ANTI-ALLERGIC AND ANTICATALEPTIC ACTIVITY OF AQUEOUS EXTRACT OF LEAVES OF AILANTHUS EXCELSA ROXB.

ANTI-ALLERGIC AND ANTICATALEPTIC ACTIVITY OF AQUEOUS EXTRACT OF LEAVES OF AILANTHUS EXCELSA ROXB.

Dinesh Kumar *, Z A Bhat , P Singh , S C Mudgade, V D Bulani  and S S Bhujbal

Department of Pharmaceutical sciences, University of Kashmir, Srinagar, India

Department of Pharmacognosy, Pad. Dr.  D. Y. Patil Institute of Pharmaceutical Science and research, Pimpri, Pune, India

ABSTRACT

The anti-allergic of an aqueous extract of the leaves of Ailanthus excelsa Roxb.(AELAq) was evaluated by using milk induced leucocytosis and eosinophilia in mice, passive paw anaphylaxis in rats models while the  anticataleptic properties was evaluated by Clonidine induced catalepsy in mice mode. The extract significantly (*p<0.05, **p< 0.01) decreased the leucocytosis and eosinophilia along with passive paw anaphylaxis in the above experimental animals respectively. The extract also significantly (**p< 0.01) decreased Clonidine induced catalepsy in mice. These results suggest that aqueous extract AELAq may have the potential therapeutic value in the treatment of allergic diseases and to produce adaptogenic properties.

INTRODUCTION: 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 ¹. Allergies occur when a hypersensitive immune system reacts to a common or unusual substance. 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 ^2-3. In Indian system of medicine Ailanthus excelsa Roxb. is used in the treatment of asthma, bronchatitis, cough, cold ^4-5 Tribals in Nilgris region traditionally used it in antifertility, fever. Studies of stem bark extracts have shown reduced labour pain, febrifuge ⁶, antispasmodic ⁷, anticancer, antimicrobial, antiamoebic, antiprotozoal activities ^8-9 and antiasthmatic activity ¹⁰. From a pharmaceutical perspective flavonoids possess a remarkable spectrum of biochemical and pharmacological activities. The leaves were reported to contain different flavonoids like kaempferol (5, 4’, 5, 7- Tetrahydroxy flavone), luteolin (3’, 4’, 5, 7-tetrahydroxy flavone), apigenin (4’.5, 7-trihydroxy flavone) ¹¹. Thus, drug development has been encouraging researchers to find strategies to treat allergic diseases and the medicinal plants have been the target of these studies and an important tool to treat immediate-type allergic response ¹².

MATERIALS AND METHODS:

Plant Material: Ailanthus excelsa Roxb. (Simarubaceae) leaves collected in Aug. 2008 from Pune-18 nearby Hindustan antibiotic Ltd, campus, India and authenticated by Regional research Institute of Ayurveda, Pune. India. A voucher specimen – 899, has been preserved in the laboratory for future reference. Leaves were dried in shade and pulverized. The powder was subjected to decoction with water as solvent. The extract was concentrated under vacuum and dried in a vacuum desiccator (yield 18%, w/w).

Animals: Albino rats (Wistar strain) and mice (musmusculus strain) of either sex weighing 150-200gm rats and 20-25gm mice respectively were used. They were housed in microlon boxes with standard laboratory diet and water ad libitum. The study was conducted after obtaining Institutional Animal Ethical Committee clearance.

Acute toxicity studies: Mice (musmusculus strain) were selected for this study. They were divided into eight groups each containing six animals. Aqueous extract of leaves of Ailanthus excelsa Roxb. was administered orally in varying doses (0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00 2.50 and 5g/kg) to these animals. They were continuously observed for 2h to detect changes in the autonomic or behavioural responses like alertness, spontaneous activity, irritability, urination, etc. Any mortality during experimentation and the following 7 days was also recorded. A group of animals treated with vehicle (distilled water) was served as control .Based on the results of preliminary toxicity testing the doses of 100, 200 and 400mg/kg p.o were chosen for further experiments.

Antiallergic and Anticataleptic studies:

Milk induced leukocytosis and eosinophilia: Mice were divided into five groups, five animals in each group. Animals belonging to group-I received distilled water (DW) 10 ml/kg, (p.o.). Animals belonging to group II, III, IV, V received boiled and cooled milk injection in dose of 4 ml/kg, (s.c.). Animals belonging to groups III, IV and V received aqueous extract of Ailanthus excelsa Roxb. in dose 100, 200 and 400 mg/kg, p. o. respectively, 1 hr before milk injection. Blood samples were collected from each mouse from the retro orbital plexus, under light ether anaesthesia. Total leukocyte count and total eosinophilia count was done in each group before drug administration and 24 hr after milk injection. Difference in Total leukocyte count and total eosinophilia count before and 24hr after drug administration was calculated ¹³.

Passive paw anaphylaxis in rats: Anti serum to egg albumin was raised in rats using aluminum hydroxide gel as an adjuvant ¹⁴. Animals were given three doses of 100 mcg of egg albumin (s.c.) absorbed on 12 mg of aluminum hydroxide gel prepared in 0.5 ml of saline on 1^st, 3^rd, 5^th day. ­On 10^th day of sensitization, 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 five groups each containing 5 animals. Animals belonging to group I served as control and were administered only the vehicle (10 ml/kg, p. o.).

Animals belonging to group II were administered Dexamethasone (0.5 mg/kg, i.p.). Whereas animals belonging to groups III, IV and V received aqueous extract of Ailanthus excelsa Roxb. in dose 100, 200 and 400mg/kg, p. o. respectively. The animals were passively sensitized with 0.1 ml of the undiluted serum into the left hind paw. The contra lateral paw received an equal volume of saline. Aqueous extract of Ailanthus excelsa Roxb. was administered 24 hr after sensitization. 1 hr. after test drug administration, the animals was challenged in the left hind paw with 10 µg of egg albumin in 0.1 ml of saline and the paw inflammation was measured by using a Plethysmometer (UGO Basile, 7140). The difference in the reading prior to and after antigen challenge represented the oedema volume and the percent inhibition of oedema 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).

Clonidine induced catalepsy in mice: Bar test was used to study the effect of test drug extracts on Clonidine induced catalepsy ^15-16. Mice were divided into five groups, five animals in each group. Animals belonging to group I served as control and were administered the vehicle (10 ml/kg, p. o.). Animals belonging to group II received standard drug Chlorpheniramine maleate (10 mg/kg, i. p.). Animals belonging to groups III, IV and V received three doses 100, 200 and 400 mg/kg p. o. respectively of aqueous extract of leaves of Ailanthus excelsa Roxb. The forepaws of mice were placed on a horizontal bar (1 cm in diameter, 3 cm above the table) and the time required to remove the paws from bar was noted for each animal. All the groups received Clonidine (1 mg/kg, s. c.), 1 hr after the test drug administration and the duration of catalepsy were measured at 15, 30, 60, 90, 120, 150 and 180 min.

Statistical analysis: The statistical analysis was performed by using one-way analysis-of-variance (ANOVA) Followed by Dunnett’s test for individual comparison of groups with control.

RESULTS:

Effect of aqueous extract of leaves of A. excelsa Roxb. (AELAq)  on milk-induced leukocytosis in mice: Subcutaneous injection of milk at dose of 4 ml/kg produced a significant (***p< 0.001) increase in the leucocytes count and eosinophiles count after 24 hr of its administration. In the groups of mice pre-treated with aqueous extract of leaves of Ailanthus excelsa Roxb. at dose (100, 200 and 400 mg/kg, p.o.), there was significant (*p< 0.05, **p< 0.01) inhibition of milk-induced Leucocytosis and eosinophilia .

Effect of AELAq on Milk-Induced leukocytosis in mice:

n= 5, values are expressed in mean ± SEM. Control = Vehicle (10 ml/kg, p. o.). Intox. = milk 4 ml/kg. AELAq100 = Ailanthus excelsa Roxb. Leaves aqueous extract (100 mg/kg, p. o.). AELAq200 = Ailanthus excelsa Roxb. Leaves aqueous extract (200 mg/kg, p.o). AELAq400 = Ailanthus excelsa Roxb. Leaves aqueous extract (400 mg/kg, p. o.) ***p< 0.001, Intox. group compared with control group using student’t, test and *p< 0.05, **p< 0.01, AELAq compared to intox. Group using Statistical analysis done by ANOVA followed by Dunnet’s test.

Effect of AELAq on Milk-Induced eosinophilia in mice:

n= 5, values are expressed in mean ± SEM. Control = Vehicle (10 ml/kg, p.o.). Intox. = milk 4 ml/kg. AELAq100 = Ailanthus excelsa Roxb. Leaves aqueous extract (100 mg/kg, p.o.). AELAq200 = Ailanthus excelsa Roxb. Leaves aqueous extract (200 mg/kg, p.o). AELAq400 = Ailanthus excelsa Roxb. Leaves aqueous extract (400 mg/kg, p.o.); ***p< 0.001, Intox. group compared with control group using student’t, test and *p< 0.05, **p< 0.01, AELAq compared to intox. Group using Statistical analysis done by ANOVA followed by Dunnet’s test.

Effect of AELAq on passive paw anaphylaxis in rats: Antiserum to egg albumin was injected 24 hr. before administration of the test drugs or standard. Egg albumin was injected after the administration of Ailanthus excelsa Roxb.  and Dexamethasone. In the vehicle treated group, egg albumin increased the paw volume in the sensitized animals, which was measurable up to the time period of 4 hrs. Pre-treatment with aqueous extract of leaves of Ailanthus excelsa Roxb. (100, 200mg/kg, p.o.) does not significantly reduced (**p< 0.01) the paw volume at 4^th hr was found to be reduced maximum i.e. 10.22% & 18.28%  respectively. Aqueous extract of leaves of Ailanthus excelsa Roxb. (400mg/kg, p.o.) significantly reduced (*p< 0.05, **p< 0.01) the paw volume at 4^th hrs and the percentage inhibition was found to be 38.17% respectively. Dexamethasone (0.5 mg/kg, i.p.) significantly reduced (**p< 0.01) the paw volume at 4^th hrs maximum and the percentage inhibition was found to be 46.77 % respectively (Table 2 & 3).

Effect of AELAq on passive paw anaphylaxis in rats:

Where, n= 5, Control = Distilled water (5 ml/kg, p. o.). Std. = Dexamethasone (0.5 mg/kg, i. p.) AELAq100 = Ailanthus excelsa Roxb. Leaves aqueous extract (100 mg/kg, p. o.). AELAq200 = Ailanthus excelsa Roxb. Leaves aqueous extract (200 mg/kg, p. o). AELAq400 = Ailanthus excelsa Roxb. Leaves aqueous extract (400 mg/kg, p. o.). Std., AELAq100, AELAq200 & AELAq400 compared with Control (ANOVA followed by Dunnett’s test), *p < 0.05, **p < 0.01..

Effect of AELAq on passive paw anaphylaxis in rats:

Where, n= 5, Control = Distilled water (5 ml/kg, p. o.). Std. = Dexamethasone (0.5 mg/kg, i. p.) AELAq100 = Ailanthus excelsa Roxb. Leaves aqueous extract (100 mg/kg, p.o.). AELAq200 = Ailanthus excelsa Roxb. Leaves aqueous extract (200 mg/kg, p. o). AELAq400 = Ailanthus excelsa Roxb. Leaves aqueous extract (400 mg/kg, p. o.). Std., AELAq100, AELAq200 & AELAq400 compared with Control (ANOVA followed by Dunnett’s test), *p < 0.05, **p < 0.01.

Effect of AELAq on Clonidine induced catalepsy in mice:

Clonidine (1 mg/kg, s. c.) produced catalepsy in mice, which remained for 2 hr. The vehicle treated group showed maximum duration of catalepsy (80.2±3.813 sec.) at 120 minute after the administration clonidine. There was significant inhibition (*p<0.05, **p<0.01) of clonidine induced catalepsy in the animals pretreated with Ailanthus excelsa Roxb. AELAq extract (100, 200, 400 mg/kg, p. o.) and the duration of catalepsy was found to be 55.40±6.33**, 49.40±3.46** and 42.20±4.42** seconds, respectively at 120 minute after the administration clonidine. Chlorpheniramine maleate (10 mg/kg, i. p.) significantly inhibited (**p< 0.01) clonidine induced catalepsy in mice at 120 minute after the administration clonidine.

Effect of AELAq on Clonidine induced catalepsy in mice:

n =5, Control = Distilled water (10 ml/kg, p.o.). Std.  = Chlorpheniramine maleate (10 mg/kg, i.p.) AELAq100 = Ailanthus excelsa Roxb. Leaves aqueous extract (100 mg/kg, p.o.). AELAq200 = Ailanthus excelsa Roxb. Leaves aqueous extract (200 mg/kg, p.o.). AELAq400 = Ailanthus excelsa Roxb. Leaves aqueous extract (400 mg/kg, p.o.). Statistical analysis done by ANOVA followed by Dunnett’s test. *p<0.05, **p<0.01, compared to control group.

DISCUSSION: Allergy and anaphylaxis are the most responsible factor for diseases like asthma, rhinitis, bronchatitis, cold, cough, pain, inflammation etc. Several medicinal properties have been attributed to the plants in the traditional system of medicine. The presence of adaptogenic properties in some plant materials is being one of them, as described to be tonics in the Ayurvedic system of medicine. According to Brahmans and Dardymov (1969) the most important characteristic of an adaptogen, is that it increases resistance to adverse influences of a wide range of factors of physical, chemical and biological nature; and its normalization action, which reveals itself irrespective of the direction of the previous pathologic shifts.

After parenteral administration of milk there is increase in TLC, and this stressful condition can be normalized by administration of an antistress or adaptogenic drug ¹⁷. Furthermore leukocytes recruited during allergic inflammation release the inflammatory mediators like cytokines, histamine, and major basic protein and promote the ongoing inflammation. This model was used to evaluate the protective effect of Ailanthus excelsa Roxb. against milk-induced leukocytosis. Eosinophilia is an abnormal increase in peripheral eosinophile count to more than 4 % of total leukocytes.

In the late phase, especially in the development of allergic asthma, eosinophiles play role as an inflammatory cell. Eosinophil secretes mediators such as eosinophile cationic protein (ECP), eosinophile 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 ¹⁸. Eosinophilia is associated with respiratory disorder, often allergic in nature together with pulmonary infiltrates that are detectable on chest films¹⁹.  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 ²⁰.  The beneficial effect of Ailanthus excelsa Roxb. could be due to either inhibition AG: AB (hypersensitivity reaction-I) i.e. Producing antiallergic and anti-inflammatory properties.

Clonidine, a α₂­adrenoreceptor agonist induces dose dependent catalepsy in mice, which is inhibited by histamine (H₁) receptor antagonists but not by H₂ receptor antagonist. It is known that Clonidine releases histamine from mast cells. Brain histamine does play a definite role in the production of the extra pyramidal motor it has been suggested that the cataleptic effect of Clonidine in the mouse be mediated by histamine (via H₁ receptors) which is released from brain mast cells in response to stimulation of α₂ adrenoreceptors by Clonidine ²¹. The extract also significantly inhibited the clonidine induced catalepsy. The inhibition of clonidine induced catalepsy by Ailanthus excelsa Roxb. may be due to the potential to antagonize H₁ receptor.

Thus, it can be concluded from the results obtained in the present investigation that aqueous extract of leaves of Ailanthus excelsa Roxb. possess significant antiallergic, anti-inflammatory, anticataleptic, adaptogenic activity, suggestive of its potential in prophylaxis and management of allergic diseases. Hence further detailed study needs to be conducted to evaluate the phytoconstituent responsible to produce above result and their clinical efficacy in the treatment of asthmatic patients.

AKNOWLEDGEMENT: We would like to acknowledge that this research was supported by Dr. D. Y. Patil IPSR, Pimpri, Pune (India) by providing the lab facility. We would also like to thank Dr. Rajesh Dabour for authentication the plant sample in R. R. I. Pune (India).

REFERENCES:

1. Biswas K, Kumar A, Babaria BA, Prabhu K, Setty SR. Hepatoprotective effect of leaves of Peltophorum pterocarpum against paracetamol Induced acute liver damage in rats. Journal of Basic and Clinical Pharmacy. 2009; 1: 1: 10- 15.
2. Benicio MH, Ferreira MU, Cardoso MR, Konno SC, Monteiro CA. Wheezing Conditions in Early Childhood: Prevalence and Risk Factors in the City of São Paulo, vol. 82. Bulletin of theWorld Health Organization, Brazil, 2004; pp. 516–522.
3. Peden DB. The epidemiology and genetics of asthma risk associated with air pollution. Journal of Allergy and Clinical Immunology. 2005; 115: 213–219 (quiz 220).
4. Nadkarni KM. Indian Materia Medica. Edn 3, Vol. I, Popular Prakashan Mumbai, 2000, pp. 56-57.
5. Anonymous: Indian Pharmacopoeia. Government of India 2, Ministry of Health and Family Welfare, Controller of Publications, New Delhi, 1996; pp. A-53-54, A-95-97, A-109.
6. Jain SP, Verma DM. Medicinal plants in folklore of North Hariyana. Natl. Acad. Sci. Lett. 1964; 47: 240–242.
7. Bhakuni DS, Dhawan BN, Mehotra BN. Screening of Indian plants for biological activity. Part II. Ind. J. Exp. Biol. 1969; 7: 250–262.
8. Ogura M, Cordell GA, Kinghorn AD, Farnsworth NR. Potential anticancer agents. VI. Constituents of Ailanthus excelsa. Lloydia. 1978; 40: 579–581.
9. Shimali M, Jain DC, Darokar MP, Sharma RP. Antibacterial activity of Ailanthus excelsa Phytother. Res. , 2001; 15: 165–166.
10. Kumar D, Bhujbal SS, Deoda RS, Mudgade SC. In-vitro and In-vivo Antiasthmatic Studies of Ailanthus excelsa on Guinea Pigs. J. Sci. Res. 2010; 2: 196-202.
11. Lavhale MS, Mishra SH. Nutritional and therapeutic potential of Ailanthus excelsa - A Review. Pharmacognosy Reviews. 2007; 1: 1: 105-113.

12. Dai Y, Hou LF, Chan YP, Cheng L, But PP. Inhibition of immediate allergic reactions by ethanol extract from Plumbago zeylanica Biological & Pharmaceutical Bulletin. 2004; 27: 429–432.
13. Vadnere GP, Somani RS, Singhai AK. Studies on antiasthmatic activity of aqueous extract of Clerodendron phlomidis. Pharmacologyonline. , 2007; 1: 487-494.
14. Gokhale AB, Saraf MN. Studies on antiallergic activity of ethanolic extract of Tephrosia purpurea Indian Drugs. 2000; 37, 5, 228-32.
15. Ferre S, Guix T, Prat G, Jane F, Casas M. Is experimental catalepsy properly measured? Pharmac Biochem Behav. 1990; 35: 753-57.
16. Taur DJ, Nirmal SA, Patil RY. Effect of various extracts of Ficus bengalensis bark on clonidine and haloperidol-induced catalepsy in mice. 2007; 3: 470-77.
17. Brekhman II, Dardymov IV. New substances of plant origin which increases non specific resistance. Ann. Rev. Pharmac1969; 9: 419-430.
18. Osama NO Joshi KR. Immunology: Immunology of Respiratory Disease. Agro Botanical Publishers, Bikaner, 1984; 321-9.
19. Ehright T, Chua S, Lim DJ. Pulmonary eosinophilic syndromes. Ann. Allergy. 1989; 62: 277-83.
20. Lee-man C, But PPH, Yue D. Antiallergic and anti-inflammatory properties of the ethanolic extract of Gleditisia sinensis. Journal Pharmacy Pharmacology. 2002; 25: 9: 1179-82.
21. Wardlaw AJ, Brightling CE, Green R, Woltmann G, Bradding P, Pavord ID. New insights into the relationship between airway inflammation and asthma. Clinical Science. 2002; 103: 201–11.
    

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