Evaluation of Anti-Asthmatic Activity in Animal Model

Evaluation of anti-asthmatic activity in animal model
INTRODUCTION
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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. 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 condition(2). According to World Health Organization,
300 million people suffer from asthma from which 180,000 people die due to it.
Asthma is characterized by the airway inflammation manifested by cough,

wheezing chest tightness, acute exacerbations, dyspnea, bronchoconstriction. 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(3). Whatever may be the cause for trigger, the end result
is nothing but smooth muscle contraction and airway inflammation.
Some other diseases of respiratory system
Emphysema
Bronchitis
Pulmonary edema
Chronic obstructive pulmonary disease
Tuberculosis
Chronic obstructive pulmonary disease - .
It is characterized by chronic and recurrent obstruction of airflow, which increases

airway resistance. Almost 30 million Americans are affected by COPD. Causes
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include air pollution, pulmonary infection, genetic factors, occupational exposure to

dust, fibres.
Cystic fibrosis is genetic disorder, caused by inheriting two defective genes for cystic
fibrosis conductance regulator (CFTR). Defective ion transport in lungs rduces the
water content. This makes it more viscous and difficult for ciliated cells to move it up
out of the lungs.
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 hyperresponsiveness 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 i.e. muscles of airways respond more
quickly to minute quantity of allergen or stimuli.
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 cells

Hyperemia epithelium to the airway and eosinophils, epithelial
Mucosal edema
surface cells involvement
Release of PGs, histamine,

Bronchoconstriction LTs, PAF
Airway inflammation
Fig. 1.1 Schematic representation for progression of the causes for asthma
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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 observed
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 asthma.
3. Occupational asthma
One type of occupational asthma is immune mediated, and other one is irreitant induced.
In immune induced, there is latency period, between workplace exposure and beginning
of symptoms. In irritant induced asthma, symptoms occur immediately after exposure.
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This can be aggravated due to repeated exposure to industrial irritants, fibres. Generally
in industries with fibre manufacturing, the employees may be at risk for this type of
asthma.
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 aspirin
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 obstruction.
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 weeks.
Symptoms in moderate asthma –
Attacks occur at least 2 times a week
Occasional emergency care may require
Symptoms in severe or adverse asthma-
Attacks occur daily
Limited physical activity
Frequent exacerbations
Symptoms of status asthmaticus

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Occurs suddenly
Emergency procedures are required
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.
Age and gender also effects for the prevalence of asthma. It is more in boys than girls.
Ration 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.
Occurance of this disease may may be impacted by environmental factors which include
aeroallergens, like dust, pollen grains, fibres.
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. 1.2 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) cells 1.
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 asthma 2. This suggests that Th1 cells may be associated with
severe disease and contribute to asthmatic cases3.
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 4. In intial
state of asthma, these cytokines help in antibody production, and in later state, they show
inflammatory responses5.
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 fluid6.
2. Mast cells
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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 airways, near
blood vessels in submucosa, spread in muscle bundles and also in bronchial luminal
space7. 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 inflammation8.
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Fig. 1.3- 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 response.
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(10). 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 airways11. Bronchial hyperresponsiveness and airway eosinophilia are
generally associated with each other12. 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 asthma13.
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 FEV11.
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 cells14.
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 respectively.
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 (2).
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.
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Various cytokines –
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.
Fig. 1.4 – Involvement of various cytokines in asthma
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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 cells.
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)
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); chemokines (CXCL8); lipids;
PAF; ROS; NO
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 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
cells.
 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).
 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 proliferation.
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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 (1).
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
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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 remodeling(2).
Fig. 1.5- 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(3). 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
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membrane(4). 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.
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 cells(3). 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 inflammation(1). 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 (5). STAT-1 has a key
role in remodeling process, Its increased level is associated with T cell accumulation in
tissue(6).
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 (7). 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
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responsible for such thickening(8). Myofibroblast play an important role in subepithelial

fibrosis. These are the phenotypic intermediate between smooth muscles and
fibroblasts(9). 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 actin(10). It also inhibit the degradation
of matrix metalloproteinase. Level of TGF β is increased in asthmatic airways and
broncho alveolar lavage in asthmatic patients than normal individuals. Matrix
metalloproteinase degrade extracellular matrix molecules (11). 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
glands(12). This may be due to desquamation of epithelial cells. This results in airway
narrowing and obstruction(13). 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 asthma(14) .
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
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cells are the consequences of this (15). This change is associated with related functional
alterations. Changes in airway microvascularization results from these alterations. This
results in airway edema(16). Overexpression of VEGF contributes mainly for increased
angiogenesis. Clinical consequences include excess delivery of inflammatory and
remodeling mediators in airways(17). Congested and enlarged mucosal blood vessels
result in more thick airway walls(18).
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OBJECTIVE
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OBJECTIVE
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
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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.
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REVIEW OF LITERATURE
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Introduction to the plant
Carica papaya
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
Genus : Carica
Botanical name : Carica Papaya Linn.
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Common names
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 3- 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, 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".
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
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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 3.2 – Leaf of Carica papaya
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.
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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
Fig. 3.3 – Plant Carica papaya bearing fruits
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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. Thereafter, natural enemies seriously reduced
the plantings. There was a similar decline in Puerto Rico about 10 years prior to the setback of
the industry in Florida. While isolated plants and a few commercial plots may be fruitful and
long-lived, plants in some fields may reach 5 or 6 ft, yield one picking of undersized and
misshapen fruits and then are so affected by virus and other diseases that they must be destroyed.
Pollination
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.
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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 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
Page 31
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.
Variable Season
Planting may be done at any time of year and local conditions determine when it is best for the
crop to come in. Papayas mature in 6 to 9 months from seed in the hotter areas of South Africa;
in 9 to 11 months where it is cooler, providing an opportunity to supply markets in the off-season
when prices are high. Seeds planted in early summer or midsummer will produce the first crop in
the second winter. Thereafter, the same plants will mature fruit from spring to early summer.
Spring fruits are apt to be sunburned because of winter leaf loss; are also subject to fruit spot and
have a low sugar content. Sunburn can be avoided by advance whitewashing of sides exposed to
the afternoon sun. Some growers manipulate the harvest season by stripping off 6 of the newly
set fruits, thus forcing the plant to bloom again and produce fruits 6 to 8 weeks later than they
normally would.
In southern Florida, plants set out in March or April will ripen their fruits in November and
December and have the advantage of a "tourist" market. July plantings will be slowed down by
winter and will not fruit for 10 months or more. Some growers advocate planting in September
and October so that the crop will be ready for harvest before the onset of the main hurricane
season. Further north in the state, papayas must be set out in March or April in order to have the
required growing season before frost.
Culture
Seeds may be planted directly in the field, or seedlings raised in beds or pots may be transplanted
when 6 weeks old or even up to 6 months of age, though there must be great care in handling and
the longer the delay the greater the risk of dehydrated or twisted roots; also, transplanting often
results in trunk-curvature in windy locations.
Experiments in Hawaii indicate that direct seeding results in deeper tap-roots, erect and more
vigorous growth, earlier flowering and larger yields.
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In Puerto Rico, it is customary to set 2 plants per hole. In El Salvador planters place 5 to 6 seeds,
separated from each other, in each hole at a depth of 3/8 in (1 cm). When the plants bloom, 90%
of the males are removed, preferably by cutting off at ground level. Pulling up disturbs the roots
of the remaining plants. If the plantation is isolated and there is no chance of cross-pollination by
males, all the seed will become female or hermaphrodite plants. Fruits should mature 5 to 8
months later.
In India, seeds are usually treated with fungicide and planted in beds 6 in (15 cm) above ground
level that have been organically enriched and fumigated. The seeds are sown 2 in (5 cm) apart
and 3/4 to 1 1/8 in (2-3 cm) deep in rows 6 in (15 cm) apart. They are watered daily and
transplanted in 2 1/2 months when 6 to 8 in (15-20 cm) high. Transplanting is more successful if
polyethylene bags of enriched soil are used instead of raised beds. Two seeds are planted in each
bag but only the stronger seedling is maintained. Transplanting is best done in the evening or on
cloudy, damp days. On hot, dry days, each plant must be protected with a leafy branch or palm
leaf stuck in the soil. Except for 'Coorg Honey Dew' and 'Solo', the plants are set out in 3's, 6 in
(15 cm) apart in enriched pits. After flowering, one female or hermaphrodite plant is retained, the
other two removed. But one male is kept for every 10 females. 'Coorg Honey Dew' and 'Solo' are
planted one to a pit and no males are necessary. Watering is done every day until the plants are
well established, but overwatering is detrimental to young plants. Double rows of
Sesbaniaaegyptiacaare planted as a windbreak.
The installation of constant drip irrigation (12 gals per day) has made possible papaya cultivation
on mountain slopes on the relatively dry island of Maui which averages 10 in (25 cm) of rain
annually.
Papaya plants require frequent fertilization for satisfactory production. In India, best results have
been obtained by giving 9 oz (250 g) of nitrogen, 9 oz (250 g) of phosphorus, and 18 oz (500 g)
potash to each plant each year, divided into 6 applications.
Because of the need to expedite growth and production before the onslaught of diseases, Puerto
Rican agronomists recommend treating the predominantly clay soil with a nematicide before
planting, giving each plant 4 oz (113 g) of 15-15-15 fertilizer at the end of the first week, and
Page 33
each month thereafter increasing the dose by 1 oz (28 g) until the beginning of flowering, then
applying .227 g per plant as a final treatment. In trials, this program has permitted 6 harvests of
green fruits for processing, each over 1 lb (1/2 kg) in weight, spanning a period of 13 months.
The roots usually extend out beyond the leaves and it is advisable to spread fertilizer over the
entire root area.
In late fertilizer applications of a crop destined for canning, nitrogen should be omitted because it
renders the fruit undesirable for processing. High nitrate content in canned papaya (as with
several common vegetables) removes the tin from the can. To avoid nitrogen deficiency at the
beginning of flowering for the next crop, 1 or 2% urea sprays can be applied.
Mechanical cultivation between rows is apt to disturb the shallow roots. judicious use of
herbicides is preferable.
Overcrowded fruits should be thinned out when young to provide room for good form
development and avoid pressure injury. Cold weather may interfere with pollination and cause
shedding of unfertilized female flowers. Spraying the inflorescence with growth regulators stops
flower drop and significantly enhances fruit set. After the first crop, the terminal growth may be
nipped off to induce branching which tends to dwarf the plant and facilitates harvesting.
However, unless the plants are strong growers, fruiting branches may need to be propped to
avoid collapse.
Renovation of Plantings
In Trinidad and Tobago, plants that have become too tall are cut to the ground and side shoots
are allowed to grow and bear. In El Salvador, after the 3rd year of bearing, the main stem is cut
off about 3 ft (1 m) from the ground at the beginning of winter and is covered with a plastic bag
to protect it from rain and subsequent rotting. Several side shoots will emerge within a few days.
When these reach 8 in to 1 ft (20-30 cm) in height, all are cut off except the most vigorous one
which replaces the original top.
Disease of the plant
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A major hazard to papayas in Florida and Venezuela is the wasp-like papaya fruit fly,
Toxotrypanacurvicauda. The female deposits eggs in the fruit which will later be found infested
with the larvae. Only thick-fleshed fruits are safe from this enemy. Control on a commercial
scale is very difficult. An important and widespread pest is the papaya web-worm, or fruit cluster
worm, Homolapalpiadalera, harbored between the main stem and the fruit and also between the
fruits. It eats into the fruit and the stem and makes way for the entrance of anthracnose. Damage
can be prevented if spraying is begun at the beginning of fruit set, or at least at the first sign of
webs.
The tiny papaya whitefly, Trialeuroidesvariabilis, is a sucking insect and it coats the leaves with
honeydew which forms the basis for sooty mold development. Shaking young leaves will often
reveal the presence of whiteflies. Spraying or dusting should begin when many adults are
noticed. Hornworms (immature state of the sphinx moth–Erinnyisobscurain Jamaica, E. elloin
Venezuela, E. alopein Florida) feed on the leaves, as do the small, light-green leafhoppers.
Other pests requiring control measures in Australia include the red spider, or red spider mite,
Tetranychusseximaculatus, which sucks the juice from the leaves. In India and on the island of
Maui, plant and fruit infestation by red spider has been a major problem. This pest and the
cucumber fly and fruit-spotting bugs feed on the very young fruits and cause them to drop. In
Hawaii, the red-and-black-flat mite feeds on the stem and leaves and scars the fruit. The broad
mite damages young plants especially during cool weather.
Indian scientists have observed that immature earthworms, Megascolexinsignis, are attracted by
and feed on rotting tissue of papaya plants. They hasten the demise of plants afflicted with stem
rot from Pythiumaphanidermatumand may act as vectors for this fungus.
Papaya ringspot virus, prevalent in Florida, the Dominican Republic and Venezuela, is
occasionally serious in the area of Oahu. It is transmitted by the same vectors. Mosaic and
ringspot viruses are the main limiting factors in papaya production in the Cauca Valley of
Colombia.
In Florida, virus diseases were recognized as the greatest threat to the papaya industry in the
early 1950's. The first signs are irregular mottling of young leaves, then yellowing with
Page 35
transparent areas, leaf distortion, and rings on the fruit. If affected plants are not removed, the
condition spreads throughout the plantation. Fruits borne 2 or 3 months after the first symptoms
will have a disagreeable, bitter flavor.
At the Agricultural Research and Education Center of the University of Florida in Homestead,
the late Dr. Robert Conover established a test plot of papayas grown from seed of 95 accessions
from a number of countries and 94 collections in Florida in the hope of finding some virus-free
strains. Most of the introductions were highly susceptible to papaya ringspot virus; local strains
showed some resistance. Highest tolerance was shown by a dioecious, round-fruited, yellow-
fleshed strain brought from Colombia by Dr. S.E. Malo several years ago. The fruits weigh 3-5
lbs (1.36-2.27 kg).
It is thought that at least 3 virus diseases are involved in papaya decline in East Africa and it has
been suggested that the diseases are spread in part by the tapping of green fruits for their latex
(the source of papain).
Bunchy top is a common, controllable mycoplasma disease transmitted by a leafhopper,

Empoascapapayae in Puerto Rico, the Dominican Republic, Haiti, and Jamaica; by that species
and E. dilitara in Cuba; and by E. stevensiin Trinidad. Bunchy top can be distinguished from
boron deficiency by the fact that the tops of affected plants do not ooze latex when pricked.
In the subtropical part of Queensland, but not in the tropical, wet climate of northern
Queensland, papaya plants are subject to die-back, a malady of unknown origin, which begins
with shortening of the petioles and bunching of inner crown leaves. Then the larger crown leaves
quickly turn yellow. Affected plants can be cut back at the first sign of the disease and if the cut
stem is covered to avoid rotting, the top will be replaced by healthy side branches. The problem
occurs mainly in the hot, dry spring after a season of heavy rains.
Anthracnose, which usually attacks the ripe fruits and is caused by the fungus
Colletotrichumgloeosporioides, was formerly the most important papaya disease in Hawaii,
Mexico and India, but it is controllable by spraying every 10 days, or every week in hot, humid
seasons, and hotwater treatment of harvested fruits. A strain of this fungus produces "chocolate
spot" (small, angular, superficial lesions). A disease resembling anthracnose but which attacks
Page 36
papayas just beginning to ripen, was reported from the Philippines in 1974 and the causal agent
was identified as Fusariumsolani.
A major disease in wet weather is phytophthora blight. Phytophthoraparasitica attacks and rots
the stem and roots of the plant and infects and spoils the fruit surface and the stem-end, inducing
fruit fall and mummification. Fungicidal sprays and removal of diseased plants and fruits
wilRoot-rot by Pythium sp. is very damaging to papayas in Africa and India. P. ultimum causes
trunk rot in Queensland. Collar rot in 8- to 10-month old seedlings, evidenced by stunting, leaf-
yellowing and shedding, and total loss of roots, was first observed in Hawaii in 1970, and was
attributed to attack by Calonectria sp. Collar rot is sometimes so severe in India as to cause
growers to abandon their plantations.
Powdery mildew, caused by Oidiumcaricae (the imperfect state of Erysiphecruciferarum the

source of mildew in the Cruciferae) often affects papaya plants in Hawaii and both plants and
fruits elsewhere. Sulfur, judiciously applied, is an effective control. Powdery mildew is caused
by Sphaerothecahumili in Queensland and by Ovulariopsispapayae in East Africa. Angular leaf
spot, a form of powdery mildew, is linked in Queensland to the fungus Oidiopsistaurica.
Corynespora leaf spot, or brown leaf spot, greasy spot or "papaya decline" (spotting of leaves
and petioles and defoliation) in St. Croix, Puerto Rico, Florida and Queensland, is caused by
Corynesporacassiicola, which is controllable with fungicides.
A new papaya disease, yellow strap leaf, similar to YSL of chrysanthemums, appeared in Florida
during the summer in 1978 and 1979.
Black spot, resulting from infection by Cercosporapapayae, has plagued Hawaiian growers since
the winter of 1952-53. It causes defoliation, reduces yield, blemishes the fruit, and is unaffected
by the hot-water dip. It can be prevented by field use of fungicides.
Rhizopusoryzae is most commonly linked with rotting fruits on Pakistan markets. R. nigricans is
the usual source of fruit rot in Queensland. Injured fruits are prone to fungal rotting caused by R.
stolonifer and Phytophthorapalmivora. Stem-end rot occurs when fruits are pulled, not cut, from
the plant and the fungus, Ascochytacaricae, is permitted entrance. This fungus attacks very
Page 37
young and older fruits in Queensland and also causes trunk rot. In South Africa, it affects cv
'Honey Gold' which is also subject to spotting by Asperisporiumcaricae on the fruits and leaves.
Both of these diseases are controllable by fungicidal sprays.
Infection at the apex by Cladospoiium sp. is manifested by internal blight. A pre-harvest fruit rot
caused by Phomopsiscaricaepapayae is troublesome in Queensland and was announced from
India in 1971. A new disease, papaya apical necrosis, caused by a rhabdovirus, was reported in
Florida in 1981.
Papayas are frequently blemished by a condition called "freckles", of unknown origin; and
mysterious hard lumps of varying size and form may be found in ripe fruits. Star spot (grayish-
white, star-shaped superficial markings) appears on immature fruits in Queensland after exposure
to cold winter winds. In Uttar Pradesh, an alga, Cephaleurosmycoidea, often disfigures the fruit
surface.
In Brazil, Hawaii and other areas, a fungus, Botryodiplodiatheobromae, causes severe stem rot
and fruit rot. Trichothecium rot (T.roseum) is evidenced by sunken spots soon covered by pink
mold on fruits in India. Charcoal rot, Macrophominaphaseoli, is reported in Pakistan.
Young papaya seedlings are highly susceptible to damping-off, a disease caused by soil-borne
fungi–Pythiumaphanidermatum, P. ultimum, Phytophthorappalmivora, and Rhizoctoniasp.,–
especially in warm, humid weather. Pre-planting treatment of the soil is the only means of
prevention.
Papayas generally do poorly on land previously planted with papayas and this is usually the
result of soil infestation by Pythiumaphanidernwtumand Phytophthorapalmivora. Plant refuse
from previous plantings should never be incorporated into the soil. Soil fumigation is necessary
before replanting papayas in the same field.
Young leaves are cooked and eaten like spinach in the East Indies. Mature leaves are bitter and
must be boiled with a change of water to eliminate much of the bitterness. Papaya leaves contain
Page 38
the bitter alkaloids,carpaine and pseudocarpaine, which act on the heart and respiration like
digitalis, but are destroyed by heat. In addition, two previously undiscovered major 1-
piperideine alkaloids, dehydrocarpaine I and II, more potent than carpaine, were reported from
the University of Hawaii in 1979. Sprays of male flowers are sold in Asian and Indonesian
markets and in New Guinea for boiling with several changes of water to remove bitterness and
then eating as a vegetable. In Indonesia, the flowers are sometimes candied. Young stems are
cooked and served in Africa. Older stems, after peeling, are grated, the bitter juice squeezed out,
and the mash mixed with sugar and salt.
Papain
The latex of the papaya plant and its green fruits contains two proteolytic enzymes, papain and
chymopapain. The latter is most abundant but papain is twice as potent. In 1933, Ceylon (Sri
Lanka) was the leading commercial source of papain but it has been surpassed by East Africa
where large-scale production began in 1937.
The latex is obtained by making incisions on the surface of the green fruits early in the morning
and repeating every 4 or 5 days until the latex ceases to flow. The tool is of bone, glass, sharp-
edged bamboo or stainless steel (knife or raxor blade). Ordinary steel stains the latex. Tappers
hold a coconut shell, clay cup, or glass, porcelain or enamel pan beneath the fruit to catch the
latex, or a container like an "inverted umbrella" is clamped around the stem. The latex coagulates
quickly and, for best results, is spread on fabric and oven-dried at a low temperature, then ground
to powder and packed in tins. Sun-drying tends to discolor the product. One must tap 1,500
average-size fruits to gain 1 1/2 lbs (0.68 kg) of papain.
The lanced fruits may be allowed to ripen and can be eaten locally, or they can be employed for
making dried papaya "leather" or powdered papaya, or may be utilized as a source of pectin.
Because of its papain content, a piece of green papaya can be rubbed on a portion of tough meat
to tenderize it. Sometimes a chunk of green papaya is cooked with meat for the same purpose.
One of the best known uses of papain is in commercial products marketed as meat tenderizers,
especially for home use. A modern development is the injection of papain into beef cattle a half-
Page 39
hour before slaughtering to tenderize more of the meat than would normally be tender. Papain-
treated meat should never be eaten "rare" but should be cooked sufficiently to inactivate the
enzyme. The tongue, liver and kidneys of injected animals must be consumed quickly after
cooking or utilized immediately in food or feed products, as they are highly perishable.
Papain has many other practical applications. It is used to clarify beer, also to treat wool and silk
before dyeing, to de-hair hides before tanning, and it serves as an adjunct in rubber
manufacturing. It is applied on tuna liver before extraction of the oil which is thereby made
richer in vitamins A and D, It enters into toothpastes, cosmetics and detergents, as well as
pharmaceutical preparations to aid digestion.
Papain has been employed to treat ulcers, dissolve membranes in diphtheria, and reduce
swelling, fever and adhesions after surgery. With considerable risk, it has been applied on meat
impacted in the gullet. Chemopapain is sometimes injected in cases of slipped spinal discs or
pinched nerves. Precautions should be taken because some individuals are allergic to papain in
any form and even to meat tenderized with papain.
Other chemical constituents
 alkaloids -- 1,300-4,000 ppm in leaves

 butanoic acid -- as much as 1.2mg/kg in fruit pulp
 methyl butanoate -- as much as 18% of the volatile components in the fruit
 carpaine -- leaves (1,000-1,500 ppm), bark, roots, and seeds
 dehydrocarpaines -- 1,000 ppm in leaves
 pseudocarpaine -- 100 ppm in leaves
 chymopapain-a and b -- latex and exudate
 flavonols -- 0-2,000 ppm in leaves
 benzylglucosinolate -- found in all parts of the plant, but highest in young leaves
 linalool -- as much as 94% of the volatile components in the frui
 cis- and trans-linalool oxide -- fruit
 alpha-linolenic acid -- 250-2,238 ppm in fruit
 nicotine
 papain -- fruit and 53,000 ppm in latex and exudate
 alpha-phellandrene -- fruit
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 tannins -- 5,000-6,000 ppm in leaves

 alpha-terpinene -- fruit
 gamma-terpinene -- fruit
 4-terpineol -- fruit
 terpinolene -- fruit
 methyl-thiocyanate and benzyl-isothiocyanate
Folk Uses
In tropical folk medicine, the fresh latex is smeared on boils, warts and freckles and given as a
vermifuge. In India, it is applied on the uterus as an irritant to cause abortion. The unripe fruit is
sometimes hazardously ingested to achieve abortion. Seeds, too, may bring on abortion. They are
often taken as an emmenagogue and given as a vermifuge. The root is ground to a paste with salt,
diluted with water and given as an enema to induce abortion. A root decoction is claimed to
expel roundworms. Roots are also used to make salt.
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.
 Medicinal Uses:
Papaya promotes cardiovascular health and protects against colon cancer.
It contains the digestive enzyme, papain and chymopapain and is commonly used to
aid digestion, and to lower inflammation and improve healing from burns, sports
injuries, trauma and allergies.
Page 41
Papaya has anti-inflammatory properties and helps alleviate the severity of asthma,
osteoarthritis, and rheumatoid arthritis.
Carpaine obtained from papaya leaves has the same effect on the heart as digitalis.
Because of its high antioxidant content, papaya helps prevent cholesterol oxidation
and is therefore helpful for the prevention of atherosclerosis, diabetic heart disease,
heart attacks or strokes.
Papaya improves the immune system and is a good choice for preventing illnesses
such as recurrent ear infections, colds and flu.
Having three servings of papaya helps reduce the risk of age-related macular
degeneration (ARMD) and improves eye-sight.
Due to its high vitamin A content it can play an important role in saving the lives of
smokers or passive smokers and lowering the risk of emphysema.
Regular consumption of both green tea and lycopene-rich fruits such as papaya can
protect against prostate cancer.
Papain is also employed to treat ulcers, dissolve membranes in diphtheria, and

reduce swelling, fever and adhesions after surgery.
Chemopapain is sometimes injected in cases of slipped spinal discs or pinched

nerves.
The fresh latex is commonly used to improve the healing process in boils, warts and
freckles; it also works as a vermifuge.
Page 42
The unripe fruit or seeds are sometimes used as a uterus irritant to cause abortion.
Smoking the dried leaves help relieve asthma.
An infusion of dried papaya leaves aids in gastrointestinal and genitourinary

ailments.
Papaya extract has anti-gram-positive bacteria properties and in high doses is

effective against gram-negative bacteria.
Papaya is believed to have rejuvenating properties and controls premature aging.
Papaya contains arginine which is essential for male fertility and also carpain, an
enzyme thought to be good for the heart.
After treatment with antibiotics the use of papaya juice will quickly help restore
normal intestinal bacteria destroyed by treatment.
Papaya peel is a good external treatment for skin wounds that do not heal quickly.
The pulp can be used as a poultice.
Papaya helps prevent constipation.
It lowers cholesterol levels, prevents urinary stones formation and parasite related
intestinal infections.
Papaya also helps stimulate milk production.
Papaya prevents nausea, morning sickness and motion sickness.
Papaya also helps prevent cataract formation, chronic obstructive pulmonary
Page 43
disease, diverticulosis, and possibly hypertension.

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
 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
Page 44
 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
 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
Page 45
Preliminary Phytochemical screening
Detection of Carbohydrates
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.
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.
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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 sod.picrate was added. Formation of yellow to orange colour
indicates the presence of cardiac glycoside.
Detection of phytosterols and triterpenes

1 Salkowski’s Test
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.
Page 47
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 flavonoids
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.
4Mineral 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 Gelatin Test
To the extract, 1% gelatin solution containing sodium chloride was added. Formation of
white precipitate indicates the presence of tannins.
TLC Profile
Page 48
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.
Distance traveled by solute

Rf =
Distance traveled by the solvent
Page 49
Materials and methods
Page 50
Materials and methods
Pharmacological Evaluation
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.
Page 51
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.
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.
Inhibition of Mast cell degranulation ( in vivo )

Page 52
Species- Wistar rats
Gender – Either
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
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 50 mg/kg 6

cromoglycate)
Test 1 100mg/kg 6
Test 2 200mg/kg 6
Page 53
Test 3 400mg/kg 6
Total 36+4=40
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.
Fig. 4.1 degranulated and intact mast cells in mesentery observed under 450X
Statistical analysis –
The results were expressed as mean ± standard error of mean. One way ANOVA was
applied. p<0.05 as considered statistically significant.
Inhibition of Mast cell degranulation (in vitro)
Page 54
Species- Wistar rats
Gender – Either
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
Studies on peritoneal mast cell degranulation induced by albumin(5)
Procedure
Saline solution was injected in the peritoneal cavity of the lightly anaesthetized wistar rats. After
giving abdominal massage, the peritoneal fluid was collected. The peritoneal fluid from 4-5 rats
was pooled together and was subjected to centrifuge at the speed of 2000 rpm for 5 minutes. The
pellet obtained was resuspended in 1 ml of saline. This suspension was divided into test tubes as
shown below.
Test tube 1- control ( no inducer)
Test tube 2 - Saline
Test tube 3 - Disodium cromoglycate 10 µ/ml
Test tube 4 - Extract of leaves of Carica papaya 50 µg/ml
Test tube 5 - Extract of leaves of Carica papaya 100 µg/ml
Test tube 6 – Extract of leaves of Carica papaya 200 µg/ml
Page 55
Each test tube, except test tube 1 was the incubated for 15 min at 37°C. Then, 0.1 ml of 1% w/v
egg albumin added into each test tube. The cells were stained with 0.1% toludine blue for 10
minutes. They are observed under microscope. (450X)
Fig. 4.2 Degranulated and intact mast cells in peritoneal fluid observed under 450X
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 .
Spasmolytic activity of isolated guinea pig ileum
Species- Guinea pig
Gender – Either
Page 56
Guinea pigs weighing between 350- 400gm procured from Haffkins , 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.
Procedue
Overnight fasted guinea pigs of either sex was 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 containing Tyrode’s solution which was sach as NaCl
0.1, NaHCO3 1.0, NaH2PO4 0.05, and Glucose 1.0 gm/lit.. Responses of contractions with
histamine 10 µg/ ml in absence and in presence of extract 50 µg/ ml, 100 µg/ ml and 200
µg/ ml was recorded.
Spasmolytic activity of isolated goat trachea
Goat trachea was obtained from nearby slaughterhouse. It is then immersed I 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 was the divided into rings and tied
together in series to form a chain. This chain was suspended in organ bath. The dose
response curve was obtained with histamine in absence and in presence of extract of
concentration 50 µg/ ml, 100 µg/ ml and 200 µg/ ml.
Page 57
Results and Discussion
Page 58
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.
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 -
5 Tannins +
Pharmacological studies-
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
Page 59
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 400 mg / kg 290.2 ± 27.61**
Values are mean ± SEM (n=5 animals) *p<0.05. **p<0.01; ns = non significant
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 PCD 1. 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 400mg/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.
Inhibition of Mast cell degranulation ( in vivo )
Page 60
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.
Observation table for Inhibition of Mast cell degranulation ( in vivo)
Results for the present studies are given in table 5.2

TREATMENT DOSE No. of INTACT % of % of
[mg/kg, p.o.] mast cells seen in the INTACT DEGRANULATED
Neubeur’s chamber mast cells mast cells
Control Saline 359.3 + 28.27*** 100 -
Disease Control Saline 55.03 44.97

198.7 + 10.61
Standard (Disodium 50 mg/kg 315.8 87.89 12.11

cromoglycate ) ±14.28***
Test 1 100 mg/kg 56.72 43.28

ns
203.8 + 12.65
Test 2 200 mg/kg 73.53 26.47

264.2 + 8.514*
Test 3 400 mg/kg 78.70 21.30

282.8 + 18.27**
Values are expressed as mean ± SEM (n = 6 animals), n.s- non significant,
Page 61
***p < 0.0001, **p<0.001, * p<0.01, compared with disease Control Group (oneway ANOVA
followed by Dunnett’s Multiple Comparisons test).
Mast cell degranulation is an important event for the initiation of the allergic cascade after
exposure of 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.
Inhibition of Mast cell degranulation ( in vitro )
Results of this study are given in table 5.3
TREATMENT DOSE No. of INTACT % of % of

[µg/ml, p.o.] mast cells seen in INTACT DEGRANULATED
the Neubeur’s mast cells mast cells
chamber
Negative Control saline 229.8 + 100 -
5.294***
Disease Control saline 119.0 + 6.763 51.78 48.22
Standard 20µg/ml 208.3 + 90.64 9.36

7.210***
Test 1 50 150.3 + 10.26ns 65.40 34.60
Test 2 100 171.7 + 16.06** 74.71 25.29
Test 3 200 173.7 + 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).
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 62
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.
Spasmolytic activity of isolated guinea pig ileum

Dose 1
Volume Concentratio Histamine Hist+Extract % % Percentage

of n in µg Response response (in maximu maximum inhibition
histamine (in mm) mm) m response
response for
for histamine+
histamine extract
0.1 ml 1 µg 30 19 69.76% 44.18% 55.52%
0.2 ml 2 µg 33 23 76.74% 53.48% 46.82%
0.4 ml 4 µg 37 29 80.04% 67.44% 32.56%
0.8 ml 8 µg 43 32 100% 74.41% 25.59%
Dose 2
Volume Concentratio Response Hist+Extract % % Percentage
of n in µg (in mm) response (in maximum maximum inhibition
histamine mm) response response
for for
histamine histamine+
extract
0.1 ml 10 µg 1.0 0.7 52.63% 36.84% 63.16%
0.2 ml 20 µg 1.4 0.7 73.68% 36.84% 63.16%
0.4 ml 40 µg 1.6 1.2 84.21% 63.15% 34.85%
0.8 ml 80 µg 1.9 1.8 100% 94.73% 5.27
Page 63
Dose 3

for for
extract
0.1 ml 10 µg 1.2 1.0 41.37% 34.48% 65.52%
0.2 ml 20 µg 1.7 1.2 58.62% 41.37% 58.63%
0.4 ml 40 µg 2.2 1.6 75.86% 55.17% 44.83
0.8 ml 80 µg 2.9 2.3 100% 79.31% 20.69%
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.
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.
Dose 1

for for
extract
0.1 ml 10 µg 2.6 2 45.61% 35.08% 64.92
0.2 ml 20 µg 3.2 2.2 56.14% 38.59% 61.41%
0.4 ml 40 µg 4.3 2.9 75.43% 50.87% 49.13%
0.8 ml 80 µg 5.7 2.9 100% 50.87% 49.31%
Page 64
Dose 2

for for
extract
0.1 ml 10 µg 2.5 1.5 55.55% 33.33% 66.67%
0.2 ml 20 µg 2.7 2.1 60% 46.66% 53.34%
0.4 ml 40 µg 3.5 2.5 77.77% 55.55% 44.45%
0.8 ml 80 µg 4.5 2.9 100% 64.44% 35.56%
Dose 3

for for
extract
0.1 ml 10 µg 2.1 1.2 50% 28.57% 71.43%
0.2 ml 20 µg 2.6 1.7 61.90% 40.47% 59.53%
0.4 ml 40 µg 3.7 2.0 88.09% 47.61% 52.39%
0.8 ml 80 µg 4.2 2.7 100% 64.28% 35.72%
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 65
Summery and Conclusion
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,
Page 66
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.
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. Degcrease 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, 200mg/kg and 400 mg/kg dose of
extract of dried leaves of carica papaya show a good spasmolytic activity, broncodilation,
and mast cell degranulation inhibition.
Page 67
Further studies are coined to establish the molecular mechanism for the anti-asthmatic
activity.
Bibliography
Page 68
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Evaluation of Anti-Asthmatic Activity in Animal Model

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Evaluation of anti-asthmatic activity in animal model

Number of cases of asthma is increasing nowadays. It has become a common complaint

Asthma is characterized by the airway inflammation manifested by cough,

Some other diseases of respiratory system

Chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease - .

It is characterized by chronic and recurrent obstruction of airflow, which increases

include air pollution, pulmonary infection, genetic factors, occupational exposure to

It is chronic pulmonary disorder accompanied by recurrent episodes of wheezing,

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 cells

Release of PGs, histamine,

4. Drug induced asthma

5. Exercise induced asthma

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

6. Cough variant asthma

Symptoms in moderate asthma –

Attacks occur at least 2 times a week

Occasional emergency care may require

Symptoms in severe or adverse asthma-

Attacks occur daily

Limited physical activity

Symptoms of status asthmaticus

Emergency procedures are required

INFLAMMATORY CELLS INVOLVED

Fig. 1.2 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.

Fig. 1.4 – Involvement of various cytokines in asthma

Cell source Released mediators

 Dendritic cells Chemokines (CCL2, CCL3, CCL4, CCL17, CCL22, CXCL8)

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

Fig. 1.5- Events and cell involved in airway remodeling in asthma.

Events in Airway remodeling

1. Epithelial layer damage

Airway remodeling is basically a repair mechanism(3). It includes changes in epithelium,

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

membrane(4). Smooth muscle mass is also increased. Pathological analysis of bronchial

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

responsible for such thickening(8). Myofibroblast play an important role in subepithelial

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

Different steps to be taken are-

1. Identification and collection of the leaves of Carica papaya

3. Phytochemical evaluation will be performed for determining the constituents of

Introduction to the plant

Scientific classification of the plant

Domain : Flowering plant

Kingdom : Plantae- plants