TITLE PAGE
Original research article
Antiallergic and antianaphylactic activity of Sida rhombifolia Linn in
management of asthma
Anupama A. Suralkar*, Kuldeep K. Gaikwad, Asha S. Jadhav, Gayatri S. Vaidya.
Center for Innovation in Pharmaceutical Education, Research and Development (CIPERD),
Padm. Dr. D.Y. Patil, Institute of Pharmaceutical Sciences & Research, Pimpri, Pune-411018
Address for correspondence*
Anupama A. Suralkar*,
Center for Innovation in Pharmaceutical Education, Research and Development (CIPERD),
Padm. Dr.D.Y.Patil Institute of Pharmaceutical Sciences and Research,
Pimpri, Pune-411 018. Maharashtra, India.
E-mail ID:anupamaas@rediffmail.com
Mobile No: +91-8390766001
ABSTRACT
Traditionally, Sida rhombifolia (SR) Linn is use in treatment of allergic diseases such as a
bronchial asthma, bronchitis, wheezing, cough, short windedness, swelling, wounds, burns,
itch, sores, eczema and skin diseases. Leaves are used in all kinds of inflammations. Leaves
are reported to contain flavonoids which display several pharmacological properties in
treating the allergic inflammations. Hence, the present study was undertaken to investigate
the effect of ethanolic extract of Sida rhombifolia (SR) (Linn.) leaves on milk induced
leukocytosis and eosinophilia in mice, compound 48/80 induced mast cell degranulation in
rats and egg albumin induced passive paw anaphylaxis in rats. In milk induced leukocytosis
and eosinophilia SR showed significant decrease in difference in no. of leucocytes and
eosinophils count (p<0.01). In compound 48/80 induced mast cell degranulation in rats SR
showed significant decrease in no. of degranulated mast cells count and significant increase
in no. of intact mast cells count (p<0.01) and also showed dose dependent increase in percent
protection of mast cells. In egg albumin induced passive paw anaphylaxis in rats SR showed
significant reduction in the paw edema volume (p<0.01) and also showed dose dependent
increase in percent inhibition of paw edema. These results suggest that SR may prove to be
potential therapeutic drug for treating allergic diseases such as allergic asthma and effect may
be due to its adaptogenic, antistress, mast cell stabilizing, inhibition of AG: AB reaction and
anti-inflammatory activities.
Key words: Sida rhombifolia, eosinophilia, mast cells, allergic asthma.
INTRODUCTION
An allergy is a hypersensitivity disorder of the immune system. (1) Allergic reactions occur
when a person's immune system reacts to common, unusual or normally harmless substances
in the environment.
(2, 3)
A substance that causes a reaction is called an allergen. These
reactions are acquired, predictable and rapid. Allergy is one of four forms of hypersensitivity
and is formally called type I (or immediate) hypersensitivity. Allergic reactions are
distinctive because of excessive activation of certain white blood cells called mast cells and
basophils by a type of antibody called Immunoglobulin E (IgE). This reaction results in an
inflammatory response which can range from uncomfortable to dangerous. Mild allergies like
hay fever are very common in the human population and cause symptoms such as red eyes,
itchiness and runny nose, eczema, hives or an asthma attack. (2)
Allergies can play a major role in conditions such as asthma. In some people, severe allergies
to environmental or dietary allergens or to medication may result in life-threatening reactions
called anaphylaxis. Food allergies and reactions to the venom of stinging insects such as
wasps and bees are often associated with these severe reactions.
(2)
Allergic diseases are a
common and increasing cause of illness, affecting between 15% and 20% of the population at
some time. They comprise a range of disorders from mild to life-threatening and affect many
organs. Atopy is the tendency to produce an exaggerated IgE immune response to otherwise
harmless environmental substances and an allergic disease may be defined as the clinical
manifestation of this inappropriate IgE immune response. (4)
The number of individuals suffering with allergic illnesses is increasing in the industrialized
as well as in large cities of developing countries. Allergies also have reached high prevalence
and incidence in all over the world. Allergy and anaphylaxis are the most responsible factor
for diseases like asthma, rhinitis, bronchitis, cold, cough, pain; inflammation etc.
(3)
Allergic
asthma is the most common type of asthma. About 90% of kids with childhood asthma have
allergies, compared with about 50% of adults with asthma. Inhaling specific substances called
allergens (allergy triggers such as pollen, mites, or molds) brings on the asthma symptoms
associated with allergic asthma. Allergic asthma, airways are hypersensitive to the allergens.
Once these allergens get into airways, immune system overreacts. The muscles around your
airways tighten (an effect called bronchospasm).The airways themselves become inflamed
and flooded with thick mucus. (5)
Asthma is a chronic inflammatory lung disease that is characterized by airway edema and
increased mucus secretion. A propensity for allergic responses, atopy, is associated with the
development of asthma. (6)
Allergic asthma, which affects an estimated 100 million individuals worldwide is caused by
chronic airway inflammation associated with IgE- synthesis and subsequent Th2 (T-helper
type-2 cell)-responses. The pathophysiological hallmark of asthma is the infiltration of
inflammatory cells, including eosinophils, neutrophils, lymphocytes and macrophages. These
cells release various inflammatory mediators, including histamine and cytokines.(7)
According to World Health Organization (WHO) statistics it is estimated that 300 million
have asthma, markedly affecting the quality of life of these individuals and their families and
negatively impacting the socio-economic welfare of society and 250,000 avoidable asthma
deaths occur in the world each year. (8)
Modern approaches includes mast cell stabilizers, leukotriene receptor antagonists, histamine
receptor blockers, β2 receptor agonists, methylxanthines and corticosteroids but clinical
evaluations of these drugs have shown incidences of relapse, side effects and drug
interactions. For examples, β2 receptor agonists like salbutamol may cause muscle tremors,
palpitation, restlessness, nervousness and throat irritation while leukotriene receptor
antagonists like montelukast and zafirlukast can cause headache and gastrointestinal
disturbances. (9, 10)
These therapeutic limitations have provided incentive anti-asthmatic drugs. Due to those side
effects, there is a need to find new anti-asthmatic compound(s) with potentially less or no
side effects. Herbal medicines are being increasingly utilized to treat a wide variety of
diseases, though the knowledge about their mode of action is relatively scanty. So there is a
growing interest regarding the pharmacological evaluation of various plants used in
traditional system of medicine.
(3)
The use of traditional medicine is expanding to newer
horizons and plants still remain as the novel source of structurally important compounds that
lead to the development of innovative drugs. Naturally occurring compounds from plants are
still used in pharmaceutical preparations in pure or extracted forms. (11)
Plants extracts, however are some of the attractive sources of new drug and have shown to
produce promising results in treatment of allergic inflammatory diseases such as asthma.
13)
(12,
It is a short-lived perennial subshrub (woody stem and herbaceous branches) commonly
growing to 60 cm, but sometimes reaching 1.5 min height. The alternate leaves are variable in
both shape and size and grows today in over 70 countries throughout the tropical, subtropical
and warm temperate regions.
(14)
It is commonly known as Huang hua mu [China], Arrow
leaf sida [English], Bala, Mahabala [India], Chikana, Sadeva [Marathi], Bariara, Swetbarela
[Hindi], Baladana [Gujarati] and Sittamutti [Madras].(15, 16, 17)
Leaves contains chemical constituents such as ascorbic-acid, beta-carotene, betaphenethylamine, calcium, carbohydrates, fat, fiber, gums, riboflavin, zinc, iron, niacin,
thiamin
and hipaphorine while phytochemical constituents such as flavonoids (& their
glycosides),alkaloids (pseudoephedrine, vascin, vasicine.), phenolic compounds, saponins,
steroids (& their glycosides), tannins, triterpenoids (& their glycosides).
(13, 15)
Roots contain
alkaloids, choline, cobalt, copper, cryptolepine, and ephedrine. Stems contain magnesium,
mucilage.
(15)
ecdysterone.
Fruits contain tannins, phenolics, alkaloids, flavonoids while seeds contain
(18, 19)
Plant is reported to possess activities such as antinociceptive and anti-
inflammatory activity,
(13)
In-Vitro antioxidant,
and hepatoprotective activity,
activity,
(24)
antigout activity,
(22)
(20)
(21)
analgesic activity,
Free radical scavanging activity,
(25)
antidirrhoeal activity,
(26)
anti-inflammatory
(23)
antiproliferative
antiarthritic activity,
(27)
nephroprotective activity (28) and hypoglycemic and hypolipidemic activity (29) etc.
Flavonoids display several pharmacological properties in treating the allergic inflammations,
acting as anti-inflammatory and antioxidant agents.
(30)
Saponins,
(31)
steroids,
(7)
tannins
(32)
and their related compounds are reported to possess anti-inflammatory activity.
Traditionally, Sida rhombifolia Linn ssp. rhombifolia commonly known as a “Mahabala” is
use in treatment of allergic diseases such as a bronchial asthma, bronchitis, wheezing, cough,
short windedness, swelling, wounds, burns, itch, sores, eczema, skin diseases, dermatosis,
tonsilitis and respiratory inflammations.
(15)
Traditionally leaves are used in all kinds of
inflammations. It removes “Tridosha”. (16)
Hence, taking into consideration the traditional claims, phytochemical constituents and
reported activities, the present study was planned to evaluate the antiallergic and
antianaphylactic activity of Sida rhombifolia Linn in management of asthma.
MATERIALS AND METHODS
Experimental animals
Swiss albino mice weighing 20-25 gm and Wistar albino rats weighing 150-250g were
housed in standard cages at room temperature 25 ± 2 °C and 50±5% relative humidity, under
a light/dark cycle of 12/12 h, for 1 week before the experiments. Animals were provided with
standard rodent pellet diet (Amrut laboratory animal feed, Sangli, Maharashtra, India) and
water ad libitum. Laboratory animal handling and experimental procedures were performed
in accordance with the guidelines of CPCSEA and experimental protocol was approved by
Institutional Animal Ethics Committee. (CPCSEA Approval No: 198/99).
Selection, procurement of plant material and preparation of extract
The leaves of Sida rhombifolia (SR) Linn (Malvaceae) were selected for study.
(33, 34)
Fresh
leaves of SR were collected from local areas of Tiruvanantpuram, Kerala, India. The
specimen was authenticated at Botanical Survey of India (BSI), Pune. The extractions of
leaves of SR were carried by soxhlation method using soxhlate’s apparatus.
(20)
In this
method 1000 gm of leaf powder was extracted with 95% ethanol. It was then filtered and
concentrated to obtain the ethanolic extract of SR. The % yield obtained from leaves was 25
gm. (25% w/w). Extract was subjected to qualitative chemical tests for the identification of
various phytoconstituents. (35)
Experimental design
Acute oral toxicity study and selection of doses
Dose was selected by using acute oral toxicity study.
(36)
The toxicity study for ethanolic
extract of leaves of was performed using rats. The animals were fasted overnight prior to the
experiment and maintained under standard conditions. To find the LD50 of ethanolic extract
of leaves of SR, six groups of rats, containing six in each group, were given SR in the doses
of 500, 1000and 2000 mg/kg orally. The animals were observed for 5 min every 30 min till 2
h and then at 4, 8 and 24 h after treatment for any behavioral changes/mortality. They were
further observed daily for 7 days for mortality. No mortality up to 7 days after treatment was
observed with the ethanolic extract of leaves of SR and therefore was found safe up to dose
of 2000 mg/kg. Accordingly 100,200 and 400 mg/kg p.o. doses were selected for rats and
mice.
Effect of ethanolic extract of Sida rhombifolia (EtOH-SR) leaves on milk induced
leukocytosis and eosinophilia in mice: (3, 31)
Procedure:
Mice were divided into five groups (n=5). Animals belonging to group I served as positive
control and was administered with only boiled and cooled milk (4 ml/kg, s.c.).Animals
belonging to group II served as standard and were administered with Dexamethasone (50
mg/kg i.p.) while animals belonging to group III to V served as test group and were received
respective doses of ethanolic extract of Sida rhombifolia and 1 hr later boiled and cooled milk
(4 ml/kg, s.c.) was administered to the same animals. After 24 hr, blood samples were
collected from all animals from their retro orbital plexus, under light ether anesthesia. Total
leukocytes and eosinophils counts were recorded in each group 24 hr after milk injection and
difference in no. of leucocytes and eosinophils count was calculated.
Effect of ethanolic extract of Sida rhombifolia (EtOH-SR) leaves on compound 48/80induced mast cell degranulation in rats: (37)
Procedure:
Rats were divided into five groups (n=5). On the 1st day of sensitization, all the animals from
each group were injected with Compound 48/80 (1mg/kg, s.c.). Animals belonging to group
II served as standard and were administered with Ketotifen fumarate (1mg/kg, p.o.). While
animals belonging to group III to group V served as test group and were administered with
respective doses of ethanolic extract of Sida rhombifolia for 15 days. On day 15th, 2 hour
after the assigned treatment, mast cells were collected from the peritoneal cavity. Ten ml of
normal saline solution was injected into peritoneal cavity and abdomen was gently massaged
for 90 second. The peritoneal cavity was carefully opened and the fluid containing mast cells
were aspirated and collected in siliconised test tube containing 7 to 10 ml of RPMI-1640
Medium (pH 7.2- 7.4).The mast cells were then washed thrice by centrifugation at low speed
(400-500 rpm) and the pallets of mast cells were taken in the RPMI-1640 medium. The mast
cell suspension (approximately 1 x 10 6 cells/ml) was challenged with 5 μg/ml of compound
48/80 solution and stained with 0.1 % toluidine blue and observed under high power
microscope (45 X).Total 100 cells were counted from different visual areas. The numbers of
intact and degranulated cells was counted and the percent protection was calculated using the
formula,
% Protection = [1-(T / C)] x 100
Where,T- No. of degranulated cells of test, C-No. of degranulated cells of control.
Effect of ethanolic extract of Sida rhombifolia (EtOH-SR) leaves on egg albumin
induced passive paw anaphylaxis in rats: (3)
Procedure:
Wistar rats were divided into five groups (n=5). Antiserum to egg albumin was raised in rats
by using aluminum hydroxide gel as an adjuvant. On 1st, 3rd and 5th day, animals were
given three doses of 250 μg of egg albumin (s.c.) adsorbed on 12 mg of aluminum hydroxide
gel prepared in 0.5 ml of saline. On 10th day of sensitization, the blood of each animal was
collected from the retro orbital plexus under light ether anesthesia. The collected blood was
allowed to clot and serum is separated by centrifugation at 1500 rpm. The animals were
passively sensitized with 0.1 ml of the undiluted serum into the left hind paw. The right hind
paw received an equal volume of saline. Animals belonging to group I served as control and
was administered with only distilled water (10 ml/kg, p.o.). Animals belonging to group II
served as standard and were administered by Dexamethasone (0.5 mg/kg, i.p.); whereas
animals belonging to group III to group V served as test group and were administered with
respective doses of ethanolic extract of Sida rhombifolia 24 hr after sensitization. One hr after
drug administration, animals were challenged by giving 10 μg of egg albumin in 0.1 ml of
saline in the left hind paw and the paw inflammation was measured by using a
Plethysmometer (UGO Basile, 7140).The difference in the reading prior to and after antigen
challenge represents the edema volume and the percent inhibition of edema was calculated by
using the formula,
% Inhibition = [1-(T / C)] x 100
T - Mean relative change in paw volume in test group,C- Mean relative change in paw
volume in control group.
Statistical analysis
The results were expressed as mean ± SEM from 5 animals. Statistical analysis done by using
one way ANOVA followed by Dunnett’s test.
(12)
p<0.05*, p<0.01** were considered
significant.
RESULTS AND DISCUSSION
Effect of ethanolic extract of Sida rhombifolia (EtOH-SR) leaves on milk induced
leukocytosis and eosinophilia in mice:
24 hr after administration of milk (4 ml/kg, s.c.), there was significant increase in eosinophil
and leukocyte count in positive control group. There was significant (p<0.01) inhibition of
milk-induced eosinophilia and leucocytosis, in group of animals treated with dexamethasone
50mg/kg, i.p. The animals treated with ethanolic extract of leaves of Sida rhombifolia at dose
of 100mg/kg, 200mg/kg and 400mg/kg, p.o. showed significant (p<0.01) inhibition of milkinduced leucocytosis respectively. The animals treated with ethanolic extract of leaves of
Sida rhombifolia at dose of 100mg/kg, p.o. showed significant (p<0.05) inhibition of milkinduced eosinophilia and at dose of 200mg/kg and 400mg/kg, p.o. showed significant
(p<0.01) inhibition of milk-induced eosinophilia respectively.
Effect of ethanolic extract of Sida rhombifolia (EtOH-SR) leaves on compound 48/80induced mast cell degranulation in rats:
Compound 48/80 induced mast cell degranulation was significantly (p<0.01) inhibited by
animals pretreated with Ketotifen fumarate (1 mg/kg, p.o) and percent protection was found
to be 69.20 %. In the groups pretreated with ethanolic extract of Sida rhombifolia at dose of
200 and 400 mg/kg, p.o. there was significant (p< 0.01) increase in no. of intact cells and
decrease in no. of degranulated mast cells. In the groups pretreated with ethanolic extract of
Sida rhombifolia at dose of 100 mg/kg, p.o., there was significant (p< 0.05) increase in no. of
intact cells and decrease in no. of degranulated mast cells and at dose of 100, 200 and 400
mg/kg, p.o. percentage protection was found to be 3.43%, 39.83% and 58.93 % respectively.
Effect of ethanolic extract of Sida rhombifolia (EtOH-SR) leaves on egg albumin
induced passive paw anaphylaxis in rats:
Antiserum to egg albumin was injected 24 hr before administration of ethanolic extract of
leaves of Sida rhombifolia and dexamethasone. In the vehicle or distilled water treated group,
egg albumin increased the paw volume in the sensitized animals, which was measurable up to
the time period of 4 hrs. Dexamethasone (0.5 mg/kg, i.p.) significantly reduced (p<0.01) the
paw volume at 0.5th, 1st, 2nd, 3rd and 4th hrs time intervals and the percent inhibition was
found to be 44.09 %, 53.39 %, 61.37 %, 65.65 % and 74.68 % respectively. Treatment with
ethanolic extract of Sida rhombifolia at the dose of 100 mg/kg, p.o. significantly reduced
(p<0.01) the paw volume at 2nd, 3rd and 4th hrs time intervals and the percent inhibition was
found to be 22.07 %, 40.66 % and 52.13 % respectively. Treatment with ethanolic extract of
Sida rhombifolia at the dose of 200 mg/kg, p.o. significantly reduced the paw volume
(p<0.05) at 0.5th hr and (p<0.01) at 1st, 2nd, 3rd and 4th hrs time intervals and the percent
inhibition was found to be 04.41%,05.26%, 38.58 %, 44.98 % and 54.87% respectively.
Treatment with ethanolic extract of Sida rhombifolia at the dose of 400 mg/kg, p.o.
significantly reduced (p<0.01) the paw volume at 0.5th, 1st, 2nd, 3rd and 4th hrs time
intervals and the percent inhibition was found to be 07.64 %, 19.55%, 44.19%, 48.98% and
60.67 % respectively.
The term adaptogen (or resistogen) was coined by N. Lazarev in the Soviet Union to classify
plants and other substances that augment non-specific resistance of the body, protecting it
from stressful factors. Adaptogens have recently been defined as natural metabolic regulators
which increase the ability of the organism to adapt to environmental factors and to avoid
damage from such factors. Stress is a common problem and a major health hazard of life. A
large proportion of all illnesses (perhaps 70-80%) are believed to occur because of stress. (38)
Cow’s milk allergy (CMA) is one of the most common food allergies in childhood and
affects 2% to 2.5% of infants. Cow’s milk contains several allergenic proteins. The most
important allergens are 4 caseins (as1, as2, b, and j) and 2 whey proteins (a-lactalbumin and
b-lactoglobulin).
(39)
It was reported that subcutaneous administration of milk produces a
marked increase in the leukocytes and eosinophils count after 24 h. Leucocytes during
asthmatic inflammation release the inflammatory mediators like cytokines, histamine, and
major basic protein, which promote the ongoing inflammation. An abnormal increase in
peripheral eosinophil to more than 4% of total leucocytes count is termed as eosinophilia. In
asthmatic patient there is increase in eosinophil count. (31)
After administration of milk there is increase in total leucocyte count, and this stressful
condition can be normalized by administration of an antistress or adaptogenic drug.
Furthermore leukocytes recruited during allergic inflammation release the inflammatory
mediators like cytokines, histamine, and major basic protein and promote the ongoing
inflammation.
(3)
In the late phase, especially in the development of allergic asthma,
eosinophils play role as an inflammatory cell. Eosinophilia is associated with respiratory
disorder, often allergic in nature together with pulmonary infiltrates that are detectable on
chest films. Immunomodulating agents are useful in the treatment of allergy by virtue of
inhibiting the antigen-antibody (AG: AB) reaction thereby inhibiting release of inflammatory
mediators. (3)
Eosinophil also have special propensity to collect in tissues in which allergic reactions occur,
such as in the peribronchial tissues of the lungs in people with asthma and in the skin after
allergic skin reactions. This is caused at least partly by the fact that many mast cells and
basophils participate in allergic reactions. The mast cells and basophils release an eosinophil
chemotactic factor that causes eosinophils to migrate toward the inflamed allergic tissue. The
eosinophils are believed to detoxify some of the inflammation-inducing substances released
by the mast cells and basophils and probably also to phagocytize and destroy allergenantibody complexes, thus preventing excess spread of the local inflammatory process.
(1)
A
blood eosinophilia is hallmark of both allergic and non allergic asthma. Eosinophils are
recruited and found to be activated during segmental allergen challenge. In the late phase,
especially in the development of allergic asthma, eosinophils secretes mediators such as
eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDNT), granulocyte
macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), and
Prostaglandin (PG), which results in epithelial shedding, bronchoconstriction and promotion
of inflammation in respiratory tract. (40)
In the present study, control group treated with milk showed significant increase in leukocyte
count, where as the group treated with Dexamethasone does not showed any remarkable
increase in leukocyte count while the group treated with Sida rhombifolia extract also does
not showed increase in leucocyte count but the decrease in leucocyte count was found to be
less effective as compared to Dexamethasone. This indicates that Sida rhombifolia extract
may have adaptogenic activity.
In the present study, control group treated with milk showed significant increase in eosinophil
count, where as the group treated with Dexamethasone does not showed any remarkable
increase in eosinophil count while the group treated with Sida rhombifolia extract also does
not showed increase in eosinophil count but the decrease in eosinophil count was found to be
less effective as compared to Dexamethasone. This indicates that Sida rhombifolia extract
may have adaptogenic activity.
Thus, the present study shows that Sida rhombifolia extract prevent the release of
inflammatory mediators by decreasing the leucocytic and eosinophilic count and which
further may contribute in the management of allergic asthma.
Compound 48/80 is a polymeric base having histamine releasing activity. Histamine release
by the amine-exchange mechanism would require access of the releaser to intracellular
binding sites of histamine. Alkalinity should enhance this process; it would favour the
passage of the compound across lipoid barriers on the mast cell membrane. Approximately
4.5 times more histamine was released at pH 8.2 than at 7.4. (41)
The pathological mechanisms involved in Type-I allergy has been explained as the
degranulation of mast cells, followed by the release of mediators such as histamine,
leukotrienes and prostaglandins from these cells . The degranulation of mast cells occurs in
response to the immunological stimuli in which the antigen– antibody reaction on the cell
surface predominates. The mast cell stabilizing effect can be attributed to a great extent to the
presence of flavonoids, as many of these compounds have been reported to possess this
activity.
(37)
Stimulation of mast cells with compound 48/80 or antiserum initiates the
activation of signal transduction pathway, which leads to histamine release. Some recent
studies showed that compound 48/80 and other polybasic compounds are able to activate G
proteins. The compound 48/80 increased the permeability of the lipid bilayer membrane by
causing a perturbation of the membrane. These results indicated that the membrane
permeability increase may be an essential trigger for the release of the mediators from the
mast cells. (42) It has been reported that agents that induce the elevation of intracellular cAMP
levels can attenuate the stimulated release of mediators from mast cells. (43)
Mast cells are the primary effector cells involved in an allergic or immediate hypersensitivity
response. Activation of mast cells occurs in response to a challenge by a specific antigen
against which the surface immunoglobulin E (IgE) is directed, or by other IgE-directed
ligands. Activated mast cells can produce histamine, as well as a wide variety of other
inflammatory mediators such as eicosanoids, proteoglycans, proteases and several proinflammatory and chemotactic cytokines such as tumor necrosis factor-α, interleukin (IL) - 6,
IL-4, IL-8, and IL-13. Among them, histamine remains the best-characterized and most
potent vasoactive mediator implicated in the acute phase of immediate hypersensitivity.
Various acute and chronic allergic responses are caused by these mediators.
(43)
Mast cell
degranulation can also be elicited by the basic secretagogues. The most potent secretagogues
include the synthetic compound 48/80 and polymers of basic amino acids. Compared with the
natural process, a high concentration of compound 48/80 induces an almost 90% release of
histamine from mast cells. Thus, an appropriate amount of compound 48/80 has been used as
a direct and convenient reagent to investigate the mechanisms of allergy and anaphylaxis.
The murine mast cell is a good experimental model for the study of compound 48/80-induced
histamine release. It is well-recognized that compound 48/80 can induce a mast celldependent, non-specific anaphylactoid reaction. The mechanism of anaphylactoid response
triggered by compound 48/80 is considered to be due to the massive release of vasoactive
amines, such as histamine, from mast cells and basophils. As noted, histamine is a typical
mediator that causes various pathophysiologic events in acute allergic reactions. (43)
In the present study, control group treated with Compound 48/80 showed more mast cell
degranulation where as group treated with Ketotifen fumarate significantly reduced the mast
cell degranulation while group treated with Sida rhombifolia extract also significantly
reduced the mast cell degranulation but reduction was found to be less as compared to
Ketotifen fumarate and the effect may be due to its mast cell stabilizing activity and thereby
reduction in release of histamine and other vasoactive amines and further array of
inflammatory cascade and which further may contribute in the management of allergic
asthma.
Egg allergy is one of the most common food allergies, affecting 1% to 2% of young children.
It is overall second only to milk allergy in prevalence and in most studies has been shown to
be the most common food allergy in children with atopic dermatitis. In addition, early
sensitization to egg is a marker of later sensitization to aeroallergens and the development of
asthma.
(44)
There is much resemblance in clinical features between inflammation and
immune reaction in the tissue. In both condition, there is local vasodilatation, increased
capillary permeability and formation of oedema due to local release of various vasoactive
amines like histamine, bradykinin, SRS-A and prostaglandins. One of the most important
approaches used in the examination of the immune pathological mechanisms of anaphylactic
and inflammatory disorder is to elicit the formation of paw oedema, injecting various
substances into the sub plantar injection of a number of substances in the hind paw of rats. (45)
Basophils, mast cells and their preformed de novo synthesized mediators, play a pivotal role
in the pathogenesis of allergic disorders. These molecules are potent vasoactive and
bronchoconstrictor agents and they modulate local immune responses and inflammatory cell
infiltration. Immunoglobulin E (IgE) mediated mast cell stimulation is an important initial
event in the development of type I allergic reactions like asthma and atopic disorders.
Clinical studies have found a close association between asthma and serum IgE levels as well
as IgE dependent skin test reactivity to allergens. Antigen challenge, in sensitized animals,
results in degranulation of mast cells, which is an important feature of anaphylaxis. The
antigen and antibody (AG: AB) reaction taking place on the surface of mast cells leading to
release of mediators. Degranulated mast cells release a number of mediators like leukotrienes,
platelet activating factor, eosinophilic chemotactic factor and eosinophil-derived neurotoxin.
The prevention of degranulation process by the extract indicates a possible stabilizing effect
on the biomembrane of mast cells. (46)
Anaphylactic allergic reaction is a life-threatening syndrome induced by the sudden systemic
release of inflammatory mediators such as histamine and proinflammatory cytokines and can
be elicited by various stimulators including compound 48/80 and IgE. IgE is prominently
related to atopic diseases such as allergy. (47)
In the present study, control group treated with egg albumin showed increase in paw edema
while group treated with Dexamethasone significantly inhibited the paw edema while group
treated with Sida rhombifolia extract also significantly inhibited the paw edema but inhibition
was found to be less as compared to Dexamethasone and the effect may be due to its
antiallergic (inhibition of AG: AB reaction) and mast cell stabilizing activity and which
further may contribute in the management of allergic asthma..
CONCLUSION
Thus, the results obtained in the present investigations indicates that ethanolic extract of
leaves of SR may prove to be useful in preventing allergic conditions and diseases such as a
asthma owing to its ability to decrease the increased eosinophilic, leucocytic count,
prevention of mast cell degranulation and inhibition of paw edema volume and the effect
may be due to adaptogenic, antistress, mast cell stabilizing, inhibition of antigen: antibody
(AG:AB) reaction and
anti-inflammatory
activity of
the extract and the presence of
phytochemical constituents such as flavonoids, alkaloids, saponins, tannins and steroids.
Thus, our studies established a significant antiallergic effect of EtOH-SR. However, further
studies are required to establish its exact mode of action and the active principles involved in
its antiallergic and antianaphylactic effect.
ACKNOWLEDGEMENT
Authors are grateful to Dr. S. S. Chitlange, Principal, Padm. Dr. D. Y. Patil, Institute of
Pharmaceutical Sciences and Research, Pimpri, Pune-411018, Maharashtra, India, for
providing laboratory facilities.
CONFLICT-OF-INTEREST
The authors declare that they have no conflict of interest.
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Table 1: Effect of ethanolic extract of Sida rhombifolia (EtOH-SR) leaves on milk
induced leukocytosis and eosinophilia in mice:
Groups
( n = 5)
I (Control)
II (Std)
III (SR 100)
IV (SR 200)
V (SR 400)
Difference in number of
leukocyte count (per cu mm)
4508.6± 48.590
837.6±24.266**
3793.6±61.845**
2287.2±28.909**
978.2±23.865**
Difference in number of
eosinophil count (per cu mm)
120.2±4.587
54.8±4.317**
104.4±2.731*
80±3.162**
70.4±4.434**
*p<0.05, **p< 0.01 when group II, III, IV and V were compared with group I.
Group-I (Positive control) = Milk 4ml/kg (s.c.).
Group- II (Std) = Dexamethasone (50mg/kg, i.p.) + Milk 4ml/kg, (s.c.).
Group-III (SR 100) = Ethanolic extract of leaves of Sida rhombifolia (100mg/kg p.o.). + Milk
4ml/kg, (s.c.).
Group- IV (SR 200) = Ethanolic extract of leaves of Sida rhombifolia (200mg/kg, p.o.) +
Milk 4ml/kg, (s.c.).
Group- V (SR 400) = Ethanolic extract of leaves of Sida rhombifolia (400mg/kg,p.o.) + Milk
4ml/kg, (s.c.).
Table 2: Effect of ethanolic extract of Sida rhombifolia (EtOH-SR) leaves on compound
48/80- induced mast cell degranulation in rats:
Groups
( n = 5)
I (Control)
II (Std)
III (SR 100)
IV (SR 200)
V (SR 400)
Mast cells (Mean ± SEM)
Intact
Degranulated
18.2±0.5831
81.8±0.5831
74.8±0.6633**
25.2±0.6633**
21±0.8944*
79±0.8944*
50.8±0.3742**
49.2±0.3742**
66.4±0.5099**
33.6±0.5099**
Percent
protection (%)
69.20
3.43
39.83
58.93
*p<0.05, **p< 0.01when group II, III, IV and V were compared with group I.
Group-I (Control) = Compound 48/80 (1mg/kg, s.c.)
Group- II (Std) = Ketotifen fumarate (1mg/kg, p.o.) + Compound 48/80 (1mg/kg, s.c.).
Group-III (SR 100) = Ethanolic extract of leaves of Sida rhombifolia (100 mg/kg p.o.). +
Compound 48/80 (1mg/kg, s.c.).
Group- IV (SR 200) = Ethanolic extract of leaves of Sida rhombifolia (200 mg/kg, p.o.) +
Compound 48/80 (1mg/kg, s.c.).
Group- V (SR 400) = Ethanolic extract of leaves of Sida rhombifolia (400 mg/kg, p.o.) +
Compound 48/80 (1mg/kg, s.c.).
Table 3: Effect of ethanolic extract of Sida rhombifolia (EtOH-SR) leaves on egg
albumin induced passive paw anaphylaxis in rats:
Groups
(n=5)
I (Control)
II (Std)
III (SR 100)
IV (SR 200)
V (SR 400)
0.5 hr
0.49±
0.005
0.27±
0.005**
0.48±
0.003
0.47±
0.005*
0.45±
0.005**
Groups
(n=5)
II (Std)
III (SR100)
IV (SR 200)
V (SR 400)
0.5 hr
44.09
2.40
4.41
7.64
Paw edema volume (ml)
(Mean ± SEM)
1 hr
2 hr
3 hr
0.53±
0.57±
0.6±
0.003
0.004
0.007
0.24±
0.22±
0.20±
0.003**
0.004**
0.005**
0.51±
0.44±
0.35±
0.005
0.005**
0.007**
0.50±
0.35±
0.33±
0.005**
0.004**
0.007**
0.42±
0.31±
0.30±
0.003**
0.003**
0.005**
Percent inhibition of
paw edema(%)
1 hr
2 hr
3 hr
53.39
61.37
65.65
3.01
22.07
40.66
5.26
38.58
44.98
19.55
44.19
48.98
4hr
0.65±
0.005
0.16±
0.005**
0.31±
0.005**
0.29±
0.002**
0.25±
0.003**
4hr
74.68
52.13
54.87
60.67
*p<0.05, **p < 0.01 when group II, III, IV, V compared with group I.
Group –I (Control) = Phosphate buffer saline (1 ml/kg p.o.) + Egg albumin (100 μg, s.c.)
Group-II (Std) = Ketotifen fumarate (1 mg/kg, p.o.) + Egg albumin (100 μg, s.c.)
Group-III (SR 100) = Ethanolic extract of leaves of Sida rhombifolia (100 mg/kg, p.o.
respectively) + Egg albumin (100 μg, s.c.)
Group-IV (SR 200) = Ethanolic extract of leaves of Sida rhombifolia (200 mg/kg, p.o.
respectively) + Egg albumin (100 μg, s.c.)
Group-V (SR 400) = Ethanolic extract of leaves of Sida rhombifolia (400 mg/kg, p.o.
respectively) + Egg albumin (100 μg, s.c.)