2 Dec 13

Evaluation of Anti-asthmatic Activity in Animal Model
1. INTRODUCTION
Page 1
Overview
Number of cases of asthma is increasing nowadays. It has become a common complaint
in pediatric and geriatric people. Growing urbanization, air pollution, environmental
tobacco smoke are the major contributing factors for increased prevalence of asthma and
its genetic predisposition. Since 1919, the prevalence is increasing from 9% to 30%
according to the statistical studies carried out by hospital in city of Bangalore 1. Inflated
morbidity due to asthma is mainly contributed by exposure of environmental tobacco
smoke during childhood, as well as adulthood. Also, local aeroallergen trigger the
hyperresponsiveness in asthmatic condition2. According to World Health Organization,
300 million people suffer from asthma from which 180,000 people die due to it.
Diverse studies suggested that house dust may be the primary cause of
development of asthma, which may trigger the exacerbation of symptoms3. Asthma is
characterized by the airway inflammation manifested by cough, wheezing chest tightness,
acute exacerbations, dyspnea, bronchoconstriction4. Triggers which take part for initiation
of asthmatic symptoms are allergens, irritants, exercise, emotional stress, viral or sinus
infections. Other substances which aggravate the condition of asthma are cigarette
smoke, pollen, molds, chalk dust, talcum powder, paints, varnishes, animal dander.
In management of asthma, two types of therapies are available, First by ‘Curative’

approach, in which medicines like Cromolyn sodium, Nedocronium sodium and steroid
are used for long duration; second by ‘Symptomatic’ approach, in which bronchodilators,
including β2 agonist like Methyl-xanthines are used4. Since ancient time, plants are the
best source of medicines. Due to lack of satisfactory success and adverse effects, patients
are in search of alternative therapy for treatment of asthma. india has about 45000 plants
having medicinal activities. From last few decades, scientist found many medicinal herbs
possessing antihistaminic, anti-allergic, anticholinergic, bronchodialatory activity.
Ayurveda offers unique insight onto comprehensive approach on management of asthma.

this approach includes the maintenance of nourishment of lungs and respiratory system
and providing a sufficient quantity of oxygen5. Herbs which are useful for the treatment
of asthma are as follows-
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i) Adhatoda vasica (Vasa) – it shows mast cell stabilizing property thus

suggesting anti anaphylactic activity6.
ii) Clerodendron serratum (Bharangi) – aqueous extract of leaves of this herb
shows bronchodialatory activity7
iii) Abrus precatorius (Gunja) – ethnolic extract of leaves of A. precatorius shows
inhibition of milk induced leukocytosis and eosinophilia8.
iv) Albizia lebeckk (Shirisha) – decoction of bark and flower exhibit inhibition of
histamine and acetylcholine induces bronchospasm in guinea pigs9.
v) Ocimum sanctum (Tulsi) – shows mast cell stabilizing activity in albino rats10.
vi) Moringa oleifera (Shigru)– this herb possess bronchodialatory, anti-
inflammatory, mast cell stabilizing activity and thus collectively having anti-
asthmatic activity11.
vii) Nyctanthes arbortristis (Parijata) – pet ether extract of this showed relaxing
property on mice and guinea pig ileum and also has mast cell stabilizing
property12.
viii) Piper nigrum (Pippali) – extract of this inhibited acetylcholine induced
contraction in isolated goat trachea13.
ix) Abutilon indicum (Atibala) – in inhibits albumin induced mast cell
degranulation in rat peritoneum, also showed a good results in carageenan
induces rat paw edema model14.
x) Ficus religiosa (Asawatha) – aqueous extract of leaves found to be effective
in curing histamine and acetylcholine induced bronchospasm15.
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2. LITERATURE REVIEW
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2.1- Review of Disease

Asthma
It is chronic pulmonary disorder accompanied by recurrent episodes of wheezing,
chest tightness, cough, breathlessness. These episodes are associated with variable
airflow obstruction which can be reversible if treated. The inflammation also
associated with airway hyper responsiveness to variety of stimuli.
It is characterized by airway inflammation, i.e. redness and swollen airway, similarly

airway obstruction i.e. tightening of the airway muscles which makes difficulty in air
movement, and airway hyperresponsiveness (AHR) i.e. muscles of airways respond
more quickly to minute quantity of allergen or stimuli16.
Common signs and symptoms –
These include coughing, wheezing, breathlessness, increased respiratory rate, chest

tightness, fatigue, agitation, increased pulse rate, inability to participate in sports.
During some episodes, the symptoms include –
 Inability to talk continually

 Retractions – increased use of chest and abdominal muscles
 Refusal to lie down – Preference for sitting position
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Exercise
Release of catacholamines
bronchodilation
Increased airflow
Airway cooling Increased lower airway

osmolarity
vasoconstriction
Movement of fluid from the Degranulation of mast

epithelium to the airway cells and eosinophils,
Hyperemia Mucosal edema surface epithelial cells
involvement
Release of PGs,
Bronchoconstriction histamine, LTs, PAF
Airway inflammation
Fig. 2.1 Schematic representation for progression of the causes for asthma
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2.1.1- Classification of asthma

1. Extrinsic asthma
2. Intrinsic asthma
3. Occupational asthma
4. Drug induced asthma
5. Exercise induced asthma
6. Cough variant asthma
1. Extrinsic asthma
Immune response associated with allergy, occurs over 3 years of age and in adults. IgE is
the main reason for this. The antigens may contain fungal spores, animal denders, pollen
grains etc. inflammatory cells involved in this asthma are T cells, B cells, Neutrophils,
eosinophils, mast cells. Their cytokines like IL-4, IL-5, IL-13, leukotrienes, IFN γ, TNF α
are also responsible. Symptoms like coughing, wheezing, chest tightness, rapid breathing
are observed17
2. Intrinsic asthma
This is also called as cryptogenic asthma and non-allergic asthma. There is no definite
immunological basis for the pathogenesis of this type of asthma, still airway
inflammation remains characteristic in this. Stimuli contain emotional stress,
infection, gastroesophageal reflux. Symptoms are almost same as in extrinsic
asthma17.
3. Occupational asthma
One type of occupational asthma is immune mediated, and other one is irritant induced.
Irritants may be animal products, plastic resins, various dusts, metals etc. In immune
induced, there is latency period, between workplace exposure and beginning of
symptoms. In irritant induced asthma, symptoms occur immediately after exposure. This
can be aggravated due to repeated exposure to industrial irritants, fibres. Generally in
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industries with fibre manufacturing, the employees may be at risk for this type of
asthma18.
4. Drug induced asthma
Approximately 10-20% of adult s are sensitive to certain drugs which may precipitate
asthmatic attack. These drugs include NSAIDs like ibuprofen, naproxen. This kind of
asthma is also generally precipitated by COX inhibitors like aspirin19
5. Exercise induced asthma
It is characterized by transient airway observation occurring after strenuous exertion. Due

to rapid airway rewarming after exercise, vascular congestion, increased permeability,
oedema leads to obstruction20.
6. Cough variant asthma
Associated with symptoms like dry and productive cough.(non-productive cough dose
not expel any mucus from respiratory tract.) it is a chronic cough which lasts for six to
eight weeks21.
2.1.2- Epidemiology
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In united states, 7% of people are suffering from asthma and 5% people in united
kingdom are affected. In last two decades prevalence is been increased by 75% . this has
become a worldwide problem for both children and adults. 235-330 million people
worldwide are affected by asthma. Approximately, 250000 people die per year from this
disease. . Since 1970s, the global prevalence, morbidity, mortality, and economic burden
of asthma have increased particular in children22.
Age and gender also effects for the prevalence of asthma. It is more in boys than girls.
Ratio for this male to female is 3:2. Children younger than age 18 are more susceptible
for the disease. This may decrease with increasing age. African and American cases with
asthma show greater morbidity and mortality. Low and middle income countries contain
80 % of mortality. In united states, plateau has been reached after 1998 with prevalence
3.8% in 2003. Asthma affect about 300 million people worldwide and it has been
estimated that a further 100 million will be affected by 2025Asthma affect about 300
million people worldwide and it has been estimated that a further 100 million will be
affected by 202523,24.
Occurrence of this disease may may be impacted by environmental factors which include
aeroallergens, like dust, pollen grains, fibres. Race is another factor affecting the
prevalence of the disease. Greater morbidity and mortality observed in Africans/
American asthmatics compared to causation asthmatics25.
2.1.3- Etiology
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There are many inflammatory cells which contribute for the etiology of asthma. The
disease is complex and multifactorial.
INFLAMMATORY CELLS INVOLVED
1. T cells
2. Mast cells
3. Eosinophils
4. Neutrophils
5. Macrophages monocytes
Fig. 2.2 Immunological cell involvement in the progression of inflammation antigen

exposure.
1. T cells-
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These are most abundant cells observed in airways of the patients with asthma.
Various cytokines are produced from T cells which perform the inflammatory
process.
CD4+ cells can differentiate into T helper 1 (Th1) cells, T helper 2 (Th2) cells
and Regulaory T (Tregs) cells. CD8+ cells develop into Cytotoxic T (Tc) cells26.
Th-1 cells
These cell produce γ interferon and IL-2 mainly. Also IL-12, IL-18, TNF-α, and
TNF-β are secreted by the same. Role of these cells is not that clear yet, bur it was
observed that the level of TNFα and interferon γ was elevated in the bronco alveolar
lavage fluid of the patients with asthma27. This suggests that Th1 cells may be
associated with severe disease and contribute to asthmatic cases28.
Th2 cells
CCR4 and CCR8 receptor are present on Th2 cells. These cells are characterized by
the secretion of IL-4, IL-5, IL-13 mainly. But the also secrete other cytokines like IL-3,
IL-4, IL-5, IL-6, IL-9, IL-10, IL-13 and GM-CSF,. They interact with B cells and
stimulate the antibody production from the same for protecting from extracellular foreign
matter like parasite etc. in the patients with asthma, IL-13 level is augmented 29. In intial
state of asthma, these cytokines help in antibody production, and in later state, they show
inflammatory responses30.
Tregs cells
These are autoimmune regulators. Treg levels were reduced in BALF. These cells
downregulate the allergic inflammation. Still the role is not that clear. According to
hypothesis, in asthmatic airway, there is suppressed airway inflammation. Recruitment of
Tregs was observed in reduced level in bronchoalveolar lavage fluid31.
2. Mast cells
In airway inflammatory cells, nearly 20% of the cells are mast cells. These cells
are found in airways at many sites like beneath the basement membrane of
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airways, near blood vessels in submucosa, spread in muscle bundles and also in
bronchial luminal space32. These are multifunctional cells and perform many
inflammatory activities. A huge aaray of mediators is released after activation of
mast cells. These cells are traditionally recognized for there role in
hypersensitivity reactions. in 1878, Paul Ehrlich firstly describe mast cell. Mast
cell houses numerous granules which contain many preformed mediators like
leukotrienes, histamine, tryptase and many cytokines. These cell have high
affinity FCεRI IgE receptors on their surface, which on cross-linking, and on
binding with IgE antibodies, activate mast cells consequently initiating it’s
degranulation. The release of cytokines and mediators occurs at this step. This is a
sufficient trigger for bronchoconstriction, airway inflammation33.
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Fig. 2.3- Mast cell degranulation due to Ag:Ab complex and IgE receptor on mast
cell
These are long lived cells and have capacity to get activated more than one time.
Mast cell activation results into release as well as de novo synthesis of mediators
such as IL-4, IL-5, thus it contributes to a continued inflammatory response34.
3. Eosionophils
These are polymorphonuclear granulocytes functions as protector against large
parasitic infection. Results of biopsies at bronchoscopy showed increased numbers of
eosinophils in the epithelium and lamina propria. Late asthmatic reactions are
characterized by eosinophilic infiltration in airways. The cellular products of
eosinophils like eosinophils peroxidase, major basic protein, eosinophil cationic
proteins are also harmful to epithelial cells of airway 35. Increased level of these cells
in peripheral blood as well as bronchalveolar lavage is the sign of more severe
asthma. By examining the number of eosinophils, the stage of asthma can be
suggested. In animal model of asthma, it was observed that, IL-4, IL-5, IL-13 are the
cytokines initiate activation and survival of eosinophils and ultimately induce
eosinophilia in airways36. Bronchial hyperresponsiveness and airway eosinophilia are
generally associated with each other37. Numbr of these cells is generally correlated
with the severity of the disease. Pro-inflammatory mediator release from activated
eosinophils is linked with mucosal damage in chronic asthma38.
4. Neutrophils
Number of neutrophils is not that increased in airway lavage of patients with mild to
moderate asthma., but in severe asthmatic conditions, the level is increased in airway
secretions. Shaw and colleagues found that high number of nutrophils and eosinophils
are associated with low prebronchodilator FEV, but only high number of neutrophils
is associated with low post-bronchodialator FEV36.
5. Macrophages monocytes
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Macrophages may traffic in airway and can be activated by allergen via low
affinity IgE antibody. This process produce many inflammatory cytokines.
Macrophages, thus have capacity to instigate a specific type of inflammatory
response by secreting a certain kind of cytokines. Increase or decrease of
inflammation is dependent on type of stimulus. Alveolar macrophages have
suppressant effect on lymphocytes, but, impaired effect is shown after allergen
exposure. These cell may also act as antigen presenting cells, thus after processing
an antigen, it can be presented to T cells39.
Cytokines
These are small molecular weight proteins which assist to the inflammatory effectors
cells for development. These are secreted by lymphocytes and macrophages. Cytokines
perform paracrine, endocrine and autocrine function as they act on adjacent cells, distant
cells and may also act on cells of origin respectively40.
Cytokines are pleiotropic in nature as they possess different biological effects on different
target cells. They are redundant, i.e. similar function can be performed by different
cytokines. Multifunctional cytokines are those which perform more than one function.
Cytokines bind to high affinity receptors on their target cells which are then internalized.
This leads to different activation/inhibition processes on enzymatic system. These effects
modify DNA and protein synthesis . Cytokines have high affinity for their receptors,
therefore, even picomolar concentration of them is able to exhibit the biological effects.
Depending upon source, these are divided into two groups, lymphokines, which are
produced by lymphocytes, and monokines, which are produced by monocytes.
Various cytokines –
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Interlukins- secreted by leukocytes.
Lymphokines- secreted by lymphocytes.
Monokines- secreted by monocytes.
Interferon- involved in the process of viral responses.
Colony stimulating factors- increase the growth of cells in media.
Chemokines- promote chemotaxis.
Cytokines have ability to induce the expressions of receptors. Some cytokines express
themselves in autocrine manner, other stimulate the production of other cytokines.
Cytokines are responsible for many inflammatory processes like airway remodeling,
bronchial hyperresponsiveness, bronchoconstriction. various interlukins involved in
asthma are as follows –
Lymphokines Cellular Effects

IL-2 Eosinophilia in vivo , Growth and differentiation of T-cells
IL-3 Eosinophilia in vivo , Pluripotential hematopoietic factor
IL-4 Eosinophil growth ↑ ;Th2 cells ↑; Th1 cells ↓, IgE↑
IL-5 Eosinophil maturation, Apoptosis ↓, IgE↑, Th2 cells ↑ BHR
IL-13 Activates eosinophils ; Apoptosis ↓; IgE↑
IL-15 Eosinophilia in vivo ; Growth and differentiation of T-cells
IL-16 Eosinophil migration ; Growth factor and chemotaxis of T-
cells (CD4+)
IL-17 T-cell proliferation ; Activates epithelial and endothelium
cells; Activates fibroblast
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Fig. 2.4 –
Pleiotropic activities of T helper type 2 (Th2) cytokines in allergic asthma.
Cyokines are small protein molecules which play an important role in persistence of
inflammation. Cytokines also regulates the expression of adhesion molecule on airway
epithelial cells and endothelial cells of circulation of the same. Interleukin-4 increases the
expression of Vascular cell adhesion molecule-1 (VCAM-1) at the site of airway
epithelial cells and endothelial cells41.
Cell source Released mediators

1. Induction phase
T cells Cytokines (IL-4, IL-5, IL-9, IL-13)- mucus hypersecretion
2. Early asthmatic reaction
Mast cells Histamine; proteases (tryptase, chymase, carboxypeptidase); proteoglycans
(heparin, chondroitin sulphate E);
prostaglandins (PGD2); leukotrienes (LTC4); cytokines (TNF-α, IL-3, IL-4, IL-5, IL-6,
IL-8, IL-16, GM-CSF);
chemokines (CCL2, CCL3, CCL11)
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Basophils Histamine; leukotrienes (cys-LTs: LTC4, LTD4, LTE4); cytokines (IL-4, IL-
13)
3. Late asthmatic reaction
 Eosinophils MBP; ECP; EDN; EP; leukotrienes (cys-LTs: LTC4, LTD4, LTE4);
cytokines (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10
 IL-11, IL-12, TNF-α, TGF-β, GM-CSF); chemokines (CXCL8, CCL3, CCL5)
 Neutrophils Leukotrienes (LTA4, LTB4); PAF; TXA2; cytokines (IL-1_, IL-6,
TNF-_, TGF); chemokine (CXCL8); proteases (elastase, collagenase, gelatinase
B); microbicidal products (lactoferrin, myeloperoxidase, lysozyme); reactive
oxygen intermediates (superoxide, hydrogen peroxide); NO
 T cells Cytokines (IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, GM-CSF);
chemokines (CCL1, CCL22)
 Macrophages Cytokines (IL-1, IL-6, IFNγ, TNFα); lipids; PAF; ROS; NO
 Dendritic cells Chemokines (CCL2, CCL3, CCL4, CCL17, CCL22, CXCL8)
 Endothelial cells ICAM-1, ICAM-2; PECAM-1; VCAM-1; selectins (E-selectin,
P-selectin)
 Airway smooth muscle cells Chemokines (CCL5, CCL7, CCL11, CCL13,
CXCL8); cytokines (GM-CSF, IL-6); prostaglandins (PGE2); ECM proteins
 Bronchial epithelial cells Cytokines (IL-6, GM-CSF); chemokines (CCL11,
CCL17, CCL22, CXCL1, CXCL6, CXCL8); ICAM-1
 Interleukin 5 plays important role in pathogenesis of asthma. It is necessary for
maturation of eosinophils.
 Interferon γ – it reduces eosinophils in bronchoalveolar lavage and airway
hyerresponsiveness (AHR).
 Platelet derived growth factor – it is secreted from platelets, macrophages,
endothelial cells, fibroblasts, airway epithelial cells and vascular smooth muscle
cells42.
 Transforming growth factor β – it is secreted from eosinophils, neutrophils,
airway smooth muscle cells mast cells and endothelial cells ultimately
responsible for the airway hyperresponsiveness (AHR). Release of TGF-β1 into
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bronchoalveolar lavage fluid has been observed after segmental allergen

challenge43.
 Epithelial growth factor – this increases the number of blood vessels in asthmatic
airways. EGF expression is increased in epithelium of patients. It increases airway
smooth muscle proliferation44.
Airway remodeling
This simply means structural changes in asthmatic lungs. Due to inflammatory responses
in airway, these structural changes occur. It is linked to bronchial hyperresponsiveness,
long-term decrease in lung function in asthmatic patients. Consequences of airway
remodeling are airway narrowing, bronchial hyperresponsiveness, airway edema, mucus
hypersecretion. Morphometry explains that, thickening of mucus layer is the cause for
bronchial hyperresponsiveness, along with narrowing of airway.
Airway remodeling is one of the central features of asthma. Remodeling is defined as

tissue injury followed by structural changes seen in airways. This ultimately leads to loss
of lung function. Airway thickening is observable as much as 50 to 300% in severe
asthmatic patients and 10 to 100 % in the cases with mild asthmatic conditions 45.
Inflammatory cells such as T cells, neutrophils, mast cells, eosinophils, macrophages,
airway smooth muscles play a major role in inflammatory reactions and airway
remodeling. Mediators like TGF-β, vascular endothelialgrowth factor (VEGF), matrix
metalloproteinase-9, Th2 cytokines (interleukin [IL]-5, IL-13, IL-4, and IL-9) are linked
to remodeling play an important role in progression of airway remodeling46.
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Fig. 2.5- Events and cell involved in airway remodeling in asthma.
Events in Airway remodeling
1. Epithelial layer damage

2. Subepithelial fibrosis
3. Goblet cell hyperplasia
Airway remodeling is basically a repair mechanism 47. It includes changes in epithelium,

lamina propria, submucosa of the airway. This review presents the mechanism, diagnosis
and treatment of airway remodeling which is one of the culprit of asthma. Persistent
inflammation and epithelial damage ultimately leads to severe condition in asthma.
The process involves changes in cellular, biochemical, and molecular components of

bronchial wall. Changes involver apoptotic activity of epithelial cells, goblet cells
hyperplasia, hypertrophy. Lamina reticularis layer become thickened. This leads to
deposition of collagenous and non-collagenous mater beneath the epithelial basement
membrane48. Smooth muscle mass is also increased. Pathological analysis of bronchial
biopsy and autopsy reveal that deposition o extracellular matrix in subepithelial layer,
submucosal glands hypertrophy, hyperplasia, increase in submucosal vessels collectively
orchestrates the event of airway remodeling.
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1. Epithelial layer damage –
It includes shedding of epithelial cells, upregulation of cytokines, several growth factors

and chemokines. Injury is associated with changes in intergrity of tight junctions in
between epithelial cells47. Bronchial biopsy results showed epithelial detatchment from
base. More apoptosis is seen in asthmatic airways than in normal ones. It can be a
consequence of ongoing inflammation45. eosinophil basic proteins and oxygen-derived
free radicals contribute for the damage process. In bronchioalveolar fluid, epithelial cells
are found in clumps suggests the detatchment of cells from basement membrane.
Consequences of epithelial layer damage are enhancement of entry of allergen due to loss
of barrier function, less degradation of inflammatory mediators 49. STAT-1 has a key role
in remodeling process, Its increased level is associated with T cell accumulation in
tissue50.
2. Subepithelial fibrosis-
Thichening of reticular basement membrane is the characteristic feature of airway

remodeling. Just below basement membrane, subepithelial fibrosis occurs in lamina
reticularis. Decreased degradation and increased increasd deposition of extracellular
matrix proteins by fibroblasts mainly contribute for this situation. This is basically due to
higher ration of tissue inhibitor metalloproteinase 2(TIMP) to matrix metalloproteinase
(MMP). This results in less degradation of ECM 51. Deposition of ECM is due to
imbalance between its synthesis and degradation. Lamina reticularis is found to be 4-5
µm in normal individual, whereas,in asthmatic patient, its thickness become 23µm.
Extracellular matrix deposition, primarily collagens I, III, and V are mainly responsible
for such thickening52.
. Myofibroblast play an important role in subepithelial fibrosis. These are the phenotypic
intermediate between smooth muscles and fibroblasts 53. They secrete ECM protein and
produce smooth muscle actin also. TGF β is the cytokine which is produced by
fibroblast, eosinophils, lymphocytes, macrophages. TGF β induces the expression of
smooth muscle actin54. It also inhibit the degradation of matrix metalloproteinase. Level
of TGF β is increased in asthmatic airways and broncho alveolar lavage in asthmatic
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patients than normal individuals. Matrix metalloproteinase degrade extracellular matrix

molecules55. TIMP 1 overexpression causes to ECM to deposite.
3. Goblet cell hyperplasia –
This is one of the important feature in asthma which shows histological changes in
peripheral airways. Proliferation of epithelial cells and hyperplasia of goblet cells are the
characteristic ones. Mucus is mainly secreted by goblet cells and submucosal glands.
Hypersecretion of mucus is the result of enlargement of goblet cells and submucosal
glands56. This may be due to desquamation of epithelial cells. This results in airway
narrowing and obstruction57. Thus., goblet cell hyperplasia results in mild, moderate and
severe asthma. Interleukin 9 and interleukin 13 play an important role in induction of
hypersecretion of mucus. IL – 4 , IL-13 also contribute for the same. Mucus occupies
higher percentage of space in airway lumen. Obstruction results from overproduction of
mucus, mucus plugging. This can be due to interaction between environmental factors or
genetic susceptibility. Hyperplasia in peripheral airways may be one of the risk factors in
deaths due to asthma58 .
Angiogenesis
Increased angiogenesis is one more characteristic of airway remodeling. Smooth muscle

cells produce vascular endothelial growth factor (VEGF), which results in increased
vascular permeability and angiogenesis. Airway edema and recruitment of inflammatory
cells are the consequences of this59. This change is associated with related functional
alterations. Changes in airway microvascularization results from these alterations. This
results in airway edema60. Overexpression of VEGF contributes mainly for increased
angiogenesis. Clinical consequences include excess delivery of inflammatory and
remodeling mediators in airways61. Congested and enlarged mucosal blood vessels result
in more thick airway walls62.
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Fig – 2.6 – Role of Angiogenesis in asthma
Involvement of Histamine in Asthma

Histamine was first identified in 1910 and recognized in 1920 as a major pathogenic
mediator of allergic disorder. It is an organic nitrogen compound involved in the
local immune responses, in the regulation of physiological function in the gut, and also
acts as a neurotransmitter. Histamine is produced by basophils and by mast cells found in
nearby connective tissues. It triggers the inflammatory response and increases the
permeability of the capillaries to W.B.Cs and some proteins, to allow them to engage
pathogens in the infected tissues.
Synthesis - Histamine is derived from the decarboxylation of the amino acid, histidine, a
reaction catalysed by the enzyme L-histidine decarboxylase. It is a hydrophilic vasoactive
amine. Once formed, histamine is either stored or rapidly inactivated by its primary
degradative enzymes, histamine-N methyltransferase or diamine oxidase63.
Pharmacological Effects of Histamine63
1. Cardiovascular system:
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a) Triple-effect on terminal vasculature (itching & pain):
i. reddening at injection site due to vasodilation

ii. wheal or disk of edema within 1 to 2 min
iii. a large, bright crimson flare or halo surrounding the wheal
b) i.v. histamine: fall in blood pressure, cutaneous flushing, over the face and upper
trunk, rise in skin temperature, intense headache.
2. Smooth muscle of bronchioles; causes contraction of nonvascular smooth muscle.

Asthmatics may experience marked bronchial constriction compared with normal
subjects.
3. Exocrine glands: potent stimulator of gastric secretion (HCl & pepsin), enhances
salivary and lacrimal gland secretion (minimal unless large doses are given), stimulates
chromaffin cells in adrenal medulla to secrete catecholamines.
4. Peripheral Nervous system: itching and pain
Mechanism of action -
Histamine exerts its actions by combining with specific cellular histamine receptors. The
four histamine receptors that have been discovered in humans and animals are designated
H1 through H4, and are all G protein-coupled receptors (GPCR).
RECEPTOR LOCATION MECHANISM FUNCTIONS ANTAGONISTS

H1 Found Gq  ileum contractio  Diphe
on smooth n nhydramine
muscle, endo  modulate circadi  Lorat
helium, an cycle adine
and Central  itching  Cetiri
Nervous zine
 systemic vasodil
System tissue
atation  Fexof
 bronchoconstrict enadine
ion (allergy-
Page 23
induced asthma)
H2 Located Gs, Ca++  speed up sinus  Raniti
on parietal rhythm dine
cells and vasc  Stimulation  Cimet
ular smooth of gastric idine
muscle cells acid secretion  Famot
 Smooth idine
muscle relaxation  Nizati
 Inhibit antibody dine
synthesis, T-cell pr
oliferation
and cytokine produ
ction
H3 Found Gi  Decrease  ABT-
on central Acetylcholine, 239
nervous Serotonin and  Cipro
system and to Norepinephrine Ne xifan
a lesser urotransmitter relea  Clobe
extent periph se in CNS npropit
eral nervous  Presynaptic auto  Thiop
system tissue receptors eramide
H4 Found Gi  mediate mast  Thiop
primarily in cell chemotaxis eramide
the basophils  JNJ
and in 7777120
the bone
marrow. It is
also found
on thymus,
small
intestine,
Page 24
spleen,
and colon.
Inhalation of allergen results into EAR i.e. early asthmatic response. This is
actually acute airway obstruction including allergen challenge, sensitization
generally followed by late asthmatic response. EAR requires early recognition,
attention and treatment of disease.
2.1.4- Diagnosis of Asthma

Clinical menifestations - middle-aged and elderly people possess more difficult
presentation of clinical menifestations. While most patients present with typical
symptoms, some present with progressively increasing cough and dyspnoea without the
characteristic day to day variability. Nevertheless, they usually have nocturnal symptoms
and these are frequently assumed to be manifestations of cardiac disease. Particular
attention should be paid to factors provoking attacks such as upper respiratory tract
infection, exercise, exposure to allergens, cold air, drugs and emotional upsets 64. Methods
for investigations measure the lung functions. The techniques like spirometry,
measurement of peak expiratory flow, immunoglobulin test.
Drugs which are used as a treatment for asthma are as follows-

 Sympathomimetics - adrenergic drugs cause bronchodilator effect through beta 2
receptors stimulationincreased cAMP formation in the bronchial muscle cell
relaxation. Also, there is an increase in cAMP level in the mast cells, which
reduces AG: AB reaction induced mediator release.
 Bronchodialators – improve the passage of air through airway. By improving
ventilation, these relieves symptoms of asthma
 Anticholinergics – Vagus nerve controls the neuronal pathway by anticholinergic
way.
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 Methylxanthines – theophylline, aminophylline increase the intracellular

concentration of CAMP, in turn results into relaxation of bronchial smooth
muscles.
 Mast cell stabilizers – these are of prophylactic value. Bronchial reactivity is
reduced after their chronic consumption. They cause an alteration in the function
of delayed Cl++ channels in the cell membranes inhibiting cellular activation. Ex.
cromoglycate
 Corticosteroids – these pass through ccell membrane, bind to specific steroid
reponse, translocate the nucleus, initiate mRNA synthesis and protein synthesis
and thus exerts effect on cellular function.
2.1.5- Some important medicinal plants having anti-asthmatic

potential –
There are many natural herbs which are useful for the treatment of asthma. natural
asthma treatment incorporates the symptomatic relief and prevent further asthmatic
attacks.
Acorus calamus is beneficial in asthma which removes catarrhal matter and phlegm in
bronchial tree. Cuminum cyminum established good relaxant effect by making breathing
easy. Cinnamomum cassia provides expectoration of fluid in lungs. Ephedra sinica
stimulate alpha and beta receptors. Glycerrhizza glabra has a good anti inflammatory
activity on lungs and many other organs.
Page 26
2.2 Review of Plant
2.2.1- Introduction to the plant
Scientific classification of the plant
Domain : Flowering plant
Kingdom : Plantae- plants
Subkingdom : Tracheobionta
Class : Magnoliopsida
Subclass : Dillenidae
Division : Magnoliophyta
Superdivision : Spermatophyta
Phyllum : Steptophyta
Order : Brassicales
Family : Caricaceae
Page 27
Genus : Carica
Botanical name : Carica Papaya Linn.
Papaya, papaw, paw paw A( Australia ), mamao ( Brazil ) Tree melon, Mamaeire.
The papaya, Carica papaya L., is a member of the small family Caricaceae . As a dual-
or multi-purpose, early-bearing, space-conserving, herbaceous crop, it is widely
acclaimed, despite its susceptibility to natural enemies.
Fig. Plant of Carica papaya
In some parts of the world, especially Australia and some islands of the West Indies, it is
known as papaw, or pawpaw, names which are better limited to the very different, While
the name papaya is widely recognized, it has been corrupted to kapaya, kepaya, lapayaor
tapaya in southern Asia and the East Indies. In French, it is papaye (the fruit) and
papayer (the plant), or sometimes figuier des Iles. Spanish-speaking people employ the
names melónzapote, lechosa, payaya (fruit), papayo or papayero (the plant), frutabomba,
Page 28
mamón or mamona, depending on the country. In Brazil, the usual name is mamao. When
first encountered by Europeans it was quite naturally nicknamed "tree melon".
2.2.2- Description
Commonly and erroneously referred to as a "tree", the plant is properly a large herb
growing at the rate of 6 to 10 ft (1.8-3 m) the first year and reaching 20 or even 30 ft (6-9
m) in height, with a hollow green or deep-purple stem becoming 12 to 16 in (30-40 cm)
or more thick at the base and roughened by leaf scars. The leaves emerge directly from
the upper part of the stem in a spiral on nearly horizontal petioles 1 to 3 1/2 ft (30-105
cm) long, hollow, succulent, green or more or less dark purple. The blade, deeply divided
into 5 to 9 main segments, each irregularly subdivided, varies from 1 to 2 ft (30-60 cm) in
width and has prominent yellowish ribs and veins. The life of a leaf is 4 to 6 months.
Both the stem and leaves contain copious white milky latex.
Fig – 2.8– Leaf of Carica papaya
Page 29
The 5-petalled flowers are fleshy, waxy and slightly fragrant. Some plants bear only
short-stalked pistillate (female) flowers, waxy and ivory-white; or hermaprodite (perfect)
flowers (having female and male organs), ivory-white with bright-yellow anthers and
borne on short stalks; while others may bear only staminate (male) flowers, clustered on
panicles to 5 or 6 ft (1.5-1.8 m) long. There may even be monoecious plants having both
male and female flowers. Some plants at certain seasons produce short-stalked male
flowers, at other times perfect flowers. This change of sex may occur temporarily during
high temperatures in midsummer. Some "all-male" plants occasionally bear, at the tip of
the spray, small flowers with perfect pistils and these produce abnormally slender fruits.
Male or hermaphrodite plants may change completely to female plants after being
beheaded.
Generally, the fruit is melon-like, oval to nearly round, somewhat pyriform, or elongated
club-shaped, 6 to 20 in (15-50 cm) long and 4 to 8 in (10-20 cm) thick; weighing up to 20
lbs (9 kg). Semi-wild (naturalized) plants bear miniature fruits 1 to 6 in (2.5-15 cm) long.
The skin is waxy and thin but fairly tough. When the fruit is green and hard it is rich in
white latex. As it ripens, it becomes light- or deep-yellow externally and the thick wall of
succulent flesh becomes aromatic, yellow, orange or various shades of salmon or red. It is
then juicy, sweetish and somewhat like a cantaloupe in flavor; in some types quite
musky. Attached lightly to the wall by soft, white, fibrous tissue, are usually numerous
small, black, ovoid, corrugated, peppery seeds about 3/16 in (5 mm) long, each coated
with a transparent, gelatinous aril.
Origin and Distribution
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Fig.2.9– Plant Carica papaya bearing fruits
Though the exact area of origin is unknown, the papaya is believed native to tropical
America, perhaps in southern Mexico and neighboring Central America. It is recorded
that seeds were taken to Panama and then the Dominican Republic before 1525 and
cultivation spread to warm elevations throughout South and Central America, southern
Mexico, the West Indies and Bahamas, and to Bermuda in 1616. Spaniards carried seeds
to the Philippines about 1550 and the papaya traveled from there to Malacca and India.
Seeds were sent from India to Naples in 1626. Now the papaya is familiar in nearly all
tropical regions of the Old World and the Pacific Islands and has become naturalized in
many areas. Seeds were probably brought to Florida from the Bahamas. Up to about
1959, the papaya was commonly grown in southern and central Florida in home gardens
and on a small commercial scale.
Pollination
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If a papaya plant is inadequately pollinated, it will bear a light crop of fruits lacking
uniformity in size and shape. Therefore, hand-pollination is advisable in commercial
plantations that are not entirely bisexual.
Bags are tied over bisexual blossoms for several days to assure that they are self-
pollinated. The progeny of self-pollinated bisexual flowers are 67% bisexual, the rest
being female.
To cross-pollinate, one or 2 stamens from a bisexual flower are placed on the pistil of a
female flower about to open and a bag is tied over the flower for a few days. Most of
such cross-pollinated blooms should set fruit. Resulting seeds will produce 1/2 female
and 1/2 bisexual plants.
By another method, all but the apical female flower bud are removed from a stalk and the
apical bud is bagged 1-2 days before opening. At full opening, the stigma is dusted with
pollen from a selected male bloom and the bag quickly resealed and it remains so for 7
days.
Plants from female flowers crossed with male flowers are 50-50 male and female.
Bisexual flowers pollinated by males give rise to 1/3 female, 1/3 bisexual and 1/3 male
plants.
Climate
The papaya is a tropical and near-tropical species, very sensitive to frost and limited to
the region between 32º north and 32º south of the Equator. It needs plentiful rainfall or
irrigation but must have good drainage. Flooding for 48 hours is fatal. Brief exposure to
32º F (-0.56º C) is damaging; prolonged cold without overhead sprinkling will kill the
plants.
Soil
While doing best in light, porous soils rich in organic matter, the plant will grow in
scarified limestone, marl, or various other soils if it is given adequate care. Optimum pH
Page 32
ranges from 5.5 to 6.7. Overly acid soils are corrected by working in lime at the rate of 1-
2 tons/acre (2.4-4.8 tons/ha). On rich organic soils the papaya makes lush growth and
bears heavily but the fruits are of low quality.
Propagation
Papayas are generally grown from seed. Germination may take 3 to 5 weeks. It is
expedited to 2 to 3 weeks and percentage of germination increased by washing off the
aril. Then the seeds need to be dried and dusted with fungicide to avoid damping-off, a
common cause of loss of seedlings. Well-prepared seeds can be stored for as long as 3
years but the percentage of germination declines with age. Dipping for 15 seconds in hot
water at 158º F (70º C) and then soaking for 24 hrs in distilled water after removal from
storage will improve the germination rate. If germination is slow at some seasons,
treatment with gibberellic acid may be needed to get quicker results.
To reproduce the characteristics of a preferred strain, air-layering has been successfully

practiced on a small scale. All offshoots except the lowest one are girdled and layered
after the parent plant has produced the first crop of fruit. Later, when the parent has
grown too tall for convenient harvesting the top is cut off and new buds in the crown are
pricked off until offshoots from the trunk appear and develop over a period of 4 to 6
weeks. These are layered and removed and the trunk cut off above the originally retained
lowest sprout which is then allowed to grow as the main stem. Thereafter the layering of
offshoots may be continued until the plant is exhausted.
2.2.3- Chemical constituents –
Young leaves of C. papaya contains mainly alkaloids like carpaine and pseudocarpaine,
piperideine alkaloids, dehydrocarpaine I and II, flavonoids, glycosides, tannins.
Carpaine -
Page 33
Carpaine occus in all green parts of C. papaya. Chemical formula is C28H50N2O4. In this,
identically substituted piperidine rings are linked together by two ester groups.
Carpaine occurs in papaya leaves in high concentration as high as 0.4% 65. It was foun to
be secondary amine, optically active, being dextro-rotatory,
According to Wall and Greshoff carpaine can be isolated by refluxing one hundred grams
of dried powder with 0.5 % acetic acid and 95 % ethanol.
Flavonoids
Flavonoids like myricetin, quercetin, kampferol, luteolin, apigenin are present in papaya
shoots nealy about 1264 mg/kg.
Page 34
Quercetin havinf chemical formula C15H10O7 shows decrease in the reactivity of trachea
smooth muscle which is precontracted by both, acetylcholine and histamine. Its
mechanism of action of bronchodialation was demonstrated by in vitro model of rat
trachea. Quercetin treatment made lower activity of Phospholipase A2 in broncoalveolar
lavage66.
Crude drug analysis shows the following results – moisture -69.7% Total ash -11.9%
Acid insoluble ash -0.8%. Alcohol soluble extractives -32.7%. volatile ether soluble
extractive -0.5%67.
2.2.4- Traditional uses -
Crushed leaves wrapped around tough meat will tenderize it overnight. The leaf also
functions as a vermifuge and as a primitive soap substitute in laundering. Dried leaves
have been smoked to relieve asthma or as a tobacco substitute. Packages of dried,
pulverized leaves are sold by "health food" stores for making tea, despite the fact that the
leaf decoction is administered as a purgative for horses in Ghana and in the Ivory Coast it
is a treatment for genito-urinary ailments. The dried leaf infusion is taken for stomach
troubles in Ghana and they say it is purgative and may cause abortion.
It also helps prevent cataract formation, chronic obstructive pulmonary disease,

diverticulosis, and possibly hypertension.
Use in many countries is as follows
 Abortifacient -- Java, Panama, Sri Lanka, and Turkey
 Amebicide -- Japan
 Arthritis and rheumatism -- Haiti and Java
 Asthma and respiration -- Mauritius, Mexico, and Philippines
 Bactericide -- India
 Cancer -- Australia and Mexico
 Cardiotonic -- Turkey
Page 35
 Colic -- Malaya
 Constipation and laxative -- Honduras, Panama, and Trinidad
 Corns and boils -- India, Malagasy, Malaya, and Philippines
 Decoagulant -- Trinidad
 Diarrhea and dysentery -- Honduras, Japan, Panama, and West Africa
 Digestive -- China, Dominican Republic, Panama, and Turkey
 Diuretic -- Trinidad
 Dyspepsia -- Mexico
 Dysuria -- Java
 Emmenagogueue -- Mexico and Turkey
 Epithelioma -- St. Vincent
 Fever -- Java and Mexico
 Flu -- Trinidad
 Fumitory -- New Caledonia
 Hypertension -- Honduras and Trinidad
 Infection -- Panama
 Intestinal disorders -- Philippines
 Kidney -- Cameroon and Honduras
 Liver -- Honduras and Turkey
 Madness -- Ivory Coast
 Milk production (increase/stimulate) -- Indonesia and Malay
 Opthalmology treatments -- Soviet Union
 Pectoral -- Mexico
 Scorpion bites -- Trinidad
 Smoothe upper respiratory tract -- Nigeria
Page 36
 Toothhache -- Cote d'Ivoire and Samoa
 Tuberculosis -- Mexico
 Tumor (Uterus) -- Ghana Indochina Nigeria
 Ulcer -- Panama
 Urology treatments -- Soviet Union
 Venereal -- Trinidad
 Vermifuge -- Haiti, Malaya, Panama, Samoa, and Turkey
 Warts -- Indonesia, Jamaica, Peru, South Africa, and Sri Lanka
2.2.5- Pharmacological activities –

Anti-inflammatory activity –
50 mg/kg, 100 mg/kg 200 mg/kg of extract has shown a good anti-inflammatory activity
in the model of carageenan induced paw edema.
Anti-arthritic activity –
4% formaldehyde used for the induction of arthritis, 25-200 mg/kg extract was used for
the activity68.
Anti-bacterial activity –
The range of MIC observed 1250 to 5000 µg/ml. Extract of leaves of C. papaya contain
some valuable antibacterial components that inhibit the growth of many gram positive
and gram negative bacteria69.
Hypoglycemic activity –
0.5 mg/kg of extract shows significant blood glucose reduction bur, on higher doses, like
10 mg/kg, in shows no significant reduction70.
Anthelmintic Activity –
It may be due to chymopapain, lysozyme present in leaves.
Page 37
Anti-tumor activity –
The effect of C. papaya leaves on proliferative response of tumor cell line and human
peripheral blood mononuclear cells. Carica papaya leaf extract can mediate a Th1 type
shift in human immune system, results suggest that the CP leaf extract may potentially
provide the means for the treatment and prevention of selected human diseases such as
cancer, various allergic disorders, and may also serve as immunoadjuvant for vaccine
therapy71.
Wound healing activity –

In rats, CP leaves shows a good activity against externally induced excised wound72.
Page 38
3. OBJECTIVES
Page 39
OBJECTIVES
Many plants are the source of many biochemical constituents like secondary metabolites
which may include flavonoids, terpenoids, tannins etc. these may exert a very good
biological activity and may serve as the medicine for the disease on which it is studied.
Asthma has become a serious public health problem, which resulted into increased
hospitalization, and deaths every year. Increased cost for the medications for this disease
necessitates the development of newer anti-asthmatics. The traditional medication
included corticosteroids have many severe side effects, and they only give the
symptomatic relief. But the main pathological cause for the disease remain untreated.
This may results into recurrence of disease progression. Flavonoids and alkaloids which
are present in the extract may heal the root-cause of this.
In the present study, it is intended to collect the plant leaves, extract with the solvent,
perform the phytochemical screening of the extract, evaluation of anti-asthmatic activity.
Different steps to be taken are-
1. Identification and collection of the leaves of Carica papaya

2. Preparation of extracts
3. phytochemical evaluation
4. evaluation of anti-asthmatic activity
Page 40
4. PLAN OF WORK
Page 41
PLAN OF WORK
1. Collection of leaves of Carica papaya (Caricaceae) from the region of thane. The
collected fresh leaves will be then dried in shade and the powdered.
2. Extraction of the plant material will be performed with the solvent 80% ethanol
using Soxhlet apparatus. Extract is stored at lower temperature.
3. Phytochemical evaluation will be performed for determining the constituents of

the extract obtained.
4. Obtained extract will be then screened for the Mast cell stabilizing activity on
wistar rats in vivo and in vitro, for anti-histaminic activity in vitro with guinea pig
ileum, and guinea pig in vivo model.
Page 42
5. PHYTOCHEMISTRY
Page 43
5.1- EXTRACTION
Collection of Plant material –

The leaves of plant Carica papaya (Family- Caricaceae) were collected from the region
thane, Maharashtra, India in the month of August and Septmber 2012. The leaves were
authenticated at the Blatter Herbarium, St. Xavier’s College, Mumbai. The fresh leaves
were washed to free them from any dust particles and contaminants, and were
subsequently dried at room temperature for 15-20 days. When the leaves were
sufficiently dry and easily crushable with hands, they were subjected to a grinder to
obtain a powdered form of the leaves. This powder was stored in an air tight container for
the further extraction process.
Around 20 % ethanol was taken in a 250 ml R.B.F (Round Bottom Flask) and was
attached to Soxhlet apparatus. 50-60 grams of the leaf powder was packed in thimble,
Solvent in RBF got evaporated and then condensed. Condensed solvent entered to the
thimble part, and through siphon arm, the content was dropped in the RBF. Cycles were
allowed to repeat for two days. The content in RBF then subjected to rotary evaporator.
The condensed extract was macerated with little quantity of water and slightly warmed.
Then it was filtered through Buchner Funnel, the liquid extract then allowed to get
condensed.
5.2 - PRILIMINARY PHYTOCHEMICAL SCREENING
Detection of Carbohydrates73
Extracts were dissolved individually in 5 ml distilled water and filtered. The filtrates
were used to test for the presence of carbohydrates.
1. Molisch’s Test
Filtrates were treated with 2 drops of alcoholic α-naphthol solution in a test tube and 2 ml
of conc. sulphuric acid was added carefully along the sides of the test tube. Formation of
violet ring at the junction indicates the presence of carbohydrates.
Page 44
2 Benedict’s test
Filtrates were treated with Benedict’s reagent and heated on water bath. Formation of
orange red precipitate indicates the presence of reducing sugars.
3 Fehling’s test
Filtrates were hydrolyzed with dil. HCl, neutralized with alkali and heated with Fehlings
A & B solutions. Formation of red precipitate indicates the presence of reducing sugars.
Detection of alkaloids
The small portions of fractions are stirred separately with a few drops of dil.HCl and
filtered and then subjected to test for alkaloids.
1 Dragendorff’s Test
Extracts were treated with Dragendorff’s reagent. Formation of orange brown precipitate
indicates the presence of alkaloids.
2 Mayer’s Test
Extracts were treated with Mayer’s reagent. Formation of cream precipitate indicates the
presence of alkaloids.
3 Wagner’s Test
Extracts were treated with Wager’s reagent. Formation of reddish brown precipitate
indicates the presence of alkaloids.
Detection of Glycosides
1. Legal Test
Extract were dissolved in Pyridine and sodium-nitropruside solution was added to it and
made alkaline. Appearance of pink or red colour indicates the presence of cardiac
glycoside.
2. Baljet Test
To a portion of extract sodium picrate was added. Formation of yellow to orange colour
indicates the presence of cardiac glycoside.
Detection of phytosterols and triterpenes

1. Salkowski’s Test
Page 45
Extracts were treated with chloroform and filtered. The filtrates were treated with few
drops of conc. sulphuric acid, shaken and allowed to stand. Appearance of golden yellow
colour indicates the presence of triterpenes.
2. Libermann Burchard’s test
Extracts were treated with chloroform and filtered. The filtrates were treated with few
drops of acetic anhydride, boiled and cooled. conc. sulphuric acid was added carefully
along the sides of the test tube. Formation of brown ring at the junction indicates the
presence of phytosterols.
3. Test for Cholesterol
Extracts were treated with chloroform and sprinkled sulphur powder on the surface of the
solution. If sulphur sinks down it indicates the presence of cholesterol.
Detection of flavonoids74, 75
1. Alkaline Reagent Test
Extracts were treated with few drops of sodium hydroxide solution. Formation of intense
yellow colour, which becomes colourless on addition of dilute acid, indicates the
presence of flavonoids.
2. Lead acetate Test
Extracts were treated with few drops of lead acetate solution. Formation of yellow colour
precipitate indicates the presence of flavonoids.
3. Shinoda Test
To the alcoholic solution of extracts, a few fragments of magnesium ribbon and conc.
hydrochloric acid was added. Appearance of magenta colour after few minutes indicates
presence of flavonoids.
4. Mineral acid Test
Extracts were treated with few drops of conc. sulphuric acid Formation of yellow orange
colour indicates the presence of flavonoids.
Detection of tannins
1. 5% FeCl3
Page 46
Extracts were treated with 1-2 drops of 5% alcoholic FeCl 3 .Formation of

Bluish black or greenish colour indicates the presence of Tannins.
2. Lead acetate Test
To 2-3 ml of alcoholic extract add Lead acetate solution white precipitation

indicates presence of Tannins.
5.3- TLC Profile

TLC profile was developed for the Fraction 4 of alcoholic extract of C.papaya which has
showed significant anti-asthmatic activity. Different solvents systems were tried for
developing a TLC Profile for the fraction no.4.
Procedure:
Slurry of silica gel G was prepared in distilled water and poured over a glass plate to
form a thin film. The prepared plates were allowed for setting (air-drying). After setting,
the plates were kept in an oven at 100 to 120º C (30 min) for activation. The fractions
were dissolved in respective solvent and spotted over an activated plate (1 cm above from
the bottom). It was then kept in previously saturated developing chamber containing
mobile phase, and allowed to run 3/4th of the height of the plate. The developed plate
was removed, air dried and observed under ultraviolet light. Mobile phase used was
Toluene : Ethyl acetate: Acetone : Formic acid
(5.2 :5: 7.5: 0.5 ). This gave good separation of quercetin Rf = 0.84
Distance traveled by solute

Rf =
Distance traveled by the solvent
Also, TLC performed for carpaine, mobile phase used was Methanol:Chloroform 5:95,
the Rf found to be 0.6
Page 47
Fig. 5.1 Spots of standard Quercetin and extract on TLC plate
Fig – 5.2 TLC plate having spot of Carpaine
Page 48
6. MATERIALS AND METHODS
Page 49
6.1- Acute Toxicity Testing
Guideline, OECD 425 (Organization for Economic Cooperation and Development) states
that, the testing laboratory should consider all available information on the test substance
prior to conducting the study. Such information will include the identity and chemical
structure of the test substance; its physical chemical properties; the results of any other in
vitro or in vivo toxicity tests on the substance; toxicological data on structurally related
substances or similar mixtures; and the anticipated use(s) of the substance.
DESCRIPTION OF THE METHOD
Selection of Animal Species

Since the preferred rodent species is the rat and because female rats are generally slightly
more sensitive than male counter-parts according to the OECD 425 guideline, five female
rats were used. The rats used were nulliparous and non-pregnant. At the commencement
of its dosing, each animal was between 8 and 12 weeks old with weight range of 250-300
gm.
Page 50
Housing and Feeding Conditions

The temperature in the experimental animal room was maintained at 22°C (± 3°C) and
relative humidity at 45-50%. Artificial lighting was done to maintain the sequence of 12
hours light and 12 hours dark cycle. The animals were housed individually. For feeding,
conventional rodent laboratory diets were used with an unlimited supply of drinking
water.
Preparation of Animals
The animals were randomly selected, marked to permit individual identification, and kept
in their cages for at least 5 days prior to dosing to allow for acclimatization to the
laboratory conditions.
PROCEDURE
Administration of Doses
Animals were fasted prior to dosing (food but not water was withheld overnight).
Following the period of fasting, the animals were weighed and the solution of extract (in
water) was administered orally. The extract was triturated for better solubility in least
possible amount of water and then was administered in a single dose to the animals by
gavage. Initially only one rat was administered the solution of extract, after 48 hr of
observation, since the rat survived remaining four rats were also given the extract. After
the administration of the extract, food was withheld for a further 3-4 hours.
Limit test and Main Test

The limit test is primarily used in situations where the experimenter has information
indicating that the test material is likely to be nontoxic. In those situations where there is
little or no information about its toxicity, or in which the test material is expected to be
toxic, the main test should be performed. Since Carica papaya has been used traditionally
to prevent and treat number of disorders and because ample data is available about the
plant ‘Limit test’ and not ‘Main test’ was conducted for judging the acute toxicity of the
plant. 100 mg/kg, 500 mg/kg and 1000 mg/kg were the dose of extract administered to
confirm the absence of acute toxicity of the extract.
OBSERVATIONS
Page 51
Animals were observed individually at least once during the first 30 minutes after dosing,
periodically during the first 24 hours (with special attention given during the first 4
hours), and daily thereafter, for a total of 14 days.
Pharmacological Evaluation
6.2- Histamine induced bronchospasm in guinea pigs (In

vivo)
Experimental animals
Animal specifications
Species- Guinea pig
Age / weight / size – 350-400 gm
Gender – Either
Guinea pigs weighing between 350- 400gm procured from Haffkins

Biopharmaceytical Corporaion Limited, Animals were maintained under standard
conditions husbandry with room temperature- 26 ± 2ºC, relative humidity of 45 –
55%, 12 h light/dark cycle, in an animal house approved by the Committee for the
Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The
animals had free access of standard diet and water and housed in a poly propylene
cages.
Procedure
Guinea pigs were divided in 5 groups containing 5 animals in each. Previous to

that., 12 animals were used for stabilization of method and dose.
Page 52
The guinea pigs fasted for 24 h were exposed to an atomized fine mist of 2%
histamine dihydrochloride aerosol (dissolved in normal saline) using nebulizer at a
pressure of 300 mm Hg in the histamine chamber (24 x 14 x 24 cm, made of
perplex glass). Guinea pigs exposed to histamine aerosol showed progressive signs
of difficulty in breathing leading to convulsions, asphyxia and death. The time
until signs of convulsion appeared is called pre-convulsion time (PCT). By
observation experience was gained so that the preconvulsion time can be judged
accurately. As soon as PCT commenced, animals were removed from the chamber
and placed in fresh air to recover. Animals were divided in five groups each
containing six animals. Ketotifen (1 mg/kg) and all fractions of alcoholic
extract(100 mg/kg, 200 mg/kg, 400 mg/kg) were administered orally 30 min prior
to exposure according to the groups
Group Dose No. of animals

disease control Saline solution 5
Standard (Ketotifen ) 1 mg/kg 5
Test 1 100 mg / kg 5
Total 25+3=28
Statistical analysis:
Results obtained were expressed as mean ± SEM. The data were analyzed using students
t-test and results were considered significant when p<0.05.
6.3-Inhibition of Mast cell degranulation ( in vivo )

Experimental animals
Animal specifications
Species- Wistar rats
Age / weight / size – 250-300 gm
Gender – Either
Page 53
Wistar rats weighing between 250- 300gm procured from Animal house of Dr. L.
H. Hiranandani College Of Pharmacy, Ulhasngar , Animals were housed at
ambient temperature 22±1º, relative humidity of 45 – 55%, 12 h light/dark cycle,
in an animal house approved by the Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA). The animals had free access
of standard diet and water and housed in a poly propylene cages.
Studies on mesenteric mast cell degranulation induced by albumin.
Procedure
Wistar rats were sensitized with 0.1 ml of 1%w/v solution of albumin intraperitoneally on
first, third, fifth and twelfth day. The extract was administered from sixth to twelfth day
orally(6).
Group I – Control
Group II- Disease control
Group III – standard - Disodium cromoglycate 10µ/ml
Group IV –Test 1- Extract of leaves of Carica papaya 100 mg/ml
Group V –Test 2- Extract of leaves of Carica papaya 200 mg/ml
Group VI – Test 3- Extract of leaves of Carica papaya 400 mg/ml
Group Dose No. of animals

control Saline solution 6
Disease control Saline solution 6
Standard (disodium 100 mg/kg 6

cromoglycate)
Test 1 200mg/kg 6
Test 2 400mg/kg 6
Test 3 400mg/kg 6
Total 36+4=40
Page 54
On twelfth day, after sensitization, rats were sacrificed and the mesentery was collected
onto the slide, followed by staining with 0.1% toludine blue solution for 10 minutes.
After staining of mast cells mesenteric pieces were then observed under light microscope
(power 450X).Percent of degranulation of the mast cells in the control group and the
treated groups were calculated by counting the number of degranulated mast cells.
Statistical analysis –
The results were expressed as mean ± standard error of mean. One way ANOVA was
applied. p<0.05 as considered statistically significant.
6.3 MAST CELL DEGRANULATION IN PERITONEAL FLUID
Animal Specifications:
Species- Wistar rats
Age / weight / size- 250-300 gm
Gender- Either sex
Wistar rats weighing between 250- 300 gm procured from Animal house of Dr. L. H.
Hiranandani College Of Pharmacy, Ulhasnagar, were housed at ambient room
temperature of 22±1º, relative humidity of 45 – 55% and 12 hr light-dark cycle, in an
animal house approved by the Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA). The animals were housed in a poly propylene cages
and were supplied with standard diet and water at regular intervals.
PROCEDURE:
Saline solution was injected intra-peritoneally into the peritoneal cavity of the lightly
anaesthetized Wistar rats. After giving abdominal massage for optimum distribution of
the injected fluid, the peritoneal fluid was collected in the centrifuge tubes placed over
Page 55
ice. Peritoneal fluid obtained from 4-5 rats was pooled together and was subjected to
centrifuge at the speed of 2000 rpm for 5 minutes. Supernatant solution was discarded
while the underlying cells (pellets) obtained were resuspended in 1 ml of saline. 0.1 ml of
this cell suspension was divided into six test tubes as shown below.
Test tube 1- 0.1 ml of the cell suspension obtained intra-peritoneally
Test tube 2 – 0.1 ml of the cell suspension obtained intra-peritoneally
Test tube 3 - 0.1 ml of the cell suspension obtained intra-peritoneally + Disodium

Cromoglycate 20 µ/ml
Test tube 4 - 0.1 ml of the cell suspension obtained intra-peritoneally + Hydro-alcoholic

Extract of leaves of C. papaya 50 µg/ ml
Test tube 5 - 0.1 ml of the cell suspension obtained intra-peritoneally + Hydro-alcoholic

Test tube 6 – 0.1 ml of the cell suspension obtained intra-peritoneally + Hydro-alcoholic

Each test tube was incubated for 15 min at 37°C. Then, 0.1 ml of 1% w/v egg albumin
was added into each test tube, except test tube no. 1 and all the test tubes were further
incubated under same conditions for 10 min. The cells were stained with 0.1% toluidine
blue for 10 minutes and were observed under microscope for stained mast cell.
Statistical Analysis:
All values were expressed as mean ± SEM. One way ANOVA was applied. The results
were considered to be statistically significant when p< 0.05.
6.4 - SPASMOLYTIC ACTIVITY OF ISOLATED GUINEA PIG

ILEUM
Animal Specifications:
Species- Guinea pig
Page 56
Age / weight / size - 350-400 gm
Gender - Either sex
Guinea pigs weighing between 350- 400gm procured from Haffkine Biopharmaceutical
Corporation Limited, animals were maintained under standard conditions with room
temperature around 26 ± 2ºC, relative humidity of 45 – 55% and 12 hr light-dark cycle, in
an animal house approved by the Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA). The animals were housed in a poly propylene
cages and were supplied with standard diet and water at regular intervals.
Procedure:
Overnight fasted guinea pigs of either sex were sacrificed by cervical dislocation method.
Ileum will be quickly dissected out and mounted in organ bath maintained at 37°C±1°C
along with continuous aeration. Organ bath contained Tyrode’s solution (NaCl 0.1,
NaHCO3 1.0, NaH2PO4 0.05, and Glucose 1.0 gm/lit.). Responses of contractions were
recorded with histamine 10 µg/ ml in absence and in presence of extract of concentrations
50 µg/ml (Test 1), 100 µg/ ml (Test 2) and 200 µg/ ml (Test 3).
6.5- SPASMOLYTIC ACTIVITY OF ISOLATED GOAT TRACHEA
Goat trachea was obtained from nearby slaughterhouse. Trachea was cleaned off
unwanted tissues and then it was cut into uniform rings. The rings were tied together in
series to form a chain. This chain was suspended in organ bath containing Kreb’s solution
(NaCl 6.9., KCl 0.35, CaCl2 0.28, MgSO4 0.28, NaHCO3 2.1, KH2PO4 0.16, Glucose 2.0
gm/lit) maintained at 37±0.5ºC. Aerator was used to continuously bubble air into the
organ tube. One end of the tissue was attached to the aerator tube by thread, while the
other end was tied to the isotonic frontal lever. The dose response curve was taken on the
kymograph paper present on the rotating drum. The dose response curve was obtained
with histamine (10 µg/ ml) in absence and in presence of extract of concentrations 50
µg/ml (Test 1), 100 µg/ ml (Test 2) and 200 µg/ ml (Test 3).
Page 57
Page 58
7. RESULTS AND DISCUSSION
The present study deals with phytochemical and pharmacological evaluation of Carica
papaya. The hydroalcoholic extract of dried leaves of Carica papaya was studied for
pharmacological activity and phytochemical investigation. Results of these evaluation are
mentioned below.
7.1- Preliminary phytochemical evaluation –
The extract was screened for phytochemical investigation. This was done to ascertain the
chemical composition of the extract. Such phytochemical tests revealed the presence of
alkaloids, glycosides, flavonoids.
Sr No. Constituents Test Result

1 Alkaloids Dragondroff’s test +
Wagner’s test
2 Flavonoids Shinoda test +
3 Glycosides Legal test +
Baljet test
4 Terpenes Salkowski test -
Page 59
5 Tannins +
Sr. No. Test Observation Inference

1 Test for tannins Blackish blue +
coloration
2 Test for Saponins Foam produced +
persists for 10 min
3 Test for Flavonoids
Shinoda test Red to pink +
coloration
Ferric chloride test Blackish green +
coloration
Lead acetate test Yellow precipitate +
formed
Alkaline reagent test/ NaOH Intense yellow color +
test which disappeared
after adding dilute
HCl
4 Test for sterols
Salkowski test Appearance of red +
color in lower layer
Liebermann-Burchard test Appearance of +
reddish brown ring
5 Test for Glycosides No colored ring at -
the interface
6 Test for Alkaloids
Wagner’s test Formation of reddish +
brown precipitate
Mayer’s test Formation of creamy +
white precipitate
Dragendorff’s reagent Formation of reddish +
brown precipitate
Hager’s test Formation of yellow +
precipitate
7 Test for carbohydrates
Molisch’s test A reddish violet ring +
Fehling’s test No formation of +
precipitate
8 Test for resins Turbidity observed +
Page 60
Pharmacological studies-
7.2- Toxicity studies –
The extract was dissolved in distilled water, and administered by oral route. The
extract was found to be safe upto 4000 mg/kg of body weight.
Results
7.3- Histamine induced bronchospasm in guinea pigs (In vivo)

Results for the present studies are given in table 5.1
Sr. No. Treatment Preconvulsion time in
seconds
1 Inducer Control 146.2 ± 20.37
2 Standard 405.8 ± 29.46**

(Ketotifen 1mg/kg)
3 CPLE 100 mg / kg 159.4± 20.65ns
4 CPLE 200 mg / kg 241.8 ± 28.31ns
5 CPLE 4000 mg / kg 290.2 ± 27.61**
Values are mean ± SEM (n=5 animals) *p<0.05. **p<0.01; ns = non significant
Page 61
450
400
Pre- Convulsion Time (sec) 350
300
250
200
150
100
50
0
Inducer Control Standard (Ketotifen)CPLE 100 mg/kg CPLE 200 mg/kg CPLE 400 mg/kg
Treatments
After exposing to histamine aerosol, animals started showing preconvulsive dyspnoea

(PCD).the time taken for the precipitation of PCD was noted. Animals in control group
showed PCD at around 146.2 seconds, whereas, animals in test groups delay in PCD1.
Preconvulsion time was found to be more in test groups. The extract administered 1 hour
before challenge with histamine produced a dose-dependent inhibition of pre-convulsive
dyspnoea. CPLE 200mg/kg shows a significant increase in preconvulsion time.
The extract at higher dose showed inhibitory effect on preconvulsive dyspnoea or
preconvulsive breathing. Latent period of convulsion in guinea pigs is prolonged when
exposed to histamine aerosol in an enclosed chamber.
During induction of bronchoconstriction, guinea pigs undergo intense smooth muscle
contraction, hypoxia, which leads to convulsion, asphyxia, death. The administration of
bronchodialators delay this occurrence of such symptoms.
The results of the study suggests that, the CPLE is may acting via dilation of bronchial
smooth muscles. This model is direct anaphylaxis model in which antigen is sprayed in
enclosed chamber. The extract administered protected the guinea pigs from preconvulsive
episodes.
7.4- Inhibition of Mast cell degranulation ( in vivo )
Page 62
The results of this study reveal that hydroalcoholic xtract of Carica papaya possesses
significant anti-asthmatic activity by inhibiting mast cell degranulation. In this, mast cell
degranulation was induced by albumin.
Mast cell degranulation in the mesentery of the rats induced by albumin was found to be
44.97% in positive control group. Addition of disodium cromoglycate inhibited
degranulation significantly (p<0.0001) when compared with disease control., and it was
reduced to 12.11%. with the doses of hydroalcoholic extract of Carica papaya, (100, 200,
400 mg/kg) lower the degranulation to 43.28, 26.47 and 21.30% respectively. Inhibition
of degranulation of mast cells was seen significantly (p<0.01) in CPLE doses. It shows
dose dependent effect. The protection given by the doses of extract was comparable with
that of disodium cromoglycate which is potent mast cell degranulation inhibitor.
Fig. 6.1 degranulated and intact mast cells in mesentery observed under 450X
Observation table for Inhibition of Mast cell degranulation ( in vivo)
Results for the present studies are given in table 5.2
Page 63
TREATMENT DOSE No. of INTACT % of % of

[mg/kg, p.o.] mast cells seen in INTACT DEGRANULATED
the Neubeur’s mast mast cells
chamber cells
Control Saline 359.3 + 28.27*** 100 -
Disease Saline 55.03 44.97

Control 198.7 + 10.61
Standard Disodium 315.8 + 87.89 12.11

cromoglycate 14.28***
50 mg/kg
Test 1 100 56.72 43.28
ns
203.8 + 12.65
Test 2 200 73.53 26.47

264.2 + 8.514*
Test 3 400 78.70 21.30

282.8 + 18.27**
Values are expressed as mean ± SEM (n = 6 animals), n.s- non significant,

***p < 0.0001, **p<0.001, * p<0.01, compared with disease Control Group (oneway
ANOVA followed by Dunnett’s Multiple Comparisons test).
Page 64
MAST CELL DEGRANULATION IN MESENTERY OF WISTAR

RATS
120
80
40
% Intact Mast Cells
0
l l e) ) ) )
tro tro at kg kg kg
Co
n o n lyc g/ g/ g/
eC og m m m
as
om 00 00 00
se cr 1 E2 4
Di LE PL LE
um (C
P
(C (C
P
di 1 2 3
si o st st st
(d Te Te Te
rd
nda
a
St
Treatments
MAST CELL DEGRANULATION IN MESENTERY OF WISTAR

RATS
50
40
30
% Degranulated Mast Cells
20
10
0
l l e) ) ) )
ro ro at kg kg kg
o nt ont lyc g/ g/ g/
C C og m m m
ase m 00 00 00
se o 1
Di cr LE E2 E4
um P PL PL
di (C (C (C
o 1 2 3
(d
is st st st
Te Te Te
rd
nda
a
St
Treatments
Mast cell degranulation is an important event for the initiation of the allergic cascade
after exposure og antigen. When allergen binds to IgE bound cell, the release of
mediators such as histamine, leucotrienes, prostaglandins, platelet activating factors. This
develops the airway inflammation and bronchoconstriction. the attempt was made to find
out whether the extract has ability to decrease the rate of disruption of mast cells. CPLE
offered significant protection against albumin induced mast cell degranulation., and this
is ultimately responsible for prevention of airway inflammation and release of mediators.
Page 65
7.5- Inhibition of Mast cell degranulation ( in vitro )

Results of this study are given in table 5.3
TREATMENT DOSE [µg/kg, No. of INTACT % of % of

p.o.] mast cells seen in INTACT DEGRANULATED
the Neubeur’s mast cells mast cells
chamber
Negative Control Saline solution 229.8 + 100 -
5.294***
Disease Control Saline solution 119.0 + 6.763 51.78 48.22
Standard Sodium 208.3 + 90.64 9.36

dicromoglycat 7.210***
e 20 µg/ml
Test 1 50 µg/ml 150.3 + 10.26ns 65.40 34.60
Test 2 100 µg/ml 171.7 + 16.06** 74.71 25.29
Test 3 200 µg/ml 173.7 + 8.894** 75.58 24.42
Values are expressed as mean ± SEM (n = 6),

n.s- non significant,
***p < 0.0001, **p < 0.001, compared with Positive Control Group (oneway ANOVA
followed by Dunnett’s Multiple Comparisons test).
Page 66
MAST CELL DEGRANULATION IN PERITONEAL FLUID (In-

vitro)
60
40
% Degranulated Mast Cells
20
0
l l e) ) ) )
tro nt
ro at kg kg kg
Co
n
Co lyc g/ g/ g/
se og m m m
a om 00 00 00
se cr 1 E2 E4
Di LE PL PL
um (C
P
(C (C
di 1 2 3
si o st st st
(d Te Te Te
rd
nda
a
St
Treatments
MAST CELL DEGRANULATION IN PERITONEAL FLUID (In-

vitro)
120
80
40
% Intact Mast Cells
0
l l e) ) ) )
tro tro at kg kg kg
Co
n o n lyc g/ g/ g/
eC og m m m
as
om 00 00 00
se cr 1 E2 4
Di LE PL LE
um (C
P
(C (C
P
di 1 2 3
si o st st st
(d Te Te Te
rd
nda
a
St
Treatments
The results of in vitro mast cell degranulation inhibition study reveal that hydroalcoholic
extract of Carica papaya possesses anti-asthmatic activity by inhibiting mast cell
degranulation. In this, mast cell degranulation was induced by albumin.
Page 67
Mast cell degranulation induced by albumin was found to be 48.22 % in positive control
group. Addition of disodium cromoglycate inhibited degranulation, and it was reduced to
9.36 %. with the doses of hydroalcoholic extract of Carica papaya, (50, 100, 200 µg/ml)
lower the degranulation significantly (p<0.01) to 34.60, 25.29 and 24.42% respectively.
The protection given by the doses of extract was comparable with that of disodium
cromoglycate which is potent mast cell degranilation inhibitor.
Mast cell play a critical role in immediate hypersensitivity. After activation of mast cells,
they show their biological effects and release the preformed mediators and de novo
synthesized mediators like histamine, leukotrienes, and other cytokines.
7.6- Spasmolytic activity of isolated guinea pig ileum
volume % inhibition
ml of histamine Test 1 (CPLE Test 2 (CPLE Test 3 (CPLE
50µ/ml) 100µ/ml) 200µ/ml)
0.1 13.46±0.96 20.05±11.54 41.15±7.572
0.2 18.09±5.713 27.08±2.083 41.33±5.925
0.4 24.46±7.038 26.67±7.143 37.50±9.282
0.8 26.77±7.787 32.59±2.390 46.15±6.454
Page 68
SPASMOLYTIC ACTIVITY OF ISOLATED GUINEA

% Inhibition of contraction induced by Histamine
PIG ILEUM
70
60
50 Test 1 (50 µg/ ml)
40 Test 2 (100 µg/ ml)2
Test 3 (200 µg/ ml)
30
20
10
0
0.1 ml 0.2 ml 0.4 ml 0.8 ml
Histamine (10 µg/ ml)
Guinea pig ileaum having H1 receptors produces graded dose related contraction after
introduction to histamine in organ bath. The present study shows the spasmolytic activity
by testing various concentrations of the extract Carica papaya, the physiological salt
solution containing extract significantly decreases the contractile effect of histamine. The
stimulation of H1 receptors produces graded dose related contraction. In the present
study, CPLE of 50µg/ml, 100 µg/ml, 200 µg/ml showed inhibition in contraction of
isolated guinea pig ileum. This indicates the extract has H1 receptor blocking activity.
7.7- Spasmolytic activity of isolated goat trachea
Contractions with several doses of histamines were checked in presence and

absence of extract. Percentage inhibition for the contraction of goat tracheal chain
was calculated. Tracheal muscle has H1, M3, β2 receptors. Stimulation of H1
receptors cause contraction of bronchiolar smooth muscles. In present study,
extracts 50 µg/ml, 100 µg/ml, 200 sµg/ml show inhibition of histamine induced
contraction.
Volume % inhibition
ml of histamine Test 1 (CPLE Test 2 (CPLE Test 3 (CPLE
50µ/ml) 100µ/ml) 200µ/ml)
0.1 12.22±7.776 16.37±9.675 27.67±5.805
Page 69
0.2 26.35±4.822 29.83±6.835 46.53±3.736

0.4 30.24±3.094 41.45±2.003 55.16±2.677
0.8 37.61±5.796 50.85±5.010 59.59±8.554
SPASMOLYTIC ACTIVITY OF ISOLATED GOAT TRACHEA

% Inhibition of contraction induced by Histamine
70
60
50
40 Test 1 (50 µg/ ml)

Test 2 (100 µg/ ml)2
30 Test 3 (200 µg/ ml)
20
10
0
0.1 ml 0.2 ml 0.4 ml 0.8 ml
Histamine (10 µg/ ml)
Approach towards this model is more useful because goat trachea is a representative if
respiratory smooth muscles. This may be more sensitive for the asthmatic attack and
anaphylaxis. This method is useful for checking the antispasmodic effect of extract on
bronchial musculature. The results show significant percent inhibition of contraction ,
which provides the data for spasmolytic activity of CPLE.
Page 70
8. SUMMARY AND CONCLUSION
Page 71
The present study shows the effect of hydroalcoholic extract of dried leaves of Carica
papaya. This study has been attempted to investigation of phytochemical screening, anti-
asthmatic activity of dried leaves of Carica papaya.
Phytochemical evaluation of extracts of the extract showed that it contains alkaloids,

glycosides, flavonoids.
From the results and discussion, it can be summarized that, dose 2 and dose 3 of the
extract of dried leaves of carica papaya inhibited the contraction induced by the inducer
or spasmogen. Similarly, it also decreases mast cell degranulation and thus depletes the
release of histamine which is one of the reason for spasmogenic response.
Histamine being a central mediator I allergic disorders, causes bronchospasm, which

causes asthma. Other inflammatory mediators also aggravate the mucus secretion, which
narrows the bronchial lumen and thus reduces the amount of air which is to be passed. In
the present study, extract used is devoid of any side effects. Outcomes of the study
showed that CPLE prolonged the preconvulsion time in the guinea pigs, following
histamine aerosols. It demonstrates that, the extract antagonizes not only the airway hyper
responsiveness but also reduces the bronchoconstriction caused by histamine.
From in vivo mast cell degranulation inhibition model, it can be concluded that, CPLE
possess significant mast cell stabilizing activity against albumin induced mast cell
degranulation in mesentery of rats. So, de novo synthesized mediators like histamine also
inhibited from its release. Which results into decreased inflammation of airways and
release of mediators.
From mast cell degranulation inhibition model, it can be concluded that, CPLE possess
significant mast cell stabilizing activity against albumin induced mast cell degranulation
peritoneal fluid collected from rats. So, the release of histamine and other mediators are
also inhibited.
Page 72
Effect on guinea pig ileum provides the data for anti spasmodic activity of extract. H1
receptors which are present onto ileum are blocked or antagonized and decrease in
contractile response was observed.
Goat trachea is a representative of respiratory smooth muscles. H1, M3, β2 receptors

present on tracheal smooth muscle which play a role in increase or decrease in contractile
response of bronchi. Decrease in contractile response in goat trachea, showed
bronchodialatory activity of the extract. This spasmolytic activity may be due to agonistic
activity on β 2 receptors. β adrenergic agonists augments bronchodilation by acting
directly on β receptors. This leads to relaxation of bronchial muscles. This also decreases
the disturbances in airflow facilitating symptomatic relief of asthma.
By summarizing the data, it can be concluded that, 100mg/kg and 200 mg/kg dose of
extract of dried leaves of carica papaya show a good spasmolytic activity, broncodilation,
and mast cell degranulation inhibition.
Further studies are coined to establish the molecular mechanism for the anti-asthmatic
activity.
Page 73
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Page 74
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44. Cerutis DR, Nogami M, Anderson JL, Churchill JD, Romberger DJ, Rennard SI
and Toews ML Lysophosphatidic acid and EGF stimulate mitogenesis in human
airway smooth muscle cells. American Journal of Physiology 1997; 273:L10-
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45. Mechanisms of Airway Remodeling in Asthma, Etsuko Tagaya and Jun Tamaoki,
Allergology International. 2007;56:331-340
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International. 2007;56:341-348.
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Chupp and Robert J. Homer, Section of Pulmonary and Critical Care Medicine,
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bronchial epithelium and fibrocytes in the pathogenesis of airway remodeling in
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49. Peter J. Branes, Pathogenesis of Asthma, British Journal of Pharmacology, 1996,
42, pp 3-10.
50. Okumura S, Sagara H, Fukuda T, Fc epsilon mediated amphiregulin production
by human mast cells increases mucin gene expression in epithelial cells, Allergy
and Clinical Immunology, 2005; (115), pp- 272-279.
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Airway Remodeling in Asthma: from benchside to clinical practice Canadian
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54. V. Batra, A. I. Musani, A. T. Hastie, Bronchoalveolar lavage fluid concentrations
of transforming growth factor (TGF)-β1, TGF-β2, interleukin (IL)-4 and IL-13
after segmental allergen challenge and their effects on α-smoothmuscle actin and
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Experimental Allergy, vol. 34, no. 3, pp. 437–444, 2004.
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Current Opinion in Pulmonary Medicine,vol. 9, no. 1, pp. 28–33, 2003.
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Page 81
10. SYNOPSIS
Page 82
SYNOPSIS OF THE THESIS TO BE SUBMITTED
TO
UNIVERSITY OF MUMBAI
FOR THE DEGREE OF
MASTER OF PHARMACY
(PARTLY BY PAPER AND PARTLY BY RESEARCH)
IN THE FACULTY OF
PHARMACOLOGY
TITLE OF THE THESIS EVALUATION OF ANTI-

ASTHMATIC ACTIVITY IN ANIMAL
MODEL
NAME OF THE CANDIDATE Chaudhari Harsha Ramesh Rajani
NAME AND DESIGNATION OF THE Mrs. Sugandha G. Chaudhari

RESEARCH GUIDE
Lecturer
PLACE OF RESEARCH Department of Pharmacology,

(DEPT/DIV/SEC) RECOGINISED BY
Page 83
THE UNIVERSITY FOR THIS Dr. L. H. Hiranandani College of

PURPOSE Pharmacy,C.H.M. Campus, Opposite
Ulhasnagar Railway Station, Ulhasnagar
– 421003.
NUMBER AND DATE OF 40/31-10-2011

REGISTRATION
DATE OF SUBMISSION OF
SYNOPSIS
SIGNATURE OF THE CANDIDATE
SIGNATURE OF THE RESEARCH

GUIDE
INTRODUCTION
There are many herbs which have been documented as the treatment for asthma. Herbal
alternatives are proven to show symptomatic relief and to alleviate the disease
progression. Many herbs like Allium cepa, Adhatoda vasica, Albizzia lebbeck,
Clerodendron phlomidis, Tinospora cordifolia, Abrus precatorias are investigated for
targeting specific pathological factors responsible for progression of asthma, these herbs
may show bronchodilation, anti-spasmodic, anti-allergic, inhibition of lipoxygenase,
cytokines, cyclooxygenase, phosphodiesterase, etc 1.
Asthma is a chronic inflammatory disease of the airway. This disease is characterized by

increased mucus production and airway hyper-responsiveness which results into
decreased air flow, narrowing of airway, and marked by recurrent episodes of wheezing,
coughing, and shortness of breath. Strenuous exertion is associated with exercise induced
asthma. Asthma may be associated with the trigger which can be allergic, environmental,
emotional or it may be due to life style factors like smoking, food 2. There are many
herbal drugs investigated which act on particular site or stage or the progression of
disease. In Ayurveda, an indigenous system of medicine in India, there are many herbs
mentioned to have an anti-asthmatic activity. Hence, there is a growing need to follow the
research and to establish the scientific basis for their activity.
Carica papaya (Caricaceae) is commonly known as papaya. It is herbaceous perennial
plant. , the papaya is believed native to tropical America, perhaps in southern Mexico and
neighboring Central America. Stem bears crown of palmate lobed leaves. Petioles are
long, hollow, pale green in colour. The leaves are useful as an anthelmintic 3,
Page 84
antibacterial4, antifungal5, anti-inflammatory6, hypoglycemic7 . Asthma is an

inflammatory disease, therefore, we are aimed to investigate anti-asthmatic activity of
leaves of Carica papaya which may exhibit the anti-asthmatic activity by some probable
mechanisms.
The present research work was performed to evaluate anti-asthmatic activity of leaves of
Carica papaya and to make an attempt to establish the mechanism of action responsible
for the same.
AIMS AND OBJECTIVES:
The main objectives of the present study were:-
1. To obtain a hydro-alcoholic extract of Carica papaya leaves.
2. To evaluate anti-asthmatic of Carica papaya leaves extract (CPLE) using various
animal models.
EXPERIMENTAL WORK:
A) Collection, authentication and extraction:
The leaves of Carica papaya were collected from the Thane region in Maharashtra and
were authenticated at the Blatter Herbarium, St. Xavier’s College, Mumbai (specimen
number PD-3755 of P.Divakar). The leaves were dried, powdered mechanically and
extracted in a Soxhlet apparatus with 80% ethanol as the solvent. The extract was stored
in an air-tight container for experimental use.
B) Preliminary phytochemical analysis: 8
Preliminary phytochemical analysis of the hydroalcoholic extract of leaves of Carica

papaya was carried out for alkaloids, flavonoids, tannins, glycosides, saponins, sterols,
carbohydrates, proteins.
C) Acute toxicity testing was done according to OECD guideline 425.
D) Pharmacological Activity Studies:

1. In vivo studies
a) Histamine induced bronchoconstriction in Guinea pigs.
Guinea pigs of body weight 350-400gm were procured from Haffkine Biopharmaceutical
Corporation Limited, and were maintained under 26 ± 2ºC, 12 h light/dark cycle.
Animals were grouped as Disease control, standard group (ketotifen fumarate
1mg/kg as standard p.o.), Test 1, Test 2, Test3 (100mg/kg, 200mg/kg, 400 mg/kg
p.o.) animals were exposed to histamine dihydrochloride 2% aerosol in histamine
chamber. Convulsions were observed in guinea pigs. Preconvulsion time was noted9,11.
b) Mast cell degranulation in mesentery of wistar rats.
Page 85
Wistar rats of body weight 250-300 gm were procured from H(S)NCB’s facility for
breeding and experimentation, Dr. L. H. Hiranandani College of Pharmacy, Ulhasnagar.
They were housed at ambient temperature 26±2º, 12 h light/dark cycle. Animals
were divided into six groups as control, disease control, standard group(Disodium
cromoglycate 50mg/kg as standard), Test 1, test 2, test 3 (100mg/kg, 200mg/kg,
400mg/kg of CPLE.)., containing 6 animals each. Wistar rats sensitized on 1 st, 3rd and 5th
day, were administered CPLE from 6th to 12th day, pieces of mesentery were collected,
stained, and mast cell numbers were observed under light microscope (power X450). 9-11.
2. in vitro studies
a) Mast cell degranulation in peritoneal fluid
Wistar rats of body weight 250-300 gm were procured from H(S)NCB’s facility for
breeding and experimentation, Dr. L. H. Hiranandani College of Pharmacy, Ulhasnagar
and were housed at ambient temperature 26±1º, 12 h light/dark cycle. Peritoneal
fluid was collected from anaesthetized wistar rats, centrifuge and pellets were collected
which is then divided into 6 test-tubes, control, disease control, standard (Disodium
cromoglycate 20 µ/ml), test 1, test 2, test 3 of CPLE. Mast cell number was observed
after staining with toludine blue9-11.
b) Spasmolytic activity on guinea pig ileum.
Guinea pigs of body weight 350-400gm were procured from Haffkine Biopharmaceutical
Corporation Limited, and were maintained under standard conditions husbandry
with room temperature- 26 ± 2ºC, 12 h light/dark cycle.
Ileum of overnight fasted guinea pig was suspended in organ tube and treated with
histamine doses in absence and presence of CPLE9. Decreased contractile responses were
observed in presence of CPLE ( 50 µg/ml, 100 µg/ml, 200 µg/ml).
Ongoing Model
c) Spasmolytic activity on isolated goat trachea.
Goat trachea will be obtained from nearby slaughterhouse, immersed in Kreb’s solution
NaCl 6.9., KCl 0.35, CaCl2 0.28, MgSO4 0.28, NaHCO3 2.1, KH2PO4 0.16, Glucose 2.0
gm/lit maintained at 37±0.5ºC. Trachea will be divided into rings and tied together in
series to form a chain, suspended in organ bath12. The chain will be treated with histamine
doses in absence and presence of CPLE ( 50 µg/ml, 100 µg/ml, 200 µg/ml) and
contractile responses will be recorded.
Statistical analysis
Page 86
The result of anti-asthmatic activity for in vivo and in vitro models are expressed as mean
± SEM. Results were significantly analyzed using one way ANOVA. P<0.05 was
considered to be significant.
RESULTS AND DISCUSSION:

In histamine induced bronchoconstriction in guinea pigs, 400mg/kg of CPLE showed
statistically significant (P<0.05) increase in preconvulsion time than disease control
group.
According to in vivo model of mast cell degranulation, the animals treated with CPLE
showed significant (P<0.05) increased number of intact mast cells and decreased number
of degranulated cells after treatment of 200mg/kg and 400 mg/kg of CPLE. In vitro
model showed spasmolytic activity on guinea pig ileum. It also decreased mast cell
degranulation in peritoneal fluid with 50 µg/ml, 100 µg/ml, 200 µg/ml of CPLE.
These experimental findings suggest that CPLE exerts anti-histaminic, spasmolytic and
mast cell degranulation inhibitory activity.
REFERENCES :
1. Ravindra G. Mali, Avinash S. Dhake, A review on herbal antiasthmatics, Oriental
Pharmacy and Exerimental Medicine(2011) 11(2):77-90
2. Alan L. Miller, ND, The Etiologies, Pathophysiology and
Alternative/Complementary Treatment of Asthma, Alterative Medicine review
2001;6(6):20-47
3. Shaziya Bi, Goyal P.K., Anthelmintic effect of Natural Plant Carica papapya
extract against the gastrointestinal nematode, Ancylostoma caninum in mice,
ISCA Journal of Biological Sciences, 2012; 1(1): 2-6
4. Rahman S., Imran M, Muhammad N. Hassan N. Chisthi A. K., Khan A. F.,
Sadozai K.S. khan S.M. Antibacterial screening of leaves and stem of Carica
papaya, Journal of medicinal plants Research 2011 5(20:) 5167-5171,
5. Pedro Chavez-Quintal, Tania Gonzalez Flores, Antifungal activity in ethanolic
extracts of Carica papaya L. cv. Maradol Leaves and seeds, Indian Journal of
Microbiology, 2011;51(1):54-60
Page 87
6. Bamidele V. Owoyele, Olubori M. Adebukola, Adeoye A. Funmilayo, et al, Anti-

inflammatory activities of ethanolic extract of Carica papaya leaves.,
Inflammopharmacology, 2008; 16:168-173
7. T. O. Fakeye, T Oladipupo, O Shownde, Effects of coadministration of extract of
Carica papaya on activity of two oral hypoglycemic agents, Tropical Journal of
pharmaceutical research, 2007;6(1):671-678
8. Prashant Tiwari, Bimlesh Kumar, Mandeep Kaur, Gurpreet Kaur, Harleen Kaur,
Phytochemical screening and extraction: A review, International Pharmaceutical
Science; 2011(1): 98-106
9. Anita Mehta, Babita Agrawal, Investigation into the mechanism of action of
Moringa oleifera for its anti-asthmatic activity;Oriental Pharmacy and
Experimental Medicine 2008; 8(1): 24-31
10. Tejas Patel, Chimkode Rajshekar, Rakesh Parmar, Mast cell stabilizing activity of
Myrica nagi bark, Journal of Pharmacognosy and Phytotherapy 2011;3(8): 114-
117,
11. Sachin Parmar, Amit Gangwal, Navin Sheth, Mast cell membrane stabilization
and anti-histaminic actions- possible mechanism of action of anti-inflammatory
action of Murraya koenigii., Journal of Current Pharmaceutical Research,
2010;2(1):21-25
12. Tamhane Adesh S., Mute Vaishali M., Takawale Harshada, et al, Preclinical
evaluation and antiasthmatic activity of cassia tora linn. Leaves, International
Journal of Research in Ayurveda and Pharmacy, 2012;3(2):273-275
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Evaluation of Anti-asthmatic Activity in Animal Model

In management of asthma, two types of therapies are available, First by ‘Curative’

Ayurveda offers unique insight onto comprehensive approach on management of asthma.

i) Adhatoda vasica (Vasa) – it shows mast cell stabilizing property thus

2.1- Review of Disease

It is characterized by airway inflammation, i.e. redness and swollen airway, similarly

Common signs and symptoms –

These include coughing, wheezing, breathlessness, increased respiratory rate, chest

During some episodes, the symptoms include –

 Inability to talk continually

Airway cooling Increased lower airway

Movement of fluid from the Degranulation of mast

2.1.1- Classification of asthma

4. Drug induced asthma

5. Exercise induced asthma

It is characterized by transient airway observation occurring after strenuous exertion. Due

6. Cough variant asthma

INFLAMMATORY CELLS INVOLVED

Fig. 2.2 Immunological cell involvement in the progression of inflammation antigen

Interlukins- secreted by leukocytes.

Lymphokines- secreted by lymphocytes.

Monokines- secreted by monocytes.

Interferon- involved in the process of viral responses.

Colony stimulating factors- increase the growth of cells in media.

Chemokines- promote chemotaxis.

Lymphokines Cellular Effects

Cell source Released mediators

bronchoalveolar lavage fluid has been observed after segmental allergen

Airway remodeling is one of the central features of asthma. Remodeling is defined as

Fig. 2.5- Events and cell involved in airway remodeling in asthma.

Events in Airway remodeling

1. Epithelial layer damage

Airway remodeling is basically a repair mechanism 47. It includes changes in epithelium,

The process involves changes in cellular, biochemical, and molecular components of

1. Epithelial layer damage –

It includes shedding of epithelial cells, upregulation of cytokines, several growth factors

Thichening of reticular basement membrane is the characteristic feature of airway

patients than normal individuals. Matrix metalloproteinase degrade extracellular matrix

Increased angiogenesis is one more characteristic of airway remodeling. Smooth muscle

Fig – 2.6 – Role of Angiogenesis in asthma

Involvement of Histamine in Asthma

Pharmacological Effects of Histamine63

a) Triple-effect on terminal vasculature (itching & pain):

i. reddening at injection site due to vasodilation

2. Smooth muscle of bronchioles; causes contraction of nonvascular smooth muscle.

RECEPTOR LOCATION MECHANISM FUNCTIONS ANTAGONISTS

2.1.4- Diagnosis of Asthma

Drugs which are used as a treatment for asthma are as follows-

 Methylxanthines – theophylline, aminophylline increase the intracellular

2.1.5- Some important medicinal plants having anti-asthmatic

2.2 Review of Plant

2.2.1- Introduction to the plant

Scientific classification of the plant

Domain : Flowering plant

Kingdom : Plantae- plants

Botanical name : Carica Papaya Linn.

Fig. Plant of Carica papaya

Fig – 2.8– Leaf of Carica papaya

Origin and Distribution

Fig.2.9– Plant Carica papaya bearing fruits