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BY INFLAMMATORY MEDIATORS AND CYTOKINES IN RESPIRATORY SMOOTH MUSCLE

A Dissertation submitted to

THE TAMIL NADU Dr.M.G.R. MEDICAL UNIVERSITY CHENNAI- 600 032

In partial fulfilment of the requirements for the award of the Degree of MASTER OF PHARMACY

IN

PHARMACOLOGY

Submitted by S.ANNIE SUSAN

261825401

Under the guidance of

Dr. S. SENGOTTUVELU M.Pharm, Ph.D., Head , Department of Pharmacology

NANDHA COLLEGE OF PHARMACY AND RESEARCH INSTITUTE ERODE- 638 052, TAMILNADU

APRIL 2020

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Success of any project depends solely on support, guidance and encouragement received from the guide and well wishers.

It gives me immense pleasure and contentment to acknowledge and thank all of those who in big ways and small have contributed for this effort.

I am highly indebted to my guide to Dr. S. Sengottuvelu M.Pharm, Ph.D., Head, Department of Pharmacology, Nandha College of Pharmacy under whose constant supervision, meticulous guidance this work has been carried out in completion. His valuable suggestions and keen interest throughout the work greatly eased my task in completing this work.

It is proud to express my sincere thanks to Dr. T. Sivakumar M.Pharm, Ph.D., Principal, Nandha College of Pharmacy, with a deep sense of gratitude for his encouragement, co-operation, kind suggestion and providing the best facilities during this work.

It is my proud privilege to express my sincere thanks to Dr. S. Haja Sherief M.Pharm, Ph.D., Professor, Department of Pharmacology, Nandha College of Pharmacy for his supportive guidance throughout my thesis work.

I am also thankful to Mrs. V. Lalitha M.Pharm., Associate Professor, Department of Pharmacology, Nandha College of Pharmacy for her suggestions and supportive guidance throughout my thesis work.

I am highly obliged to thank honourable Thiru V. Shanmugan B.Com., Chairman and Mr. S. Nandhakumar Pradeep M.B.A., Secretary, Nandha College of Pharmacy for

providing me the required infrastructure to carry out my studies.

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S.Prabhakaran, M.Latha, E. Manickavalli who helped me in all possible ways throughout the entire course time.

Reg.Number: 261825401 II-M.Pharm, Department of Pharmacology Nandha College of Pharmacy

Erode-52 Place:

Date:

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Dr. S. Sengottuvelu M.Pharm, Ph.D., Head, Department of Pharmacology,

Nandha College of Pharmacy, Erode.638 052

GUIDE CERTIFICATE

This is to certify that the work embodied in the thesis entitled “ROLE OF CURCUMIN AND VASICINE ON IMMUNE RESPONSE INFLUENCED BY INFLAMMATORY MEDIATORS AND CYTOKINES IN RESPIRATORY SMOOTH MUSCLE” submitted to The Tamilnadu Dr. MGR. Medical University, Chennai was carried out by Ms. S. Annie Susan (261825401) in the Department of Pharmacology, Nandha College of Pharmacy, Erode-52. In the partial fulfilment for the award of degree of Master of Pharmacy in pharmacology and my direct supervision and guidance.

This work is original and has not been submitted in part or full for any other degree or diploma in any university.

Place: Erode Dr. S. Sengottuvelu M.Pharm, Ph.D.

Date:

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S.NO TITLE Page No

1 INTRODUCTION

2 LITERATURE REVIEW

3 AIM AND OBJECTIVE

4 PLAN OF WORK

5 DRUG PROFILE

6 MATERIALS AND METHODS

7 RESULTS

8 DISCUSSION

9 CONCLUSION

10 REFERENCES

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S.No FIGURES PAGE NO

1 Release of inflammatory mediators and cytokines in response

to immune system 11

2 Effect of Curcumin and Vasicine on Total WBC count

41 3 Effect of Curcumin and Vasicine on differential WBC count

41 4 Effect of Curcumin and Vasicine on Serum IgE Concentration

43 5 Histopathological Evaluation of Lung using Haematoxylin and

Eosin staining. (H&E) 45

6 Histological scoring of Haematoxylin and Eosin stained Lung

Section 47

7 Histopathological Evaluation of Periodic Acid Shiff Staining

(PAS) 48

8 Histological scoring of stained Periodic Acid Shiff Staining

Lung Section 50

9 Dose Response Curve of Histamine in presence and absence of

Curcumin and Vasicine in Guinea pig tracheal chain 52 10 Effect of Curcumin and Vasicine on Histamine induced

contraction on isolated Guinea pig tracheal chain preparation 53 11 Dose Response Curve of Histamine in presence and absence of

Curcumin and Vasicine in Guinea pig ileum 56 12 Effect of Curcumin and Vasicine on Histamine induced

contraction on isolated Guinea pig ileum preparation 57

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S.No TABLES PAGE NO

1 Effect of Curcumin and Vasicine on Total and differential

WBC count 40

2 Effect of Curcumin and Vasicine on serum IgE concentration

43 3 Histological scoring of Haematoxylin and eosin stained Lung

Section 46

4 Histological scoring of stained Periodic Acid Shiff Staining

Lung Section 49

5 Effect of Curcumin and Vasicine on Histamine induced

contraction on isolated Guinea Pig Trachea preparation 51 6 Percentage inhibition response of different antagonist in

Histamine induced contraction on isolated Guinea Pig Trachea preparation.

51

7 Effect of Curcumin and Vasicine on Histamine induced

contraction on isolated Guinea pig ileum preparation 55

8 Percentage inhibition response of different antagonist in histamine induced contraction on isolated guinea pig ileum

preparation

55

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Department of Pharmacology, Nandha College of Pharmacy Page 1

INTRODUCTION

Inflammation is the body’s response to infection, trauma, and hypersensitivity. The inflammatory response is complicated and involves a variety of mechanisms to defend against pathogens and repair tissues. In the Lungs, inflammation is commonly caused by pathogens or by exposure to toxins, pollutants, irritants, and allergens. During inflammation, several types of inflammatory cells are activated. Each release cytokines and mediators to alter activities of other inflammatory cells. Orchestration of these cells and molecules lead to evolution of inflammation.

Clinically, acute inflammation is observed in pneumonia and acute respiratory distress (ARDS), whereas chronic inflammation is characterized by asthma and chronic obstructive pulmonary disease (COPD). Because the lung is the vital organ for gas exchange, more inflammation is life threatening. Whenever the lung is continually exposed to harmful pathogens, an immediate and intense defense action is required to eradicate the invaders as early as possible. A delicate balance between inflammation and anti-inflammation is crucial for lung homeostasis. [1]

INFLAMMATORY MEDIATORS AND CYTOKINES CELLS

EOSINOPHILS

Allergen inhalation results in a considerable increase in eosinophil count in bronchoalveolar (BAL) fluid with a decrease in peripheral eosinophil counts with the appearance of eosinophil forerunner in the circulation. Recruitment of eosinophils to airways is mediated by interleukin (IL)-13, histamine, prostaglandin type 2, and chemokines, such as RANTES (regulated on activation T-cell expressed and secreted), eotaxins, and macrophage chemotactic protein (MCP)-4, expressed in epithelial cells. [2, 3]

NEUTROPHILS

Neutrophils are broadly observed in the airways and sputum of patients with airway inflammation, chiefly during acute exacerbations of asthma and in some patients with long- lasting or corticosteroids dependent or insensitive to inhaled steroids. They are enlisted through Th17 pathways and lead to increased concentrations of IL-8 in sputum, which in turn may be due to the increased level of oxidative stress. Neutrophils contribute to BHR and airway

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Department of Pharmacology, Nandha College of Pharmacy Page 2 inflammation through the release of mediators like PAF, thromboxanes, and leukotrienes and tissue damage through secretion of proteases and oxygen radicals. [4-6]

MACROPHAGES

Macrophages, derived from blood monocytes, develop inflammatory process through production of a variety of cytokines, after being stimulated by allergen via low-affinity IgE receptors (FceRII). Macrophages may both increase and decrease inflammation, depending on the stimulus. Alveolar macrophages have a suppressive effect on lymphocyte function, but this may get impaired in asthma after allergen exposure. Macrophages secrete an anti-inflammatory protein IL-10 which is decreased in alveolar macrophages from patients with asthma.

Macrophages may, therefore, play a crucial anti-inflammatory role, by avoid the development of allergic inflammation. [7-10]

MAST CELLS

Mast cells are central to the progression of type I hypersensitivity reaction. Mast cells are bone marrow-derived cells extensively distributed in the body predominantly near blood vessels, subepithelial cells and nerves, mucosal lining of the gut, and upper and lower respiratory tract.

Mast cells encompass membrane bound granules filled with biologically active mediators. After re-exposure, mast cells get stimulated by cross-linking of high-affinity IgE Fc receptors present on mast cell surface or by stimuli such as C5a and C3a (anaphylatoxins) and release a wide variety of mediators that result in acute bronchospasm or perpetuate underlying inflammation through cytokines. Mast cells are an important source of histamine, cysteinyl leukotrienes, prostaglandins, cytokines, and platelet-activating factor, after getting stimulated by binding of stem cell factor to the surface receptor c-kit, IgE cross-linking, or binding of tyrosine kinase, and the process is called degranulation of mast cells. [11-13]

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Department of Pharmacology, Nandha College of Pharmacy Page 3 T-LYMPHOCYTES

Several types of T-lymphocytes (especially, Th1, Th2, Th9, and Th17) play an crucial role in coordinating the inflammatory response through release of a number of cytokines.

Traditionally, Th2 cells have been thought to predominate, with distinctive raised levels of IL-4, IL-5, and IL-13. High proportion of TH1 cells that can progress under the influence of IL-18 and interferon γ (IFN-γ) correlate with further management of IFN-γ is also found in some respiratory disorders. Th17 cells, expressing IL-17, also play an unusual role in asthmatic patients. Th17 are CD4-positive T cells and result in neutrophils influx. Th9 levels are raised in people with atopy cells, secrete IL-9, and promote allergic responses, probably through stimulation of mast cells. T-regulatory cells, characterized by secretion of transforming growth factor β (TGF-β) and IL-10, are thought to be important because of their role in blunting atopic responses. [15]

B-LYMPHOCYTES

B cells are important in asthma correlate with atopy because they produce IgE. Their survival is supported by IL-5 and a B-cell-activating factor. B cells need to bind to T cells under the influence of IL-4 or IL-13. Secreted IgE are chiefly bound through the high-affinity Fc receptors on mast cells and basophils, and when cross-linked by aeroallergen, it causes these cells to degranulate and release their mediators. [16]

INNATE LYMPHOID CELLS

ILCs are a family of immune cells that are defined by several features including the absence of recombination-activating gene (RAG)-dependent rearranged antigen receptors, their lymphoid morphology, as well as lack of myeloid phenotypic markers and are therefore called cell lineage marker-negative (Lin−) cells. These ILCs are present in the skin, adipose tissues, mesenteric lymph nodes, tonsils, and spleen and mediate inflammatory pathways in various diseases of the lungs and skin. ILCs are classified into three groups according to their transcription factors and cytokine production profile that resembles T-helper (TH) cell subsets.

Among these cells, group 2 innate lymphoid cells (ILC2s) are known to play a crucial role in pathogenesis of type 2 inflammatory diseases of the lungs and skin such as asthma and atopic dermatitis. They have the capacity to produce type 2 (TH2) cytokines and interact with both

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Department of Pharmacology, Nandha College of Pharmacy Page 4 immune and non immune cell populations in the local tissue environment. ILC1s produce TH1 inflammatory cytokines, particularly IFN-γ and tumor necrosis factor (TNF-α). They play their important role in the pathogenesis of chronic obstructive pulmonary disease (COPD) and human inflammatory bowel (IBD). ILCs generally differentiate into macrophages and granulocytes while stimulating eosinophils and producing Th2 cytokines. Stimulation of mast cells and release of mediators in allergic asthma. [18-20]

AIRWAY EPITHELIAL CELLS

Airway epithelial cells play an important role in mucociliary clearance signaling through special receptors Toll-like receptor 4 expressed on epithelial cells activated by allergic and infectious triggers. These cells form barrier against mechanical stress, oxidant stress, allergens, pollutants, infectious agents, and leakage of endogenous solutes. In asthma, epithelial cell- derived cytokines and chemokines (including IL-25, IL-33, thymic stromal lymphopoietin [TSLP], and granulocyte-macrophage colony-stimulating factor [GM-CSF]) signal effector cells (including basophils, eosinophils, mast cells, and lymphocytes) and dendritic cells are of concern in developing characteristic asthmatic immune response patterns to various types of allergic stimuli. [21]

DENDRITIC CELLS

Like airway epithelial cells, pulmonary dendritic cells are also directly disclosed to the external environment. These dendritic cells act as antigen-presenting cells and are directly activated by allergens or infectious agents directly after binding with recognition receptors or indirectly activated by airway epithelial cells (by mediators such as IL-25, IL-33, GM-CSF);

dendritic cells can recruit eosinophils in allergen-presenting regions. Dendritic cells are also found to effect T-cell differentiation and generate Th2 response commonly seen in atopic asthma. [22]

ADHESION MOLECULES

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Department of Pharmacology, Nandha College of Pharmacy Page 5 These molecules promote infiltration of inflammatory cells at the site of inflammation, enrollment of leukocytes from vascular lumen to tissues, and cell stimulation. Adhesion molecules are up regulated in allergic inflammation and play a important role in pathogenesis inflammation. More than 35 adhesion molecules have been found, for example, integrins, immunoglobulin supergene family, selectins, and carbohydrate ligands including ICAM-1 and VCAM-1. [24]

INFLAMMATORY MEDIATORS

A number of mediators that account for pathophysiological features of allergic diseases.

Mediators such as histamine, PG, leukotrienes, and kinins contract airway smooth muscle, increase microvascular leakage, increase airway mucus secretion, and captivate other inflammatory cells.

HISTAMINE

Histamine was the first mediator known to be involved in pathophysiology of asthma.

Histamine is integrated and released by mast cells and basophils in the airways. Histamine causes mucus secretion and bronchoconstriction which is partly mediated by vagal cholinergic reflex. Histamine also acts as a chemoattractant for eosinophils and stimulates eosinophils. [25]

LEUKOTRIENES

The cysteinyl leukotrienes, LTC4, LTD4, and LTE4, are eicosanoids derived from arachidonic acid by 5-LOX (lipoxygenase) pathway. They are powerful constrictors of human airway and have been reported to increase AHR and play a crucial role in asthma. They constitute the slow-reacting substance of anaphylaxis. Potent LTD4 antagonists protect (by 50%) against exercise- and allergen-induced bronchoconstriction, implying that leukotrienes contribute to bronchoconstrictor responses. [26]

PLATELET-ACTIVATING FACTOR

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Department of Pharmacology, Nandha College of Pharmacy Page 6 Platelet-activating factor (PAF) is a potent inflammatory mediator that mimics many features of asthma, including eosinophil recruitment and stimulation and introduction of AHR, plasma exudation, and mucus hypersecretion. The high level of lysoPAF (metabolite of PAF) is evaluated in BALF of patients with allergic asthma. [27]

PROSTAGLANDIN

Prostaglandins are produced from arachidonic acid by cyclooxygenase (COX) pathway.

Increased concentration of PGF2, PGD2, and thromboxane B2 in bronchoalveolar (BAL) fluid.

When inhaled, they cause bronchoconstriction and increase airway responsiveness to spasmogen.

[28]

PROTEASES

Tryptase is a mast cell serine protease and plays a role in hemostasis, mucus secretion, and vascular permeability. Elevated levels of tryptase have been found in BAL fluid and sputum of patients after allergen challenge. Elevated levels of MMP-9 (metalloproteinase-9), a protease released by eosinophils and alveolar macrophages, are found in bronchoalveolar fluid. [29]

KININS

Kinins are vasoactive peptides secreted from kininogens by the action of kininogenase during the inflammatory response. Bradykinin is an important kinin that has many effects on airway functions mediated by direct activation of B2 receptors of airway smooth muscles.

Bradykinin activates alveolar macrophages to release LTB4 and PAF and stimulates nociceptive nerve fibers in the airways of asthmatic patients only which may mediate cough and chest tightness appearance features of asthma. [30]

CYTOKINES

Cytokines are extracellular signaling proteins secreted by almost every cell under certain conditions and play a important role in orchestrating all types of inflammatory response in respiratory disorders. They act on target cells to cause a wide range of cellular functions like activation, proliferation, chemotaxis, immunomodulation, release of inflammatory mediators, growth and cell differentiation, and apoptosis. In contrast to acute and subacute inflammatory

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Department of Pharmacology, Nandha College of Pharmacy Page 7 responses, cytokines play a important role in maintaining chronic inflammation in allergic diseases. The important cytokines in asthma are lymphokines secreted by T-lymphocytes: IL-1β, IL-3, IL-4, IL-5, IL-6, IL-9, IL-13, TNF-α, etc. where IL-3 is reported to be important for the survival of mast cells in tissues, but IL-4 plays an crucial role in switching B-lymphocytes to produce IgE and expression of VCAM-1 on endothelial cells. IL-5 plays a critical role in differentiation, survival, and priming of eosinophils, thus contribute to eosinophilic inflammation, and present in BAL fluid. Airway macrophages are important source of IL-1β, TNF- α, and IL-6 which act on epithelial cells to release GM-CSF, IL-8, and RANTES and amplify the inflammatory response leading to influx of secondary cells like eosinophils. [31]

PROINFLAMMATORY CYTOKINES

IL-9 and IL-13 are considered as proinflammatory cytokines. IL-9 is known to stimulate proliferation of activated T cells, agument IgE production from B cells, developing proliferation and differentiation of mast cells, up regulating the α-chain of the FcεRI receptor, and activating CC chemokine expression in lung epithelial cells commiting in allergen-induced airway changes.

IL-13 is present in increased amounts in asthmatic airways and possesses biological activities similar to IL-4. Unlike IL-4 which is central to development of Th2 cells during primary sensitization, IL-13 release is more important during secondary antigen exposure. Another group of proinflammatory cytokines are TNF-α that help in leukocyte recruitment through upregulation of adhesion molecules on vascular endothelial cells and activation of cytokine and chemokine synthesis airway hyper-responsiveness and pathogenesis of airway remodeling. [32]

IMMUNOMODULATORY CYTOKINES

IL-10, IL-12, IL-18, and interferon gamma (IFN-γ) are known as immunomodulatory cytokines. IL-10 is a pleiotropic cytokine that has the potential to down regulate both Th1- and Th2-driven inflammatory processes and beneficial effect on airway remodeling. IL-12 is released by antigen-presenting cells and is known to play an important role in Th1/Th2 differentiation during primary antigen presentation. IL-18 is secreted by macrophages and IFN-γ is reported to IL-12 and IL-18 act synergistically for inducing IFN-γ and inhibiting IL-4-dependent IgE synthesis as well as inhibiting allergen-induced airway hyperresponsiveness. Balance between Th1 and Th2 cells is thought to be determined by locally released cytokines, such as IL-12,

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Department of Pharmacology, Nandha College of Pharmacy Page 8 which favor emergence of Th1 cells; contrary to this, IL-4 and IL-13 favor the growth of Th2 cells. [33]

CHEMOKINES

Chemokines are chemotactic cytokines responsible for recruitment of inflammatory cells in the airways. Chemokines have been categorized into two main groups, (a) CXC (α-type) and CC (β-type) chemokines, and exert their effects through G-protein-coupled chemokine receptors (CCR). Exacerbation of asthma leads to the synthesis and release of a number of chemokines.

Increased expression of eotaxin, eotaxin-2, MCP-3, MCP-4, and CCR3 in the airways of asthmatic patients is found, and this can be correlated to increased AHR. [34]

TACHYKININS

Tachykinins are neuropeptides derived from preprotachykinins (PPTs). They are released by sensory nerves of airways and stimulate mucus secretion, plasma exudation, neural activation, bronchoconstriction, and structural changes. These peptides stimulates macrophages and monocytes to release inflammatory cytokines, IL-6. Higher concentration of a tachykinin, substance-P (SP), has been found in BALF of asthmatic lungs. [35]

ENDOTHELINS

Endothelins are peptide mediators secreted via endothelin-converting enzyme (ECE) through mRNA present in airway epithelial cells and regulated by a number of proinflammatory cytokines in asthma. The biological effects of endothelins are mediated by two receptors: ETA and ETB. Endothelins are potent. Release of mediators after allergen exposure to airway epithelia. bronchoconstrictors and induce airway smooth muscle cell proliferation and fibrosis and play an important role in chronic inflammation of asthmatic airways. After the allergen challenge, endothelins (ETs) are secreted de novo. Higher levels of endothelin-1 are found in the sputum of asthmatic patients. [36]

NEURAL MEDIATORS

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Department of Pharmacology, Nandha College of Pharmacy Page 9 Several nonadrenergic-noncholinergic (NANC) nerves and neuropeptides have been identified in the respiratory tract. Airway nerves may also release neurotransmitters that have inflammatory effects such as substance P (SP), neurokinin A, and calcitonin gene-related peptide, may be released from sensitized inflammatory nerves in the airways, and perpetuate the ongoing inflammatory response. Thus, chronic asthma may be associated with increased neurogenic inflammation, which may provide a mechanism for prolonging the inflammatory response even in the absence of initiating inflammatory stimuli.

ANTIBODIES

Antibodies are protein molecules released by immune system in response to foreign bodies, allergens. Five classes of antibodies, namely, IgM, IgG, IgA, IgD, and IgE are known. Of these IgE is the predominant antibody in asthma in humans. IgE is the antibody responsible for all types of allergic reaction and pathogenesis of allergic asthma and development of inflammation in the human body. Elevated levels of IgE are found in bronchial asthma. [37]

OXIDATIVE STRESS

The increased level of oxidative stress found in airways of people with allergic asthma activates circulatory inflammatory cells, such as macrophages and eosinophils. Activated inflammatory cells produce more number of reactive oxygen species causing increased concentrations of 8-isoprostane (a product of oxidized arachidonic acid) and ethane (a product of oxidative lipid peroxidation) in exhaled breath of asthmatic patients. Increased oxidative stress can be related to disease severity and may amplify the inflammatory response and reduce responsiveness to corticosteroids, particularly in severe disease and during exacerbations.

Mechanism underlying the role of oxidative stress in asthma severity may be due to reaction of superoxide anions with nitric oxide (NO) forming reactive radical peroxynitrites that may modify several target proteins. [38]

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Department of Pharmacology, Nandha College of Pharmacy Page 10 NITRIC OXIDE (NO)

Measurement of the level of NO in exhaled air of asthmatic patients is increasingly being used as a noninvasive way of monitoring the inflammatory process. NO is produced by NO synthase, but in epithelial cells of asthmatic patients, the enzyme inducible of NO synthase (iNOS) is present. Recent studies report the higher level of NO in the exhaled air of patients with asthma than the level of NO in the exhaled air of normal subjects. The combination of increased oxidative stress and NO may lead to the formation of the potent radical peroxynitrite that may result in nitrosylation of proteins in the airways. Since NO is a potent vasodilator, this may increase plasma exudation in airways, and it may also amplify the Th2-mediated response. [39]

PHATHOPHYSIOLOGICAL ROLE OF INFLAMMATORY MEDIATORS AND CYTOKININE ON RESPIRATORY SMOOTH MUSLES

Dendritic cells are antigen-presenting cells (APCs), which stimulate naïve T cell proliferation. Dendritic cells and macrophages are the first line of defense in recognizing various pathogens. Originating in bone marrow, dendritic cells reach tissues through blood circulation and, in the lung, reside in and below the airway epithelium, the alveolar septa, pulmonary capillaries, and airway spaces. Once the dendritic cell identifies, ingests, and processes an antigen, it migrates to the lymph nodes and presents the antigen to resident T cells, inducing the immune response. [40]

Macrophages reside in the airways, alveoli, and lung interstitium, or migrate into the lung microvasculature. Their role is essential in modulating acute and chronic inflammatory responses, but although macrophages can proliferate within the lung, their number is not adequate to fight infection. Macrophage function is augmented by dendritic cells. Together they

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Department of Pharmacology, Nandha College of Pharmacy Page 11 Figure 1: Release of inflammatory mediators and cytokines in response to immune system

are capable of phagocytosing bacteria, particulates, and apoptotic cells. However, macrophages are the main source of cytokines, chemokines, and other inflammatory mediators that propagate or suppress the immune response. Following an insult, macrophages and epithelial cells secrete chemokines and cytokines, promoting neutrophil accumulation and local inflammation. [41]

Neutrophils provide second-line defense. They are the first cells to be recruited to sites of infection or injury, and attack fungi, protozoa, bacteria, viruses, and tumor cells. During pulmonary infection, neutrophils migrate out of the pulmonary capillaries and into the air spaces. After phagocytosis, neutrophils kill ingested microbes with reactive oxygen species,

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Department of Pharmacology, Nandha College of Pharmacy Page 12 antimicrobial proteins (bactericidal permeability-inducing protein and lactoferrin), and degradative enzymes (elastase). Deficits in neutrophil quantity (neutropenia) and quality (chronic granulomatous disease) predispose patients to opportunistic lung infections. [42-44]

Lymphocytes are found throughout the airway and lung parenchyma. There are two major populations of lymphocytes: thymus-dependent T cells and bone marrow-dependent B cells. T lymphocytes provide cell-mediated immunity, while B lymphocytes produce humoral immune responses by synthesizing antibodies (immunoglobulins). T lymphocytes have two major subsets: CD4+ and CD8+. CD4+ T lymphocytes are also known as helper T cells, which are further subdivided into Th1 and Th2, with different cytokine profiles. Th1 cells drive cellular immunity. Th1 cytokines (interferon gamma, TNF-α) produce the pro-inflammatory responses to fight viruses and other intracellular parasites, and to eliminate cancer cells. Excessive pro- inflammatory responses ca n lead to uncontrolled tissue damage. Th2 cells drive humoral immunity to up regulate antibody production to fight extracellular organisms. Th2 cytokines (IL- 4, IL-5, IL-9, and IL-13) promote IgE and eosinophilic responses in atopy. Excessive Th2 responses will counteract the Th1-mediated anti-microbial actions. Optimally, a balanced Th1 and Th2 response is suited to the immune challenge, and a dysregulated response is linked to a variety of chronic inflammatory conditions like asthma and chronic bronchitis.CD8+ T cells are mainly cytotoxic T cells. They secrete molecules that kill infected cells and tumor cells. In addition, there is a natural killer cell (NK cell) subset of T cells with no antigen-specific receptors. Another subset of T cells, named NKT cells, which have the properties of NK cells, are important in combating bacteria, protozoa, and viruses. Furthermore, there are regulatory T cells, which suppress the other lymphocytes. During the immune response, some antigen- activated B cells and T cells differentiate into memory cells, producing long-lasting immunity.

(Figure-1) [44-46]

Eosinophils, the least common white blood cells, are often associated with parasite infections, allergic diseases (such as asthma), chronic lung inflammatory states, and hypereosinophilic syndrome. The eosinophil is an important source of major basic proteins, lipid mediators, cytokines, and growth factors, and also secretes mast cell stem cell factor, essential for mast cell growth, activation, chemotaxis, and degranulation.

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Department of Pharmacology, Nandha College of Pharmacy Page 13 Although the inflammatory cells take center stage, epithelial, endothelial and mesenchymal cells also participate in the inflammatory process. [47]

ASTHMA

Asthma is an inflammatory disease of the airways characterized by recurrent reversible airway obstruction and hyper responsiveness to non-specific stimuli such as air pollutants, cold air, exercise, allergens and infections. Each of these stimuli evokes airway narrowing indirectly through the release of mediators from a variety of effector cells, including inflammatory cells and epithelial cells.

Some of the different inflammatory cells involved in asthma are predominant in asthmatic inflammation. Cumulative findings support the notion that T- helper 2 (Th 2) cells, B cells, mast cells and eosinophils contribute to the chronic inflammation of the airways and that mediators such as histamine, prostaglandins and leucotrienes contract airway smooth muscle, increase microvascular leakage, increase airway mucus secretion and attract other inflammatory cells. Asthma is also a chronic inflammatory disease, with inflammation persisting over many years in most patients. 10 Inhaled β2 adrenergic receptor agonists and corticosteroids have represented the mainstay of the therapeutic management of asthma for at least 25 years. Beta-2 adrenoceptor agonists, which inhibit bronchoconstriction, would provide little more than symptomatic relief, while the anti-inflammatory effect of glucocorticoids may affect disease progression. [48]

PREVALANCE AND OCCURRENCE

There are >300 million people in the world who are affected by asthma, making it one of the most common chronic diseases. Although the prevalence of asthma is greatest in countries with a high gross domestic product, the disease is recognized worldwide. In the lowest income and most rural countries, the prevalence of asthma tends to be ≤1%, far lower than the 10%

usually seen in developed western countries. Within populations of a given gross domestic product, the prevalence of asthma follows an urban–rural gradient and a weak latitudinal

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Department of Pharmacology, Nandha College of Pharmacy Page 14 gradient, that is, there is greater disease prevalence with greater distance from the equator and asthma is more common in urban areas. Despite the low prevalence of asthma in low-income and middle-income countries, under diagnosis and misdiagnosis together with inadequate treatment in these regions leads to considerable, and potentially avoidable, disease morbidity and mortality.

The prevalence of asthma has increased in many parts of the world over the past few decades, and, until recently, asthma prevalence was increasing on a year by-year basis in developed western countries. The cause of the epidemic that began in the late 1970s is unclear, but the rise in asthma prevalence is consistent with a rise in other immune-mediated diseases, such as type 1 diabetes mellitus, inflammatory bowel disease and multiple sclerosis.

Immunological factors, age and sex all influence the development of asthma. The disease is closely linked to the presence of immediate hypersensitivity, and 50% of children who are diagnosed with asthma by 3 years of age and 80% of those diagnosed by the time they are 6 years of age are atopic — that is, they are genetically predisposed to allergic hypersensitivity.

The prevalence of wheezing exceeds the prevalence of asthma in children up to 6 years of age.

This observation suggests that factors other than asthma, such as physician diagnostic bias and lower respiratory tract infections, can drive the onset and persistence of wheezing. Asthma is not constant across the life course of the patient, and patients can experience periods of remission and the onset of new asthma. Whereas asthma is more common in boys than girls in early childhood, throughout puberty and early adulthood, boys experience asthma remission at a higher rate than in girls. In addition, girls acquire asthma more often than boys in this age period.

Consequently, the sex ratio of asthma during childhood reverses in adolescence and in young adulthood. The reasons for this variability in asthma across the lifecourse of the patient are not clear, but evidence is mounting in support of a key role for hormones in this process.

Furthermore, asthma remission during adolescence is associated with lower initial airway hyper responsiveness and greater gain in the function of small airways compared with asthma that begins after childhood18. The reasons for this variability between early onset and late-onset asthma are probably complex, but differing environmental exposures (exposome) — including those that occur in occupational settings — are thought to be important. The heterogeneity of asthma can pose challenges for epidemiological research. Cross-sectional studies of asthma can be difficult to interpret given that both recall bias and the fact that most patients with asthma will

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Department of Pharmacology, Nandha College of Pharmacy Page 15 have had a varied disease course. As such, at any one time, individuals at different stages of their disease and with different pathophysiological mechanisms for their asthma might be affected.

The exception to this is severe asthma, which usually has an onset in early childhood, is associated with multi-allergen sensitization and persists across the life course. In addition, new asthma in older adults tends to be more severe from the onset than asthma that develops in younger age groups. Finally, asthma might co-occur with chronic obstructive pulmonary disease (COPD) to cause asthma COPD overlap syndrome. The prevalence of this overlap is substantial.[49]

ROLE OF INFLAMMATORY MEDIATORS AND CYTOKINE IN ASTHMA AIRWAY INFLAMMATION

Airway inflammation is a prominent feature of asthma T2-type inflammation occurs in

>80% of children and in the majority of adults with asthma in association with sensitization to environmental allergens, such as those from dust mites, fungi, pets and pollens. This sensitization is often associated with other clinical manifestations of atopy such as atopic dermatitis (eczema), allergic rhinoconjunctivitis and food allergy. The inflammatory infiltrate that accompanies T helper 2 (TH2) lymphocyte responses is mainly composed of eosinophils but also includes mast cells, basophils, neutrophils, monocytes and macrophages. Cellular activation and release of inflammatory mediators in asthma is evidenced by mast cell degranulation and eosinophil vacuolation. The majority of mucosal mast cells in mild-to-moderate allergic-type asthma are of the TH2 cell-dependant tryptase-expressing type (MCT). In the more intractable forms of asthma, mast cells containing both tryptase and chymase (MCTC) predominate, which are more dependent on stem cell factor (also known as KIT ligand) for their survival than are MCT cells.

The principal role of T cells in asthmatic airways is in controlling the inflammatory cell profile. Whereas the activity of TH2 CD4+ lymphocytes predominate in classic allergic-type asthma, roles for a range of other T cells in different asthma subtypes have been described, including the association of TH1 cells and TH17 cells with neutrophilic asthma. In eosinophilic allergic asthma, and potentially non-allergic asthma, the initiation of T2-type immune responses

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Department of Pharmacology, Nandha College of Pharmacy Page 16 occurs through secretion of the epithelial cell-derived cytokines IL-25, IL-33 and thymic stromal lymphopoietin (TSLP). These cytokines induce a new innate lymphoid subset (nuocytes, a type of group 2 innate lymphoid cells (ILC2s)) to produce the T2-type cytokines IL-5, IL-9 and IL-13 Allergen sensitization also requires an interaction between specialized antigen-presenting airway dendritic cells (DCs) and T cells. This mechanism involves processing of allergen into small peptides and the selective major histocompatibility complex (MHC) class II presentation of these processed peptides to the T cell receptors of naive T cells. Effective allergen signalling also requires co-stimulatory interactions between DCs and T cells that take place in local lymphoid collections, resulting in T cell differentiation into TH2-type T cells. These TH2-type T cells secrete the pro-allergic cytokines, IL-3, IL-4, IL-5, IL-9, IL-13 and granulocyte–macrophage colony-stimulating factor (GM-CSF), which in turn leads to the IgE, mast cell and eosinophilic responses that are characteristic of allergic asthma.

Many of the asthma-related allergens — such as those from dust mite, cockroach, animal and fungal sources — exhibit enzymatic properties that enable them to penetrate the epithelial barrier and directly interact with mucosal DCs. During this time, quiescent DCs transform to express an array of cell adhesion and co-stimulatory molecules. These molecules are recognized by naive T cells, which interact with DCs to create an immunological synapse that facilitates allergen presentation. Whereas a minority of allergen-specific TH2 cells migrate to the B cell follicle to initiate immunoglobulin class switching from IgM to IgE, others relocate to the airway mucosa, under the influence of chemoattractants, to elicit the T2-type inflammatory response and the associated coordinated secretion of pro-allergic cytokines.

Once sensitized, further exposure of the airways to allergen results in a mast-cell-driven early-type bronchoconstrictor response (EAR) that lasts for 5–90 minutes and involves IgE- dependent release of histamine, prostaglandin D2 and the leukotriene C4 (LTC4 ), which is subsequently converted to LTD4 and LTE4. The EAR is followed by a late-phase response (LAR) that evolves over 3–12 hours, and is linked to infiltration and activation of leukocytes (especially eosinophils) with further LTC4 generation, TH2 cytokine release from mast cells and T cells and an increase in airway responsiveness.

AIRWAY REMODELLING

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Department of Pharmacology, Nandha College of Pharmacy Page 17 In asthma, the airway wall thickens in proportion to disease severity and duration. This remodelling involves an increase in airway smooth muscle, thickening of the subepithelial reticular lamina, matrix deposition throughout the airway wall, angiogenesis, neuronal proliferation and epithelial mucous metaplasia — a process that involves the appearance of mucous cells in new areas of the airways and increased production of mucus. These events are thought to underlie airway hyper-responsiveness, whereas mucus forms plugs that can extend into the small airways and lead to air` trapping and hyperinflation. In addition, epithelial goblet cell metaplasia results from the actions of IL-4, IL-9 and IL-13, as well as from the secretion of growth factors such as members of the epidermal growth factor family, which cause epithelial cell stress and injury. [50]

CURRENT TREATMENT OF ASTHMA

There are two main categories of anti asthma drugs: the bronchodilators and the anti - inflammatory agents.

BRONCHODILATORS DRUGS

Three types of bronchodilators are used to treat asthma, namely the β2- adrenoceptor agonists, the xanthines and the muscarinic receptor antagonists. Drugs that act as selective β2- adrenoceptor agonists are the first line bronchodilator agents for the treatment of asthma. The main actions of β2- adrenoceptor agonists are bronchial smooth muscle relaxation, inhibition of mediators released from inflammatory cells, inhibition of cholinergic neurotransmission, reduced vascular permeability and increased mucociliary clearance. Considering their duration of action following inhalation of conventional doses, β2- agonists can be divided into three broad groups:

(a) the catecholamines, such as rimiterol, which have a very short action of 1-2 h;

(b) those described as short acting, such as salbutamol and terbutaline, which are active for 3-6 h, although fenoterol may be slightly shorter acting and

(c) the long-acting βagonists salmeterol and formoterol, which cause bronchodilation for at least 12 h.

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Department of Pharmacology, Nandha College of Pharmacy Page 18 Finally, the β2- adrenoceptor agonists are usually given by inhalation of aerosol, powder or nebulised solution, but some may also be given orally or by injection. 11 The xanthine bronchodilator group constitutes three pharmacologically active, naturally occurring methylxanthines, viz. theophylline, theobromine and caffeine. Theophylline (1, 3- dimethylxantine) is the most frequently employed in clinical medicine and can also be used as theophylline ethylenediamine, known as aminophylline. Theophylline has bronchodilator action, but it is rather less effective than the β2- adrenoceptor agonists. The xanthine drugs are usually given orally in sustained-release preparations. Aminophylline can also be given by slow intravenous injection of a loading dose followed by intravenous infusion. Finally, the main muscarinic receptor antagonist that is specifically used in asthma is ipratropium bromide. It relaxes bronchial constriction caused by parasympathetic stimulation, which occurs particularly in asthma induced by irritant stimuli and in allergic asthma. Ipratropium bromide inhibits the augmentation of mucus secretion that occurs in asthma and may increase the mucociliary clearance of bronchial secretions. Generally, ipratropium bromide is given by aerosol inhalation.

ANTI-INFLAMMATORY AGENTS

Two different types of anti-inflammatory drugs are used in the treatment of asthma: the glucocorticoids that are mainly used in chronic conditions and sodium cromoglycate, which is thought to reduce bronchial hyper-reactivity. Steroids are the most effective therapy currently available for asthma and inhaled glucocorticoids have become the mainstay of therapy for patients with chronic disease. Glucocorticoids are highly effective because they block many of the inflammatory pathways activated in asthma. They exert their effects by binding to 12 glucocorticoids receptors (GRs), which are localized in the cytoplasm of target cells and therefore may control inflammation by inhibiting many aspects of the inflammatory process. The main glucocorticoid compounds used are beclomethasone dipropionate, budesonide and fluticasone propionate, which are given by inhalation with a metered dose inhaler, the full effect being attained only after several days of therapy. Cromoglicate and the related drug nedocromil sodium are not bronchodilators. They do not have any indirect effects on smooth muscle, nor do they inhibit the actions of any of the known smooth muscle stimulants. If given prophylactically, they can reduce both the immediate and the late-phase asthmatic responses and reduce bronchial hyper-reactivity. They are effective in antigen-induced, exercise-induced and irritant-induced

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Department of Pharmacology, Nandha College of Pharmacy Page 19 asthma. Cromoglicate is only given by inhalation, either as an aerosol, as a nebulised solution or in powder form. [51]

OTHER DRUGS

Leukotriene inhibitors and phosphodiesterase 4 inhibitors are the new classes of drugs for the treatment of asthma. Leukotriene modifiers are an entirely new class of asthma treatment, which have entered clinical practice in 1996-7 in several countries including Britain, Japan, and the United States. Leukotriene synthesis inhibitors and cysteinyl leukotriene receptor antagonists constitute the two types of leukotriene modifier. Both are used to block the bronchoconstrictor and pro-inflammatory activity of cysteinyl leukotrienes within the asthmatic airway. The leukotriene receptor antagonists include zafirlukast and montelukast; zileuton is the only leukotriene synthesis inhibitor. In the treatment of asthma, randomized controlled trials have shown leukotriene inhibitors to be more effective than placebo but less effective than inhaled corticosteroids or long-acting beta2 agonists. Although their antiinflammatory effects are likely to be less pronounced than those of high dose 13 corticosteroids, their excellent side effect profile and their availability as oral drugs are likely to ensure that compliance with treatment is substantially better than for inhaled corticosteroids. While interrupting the leukotriene pathway offers a new opportunity for treating asthma, the position of such drugs in the asthma armamentarium has not yet been firmly established. Further effectiveness studies are needed to determine the true value of this oral anti-asthma treatment. [52]

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Department of Pharmacology, Nandha College of Pharmacy Page 20 PHYTONUTRIENTS AS THERAPEUTIC AGENTS

The vast and versatile pharmacological effects of medicinal plants are basically dependent on their phytonutrients. Generally, the phytonutrients of plants fall into two categories based on their role in basic metabolic processes, namely primary and secondary metabolites Primary plant metabolites are involved in basic life functions, therefore, they are more or less similar in all living cells. On the other hand, secondary plant metabolites are products of subsidiary pathways as the shikimic acid pathway. In the course of studying, the medicinal effect of herbs is oriented towards the secondary plant metabolites. Secondary plant metabolites played an important role in alleviating several ailments in the traditional medicine and folk uses. In modem medicine, they provided lead compounds for the production of medications for treating various diseases from migraine up to cancer Secondary plant metabolites are classified according to their chemical structures into various classes.

This secondary metabolite play a positive role by maintaining and modulating immune function to prevent specific diseases. Being natural products, they hold a great promise in clinical therapy as they possess no side effects that are usually associated with chemotherapy or radiotherapy. They are also comparatively cheap and thus significantly reduce health care cost.

Secondary metabolite are the plant nutrients with specific biological activities that support human health. Some of the important bioactive secondary metabolite include polyphenols, terpenoids, resveratrol, flavonoids, isoflavonoids, carotenoids, limonoids, glucosinolates, phytoestrogens, phytosterols, anthocyanins, ω-3 fatty acids, and probiotics. They play specific pharmacological effects in human health such as anti-microbial, anti-oxidants, anti- inflammatory, antiallergic, anti-spasmodic, anti-cancer, anti-aging, hepatoprotective, hypolipidemic, neuroprotective, hypotensive, diabetes, osteoporosis, CNS stimulant, analgesic, protection from UVB-induced carcinogenesis, immuno-modulator, and carminative. [53]

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Department of Pharmacology, Nandha College of Pharmacy Page 21 CURCUMIN

Curcumin is a yellow coloured polyphenolic pigment obtained from powdered rhizomes of Curcuma longa belonging to family-Zingiberaceae, commonly known as turmeric, a medicinal plant widely used in traditional Indian and Chinese medicine

Curcuma longa has a 25 years(is it only 25 years) old medical history. Ayurveda, Siddha, Unani and Chinese systems of medicine recommend curcumin for large number of diseases and disorders. Traditional Indian systems of medicine use the powder against disorders like diabetic wound, cough, hepatic disorders, rheumatic disorders etc.Traditional Chinese medicine uses curcuma in diseases associated with abdominal pain, amenorrhea, dysmenorrhea, distending or pricking pain in the chest and abdomen. It is also applied topically for ulcers, wounds, eczema, and inflammations

PHARMACOLOGICAL ACTIVITES OF CURCUMIN

Various pharmacological actions of curcumin have been studied by various researchers worldwide. Curcumin has been shown to possess several pharmacological actions including antiinflammatory, anticancer, antioxidant and antimicrobial effects. Curcumin has demonstrated chemo preventive properties, suppressing the tumorigenic activity of a wide variety of carcinogens in several kinds of cancer. In culture cell and animal studies, curcumin has been shown to exhibit antiproliferative, antiinvasive, and anti angiogenic properties. Curcumin has also demonstrated its usefulness for the treatment of other diseases such as Diabetes, Alzheimer’s disease, Parkinson’s disease, and Arthritis. [54]

VASICINE

Vasicine is a quinazoline type alkaloid mainly obtained from the plant Adhatoda vasica (zeylanica)/Justicia adhatoda (Acanthaceae) . Few of the main chemical constituents of this plant are vasicine (derived from leaves), 2'-hydroxy-4- glucosyloxychalcone, vasicol (from leaves), vasicinone (from leaves, stem and roots), vasicinol (contained in stem and roots), and deoxyvasicinone (from leaves)

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Department of Pharmacology, Nandha College of Pharmacy Page 22 The herb Adathoda vasica is an old plant known for its promising therapeutic action against respiratory disordes. The juice of leaves is a cure for diahhrea, dysentery and glandular tumor. It has been traditionally used in the management of allergic conditions and bronchial asthma. Research carried out over the last three decades reflects the fact that this plant’s rich in alkaloid possess respiratory stimulant activity. It aid in curing common cold, bronchitis, influenza, cramp and dry cough, pertussis, hay-fever, asthma and sinusitis.

PHARMACOLOGICAL ACTIVITY OF VASICINE

Various pharmacological actions of Vasicine have been studied by various researchers worldwide. Vasicine has been shown to possess several pharmacological actions including anti- inflammatory and antioxidant activity, Genoprotective role, Hepatoprotective, Antitussive and bronchodilatory action, Muscle relaxant activity, Antiulcer, Antibacterial, Antihelmenthic, Antidiarrheal and Abortifacient. [55]

COMBINATION THERAPY

The “one drug, one target, one disease” approach has for some time remained the conventional pharmaceutical approach to the development of medicines and treatment strategies.

However, over the last decade, this mono-substance therapy model has gradually shifted toward the adoption of combination therapies, in which multiple active components are employed. This paradigm shift has been partly driven by its limited effectiveness in chronic diseases, treatment resistance, and side effects of synthetic mono-drugs.

Generally, synergy is defined as the interaction of two or more agents to produce a combined effect greater than the sum of their individual effects. In medicinal research field, however, the understanding of synergy is complicated. Classified the concept of synergy broadly into two main categories based on the mode of actions pharmacodynamic and pharmacokinetic synergy. The first type of synergy describes two or more agents that work on the same receptors or biological targets that result in enhanced therapeutic outcomes through their positive interactions. The second type of synergy results from interactions between two or more agents during their pharmacokinetic processes (absorption, distribution, metabolism and elimination) leading to changes of the agents quantitatively in the body and hence their therapeutic effects. [56]

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Department of Pharmacology, Nandha College of Pharmacy Page 23

LITERATURE REVIEW

Tao Zhu et al., (2019) Aimed to study the value of curcumin in asthmatic airway inflammation and mucus secretion and its underlying mechanism. The results indicate that curcumin attenuated OVA-induced airway inflammation and mucus hypersecretion in mice and suppressed OVA- and IL-4-induced upregulation of MCP-1 and MUC5AC both in vivo and in vitro, most likely through a PPARγ-dependent NF-κB signaling pathway. [57]

Tiechao Jiang et al., (2019) investigated the protective effect of vasicine (VAS) against myocardial infarction in rats, and its mechanism. VAS suppressed apoptosis when tested on animals suffering from ISO-induced MI, by decreasing the expression of cleaved Caspase-3 and Bax while increasing the expression of Bcl-2. [58]

Mumtaz Guran et al., (2019) studied the combinatory anti-inflammatory interactions between Quercetin (Q) and Curcumin (C) along with their combined antimicrobial activity against MRSA. Results suggest that combining low concentrations of Q and C yield similar or better anti-inflammatory effectiveness when compared to treatment with each agent alone.

Moreover, they co-operate synergistically in the context of antimicrobial activity, with an increased effectiveness when compared to Q or C alone at high concentrations. [59]

Asha Kumari et al., (2018) studied the antiashmatic activity of curcumin on Balb/c mice which was exposed to antigen (ovalbumin) and LPS simultanously. Curcumin well known for its anti-infammatory potential, was administered through intranasal route 1 h before LPS and OVA (ovalbumin) exposure to evaluate its efficacy against airway structural changes. Result shows Intranasal curcumin pretreatment had signifcantly suppressed infammatory mediators and airway remodeling protein. [60]

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Department of Pharmacology, Nandha College of Pharmacy Page 24 Cong Lan et al., (2018) aimed to explore whether Curcumin has a preventive effect on stroke. Result showed that administration of Curcumin significantly delayed the onset of stroke and increased the survival of SHRsp, which was ascribed to decreased ROS and improved endothelial dependent relaxation of carotid arteries. In the presence of UCP2 inhibitor genipin, both Curcumin-mediated decrease of ROS and increase of NO production were blocked. [61]

Farzaneh Shakeri et al., (2017) evaluated the effect of Curcuma longa (C. longa) and Curcumin on total and differential WBC count and oxidant, antioxidant biomarkers, in rat model of asthma. Antioxidant and anti-inflammatory effects of C. longa extract and its constituent Curcumin in animal model of asthma was observed which suggest a therapeutic potential for the plant and its constituent on asthma. [62]

Adriana Bulboac et al., (2017) aimed to investigate the analgesic and antioxidative stress effects of Curcumin (CC) in experimental migraine induced by Nitroglycerin (NTG) on rats, compared with Indomethacin (ID) and Propranolol (PP) treatments. The group pretreated with Curcumin proved significantly smaller number of flinches and shakes compared with both NTG + PP and NTG + ID. Hence, the study demonstrates a superior activity of Curcumin not only versus control, but also versus Propranolol and Indomethacin.[63]

Renata Czekaj et al., (2017) determined the effect of Curcumin against gastric haemorrhagic lesions induced by 75% ethanol and alterations in gastric blood flow (GBF) in rats with cyclooxygenase-1 (COX-1) and COX-2 activity inhibited by indomethacin, SC-560 or rofecoxib, inhibited NO-synthase activity, capsaicin denervation and blockade of TRPV1 receptors by capsazepine and plasma gastrin levels. Curcumin-induced protection against ethanol damage involves endogenous PG, NO, gastrin and CGRP released from sensory nerves due to activation of the vanilloid TRPV1 receptor. This protective effect can be attributed to the inhibition of HIF-1α and Cdx-2 expression and the activation of HO-1 and SOD 2 expression. [64]

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Department of Pharmacology, Nandha College of Pharmacy Page 25 Arvinder Kaur et al., (2016) studied an alkaloid-vasicine has been isolated from the most bioactive n-butanol fraction of Justicia adhatoda and analysed for antioxidant, antimutagenic (Ames assay), and anticarcinogenic potential. Overall studies shows that vasicine contains antioxidant, antimutagenic as well as anicancerous effect.[65]

Matthew et al., (2016) reviewed the experimental and pharmacologic trials that demonstrated the efficacy of Curcumin as an anti-inflammatory agent. This is a review article written with the objective to systematically analyze the wealth of information regarding the medical use of Curcumin, the “curry spice”, and to understand the existent gaps which have prevented its widespread application in the medical community. [66]

Wei Liua et al., (2015) evaluated the antitussive, expectorant, and bronchodilating effects of the quinazoline alkaloids (±)-vasicine (VAS), deoxyvasicine (DVAS) (both isolated from the alkaloid fraction of APP) and (±)-vasicinone (VAO) (synthesized from VAS).

Bronchodilation tests showed that VAS, VAO, and DVAS prolonged the pre-convulsive time, whereas aminophylline prolonged the pre-convulsive time more when compared with pretreatment. [67]

Rachana et al., (2014) investigated the protection against cytotoxicity due to tobacco smoke by Adhatoda vasica and Vasicine. The antioxidant potential of AVE was analyzed through in vitro assays. The protective effect of Adhatoda vasica extract (AVE) and vasicine were analyzed in TSE treated group through MTT assay and microscopic analysis. A dose dependent increase in reducing power of AVE was observed. Treatment of A549 & THP-1 cell lines with 1-2 µg/ml (AVE) & 0.01-0.02 µg/ml (Vasicine) respectively for 3 hrs maintained the cell viability. Approximately 50% cell death was observed at 2% & 5% TSE on 24 hrs exposure. Pre-treatment of cell lines with AVE & Vasicine (2µg/ml & 0.02 µg/ml) respectively could overcome the toxic effect of TSE. [68]

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Department of Pharmacology, Nandha College of Pharmacy Page 26 Preeti et al., (2014) investigated the effects of intranasal Curcumin in chronic asthma where animals were exposed to allergen for longer time. Intranasal Curcumin administration prevented accumulation of inflammatory cells to the airways, structural alterations and remodeling associated with chronic asthma like peribronchial and airway smooth muscle thickening, sloughing off of the epithelial lining and mucus secretion in Ovalbumin induced murine model of chronic asthma. [69]

Asha Kumari et al., (2014) aimed to investigate effects of intranasal Curcumin on LPS- induced ALI in mice where Curcumin (10 mg/kg, intranasal (i.n.) was given an hour before LPS exposure. The results showed intranasal Curcumin could be a novel therapeutic strategy for LPS- induced ALI by directly targeting the lungs and enhancing anti-oxidant levels. [70]

Chunhua Ma et al., (2013) aimed to determine the protective effects and the underlying mechanisms of Curcumin on Ovalbumin (OVA)-induced allergic inflammation in a mouse model of allergic asthma. The results in vivo show Ovalbumin-induced significantly broke Treg/Th17 balance; Curcumin treatments markedly attenuated the inflammatory in asthma model by regulating Treg/Th17 balance. [71]

Rashmi Pa et al., (2012) evaluated the adathoda by determination of the diameter of zone of inhibition against bacteria and fungi. 25g ml-1 concentration was used to check the antimicrobial activity of plant extracts and vasicine. The study revealed that J. adhatoda has broad spectrum of antimicrobial activity and a potential source of antimicrobial agents that could be useful for chemotherapy and control of infectious diseases. [72]

El-Sayed et al., (2011) study was designed to compare the inhibitory effects of thymoquinone (TQ) and Curcumin (CMN) on the biological changes associating asthma. These results suggest that TQ is more potent in inhibiting the inflammatory changes associating asthma.

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Department of Pharmacology, Nandha College of Pharmacy Page 27 On the other hand, CMN was a less potent inhibitor of all measured parameters, despite its superior inhibitory effect on TNF-α mRNA levels. [73]

Karaman et al., (2011) amied to study the inflammatory activity of Curcumin using murine mouse model. Finally the study demonstrated that Curcumin administration alleviates the pathological changes of chronic asthma. Curcumin might be a promising therapy for asthma in the future. [74]

Dong Oh Moon et al., (2008) studied the Curcumin contributes to anti-inflammatory activity in the murine asthma model and lung epithelial cell A549 through suppression of nitric oxide (NO). These findings show that Curcumin may be useful as an adjuvant therapy for airway inflammation through suppression of iNOS and NO. [75]

Srinivasarao et al., (2006) Investigated the antioxidant and anti-inflammatory potential of vasicine isolated from leaves of the Adhathoda vasica in murine model of asthma. After treatment with vasica significant decrease in lipid peroxidase and similarly significant increase in antioxidant superoxide dismutase, catalase, glutathione peroxidase and retered glutathione was recorded.[76]

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Department of Pharmacology, Nandha College of Pharmacy Page 28

AIM AND OBJECTIVE

AIM

To evaluate the combined effect of Curcumin and Vasicine using In-vivo ova-induced allergic airway inflammation mouse model and In-vitro Guniea pig ileum and trachea

preparation.

OBJECTIVE

Curcumin is a polyphenolic compound also known as diferulayl methane, is an active compound from the golden spice turmeric (Curcuma longa). It is a highly pleiotropic molecule that exhibit good anti-inflammatory activity,Which is proved to treat allergic condition in asthma.

Vasicine is an Alkaloid obtained from the plant Adathoda Vasica. It is an ancient drug which is used in the treatment of respiratory disorder. It is used as a mycolytic, bronchodilator and also contains anti-inflammatory activity. Both the constituents show great result in treating asthma through their anti-inflammatory activity.

Our study is mainly focuses on combining both the constituents together to find the synergetic effect. Anti-inflammatory effect of both the drugs is believed to be synergetic and increase the anti-inflammatory activity compared to the activity produced by single constituents.

Hence based on the observation, the main objective of the study is,

 To evaluate the Anti-asthmatic activity of Curcumin and Vasicine combination by following in-vitro and in-vivo methods.

1. In-vivo ova-induced allergic airway inflammation mouse model 2. In-vitro Guniea pig ileum preparation.

3. In-vitro Guniea pig tracheal chain preparation.

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Department of Pharmacology, Nandha College of Pharmacy Page 29

PLAN OF WORK

PHARMACOLOGICAL EVALUATION

In-vivo Study

Evaluation of Anti-asthmatic activity using suitable In-vivo method

 Ova-Induced Allergic Airway Inflammation Mouse Model Parameters:-

1. Lung pathology and Remodeling

 Haemotoxylin and eosin staining

 Periodic acid-Schiff staining (PAS)

2. Total and differential counts in the blood and lung lavage 3. Total IgE level in serum.

In-vitro Study

Evaluation of Anti-histaminic activity using In-Vitro study

 Isolated Guinea pig tracheal chain preparation

 Isolated Guinea pig ileum preparations

STATISTICAL EVALUATION

The data were analyzed using one way analysis of variance (ANOVA) followed by Dunnett’s “t” test. P<0.05was considered as significant.

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Department of Pharmacology, Nandha College of Pharmacy Page 30

DRUG PROFILE

CURCUMIN[77]

Synonyms: Curcumin, Diferuloylmethane, Natural yellow 3, Turmeric yellow

IUPAC Name: (1E, 6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione

Structure:

Chemical Formula:C21H20O6 Molecular Weight: 368.4 g/mol Melting point: 356 to 361 °F Log P: 3.29

H bond donor: 2 H bond acceptor: 6

Appearance: Yellow colored powder

Solubility: Very soluble in ethanol, acetic acid

Stability: Stable under recommended storage conditions.

Description: Curcumin, also known as diferuloylmethane, is an active component in the golden spice turmeric (Curcuma longa). It is a highly pleiotropic molecule that exhibits antibacterial, anti-inflammatory, hypoglycemic, antioxidant, wound-healing, and antimicrobial activities. Due

References

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