PATHOPHYSIOLOGY OF ASTHMA: Pathologically, asthma is characterized by the accumulation of eosinophils and CD₄+ lymphocytes in the submucosa, mucous gland hyperplasia, thickening of subepithelial collagen layer, sub mucosal matrix deposition, mast cell degranulation and hypertrophy and hyperplasia of the airway smooth muscle. The pathophysiology of asthma involves the development of acute and chronic inflammation in airway narrowing by producing increased vascular permeability, edema and airway smooth muscle contraction. The histopathologic characteristic of asthma include shedding of airway epithelium, sub-basement membrane fibrosis, hypertrophy of air way smooth muscle, excessive secretion of mucus and a multicellular inflammation involving activated mast cells, eosinophils, neutrophils, macrophages, basophils, and lymphocytes.

It is observed those patients that have died due to asthma showed considerable increase in thickness of the airway wall throughout the bronchial tree, partly as a result smooth muscle hypertrophy(Wardlaw et al., 2002)¹.

The diagnosis of asthma involves a detailed history, Physical examination, Chest radiography, Full blood count (Oliver et al., 2003)², Skin test (Oliver et al., 2003)², Spirometry, IgE specific test, Psychiatric evaluation (Ryland, 2000)³.

Different types of drugs involved in the treatment are- Bronchodilator, Mast cell stabilizers, Anti-inflammatory, Antihistaminic.

According to Ayurveda Tamarindus indica linn., is used in asthma, bronchitis, leprosy, tuberculosis, wounds and ulcers. Leaves are used to treat asthma and liver complaints in Dominican Republic.

MATERIALS AND METHODS:

Drugs used: Histamine diphosphate, Clonidine, Dexamethasone, Chlorpheniramine maleate, Egg albumin, Sodium cromoglycate, RPMI buffer medium 1640.

Instruments: Histamine chamber, Plethysmometer, Cooling centrifuge, Vacuum distillation unit.

Experimental animals: Male albino rats of Wistar strain(120-180 gm), albino Swiss mice(20-25 gm), Dunkin-Hartley guinea pigs(350-400 gm).

Extraction process: After 10 days of drying under shed, the seeds were powdered using a mixer. The powder was sieved by 40 sieve. The methanolic extract was prepared by maceration method. 500 gm of dried powder was extracted with 3500 ml methanol (1:7) for 72 hours. The extract was concentrated and dried(yield- 8%w/w).

Toxicity study and dose selection: Acute toxicity study for the methanolic extract was carried out on mice according to OECD guidelines (2004). Three mice were fasted over night and maintained with water ad libitam. Each animal received single dose of methanolic extract of Tamarindus indica Linn., (5000 mg/kg). After administration of the test compound animals were observed individually and continuously for 30min, 2hours, and 24 hours to detect changes in the autonomic or behavioral response and also for the tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma were observed during 14 days. Therefore 1/10^th of the maximum tolerable doses i.e.,- 250, 500, and 1000mg/kg were taken for the further studies.

Preliminary pharmacognostical testing:

S. No	Test	Observation	Inference
1	Dragendroff’s test	Orange brown precipitate	Alkaloid present
2	Foam test	Persistent foam	Saponin present
3	Extract+ HCL + magnesium turnings	Slight pink colour	Flavonoid present
4	Extract +5% FeCl₃	Slight yellow colour	Tannin Present
5	Brontragger’s test	Rose pink to red	Glycoside present
6	Keller Killani test	Reddish brown color	Glycoside present
7	Grignard reagent	Brick red or maroon	Glycoside present

Preparation of drug solution: The extract was suspended in physiological salt solution/5% gum acacia solution. Chlorpheniramine maleate was dissolved in distilled water. Clonidine, Histidine, Sodium cromoglycate and Dexamethasone were dissolved in physiological saline.

Fresh drug solutions were prepared for each days’ work. The solutions were kept in air tight chamber bottles and stored at room temperature till use.

Volume of drug solution was calculated based on the body weight of the animal

SCREENING MODELS:

Effect of test extracts on histamine and acetylcholine induced contraction by isolated goat tracheal chain preparation:

This method is used for the study of action of antispasmodic drugs on the tracheal musculature. Although, the method is known for its stability in the study of antispasmodic drugs in general, emphasis is given on its use in the testing of bronchodilators. This is because of the close anatomical and physiological association, which exists between tracheal and bronchial musculature (Castillo and De-beers, 1947)⁴. In isolated goat tracheal preparation there is preponderance of H₁excitatory and a scanty population of H₂ inhibitory receptors (Kulshrestha et al, 1983)⁵, (Nagchoudhari and Lahari, 1974)⁶. They also found that both goat tracheal preparation and strip preparation were suitable for screening spasmogenic activity on respiratory smooth muscle and goat tracheal chain is easier to handle and prepare and is also much sensitive than guinea pig tracheal chain.

(Ghosh, 1984)⁷, Isolated adult goat tracheal tissue was obtained immediately after slaughter of the animal. Trachea was then cut into individual rings tied together in series to form a chain. It was suspended in bath containing kreb’s solution of following composition {conc. In gm/litre: Nacl-6.9, KCl-0.35, CaCl₂-0.28, MgSO₄-0.28, NaHCO₃-2.1, KH₂PO₄-0.16, Glucose-1.0}. It was continuously aerated maintained at 37±^oc. One end of the tracheal chain was attached to an S-shaped aerator tube and other attached to isotonic frontal writing lever to smoked drum (magnification is 10-12 folds). The tissue was allowed to equilibrate, for 45min under a load of 400mg.

For Histamine- A dose response curve for histamine was taken in variant molar concentrations by maintaining 15min time cycle. After obtaining a dose response curve of histamine on trachea (n=6), the test drug was added to reservoir and the same doses of histamine were repeated. The test drug extract was dissolved itself in the physiological salt solution. Graph of percentage of maximum contractile response on ordinate and negative logarithm of molar concentration of histamine abscissa was plotted to record response curve of histamine in absence and in presence of test drug.

For Acetyl choline- A dose response curve for Ach was taken in variant molar concentrations by maintaining 15min time cycle. After obtaining a dose response curve of Ach on trachea (n=6), the test drug was added to reservoir and the same doses of Ach were repeated. The test drug extract was dissolved itself in the physiological salt solution. Graph of percentage of maximum contractile response on ordinate and negative logarithm of molar concentration of Ach abscissa was plotted to record response curve of Ach in absence and in presence of test drug.

BRONCHIAL HYPER ACTIVITY IN GUINEA PIG:

In this model histamine was applied as aerosol (0.2%), produced by an ultrasound nebulizer , causes severe bronchoconstriction in guinea pig that causes asphyxia and death. Bronchodilator can delay the occurrence of these symptoms. Time required for appearance of preconvulsive dyspnea (PCD) was recorded for each animal (Singh et al., 1990)⁸.

(Tripathi and Das, 1977)⁹, Fastened guinea pigs were randomly divided into 4 groups, containing 5 animals. Group-1 received Chlorpheniramine maleate (2mg/kg,p.o.). Group-II,III and IV received methanolic extract of Tamarindus indica Linn (217.5, 435 and 870 mg/kg p.o.) .Prior to drug treatment each animal was placed in the histamine chamber and exposed to 0.2%histamine aerosol. The time for preconvulsion dyspnea (the time for aerosol exposure to the onset of dyspnea leading to the appearance of convulsion) was noted. As soon as PCD commenced, animals were removed from the chamber and placed in fresh air to recover. This time for PCD was taken as basal value. Guinea pigs were allowed to recover from dyspnea for 4 hours.

After4hours, the animals of group II, III, IV were administered with the test drug extract and group–I received Chlorpheniramine maleate. These animals were again subjected to histamine aerosol later at interval of 1hr, 4hr, and 24 hr of drug administration and time for PCD was determined. The protection offered by treatment was calculated by using the following formula, (Mitra et al, 1999)¹⁰.

% Protection= (T2-T1)/T2× 100

Where, T1= the mean time for PCD before administration of test drug. T2= the mean time for PCD after administration of test drug at 1hr, 4hr, and 24hr.

CLONIDINE INDUCED CATALEPSY IN MICE:

Clonidine, an alpha2 –adrenoreceptor agonist, induces dose dependent catalepsy in mice, which is inhibited by histamine H1 receptor antagonist but not by H2 receptor antagonist (Jadhav et al, 1983)¹¹. Histamine acts as a modulator of presynaptic catecholamines processes in the CNS by causing depletion of the transmitter stores in the nerve terminals, (Muley et al., 1983)¹².

Schwartz (1987)¹³ identified histamine containing mast cells in brain. It has been suggested that the cataleptic effect of clonidine in the mouse be mediated by histamine (via H1 receptor), which is released from the brain mast cells in response to stimulation of α₂–adrenoreceptor by clonidine (BalsaraJ.J.1983)¹⁴.

(Ferre et al, 1990)¹⁵, Bar test was used to study the effect of the test drug extract on clonidine induced catalepsy. Mice were divided into 5 groups, 5 animals in each group. Animals belonging to Group I serve as control and were administered the distilled water (10ml/kg, p.o.). Animals belonging to Group II received standard drug Chlorpheniramine maleate (10ml/kg, i.p.). Animals belonging to Group III, IV, V received three doses i.e., 250, 500 and 1000mg/kg p.o., respectively of methanolic extract. All the groups received clonidine (1mg/kg s.c.,) 1 hr after the test drug administration and the duration of catalepsy was measured at 15, 30, 60, 90, 120, 150 and180 min. the fore paws of mice were placed on horizontal bar (1 cm in diameter, 3 cm above the table) and the time required to remove the paws from the bar was noted for each animal.

MAST CELL DEGRANULATION:

In the mast cell granules the histamine concentration has been calculated to be around 0.3m. (Uvnas, 1969)¹⁶. The clonidine act through the dynamic expulsion of granules without causing any damage to the cell wall. Sodium cromoglycate a standard mast cell stabilizer prevents degranulation of mast cells by raising the cyclic adenosine monophosphate (Geetha et al., 1981)¹⁷.

(Lakadwala et al., 1980)¹⁸, Rats were divided in five groups, five animals in each group.7 day drug treatment schedule was followed. Group I was administered distilled water (10ml/kg p.o.). Group II was administered Sodium cromoglycate (50mg/kg i.p.,). Group III, IV and V were administered methanolic extract 175, 350, and 700 mg/kg p.o. respectively. On 7^th day each animal was injected 10mg/kg, o.9% saline solution in the peritoneal cavity. After gently massage, peritoneal fluid was collected after 5 min. and transferred into siliconised test tubes containing 7-10ml of RPMI 1640 buffer medium(PH-7.2-7.4). This solution was centrifuged at 4000-5000 RPM. Pellet of mast cells was washed with 7-10ml of RPMI buffer medium twice by centrifugation, discarding supernatant. These cells were challenged with clonidine(50µg), incubated at 37˚c in a water bath for 10 min. Followed by staining with 1%toluidine blue and observed under microscope (45X). Total 100 cells were counted from different visual area. Percent protection against degranulation was calculated.

MILK INDUCED EOSINOPHILIA AND LEUCOCYTOSIS IN MICE:

Ayurveda provides a number of herbs for the treatment of asthma and herbal formulations used for the treatment of asthma include some antistress (nervine support ) herbs to enable adaptation to stress, since excessive stress or nervous debility may aggravate the symptoms of asthma. After parenteral administration of milk there is a increase in TLC, and this stressful condition can be normalized by administration of an antistress or adaptogenic drug (Brekhmen and Dardymov, 1969)¹⁹. Furthermore leukocytes recruited during asthmatic inflammation release the inflammatory mediators like cytokines, histamine, and major basic protein.

Eosinophilia is an abnormal increase in peripheral eosinophil count to more than 4% of total leucocytes (Brigden, 1999)²⁰. In the late phase, especially in the development of the allergic asthma, eosinophil play role as an inflammatory cell. Eosinophil secretes mediators such as Eosinophil cationic protein(ECP), Eosinophil derived neurotoxin (EDNT), granulocyte macrophage colony stimulating factor (GMCSF), tumour necrosis factor(TNF), and prostaglandins(PG), which results in epithelial shedding, bronchoconstriction and promotion of inflammation in respiratory tract (Brigden,1999)²⁰. Eosinophil is associated with respiratory disorder, often allergic in nature together with pulmonary infiltrates that are detectable on chest films (Ehright et al, 1989)²¹. It was also demonstrated that parenteral administration milk produces a marked and significant increase in the leucocytes/eosinophils count after 24 hour of its administration (Bhargava and Singh, 1981)²².

In this model milk in a dose of 4mg/kg was administered subcutaneously and eosinophil count was taken before and after administration of milk was calculated (Ghai, 1987)²³.

(Brekhman, 1969)¹⁹ Mice were divided into 5 groups with 5 animals in each group. Animals belonging to group-1 received distilled water (10ml/kg, p.o.). Animals belonging to group II, III, IV, V received boiled and cooled milk injection in dose of 4ml/kg, (s.c). Animals belonging to group III, IV, V received methanolic extract in dose 250, 500 and 1000 mg/kg p.o. respectively, 1hour before milk injection. Blood samples were collected from each mouse from the retro orbital plexus, under light ether anesthesia. Total leukocyte and eosinophils count were determined for each group before drug administration and 24 hours after milk injection. Difference in total leukocyte and eosinophils count before and 24hours after drug administration were calculated.

PASSIVE PAW ANAPHYLAXIS IN RATS:

In this method antibodies against egg albumin were raised in rats. Animals were sensitized by injecting these antibodies. 24 hours after sensitization, the extract was administered. After 1hr of extract administration animals were challenged with egg albumin. This model was used to evaluate the protective effect of extract against allergen-induced passive paw anaphylaxis and thus to study the effect of extract on AG:AB reaction mediated inflammatory response.

(Gokhale et al., 1996)²⁴Antiserum to egg albumin was raised in rats using aluminum hydroxide gel as an adjuvant. Animals were given three doses of 100µg of egg albumin (S.C.,) absorbed on 12mg of albumin hydroxide gel prepared in 0.5ml of saline on 1^st, 3^rd, 5^th day. On 10^th day of sterilization, the blood was collected from the retro orbital plexus. The collected blood was allowed to clot and the serum was separated by centrifugation at 1500 rpm. Animals were divided into 5 groups each containing 5 animals. Animals belonging to group-1 served as control and were administered only the distilled water (5ml/kg p.o.). Animals belonging to group-II were administered dexamethasone (0.5 mg/kg i.p.,). Animals belonging to group III, IV, and V received methanolic extract of drug (175, 350, and 700 mg/kg, p.o.,) respectively. The animals were passively sensitized with 0.1 ml of the undiluted serum into the left hind paw. The counter lateral paw received an equal volume of saline. The test drug extract/dexamethasone was administered 24 hours after sensitization. One hour after drug administration, the animals were challenged in the left hind paw with 10µg of egg albumin in 0.1ml of saline and the paw inflammation was measured by using a plethysmometer (UGO basile, 7140). The difference in the reading prior and after antigen challenge represented the edema volume and the % inhibition of edema was calculated by using following formula.

% Inhibition = 1-(v_t/v_C) × 100

Where v_t = mean relative change in paw volume in test group.

v_c = mean relative change in paw volume in control group.

STATISTICAL ANALYSIS: All observations were presented as mean ± SEM. The data was analyzed by student‘t’ test or one way ANOVA followed by Dunnett test. P ≤ 0.05 was considered as significant.

RESULTS:

Pharmacognostical evaluation of methanolic extract shown the presence of Alkaloids, Saponins, Flavonoids, Tannins and Glycosides.

Isolated goat tracheal chain preparation:

In the study, histamine and Ach produced dose dependent contraction of goat tracheal chain preparation. The modified physiological salt solution containing methanolic extract of Tamarindus indica Linn (200µg/ml) significantly inhibited (p<0.01) the contractile effect of histamine (Table-1) and Ach (Table-2).

Table -1 Effect of methanolic extract of Tamarindus indica Linn (200 µg/ml) on Histamine induced contraction of isolated goat tracheal chain preparation

SR. NO	Log Molar concentration of Ach	Control Group % maximum contraction	Test group % maximum contraction
1	6.61	19.9 0.90	10.25±0.61**
2	6.31	43.06 1.61	21.11±0.84**
3	6.01	63.51 1.28	31.88±0.83**
4	5.71	74.45 0.89	43.45±1.14**
5	5.40	85.0 1.13	48.05±0.85**
6	5.10	98.45 1.24	56.03±2.11**
7	4.80	100.00 1.56	68.55±1.97**
8	4.50	100±1.42	75.51±1.56**
9	4.20	--	98.20±1.68**
10	3.90	--	100.00±1.36**
11	2.60	--	100.00±1.25**

Values in Mean ± SEM. n = 6

Control = D.R.C of Histamine in absence of Tamarindus indica Linn extract. Test = D.R.C of Histamine in presence of Tamarindus indica Linn extract (200 µg/ml). Statistical analysis done by using student ‘t’-test. *p<0.05, **p<0.001, significantly different from control.

Table -2 Effect of methanolic extract of Tamarindus indica Linn (200 µg/ml) on ACh induced contraction of isolated goat tracheal chain preparation.

SR. NO	Log Molar concentration of Ach	Control Group % maximum contraction	Test group % maximum contraction
1	6.61	17.2±0.90	9.12±0.76**
2	6.31	38.15±1.65	19.21±0.85**
3	6.01	57.45±1.21	30.32±0.97**
4	5.71	68.32±0.73	35.18±1.21**
5	5.40	77.54±1.05	39.34±1.72**
6	5.10	86.71±1.32.	50.65±1.67**
7	4.80	100±0.00	65.21±1.10**
8	4.50	100±0.00	73.35±1.23**
9	4.20	--	96.34±1.44**
10	3.90	--	100.00±1.36**
11	2.60	--	100.00±1.25**

Values of Mean ± SEM. n= 6

Control = D.R.C ACh. of absence of Tamarindus indica Linn extract.

Test = D.R.C of ACh. In presence of Tamarindus indica Linn extract (200 µg/ml).Statistical analysis done by using student ‘t’-test.

*p<0.05, **p<0.001, significantly different from control.

Table 3: Effect of Tamarindus indica Linn extract against histamine induced bornchoconstriction in guinea pigs.

Group	Latent period of convulsion (in sec) Mean ± SEM
Group	Before	After 1 Hr	4 Hr	24 Hr
I	44.6± 1.66	***71.2 ±1.65	***87.6 ±1.56	*54.0 ±1.44
II	49.0± 2.68	**58.6 ±5.29	**81.0 ±1.00	45.4 ±1.67
III	52.4± 1.69	**69.0 ±2.50	**84.4 ±1.77	48.0± 2.21
IV	41.4± 1.36	**71.2 ±2.47	**86.55 ±2.90	*50.8 ±2.35

Values in Mean ±SEM

Group – I = chlorpheniramine maleate (2 mg/kg, p.o) ;

Group – II = Tamarindus indica Linn extract (217.5 mg/kg, p.o)

Group-III= Tamarindus indica Linn extract (435 mg/kg, p.o) ;

Group–IV= Tamarindus indica Linn extract (870 mg/kg, p.o)

Statistical analysis done by using studentʽtʼ-test.

*p<0.05, **p<0.01,***p<0.001

Table 4 :Percent protection against histamine induced Bronchoconstriction in guinea Pig

Groups	% Protection
	1 Hr	4 Hr	24 Hr
I	39.76	50.65	21.17
II	19.08	41.00	11.31
III	25.41	45.52	14.36
IV	43.45	49.22	19.87

Values in Mean ±SEM

Where n=5

Group – I = chlorpheniramine maleate (2 mg/kg, p.o)

Group – II = Tamarindus indica Linn extract (217.5 mg/kg, p.o)

Group -III = Tamarindus indica Linn extract (435 mg/kg, p.o)

Group –IV = Tamarindus indica Linn extract (870 mg/kg, p.o)

Table 5: Effect of extract on clonidine induced mast cell degranulation in rats

Group	Treatment	Intact	Disrupted	%protection
I	Control	22.8± 0.92	79.2± 0.92	-
II	Sodium cromoglycolate (50 mg/kg,i.p.)	72.2± 1.80**	29.8± 1.80**	71.42
III	Extract (175 mg/kg, p.o.)	54.2± 1.85**	47.8± 1.81**	48.92
IV	Extract (350 mg/kg, p.o.)	60.1± 1.72**	41.9± 1.72**	61.74
V	Extract (700 mg/kg, p.o.)	68.2± 1.36**	33.4± 1.36**	68.42

Group I= Distilled water (5 ml/kg, p.o.)

Group II, III, IV, V compared with Group-I (ANOVA followed by Dunnett test) **p<0.01.

Histamine induced bronchoconstriction in guinea pig:

Guinea pigs when exposed to histamine aerosol showed signs of progressive dyspnea leading to convulsions. The methanolic extract of Tamarindus indica Linn (217.5mg/kg, 435mg/kg and 870 mg/kg p.o.,) significantly prolonged (p<0.01) the latent period of convulsions as compared to control following exposure to histamine aerosol at 1^st and 4^th hour. Seed extract (870mg/kg, p.o.) showed significant (p<0.05) action at 24^th hour also.

Table 7: Effect of Tamarindus indica Linn on milk induced leucocytosis in mice

Group	Difference in no. of Leucocytes (per cu mm)
I	85.4±2.97
II	4688±16.74*
III	2930±34.84**
IV	2247±12.81**
V	1734±14.51**

Values of Mean ± SEM

Where n= 5

Group 1 = Distilled water (10 ml/kg, .p.o.,) .

Group II = Distilled water (10 mg/kg, p.o) + Milk (4 ml/kg, s.c.,) .

Group III = Tamarindus indica Linn extract (250 mg/kg,p.o) +Milk (4 ml/kg , s.c.,) .

Group IV = Tamarindus indica Linn extract (500 mg/kg,p.o)+ Milk (4 ml/kg , s.c.,) .

Group V= Tamarindus indica Linn extract (1000 mg/kg,p.o)+ Milk (4 ml/kg , s.c.,) .

Statistical analysis done by using studentʽtʼ-test (Group II were compared with Group I) ANOVA followed by Dunnett test (Group III, IV and V compared Group II).

*p<0.001, **p<0.01, significantly different from control.

Mast cell degranulation:

Clonidine induced mast cell degranulation was significantly (p<0.01) inhibited by sodium cromoglycate (50mg/kg i.p.) and percent protection was found to be 71.42%. In the groups treated with methanolic extract of Tamarindus indica Linn (175 mg/kg, 350mg/kg, 700mg/kg p.o.) there was significant protection (p<0.01) of mast cells and the percent protection was 47.92, 60.74, and 67.42% respectively.

Clonidine induced catalepsy in mice:

Clonidine induced catalepsy in mice, which remained for 2 hr. the vehicle treated group showed maximum duration of catalepsy (235.6±15.34 sec) at 120 min after the administration of clonidine. There was significant inhibition (p<0.05) of clonidine induced catalepsy in animals pretreated with Tamarindus indica Linn extract (250 mg/kg, 500 mg/kg, 1000mg/kg, p.o.) and the duration of catalepsy was found to be 171.8±11.9, 153.6, and 141.6±5.13 seconds respectively at 120 min after the administration of clonidine. Chlorpheniramine maleate (10 mg/kg, i.p.,) significantly inhibited (p<0.01) catalepsy in mice at 120 minutes after the administration of clonidine.

Table 8: Effect of Tamarindus indica Linn on Milk induced eosinophilia in mice.

Group	Difference in no. of Eosinophils Per cu mm (Mean + SEM)
I	22.2 +2.05
II	165.4 +7.092***
III	142.4 +3.98*
IV	130.4+3.70**
V	102.2+ 3.63**

Values in Mean +SEM

n=5

Group I =Distilled Water (10 ml/kg,p.o.,)

Group II =Distilled Water (10 ml/kg,p.o.)+Milk (4ml/kg, s.c.,)

Group III = Tamarindus indica Linn extract (250 mg/kg,p.o.) + Milk (4ml/kg, s.c.,)

Group IV= Tamarindus indica Linn extract (500mg/kg, p.o.) + Milk (4ml/kg, s.c.)

Group V= Tamarindus indica Linn extract (1000 mg/kg,p.o.) + Milk (4ml/kg, s.c.)

Statistical analysis done by using studentʽtʼ-test (Group II were compared with Group I) ANOVA followed by Dunnett test (Group III, IV and V compared Group II).

***p<0.001, **p<0.01, *p<0.05 significantly different from control.

Milk induced leucocytosis in mice:

Sub cutaneous injection of milk at doses of 4mg/kg produced a significant (p<0.001) increase in the leukocyte count after 24 hour of its administration. In the group of mice pretreated with methanolic extract of Tamarindus indica Linn at dose of 250mg/kg, 500mg/kg, 1000mg/kg p.o, there was significant (p<0.01) inhibition of milk induced leucocytosis.

Milk induced Eosinophilia in mice:

Injection of milk (4ml/kg, s.c.,) produced a significant increase (p<0.001) in the total eosinophil count. In the groups pretreated with extract at the dose of 250 mg/kg, 500mg/kg, 1000mg/kg, p.o., there was significant (p<0.01) inhibition of milk included eosinophilia and the eosinophil count was 141.4±3.98, 129.4±3.70, and 101.2±3.63 respectively.

Passive paw anaphylaxis:

Anti serum to egg albumin was injected 24 hr before administration of the test extract/Dexamethasone. Egg albumin was injected after administration of Tamarindus indica Linn extract and dexamethasone. In the vehicle treated group, egg albumin increased the paw edema volume in the sensitized animals, which was measurable up to time period of 4 hours. Dexamethasone significantly reduced (p<0.01) the paw edema volume at 0.5,1,2,3 and 4hrs time intervals and the percentage inhibition was 45.8%, 52.2%, 57.1%, 62.5% respectively.

DISCUSSION:

Asthma is a heterogeneous disorder immunologically, immunologically, and biochemically and its etiology is multifactorial. The present study was planned to evaluate the action of Tamarindus indica Linn on various aspects of asthma using various invitro and in vivo models.

Elevated IgE levels in extrinsic asthma can range from 62-87% (Cua-Lim, 1984)²⁵. The major goal in the asthma therapy is to treat acute exacerbation, which is mainly due to immediate hypersensitivity reaction and inflammation. Histamine is one of the important mediators of allergy and has been shown to activate action potentials in intra pulmonary vagal afferents(Gokhale&Saraf,200)²⁴. Targeting histamine becomes part of anti histaminic therapy (Kulakarni, 2005)²⁶.

Both goat tracheal chain and strip preparation are suitable for screening the activity of a drug on respiratory smooth muscles (Nag chaudhari and Lahiri,1974)⁶. The goat tracheal muscle has H₁,H₃, and β₂ receptors. The stimulation of H₁/H₃receptors causes contraction of bronchial smooth muscle (Kulshrestha et al., 1983)⁵. In the present study, Tamarindus indica Linn extract significantly inhibited the histamine and acetylcholine induced contraction indicating antihistaminic and antimuscarinic activity.

The guinea pigs exposed to histamine aerosol showed signs of progressive dyspnoea leading to convulsions. In the present study the extract significantly prolonged the latent period of convulsions.

Therefore the results of present study indicates the utility of Tamarindus indica Linn in the treatment of asthma and bronchitis by virtue of its H₁-receptor blocking or bronchodilating activity.

Immunomodulating agents are useful in the treatment of asthma by virtue of inhibiting the treatment the antigen-antibody (AG:AB) reaction there by inhibiting release of inflammatory mediators. The beneficial effect of extract in passive paw anaphylaxis study (Mengi et al., 2003)²⁷ could be due to either inhibition of AG:AB/antihistaminic activity.

Clonidine induces catalepsy via H₁ receptor. The prior treatment with the extract significantly inhibits the catalepsy, which may be due to its H_1-antagonostic activity.

Adaptogens increase the organism resistance to various adverse effects of a physical, chemical, and biological nature (Brekhman et al.,1969)¹⁹. Adaptogenic nature of the extract was demonstrated by using milk induced leukocyte method (Bhargava and Singh, 1981)²².

The mast cells contain basophil granules literally loaded with active substances which, if allowed to escape themselves/via enzymatically formed products, cause vascular and other tissue reaction similar to those characteristic of inflammatory process (Uvnas,1969)¹⁶.

In the present study, the groups of animals pre treated with methanollic extract resulted in reduction in degranulation of mast cells and offered significant protection when challenged with clonidine indicating mast cell stabilizing activity.

CONCLUSION:

Thus, it can be concluded from the results obtained in the present investigation that Tamarindus indica Linn possess significant anti asthmatic activity. The anti asthmatic activity of methanolic extract of seed of Tamarindus indica Linn can be attributed to bronchodilating, antihistaminic (H₁-antagonist), antiallergic, anti-inflammatory, mast cell stabilizing and adaptogenic activity, suggestive of its potential in prophylaxis and management of asthma.

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Received on 09.02.2011

Accepted on 06.03.2011

Research J. Pharmacology and Pharmacodynamics. 3(3): May –June, 2011, 115-122

Group	Duration of catalepsy (Sec) at Mean ± SEM
	15 min	30 min	60 min	90 min	120 min	150 min	180 min
I	29.4±1.91	110.6±2.07	68.4±20.57	202.1±8.21	236.6±15.34	217.8±7.76	198.6±14.14
II	15.0±0.70*	23.6±0.67**	66.4±10.7**	72.8±1.72**	89.8±13.1**	95.8±6.46**	108.8±5.77**
III	22.2±2.13*	98.0±1.14**	109.6±3.58**	138.2±7.72**	172.8±11.9**	153.8±6.74**	147.4±5.93**
IV	21.4±0.87*	93.8±2.08**	99.0±12.4**	130.8±11.3**	154.6±16.5**	142.0±5.37**	132.4±4.10**
V	18.0±1.58*	88.4±4.20**	63.8±10.8**	124.8±14.6**	142.6±5.13**	133.2±3.83**	127.2±3.98**