Evaluation of antiasthmatic activity of ethanolic extract of Elephantopus scaber L. leaves.

AIMS: The aim of study was to evaluate the scientific basis for the traditional use of Elephantopus scaber leaves.
MATERIALS AND METHODS: In the present study, ethanol extract of Elephantopus scaber leaves was evaluated for preliminary phytochemical screening and antiasthmatic activity using histamine and acetylcholine-induced bronchospasm, mast cell degranulation and histamine induced constriction on isolated guinea pig tracheal chain at different dose levels. Student's t-Test and Dunett's test were used for statistical analysis.
RESULTS: The result of present investigation showed that the ethanolic extract of E. scaber significantly (P<0.001) decreased the bronchospasm induced by histamine, acetylcholine and protected mast cell degranulation as compared to control groups. It also decreased the histamine induce constriction on isolated guinea pig trachea in dose-dependent manner. Phytochemical studies revealed the presence of steroids, saponin, flavonoids, and phenolic compounds in the extract.
CONCLUSIONS: The present study concludes that the antiasthmatic activity of ethanolic extract of E. scaber leaves may be due to the presence of flavonoids or steroids. Antiasthmatic action of the E. scaber could be due to its antihistaminic, anticholinergic and mast-cell-stabilizing property.

Introduction

Asthma is one of the most common disorders characterized by airway inflammation. It can be caused by various factors like allergens, drugs, respiratory infection, dust, cold air, exercise, emotions, occupational stimuli, chemicals, histamine, etc.

Elephantopus scaber Linn. Popularly known as Elephant's foot, (Asteraceae) is a scabrescent aromatic herb distributed in the moist deciduous forests of central Western Ghats. As per the traditional claims, roots were used as an antipyretic, cardiotonic and diuretic.[1] Decoction of roots and leaves is used as emollient and given in dysuria, diarrhea, dysentery and in stomachic pain.[2] The aqueous extract of leaves is applied externally to treat eczema and ulcers.[3] Phytochemically the plant has been reported to contain sesquiterpenes lactones deoxyelephantopin, isodeoxy-elephantopin and scabertopin.[4] It also contains epifriedelinol, lupeol and stigmasterol.[5] The pharmacological properties of the leaf extracts have been evaluated for diuretic,[6] anti-inflammatory,[7] and hepatoprotective properties.[8] On the basis of above-mentioned scientific evidences, the present study was undertaken to investigate the effect of the leaf extract of E. scaber L. for its antiasthmatic activity.

Materials and Methods

Plant Material

Leaves of E. scaber were collected from local area of Bhopal, India. The species was identified and authenticated at the Department of Botany, Dr. H. S. Gour Central University, Sagar (M.P), where a plant specimen was deposited.

Preparation of Extract

The dried leaves were reduced to coarse powder and macerated with ethyl alcohol for 48 hours and filtrate was evaporated under reduced pressure to obtain dried extract (extractive value 9.28% w/w). A qualitative preliminary phytochemical tests were performed to find the presence of various phytochemicals in the extract.[9] For antiasthmatic evaluation, extract was dissolved in distilled water prior to its use.

Chemicals

Histamine dihydrochloride, acetylcholine chloride, ketotifen, compound 48/80 were purchased from Sigma-Aldrich Chemical Co., USA. Egg albumin and other chemicals were purchased from Himedia Laboratories Pvt. Ltd., India. All the other chemicals were of analytical grade.

Phytochemical Screening

Preliminary phytochemical tests were performed on ethanolic extract of E. scaber for the presence of various phytoconstituents as per described methods.[10]

Experimental Animals

Wister rats (175-200 g) and guinea pigs (400-600 g) of either sex housed in standard conditions of temperature (22 ± 2°C), relative humidity (55 ± 5%) and light (12 hrs light/dark cycles) were used. They were fed with standard pellet diet and water ad libitum. The experimental protocol was approved by Institutional Animal Ethical Committee as per the guidance of CPCSEA, Ministry of Social Justice and Empowerment, Government of India. (Protocol No. 379/01/ab/CPCSEA).

Acute Toxicity Testing

No mortality and the sign of toxicity were observed at the dose of 6000 mg/kg.[6] Dose selected for antiasthmatic evaluation was 250 mg/kg and 500 mg/kg.

Histamine and acetylcholine induced bronchospasm in guinea pigs.[11]

Guinea pigs of either sex were divided into two groups of six animals each and exposed to 0.1% w/v of histamine dihydrochloride aerosol in histamine chamber. The progressive dyspnea was observed in animals when exposed to histamine aerosol. The end point, preconvulsion dyspnea (PCD) was determined from the time of aerosol exposure to the onset of dyspnoea leading to the appearance of convulsion. As soon as PCD commenced, the animals were removed from chamber and placed in fresh air. PCD of this time was taken as day 0 value. Both groups of guinea pigs were given ethanolic extract of E. scaber at the dose of 250 mg/kg, and 500 mg/kg, p.o. respectively, once a day for 7 days. On the 7 day 2 h after the last dose, the time for the onset of PCD was recorded as on day 0. Same procedure was followed in another set of animals (n = 6) for acetylcholine induce bronchospasm study using 0.5% acetylcholine chloride. The percentage increased in time of PCD was calculated using following formula.[12]

Percentage increased in time of PCD = (1-T1/T2) × 100

where T = time for PCD onset on day 0, T = time for PCD onset on day 7

Mast Cell Degranulation Studies.[13]

Albino rats of either sex were divided into 6 groups of six animals each and sacrificed by cervical dislocation. The animals were immediately injected with 15 ml of prewarmed (37°C) buffered salt solution (NaCl 137 mM; KCl 2.7 mM; MgCl2; 1 mM; CaCl2 0.5 mM; NaH2PO4 0.4 mM; Glucose 5.6 mM; HEPES 10 mM) into the peritoneal cavity, and massaged gently in this region for 90 s, to facilitate cell recovery. A midline incision was made and the peritoneum was exposed. The pale fluid was aspirated using a blunted plastic Pasteur pipette, and collected in a plastic centrifuge tube. The fluid was then centrifuged at 1000 rpm for 5 min, and the supernatant was discarded to reveal a pale cell pellet. The cell pellets were resuspended in fresh buffer and recentrifuged.

The peritoneal cell suspension divided in six parts viz. – ve control, +ve control, reference standard (Ketotifen 10 μg/ml) and ethanolic extract of E. scaber at different concentration i.e., 600, 800, 1000 μg/ml, each containing 0.1 ml of cell suspension and incubated at constant temperature 37°C in water bath for 15 min. Then 0.1 ml of compound 48/80 was added in all samples except in – ve control group and suspensions were further incubated for 10 min at 37°C. The cells were then stained with 10% of toludine blue solution and observed under the higher magnification by microscope. The percent granulated and degranulated mast cells were counted in each group.

Guinea Pig Tracheal Chain Preparation.[14]

Guinea pigs of either sex (200-500 g) were divided into 4 groups. Each group contains six animals and were allowed to starve overnight and free access to water. The animals were killed by a blow on the head and exsanguinated. The isolated trachea was mounted in a 30-ml organ bath containing Tyrode solution, maintained at 37 ± 1°C and gassed with air. The tissue was equilibrated for 45 min during which the bath solution was replaced every 10 min. At the end of the equilibration period, histamine (0. 5 μg/ml)-induced contraction as well as effect of extract (up to 1000 μg/ml) was recorded. A drug tissue contact time of 1 min was maintained. The percent response of each groups were calculated from the height of the peaks obtained.

Statistical Analysis

The results of various studies were expressed as Mean ± SEM and analyzed using one-way ANOVA followed by Student's t-Test to find out the level of significance. Data were considered statistically significant at minimum level of P<0.001.

Results

Preliminary qualitative phytochemical screening of ethanolic extract of E. scaber showed the presence of flavonoids, phenols, steroids, tannins, terpenes, xanthoproteins and sugar.

E. Scaber significantly (P<0.001) increased the preconvulsive dyspnoea time following exposure to histamine and acetylcholine aerosols induced bronchospasm in guinea pigs [Table 1]. It also inhibited in vitro rat peritoneal mast cell degranulation significantly (P<0.001) induced by compound 48/80 as compared to base line value [Table 2]. The isolated guinea pig tracheal chain preparation showed dose dependent significant (P<0.001) inhibition of the contraction of tracheal muscles induced by histamine as compared to control group [Table 3].

Table 1

Effect of ethanolic extract of Elephantopus scaber (p.o., for 7 days) on histamine and acetylcholine-induced bronchospasm in guinea pigs

Table 2

Effect of ethanolic extract of Elephantopus scaber on induced mast cell degranulation in albino rats

Table 3

Percent inhibition of Elephantopus scaber on histamine induced contraction on isolated guinea pig trachea

Discussion

Bronchodilating effect of ethanolic extract of E. scaber was evaluated by observing its effects on the time of PCD. In our study we found that the time of occurrence of PCD was significantly increased suggestive of bronchodilating activity of E. scaber against spasmogens.

Mast cell degranulation is important in the initiation of immediate responses following exposure to allergens.[15] An attempt was made to find out whether ethanolic extract of E. scaber has any effect on the rate of disruption of mast cells following exposure to compound 48/80, an agent which causes histamine release.[16] The present study, E. scaber offered significant protection against Compound 48/80 induced mast cell degranulation by stabilizing it, which is responsible for the decreasing airway inflammation by preventing release of various inflammatory mediators.

The relaxant effects of ethanolic extract of E. scaber on tracheal chains of guinea pigs might be produced by different mechanisms including stimulation of β-adrenergic receptors,[1718] inhibition of histamine H1 receptors,[19] or an anticholinergic property of this plant.[20] The relaxant effects of all concentrations of the extract of E. scaber obtained were significantly lower than control group. These findings suggest probable b-adrenergic stimulatory, muscarinic and/or histamine H1 blocking properties of the plant extract.

Phytochemical screening of E. scaber showed presence of lignans, flavanoids, steroids and sugar derivatives etc.[21] Lignans are known to posses various biological activities including antibacterial, antioxidant, anticancer, spasmolytic and anti-inflammatory effects.[22] Antiasthmatic activity of E. Scaber may be due to presence of the above constituents.

Our data suggest that the ethanolic extract of the leaves of E. scaber possesses significant antianaphylactic, anticholinergic and antihistaminic activity. However, further studies are needed to establish molecular mechanism and to isolate and characterize the active principles, which are responsible for the antiasthmatic action.