-Tang (DCT) is a traditional Chinese herb formula used for the treatment of cough, wheezing, and chest tightness, developed about four hundred years ago during the Ming dynasty. This formula contains nine herbs including Radix glycyrrhizae, Tuber pinellia, Gingko bilboae, Herba ephedrae, Flos tussilaginis farfarae, Cortex mori albae radicis, Fructus perilla frutescens, Semen pruni armeniacae, Radix scutellariae baicalensis [263]. In OVA-induced pig asthma model, DCT given orally to animals 30 min before antigen challenge inhibits the antigen induced immediate asthmatic responses. If it is given together with sensitization, immediate and late asthmatic responses are all suppressed. In addition, DCT relaxes trachea contracted with carbachol. The effects are attributed to decreased eosinophil infiltration to airway [263].
Randomized double-blind, placebo-controlled study to assess the effect of DCT on airway hyperreactivity in children with mild to moderate persistent asthma has shown that the FEV1 is significantly increased in DCT group (196%, p=0.034), but not in placebo control group. Compared to placebo control group, total clinical/medication score shows improvement in the DCT group (p=0.004). No side effects have been reported [296]. These results suggest that DCT Using Old Solutions to New Problems -Natural Drug Discovery in the 21st Century might be effective in treating asthma in children. Larger sample size and wider population are required in further investigations.

STA-1 and STA-2
STA is a combination of mMMDT and another Chinese herb formula Liu-Wei-Di-Huang-Wan (LWDHW), which is also used by Chinese as an anti-cough agent. LWDHW contains six herbs including Rehmannia root, Alisma rhizome, Dioscorea rhizome, Poria, Hoelen, Moutan root bark, Shanzhu yu. The formula for STA-1 and 2 are the same while the only difference being in the preparation of LWDHW is different [265]. In a mouse asthma model induced by intraperitoneally administrated dermatophagoides pteronyssinus group 5 allergen (Der p 5), oral STA-1 treatment during sensitization suppresses Der p 5-specific IgE production from animals in response to inhaled Der p 5 challenge. In addition, eosinophil and neutrophil airway infiltration, and airway hyperreactivity are all significantly reduced in STA-1 group compared to control animals [266]. The efficacy and side effects of STA-1 and STA-2 on childhood asthma treatment have been evaluated in a randomized, double-blind, placebo-controlled study. The herbs and placebo provided to pediatric patients with mild to moderate asthma reduces symptom scores, serum steroid concentration, total IgE, and allergen-specific IgE levels and improves FEV1 in the STA-1 group. STA-2 does not show protective effects. No severe side effects were reported [297]. These results indicate that STA-1 might be a valuable formula for childhood asthma, especially subjects induced by dust mite antigen. However, there is not enough evidence to draw a concrete conclusion. It is worthwhile to evaluate their potential use as immunotherapy as well.

Anti-Asthma Herbal Medicine Intervention (ASHMI)
ASHMI is a relatively new formula developed by a group of Chinese researchers and physicians, which is an extract from three herbs: Radix glycyrrhizaen prednisone, Radix sophorae flavescentis, and Ganoderma [267]. In OVA-induced asthma, oral ASHMI treatment before and during OVA sensitization and challenge reduces AHR represented by time-integrated change in peak airway pressure. Eosinophil infiltration in BALF, lung inflammation, OVA-specific IgE production, and level of IL-4, IL-5, and IL-13 in lung and splenocyte cultures are significantly lower in ASHMI treated mice, whereas IFN-γ production is increased [267,268]. A 6-week treatment of ASHMI beginning 24 hr after the first OVA challenge in mice reduces early phase response by decreasing histamine, leukotriene C, and OVA-specific IgE levels, and suppresses late phase responses by decreasing eosinophil count and Th2 cytokines in BALF. In addition, it relieves contraction of murine tracheal rings by increasing the production of PGI 2 [269]. These results suggest that ASHMI inhibits asthmatic inflammation and airway muscle contraction, primarily by inhibiting Th2 cell function and might be suitable for treating antigen-induced asthma in both young and old subjects.
In clinical trial, ASHMI has been shown to improve lung function indicated by increased FEV1 and peak expiratory flow. Clinical symptom scores, use of β2-bronchodilators, serum IgE level, serum IL-5, IL-13 concentrations are all reduced, and some effects are even better than prednisone. During the study no adverse effect were recorded [298]. These results indicate the effectiveness of ASHMI on treating asthma in both young and old adult patients. More adequately powered investigations are needed to evaluate ASHMI's effect on asthma.

n-3 polyunsaturated fatty acids
PUFA are a group of fatty acids with more than two carbon-carbon double bonds. There are three types of PUFA, n-3, n-6 and n-9, with their names based on the position of first double band from methyl end in their chemical structures. Currently, many studies have focused on n-3 and n-6 PUFA because EPA (20:4 n-3), Dihomo-γ-Linolenic acid (DGLA, 20:3 n-6) and Arachidonic acid (AA, 20:4 n-6) in cell membrane can be metabolized and become eicosanoid precursors, which are important modulatory autocrine molecules. Eicosanoids include prostaglandins, leukotrienes, thromboxanes, resolvins, lipoxins, are signal molecules that exert complex effects on health. They can modulate inflammation, fever, blood pressure, the immune system, etc. Eicosanoids can be made by oxidation of twenty carbon n-3 (EPA) and n-6 (DGLA, AA) PUFA. Eicosanoids from AA are proinflammatory, while those from EPA and DGLA are less so. There is competition between n-3 PUFA and n-6 PUFA in oxidation in terms of cyclooxygenase and lipoxygenase, which are critical enzymes for eicosanoid generation. AA is the predominant n-6 PUFA in body. In general, n-3 and n-6 are hypothesized to be beneficial and detrimental respectively [299,300]. Fish, fish oil, krill, mussel and seal oil are natural sources of n-3 PUFA.
The major n-3 PUFA are listed in Table 12. In mammals, including humans, n-3 PUFA cannot be synthesized de novo. Therefore they must be absorbed through the diet or produced from α-Linolenic acid (ALA), which is an essential fatty acid. Among them, the health beneficial effects of EPA and DHA (22:6 n-3) are well documented in a broad range of health and disease conditions. The consumption of EPA and DHA are associated with lower risk of cancer, hyperlipidemia, and cardiovascular disease, high blood pressure, and neurodegenerative diseases [301][302][303][304]. Their regulating function on immune system was also well known and are involved in activation of immune cells like of T cells, B cells, mast cells and basophils [305,306].
In recent decade, the relationship between n-3 PUFA and inflammatory diseases has been investigated in many studies. In a study conducted in rheumatoid arthritis patients, significant improvement in symptoms have been reported after 3 month fish oil supplementation in terms of tender joint count and duration of morning stiffness [307]. Besides reduction in the production of proinflammatory eicosanoids by competition with n-6 PUFA, n-3 PUFA has been found to be effective in inhibiting the synthesis of proinflammatory cytokines. In fat-1 transgenic mice, which have a much lower n-6:n-3 PUFA in tissues because they are genetically modified to possess the ability to convert n-6 PUFA to n-3 PUFA, serum proinflammatory cytokines, including TNF, IL-1β, and IL-6 are lower. [280]. DHA and ALA also reduce the mRNA expression of IL-1β, IL-6 in a cerulein-induced pancreatitis model. They inhibit the activation of AP-1, suppress DNA fragmentation and decrease mRNA expression of apoptotic genes including p53, Bax and apoptosis-inducing factor in hydrogen peroxide-treated pancreatic acinar cells [308]. A randomized, double-blind human study has confirmed their suppressing effect on production of proinflammatory cytokines [309]. It has been shown that n-3 fatty acids can alleviate inflammatory process by modulating cell signalling pathways in immune cells, such as T cell receptor pathway and cytokine receptor pathways. [310][311][312] There have been a number of clinical studies that have shown n-3 PUFA's potentially protective effects on asthma, especially on childhood patients. There is a positive association between the n-6:n-3 PUFA in diet and risk for asthma [313]. A randomized, double-blind, placebocontrolled 3-year study on effect of n-3 PUFA supplementation on asthma has found that high n-3 PUFA diet intervention significantly reducesd the prevalence of cough in atopic children, suggesting that n-3 PUFA may be effective in preventing the development of asthma in early childhood [314]. In a cohort study on the relations between fish/cod oil intake and asthma, results have shown that adults with low fish intake frequency (less than weekly) have increased risk to have asthma [315]. Another randomized double-blind study with 5-weeks n-3 PUFA supplementation has reported a significant decrease in exhaled NO from asthma patients challenged with mite allergen. Serum eosinophils count and ECP, and the production of CysLTs from isolated leukocyte stimulated with mite antigen are also reduced [194]. Overall, n-3 PUFA might be a promising remedy agents for allergic diseases like asthma but the mechanism remains to be elucidated.

Conclusion
The prevalence of asthma is becoming the mortality and morbidity pandemic of the 21 st century. The cost of in quality of patient's lives and economic burden of treatment is continuing to grow at pace unmatched in our current health system. It is impossible to enter public classroom now without seeing a young sufferer of this condition and any trip to the emergency department will show how dangerous this disease can be. As the incidence and severity of the disease continues to rise, medical research is continuing to search new treatment strategies. While many treatments currently exists those reserve for the severest of conditions carry their own inherent risk which may match the severity of disease itself. It is for these reasons alone that health care professionals are now examining the traits of our ancestors in time when this epidemic was less severe to determine if their medicines and practices hold the answer for the next treatment strategy. By combining the scientific knowledge at the molecular and clinical level and the resources of past it might hold the answer to breathless pandemic of the 21 st century.