**3. Common molecular targets used in current asthma therapy**

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 β2-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

**Figure 1.** AHR and bronchial inflammation starts with the inhalation of an allergen. 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 sen‐ sitizing them to future allergen exposures [30].

are not improved with current therapies. Nutraceuticals is a very general term which encom‐ passes 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.

#### **3.1. 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.

**Figure 2.** The EAR occurs within 30 min of allergen exposure and is principally initiated by mast cell and basophil acti‐ vation [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 intracel‐ lular 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 fac‐ tors 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 media‐ tors 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].

#### *3.1.1. Lipid mediator blockers*

are not improved with current therapies. Nutraceuticals is a very general term which encom‐ passes 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

**Figure 1.** AHR and bronchial inflammation starts with the inhalation of an allergen. 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 sen‐

YY <sup>Y</sup>

IgE

Naive T cell Th2 cell

IL-4 IL-13

B cell

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

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

used to treat allergy and asthma currently.

sitizing them to future allergen exposures [30].

Dendritic cell

Mast cell

MHC class II

Allergen exposure

280 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

Fc RI receptor

TCR

many of these inhibitors have been developed.

involved.

**3.1. Anti-mediator agents**

Montelukast is a current FDA approved drug used in asthma treatment and serves a proto‐ typical drug for Lipid Mediator Blocking class of drugs. Its mechanism of actions works through the blocking of the CysLT receptor for leukotriene D4 which reduces bronchocon‐ striction and inflammation. Zileuton, a related drug in the same class, is a 5-lipoxygenase inhibitor which blocks the synthesis of cysLTs and leukotriene B4. 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. Antag‐ onists of the prostaglandin D2 receptors DP1 and CRTH2 reduce inflammation in a murine model of asthma, possibly by inhibiting prostaglandin synthesis [49, 50]. Antagonist of the leukotriene B4 receptor BLT1 (R05101576) prevents airway inflammation and AHR in animal models and non-human primates [51]. Quercetin and luteolin, flavonoids found in fruits, vegetables and wine, inhibit the release of leukotrienes and PGD*2* from human cultured mast cells [52]. Table 4 summarizes phytochemicals that act on pathways related to the synthesis of lipid mediators in allergic inflammation.

**Figure 3.** The LAR typically develops 2-6 hr following allergen challenge, often peaks after 6-9 hr, and has a more se‐ vere and prolonged phase. In general, allergen activated mast cells release various *de novo* synthesized cytokines, che‐ mokines and growth factors, which are released more slowly than granule-associated mediators [34]. Thus, LAR is sustained by *de novo* synthesized mast cell-derived mediators which recruit inflammatory cells to the airways several hours after allergen challenge. These recruited cells include effector cells, such as eosinophils, basophils, neutrophils, macrophages, T cells, and DCs [34, 35]. These inflammatory cells are activated when they reach the airway and pro‐ duce a vast array of inflammatory mediators that act on specific receptors and exacerbate airway inflammation and airway remodeling. Eosinophils are the central effector cells in the LAR [36], and are present not only in the airway wall [37] but are also found in large numbers in the sputum and bronchoalveolar lavage fluid (BALF) [38]. Eosinophils are a rich source of granule basic proteins (EBP), such as major basic proteins (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), and can also generate lipid-mediators (prosta‐ glandins and cysteinyl leukotrienes), cytokines (such as TNF, TGF-β, IL-4 and IL-13) and chemokines [39]. These eosino‐ phil-derived products promote some of the pathophysiological hallmarks of asthma such as AHR [40]. The activation of peripheral blood neutrophils during allergen challenge results in their intravascular migration, adhesion to the en‐ dothelium, and migration to the site of inflammation and can be responsible for significant damage. Nocturnal asth‐ ma is associated with high levels of neutrophils, which correlate with the severity of the disease [41]. Furthermore, in a small number of patients who died of sudden-onset asthma, the predominant cell type in the sputum is the neutro‐ phils, not eosinophils [42]. Neutrophils also predominate more frequently in the sputum of patients with acute exacer‐ bations of asthma, mostly associated with respiratory tract infection [43]. T cells are not only important during the induction phase, but play also a very important role during ongoing inflammation. Th2 cells and their cytokines are crucial for promoting acute hypersensitivity responses, and for maintaining the state of chronic and relapsing eosino‐ phil-predominant inflammation that is characteristic of chronic allergic inflammation. Elevated levels of CD4+ T cells are observed in the bronchial mucosa of biopsy samples, BALF and sputum from patients with asthma [44]. In a major‐ ity of studies, T cells found in asthmatic patients express cytokines or transcription factors characteristic of Th2 cells, especially IL-4, IL-5, IL-9 and IL-13 [45].


**Table 2.** Mast cell-derived proinflammatory mediators

Th2 cell

TNF

muscle cells Blood

MMPs

Airway smooth

especially IL-4, IL-5, IL-9 and IL-13 [45].

Mast cell

IL-3

TNF IL-13

IL-13, IL-5

Epithelial cells

Neutrophil Eosinophil Basophil

IL-8, TNF GM-CSF

IL-9

GM-CSF IL-13, IL-5

IL-13

282 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

vessel

IL-13 CysLTs

CysLTs EBPs

Bronchoconstriction Vasodilation Mucus production

**Figure 3.** The LAR typically develops 2-6 hr following allergen challenge, often peaks after 6-9 hr, and has a more se‐ vere and prolonged phase. In general, allergen activated mast cells release various *de novo* synthesized cytokines, che‐ mokines and growth factors, which are released more slowly than granule-associated mediators [34]. Thus, LAR is sustained by *de novo* synthesized mast cell-derived mediators which recruit inflammatory cells to the airways several hours after allergen challenge. These recruited cells include effector cells, such as eosinophils, basophils, neutrophils, macrophages, T cells, and DCs [34, 35]. These inflammatory cells are activated when they reach the airway and pro‐ duce a vast array of inflammatory mediators that act on specific receptors and exacerbate airway inflammation and airway remodeling. Eosinophils are the central effector cells in the LAR [36], and are present not only in the airway wall [37] but are also found in large numbers in the sputum and bronchoalveolar lavage fluid (BALF) [38]. Eosinophils are a rich source of granule basic proteins (EBP), such as major basic proteins (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), and can also generate lipid-mediators (prosta‐ glandins and cysteinyl leukotrienes), cytokines (such as TNF, TGF-β, IL-4 and IL-13) and chemokines [39]. These eosino‐ phil-derived products promote some of the pathophysiological hallmarks of asthma such as AHR [40]. The activation of peripheral blood neutrophils during allergen challenge results in their intravascular migration, adhesion to the en‐ dothelium, and migration to the site of inflammation and can be responsible for significant damage. Nocturnal asth‐ ma is associated with high levels of neutrophils, which correlate with the severity of the disease [41]. Furthermore, in a small number of patients who died of sudden-onset asthma, the predominant cell type in the sputum is the neutro‐ phils, not eosinophils [42]. Neutrophils also predominate more frequently in the sputum of patients with acute exacer‐ bations of asthma, mostly associated with respiratory tract infection [43]. T cells are not only important during the induction phase, but play also a very important role during ongoing inflammation. Th2 cells and their cytokines are crucial for promoting acute hypersensitivity responses, and for maintaining the state of chronic and relapsing eosino‐ phil-predominant inflammation that is characteristic of chronic allergic inflammation. Elevated levels of CD4+ T cells are observed in the bronchial mucosa of biopsy samples, BALF and sputum from patients with asthma [44]. In a major‐ ity of studies, T cells found in asthmatic patients express cytokines or transcription factors characteristic of Th2 cells,

membrane-bound antioxidant employed by cells. Its main antioxidant function is protection against lipid peroxidation. There is an interaction between Vitamin E and other nutrients, particularly selenium and vitamin C in the antioxidant role. Vitamin E is found in fruits (tomato, mango, and papaya), green leafy vegetables (lettuce, spinach, turnip, and beet), nuts and nut oils (almonds and hazelnuts), vegetable oils (wheat germ oil, sunflower oil, and safflower oil), meat, and poultry.

Flavonoids

Isoflavones

Flavonoids constitute the most important single group of polyphenols of low molecular weight polyphenolic secondary plant metabolites, with more than 8,000 compounds described. They are found in fruits, vegetables, nuts, seeds, stems, flowers, roots, tea, wine, and coffee and are common substances in our daily diet. Their structure is a heterocyclic hydrocarbon, chromane, and substitution of its ring C in position 2 or 3 with a phenyl group (B-ring) results in flavans or isoflavans. An oxo-group in position 4 leads to flavanones and isoflavanones. The presence of a double bond between C2 and C3 provides flavones and isoflavones. An additional double bond in between C1 and C2 makes these compounds colourful anthocyanidins. Based on their structure, flavonoids are categorized into eight groups:

Resveratrol

HO

OH

membrane-bound antioxidant employed by cells. Its main antioxidant function is protection against lipid peroxidation. There is an interaction between Vitamin E and other nutrients, particularly selenium and vitamin C in the antioxidant role.

Vitamin E is found in fruits (tomato, mango, and papaya), green leafy vegetables (lettuce, spinach, turnip, and beet), nuts and nut oils (almonds and hazelnuts), vegetable oils (wheat germ oil, sunflower oil, and safflower

Flavonoids constitute the most important single group of polyphenols of low molecular weight polyphenolic secondary plant metabolites, with more than 8,000 compounds described. They are found in fruits, vegetables, nuts, seeds, stems, flowers, roots, tea, wine, and coffee and are common substances in our daily diet. Their structure is a heterocyclic hydrocarbon, chromane, and substitution of its ring C in position 2 or 3 with a phenyl group (B-ring) results in flavans or isoflavans. An oxo-group in position 4 leads to flavanones and isoflavanones. The presence of a double bond between C2 and C3 provides flavones and isoflavones. An additional double bond in between C1 and C2 makes these compounds colourful anthocyanidins. Based on their structure, flavonoids are categorized into eight groups:

oil), meat, and poultry.

CH3

OH

H3C

**Antioxidants**

Vitamin D

Flavonoids

HO

D

CD2

O

O Flavones

O

O Isoflavones

CH3

H

H3C

284 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

H

OH

Resveratrol is a stilbenoid, a type of natural phenol, and a phytoalexin produced naturally by several plants when under attack by pathogens such as bacteria or fungi. Resveratrol is found in the skin of red grapes and in other fruits. It is sold as a nutritional supplement derived primarily from Japanese knotweed.

Selenium is derived from both vegetable and animal products, particularly seafood, liver, and cereals. As a member of the sulfur family of elements, it shares several chemical properties with sulfur, including valence states and the ability to form covalent bonds with carbon. It is unique among antioxidants in that it exerts its biological effects through direct incorporation into proteins (selenoproteins) as the amino acid selenocysteine. Some selenoproteins that have been characterized as important antioxidant enzymes include GPX-1, GPX-4, thioredoxin reductase-1 and thioredoxin reductase-2, and selenoprotein P. The selenium-dependent enzyme, GPX recycles glutathione, reducing lipid peroxidation by catalyzing the reduction of peroxides, including hydrogen peroxide.

Oats contain unique, low-molecular-weight, soluble phenolic compounds called avenanthramides (Avns), which are not present in other cereal grains. These compounds are antipathogens (phytoalexins), which are produced by the plant in response to exposure to pathogens

Selenium

e

such as fungi. Avns are conjugates of a phenylpropanoid with anthranilic acid or 5 hydroxy anthranilic acid. More than 20 different forms of Avns are present when extracted from oats, and the three major forms are A, B, and C.

**Table 3.** Some of the dietary nutraceuticals indicated in asthma prevention.



**Phytochemicals**

4,3'-dihydoxy-5'methoxystilbene

**Table 3.** Some of the dietary nutraceuticals indicated in asthma prevention.

286 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

inhibits Cyclooxygenase 1

Apigenin Inhibits Cyclooxygenase 2, 12-Lipoxygenase, 15-Lipoxygenase Artonin E Inhibits Cyclooxygenase 1, 5-Lipoxygenase, 12-Lipoxygenase, 15-

Baicalein Inhibits Cyclooxygenase 1, Cyclooxygenase 2, 5-Lipoxygenase, 12- Lipoxygenase, 15-Lipoxygenase

Fisetin Inhibits Phospholipase A2, 5-Lipoxygenase, 12-Lipoxygenase, 15-

Lipoxygenase

Lipoxygenase

**Flavonoids Target/Function**

4,3',5'-trihydroxystilbene Inhibits Cyclooxygenase 1

4-hydoxy-3'5'-dimethoxystilbene Inhibits Cyclooxygenase 1 Acacetin Inhibits Cyclooxygenase 1

Buddledin A Inhibits Cyclooxygenase 1

Chrysol Inhibits 5-Lipoxygenase

Daidzein Inhibits Cyclooxygenase 2 Epicatechin Inhibits 5-Lipoxygenase Epigallocatechin Inhibits 5-Lipoxygenase

Flavone Inhibits 5-Lipoxygenase Gambogenic acid Inhibits Cyclooxygenase 2 Genistein Inhibits Cyclooxygenase 2

Andanthoflavone Inhibits 12-Lipoxygenase, 15-Lipoxygenase

Bicalin Inhibits Cyclooxygenase 1, Cyclooxygenase 2

Chrysin Inhibits Cyclooxygenase 1, 5-Lipoxygenase

Cirsiliol Inhibits 5-Lipoxygenase, 12-Lipoxygenase

such as fungi. Avns are conjugates of a phenylpropanoid with anthranilic acid or 5 hydroxy anthranilic acid. More than 20 different forms of Avns are present when extracted from oats, and the three major

forms are A, B, and C.


**Table 4.** Phytochemical inhibitors of lipid mediators

#### *3.1.2. 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 inflam‐ mation [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 mecha‐ nism 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 (Mepoli‐ zumab 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.

Curcumin Inhibits Phospholipase A2, Cyclooxygenase 1, Cyclooxygenase 2, 5-

Lipoxygenase

Ginkgetin Inhibits Phospholipase A2, 5-Lipoxygenase

n-3 PUFA Inhibits Cyclooxygenase 2 and 5-Lipoxygenase

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 inflam‐ mation [53], therefore, natural products that specifically target cytokines or their receptors

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 mecha‐ nism 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

Diphyllin acetapioside Inhibits 5-Lipoxygenase EGCG Inhibits Cyclooxygenase 2 Eugenol Inhibits 5-Lipoxygenase Gingerol Inhibits 5-Lipoxygenase

288 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

Hydroxytyrosol Inhibits 5-Lipoxygenase Hyperforin Inhibits 5-Lipoxygenase Medicarpin Inhibits 5-Lipoxygenase Ohenethyl ferulate Inhibits Cyclooxygenase 2 Onosmins A and B Inhibits 5-Lipoxygenase Panaxynol Inhibits 5-Lipoxygenase Phenethyl ferulate Inhibits 5-Lipoxygenase Quercetagetin-7-O-beta-O-glucoside Inhibits 5-Lipoxygenase Rosmarinic acid Inhibits 5-Lipoxygenase Rosmarinic acid methylester Inhibits 5-Lipoxygenase Rosmarol Inhibits Cyclooxygenase 20

**Table 4.** Phytochemical inhibitors of lipid mediators

have the potential to be effective asthma treatments.

*3.1.2. Cytokines blockers*

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.




**Phytochemicals**

Kaempferol

Luteolin

Myricetin

Naringenin

Resveratrol

**Anti-oxidants Target/Function and Effective Concentration(s) References**

Impairs Th2 cytokines production (IL-5 and IL-13) in OVA-sensitized mice. Suppresses the release of IL-4 and TNF in RBL-2H3 cells and macrophages. Inhibits IgE-mediated TNF and IL-6 release in hCBMCs.

macrophages (40 μM). [66]

macrophages (40 μM). [66]

Inhibits TNF secretion in LPS-activated macrophages. [74, 75]

μM). [80]

release from hCBMCs. [75]

Inhibits increase in Th2 cytokines (IL-4 and IL-5) in plasma and BALF in asthmatic mouse model (30 mg/kg body wt.). Inhibits TNF induced GM-CSF and VEGF release in HASM cells. Inhibits PMA- and A23187 induced TNF and IL-6 release in HMC-1 cells. Decreases production of IL-1β in lung tissue of mice with LPS-induced acute lung injury (1

[57, 67-73]

[75, 76]

[77-79]

[68, 81-86]

Reduces the levels of TNF and IL-1β in LPS-stimulated macrophages (8 & 16 μM). Inhibits induction of TNF, IL-6 and GM-CSF in HMC-1 cells (10 & 50 μM). Inhibits Th2 cytokines (IL-4, IL-5 and IL-13) expression in murine asthma model (50 & 100 mg/kg body wt.). Inhibits myelin basic protein-induced IL-6, TGF-β1, and TNF release in hCBMCs (10 & 100 μM). Decreases TNF (IC50 7.9±4.6 μM) and IL-1β (IC50 5.1±0.4 μM) in PBMCs. Reduces IL-4 and IL-5 levels in BALF of murine asthma model (0.1 mg/kg body wt.). Inhibits antigen-IgE-mediated TNF (IC50

5.8 μM) and IL-4 (IC50 3.7 μM) production in RBL-2H3 cells.

Inhibits TNF (30 μM) and IL-6 (30 μM) production in HMC-1 cells. Inhibits IgE-mediated TNF (10 & 100 μM) and IL-6 (1, 10 & 100 μM)

Suppresses Th2 cytokines production from CD4 T cells (0.8 mg/kg body wt.). Reduces IL-4 (25, 50 & 100 mg/kg body wt.) and IL-13 (50 &

100 mg/kg body wt.) levels in BALF of murine asthma model. Suppresses LPS-induced IL-1β (10, 25 & 50 μg/mL), IL-6 (5, 10, 25 & 50 μg/mL), and TNF (25 & 50 μg/mL) production in macrophages and

Morin Inhibits IgE-mediated TNF and IL-6 release in hCBMCs (10 & 100 μM).

release from hCBMCs.

human whole-blood samples.

Pedalitin Inhibits TNF and IL-12 production in LPS-activated macrophages (40

Quercetin Inhibits IgE-mediated TNF (10 & 100 μM) and IL-6 (1, 10 & 100 μM)

Isoliquiritigenin Inhibits the release of TNF and IL-6 in activated inflammatory cells.

Kuraridin Suppresses expression of TNF and IL-1β in LPS-stimulated

290 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

Kurarinone Suppresses expression of TNF and IL-1β in LPS-stimulated


**Table 5.** Phytochemical inhibitors of proinflammatory cytokines

#### *3.1.3. Chemokines and chemokine receptors blockers*

Chemokines (CC) and their receptors (CCR) play a crucial role in the recruitment of inflam‐ matory 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.



**Phytochemicals**

n-3 PUFA

in Table 6.

**Phytochemicals**

Apigenin

Baicalein

Baicalin

**Anti-oxidants Target/Function and Effective Concentration(s) References**

Helenalin Inhibits TNF and IL-6 secretion by ASMCs (1 μM). [113]

EPA and DHA Lower BALF concentration of pro-inflammatory cytokines IL-1α, IL-2, IL-5, IL-9, IL-13, G-CSF and RANTES.

Chemokines (CC) and their receptors (CCR) play a crucial role in the recruitment of inflam‐ matory 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

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

**Flavonoids Target/Function and Effective Concentration(s) References**

Suppresses the production of MDC and IP-10 in THP-1 cells (10-6 and 10-5 M). Inhibits release of LPS-induced MCP-1 in J774.2 macrophages (10 & 30 μM). Inhibits production of IL-8 in HMC-1

Inhibits IL-8 and MCP-1 release in activated human mast cells. Inhibits eotaxin production in human dermal fibroblasts (10 μg/

Reduces IL-8 levels in plasma and BALF in cigarette smoke-induced COPD rat model. Inhibits eotaxin production in human dermal

EPA (120 μM) suppress TNF and IL-1β expression and production in LPS-stimulated alveolar macrophages from asthmatic patients

macrophages (0.1, 0.5 & 1 μM). [111, 112]

Ref?

[58, 120, 121]

[59, 122]

[60, 122]

progenitor cell infiltration into the

Costunolide Inhibits production of TNF, IL-1β and IL-6 by LPS-stimulated

292 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

**Table 5.** Phytochemical inhibitors of proinflammatory cytokines

antibody inhibits allergen-induced eosinophilia and CD34+

cells.

mL).

fibroblasts (10 μg/mL).

*3.1.3. Chemokines and chemokine receptors blockers*


**Table 6.** Phytochemical inhibitors of chemokines

#### *3.1.4. Miscellaneous: IgE, histamine, enzymes*

**Phytochemicals**

**Polyphenols**

Caffeic acid

EGCG

Gallic acid

Rosmarinic acid

**Flavonoids Target/Function and Effective Concentration(s) References**

Tectorigenin Inhibits MCP-1 expression in endothelial cells. [138]

Decreases IL-8 release in chitinase-activated human airway epithelial cells (1 μM). Inhibits production of eotaxin in human lung fibroblast cells (1, 10 & 100 μM). Inhibits IP-10 expression in allergic patients' DCs (10 μM). Decreases expression of CXCR4 receptor on CD4 T cells (20 μg/mL). Suppresses IL-8 levels in asthmatic patients

Inhibits IL-8 release in TNF-stimulated human keratinocytes (0.5, 1, 5 & 10 μM). Reduces airway inflammation in asthmatic mouse model by binding to chemokines CXCL9, CXCL10 and CXCL11 (10 & 100 μM). Decreases MCP-1 and CCR2 expression on THP-1 cells (100 μM). Attenuates production of MIP-2 in lungs of mice with LPSinduced acute lung injury. Reduces expression of MCP-1 and IL-8 in HMC-1 cells (100 μM). Inhibits neutrophil migration by suppression of CINC-1 production (15, 50 & 150 μg/mL). Downregulates cigarette smoke-induced IL-8 release from bronchial epithelial cells.

Inhibits production of IL-8 and TARC (5 & 10 μg/mL) in neutrophils and keratinocytes respectively. Inhibits eotaxin and RANTES in pleural lavage fluid of allergen-challenged mouse model (100 mg/kg body wt.). Inhibits IL-8 release in HMC-1 cells (10 μM).

Inhibits LPS-induced production of MCP-1 and MIP-1α in bonemarrow derived DCs (100 μM). Reduces diesel exhaust particlesinduced MIP-1α, MCP-1 and KC expression in mice lung (4.6 μg/kg body wt.). Inhibits expression of eotaxin in lungs of mite antigensensitized mice (1.5 mg/kg body wt. PO). Inhibits expression of CCL11 and CCR3 genes induced by TNF in human dermal fibroblast

Helenalin Inhibits eotaxin and RANTES secretion in ASMCs (1 μM). [113]

mg/kg body wt.).

294 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

(13% soln.).

cells.

**Table 6.** Phytochemical inhibitors of chemokines

Wogonin Suppresses mite antigen-induced TARC expression in human

Curcumin Inhibits tryptase-induced IL-8 release in eosinophils (25 μM).

Inhibits IL-8 production in THP-1 cells (0.1, 1, 10 & 100 μg/mL). Attenuates C5a-induced MIP-1α production in mouse model (1

keratinocytes (250 ng/mL). [139]

[89, 90, 140-143]

[97, 99, 125, 144-147]

[148-150]

[102, 107, 110, 151]

The activation of mast cells involves the cross-linking of IgE bound to the FCεRI 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.



**Table 7.** Phytochemical inhibitors of beta-hexosaminidase enzyme

#### **3.2. 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.

#### *3.2.1. 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 phosphoi‐ nositol 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 macro‐ phages, suggesting the usefulness of PI3K inhibitors in asthma [172].

#### *3.2.2. Transcription factor inhibitors*

**Phytochemicals IC50 References** Medicarpin >100 μM [155] Rosmarinic acid ? [160]

Celastrol ? [163] Costunolide 34 μM [164]

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 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 phosphoi‐ nositol 3-kinase (PI3K) signaling pathways have all been proposed to have a role in the

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*

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,

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.

**Table 7.** Phytochemical inhibitors of beta-hexosaminidase enzyme

296 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

**3.2. Inhibitors of intracellular signaling pathways**

*3.2.1. Protein kinase inhibitor*

pathogenesis of asthma.

bronchial asthma model [166].

ERK and JNK [168].

**Terpenes**

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, Poly‐ phenols 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].

#### **3.3. 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 offcina‐ lis* [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.



**Table 9.** Phytochemical inhibitors of oxidative stress

interference RNA promises a novel approach for asthma treatment [178]. Polyphenols, such as apigenin and quercetin, ameliorate asthma symptoms, and suppress the translocation of

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

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

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 offcina‐ lis* [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.

**Phytochemicals Function and effective concentration(s) References**

Apigenin Suppresses LPS-induced NO production in RAW264.7 macrophages [191]

Fisetin Inhibits TNF-induced ROS production in HEK cells [123]

RAW264.7 macrophages [192]

RAW264.7 macrophages [74]

Baicalein Inhibits LPS-induced NO production and iNOS expression in

Chrysin Inhibits NO production (IC50 7.50±1.84 μM) in LPS-activated

GATA-3 in the cytosol of lung tissue of OVA-sensitized and -challenged mice [179].

298 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

treatment strategy [182].

PPARγ activation [185].

**Flavonoids**

**3.3. Inhibitors of oxidative stress**

#### **4. 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.



**Table 10.** *In vivo* and *in vitro* models for asthma studies
