Oriental Pharmacy and Experimental Medicine 2008 8(1), 24-31 Investigation into the mechanism of action of Moringa oleifera for its anti-asthmatic activity Anita Mehta* and Babita Agrawal Department of Pharmacology, L.M. College of Pharmacy, Ahmedabad - 3800009, India In the present investigation, we studied the effect of alcoholic extract of Moringa oleifera (M.
oleifera) seed kernels on various experimental models of bronchial asthma. Significant (P < 0. 05)increase in preconvulsion time was observed due to pretreatment with M. oleifera when theguinea pigs were exposed to either acetylcholine (Ach) or histamine aerosol. This bronchodilatingeffect of M. oleifera was comparable to ketotifen fumarate. Spasmolytic effect of M. oleifera was alsoobserved by dose dependent inhibition of ideal contractions induced by Ach, 5HT, histamine andBaCl2. Alcoholic extract of M. oleifera produced significant dose dependent protection by eggalbumin and compound 48/80 induced mast cell degranulation. Pretreatment with alcoholicextract of M. oleifera also decreased carrageenan induced rat paw edema, which was comparableto that of standard diclofenac sodium. Minimum inhibitory concentration for alcoholic extract ofM. oleifera was low as compared to cold-water extract and hot water extract when antimicrobialactivity was tested against various respiratory pathogens like Escherichia coli (E. coli), Staphylococusaureus (S. aureus) and pseudomonas aeruginosa (P. aeruginosa). Our data suggest that antiasthmaticactivity of M. oleifera seed kernels may be due to its bronchodilator, anti-inflammatory, mast cellstabilization and antimicrobial activity.
Key words: Bronchial asthma; Moringa oleifera; Bronchodilator; Anti-inflammatory; Mast cellstabilization index and requires monitoring of drug levels(Nasser and Rees, 1993; Stoloff, 1994). Adverse Bronchial asthma is a chronic respiratory disorder effects of corticosteroids include fluid retention, affecting a large proportion of population increased cell mass, increased appetite, weight throughout the world. The currently used drugs gain, osteoporosis, capillary fragility, hypertension, for the treatment of this disease in modern peptic ulceration, diabetes, cataract, and phychosis medicine are far from satisfactory as they provide (Dajani et al., 1981). Hence Ayurveda has only symptomatic relief, produce several adverse recommended number of drugs from indigenous effects and may lose effectiveness on continued plants sources for the treatment of bronchial use. Muscle tremor and hypokalemia are major asthma and other allergic disorders and have been adverse effects of β2 agonists (Haalboom et al., 1985; successful in controlling the disease as well. Large Nelson, 1986). Theophyline has narrow therapeutic numbers of medicinal plant preparations havebeen reported to possess anti-asthmatic effects.
*Correspondence: Anita Mehta, Department of Moringa oleifera (M. oleifera) is a small or medium Pharmacology, L.M. College of Pharmacy, Ahmedabad -3800009, India. E-mail: [email protected] sized tree, cultivated throughout India. The tender 2008 Kyung Hee University Press Investigation into the mechanism of action of Moringa oleifera for its anti-asthmatic activity pods are esteemed as a vegetable. Seeds are used as light/dark cycle. Animals had access to standard purgative, antipyretic and anti inflammatory (Warrier pallet diet and water given ad libitum. The et al., 1997). The plant possesses antimicrobial activity protocol of the experiment was approved by the (Caceres et al., 1991), while the seeds reportedly institutional animal ethical committee as per the have antispasmodic, anti inflammatory and diuretic guidance of the Committee for the purpose of activity (Caceres et al., 1992). Its leaves and fruits Control and Supervision of Experiments on are edible, rich in ascorbic acid. The plant is also Animals (CPCSEA), Ministry of Social Justice and reported to elicit good clinical response in children Empowerment, Government of India. suffering from upper respiratory tract infectionand skin infection. It has been reported that Studies on Acetylcholine and Histamine induced alkaloid from the plant closely resembles ephedrine bronchospasm in guinea pigs in action and useful in treatment of asthma.
Guinea pigs of either sex weighing 350 - 500 g were Alkaloid Moringine relaxes bronchioles (Kirtikar selected and randomly divided into six groups and Basu, 1975). In the present study, we have each containing six animals. The drugs were investigated the anti-asthmatic potential of M. oleifera administered orally in 0.5% sodium carboxymethyl on various experimental models like Bronchodilating, cellulose (CMC). The single dose treatments were mast cell stabilizing, anti inflammatory and given one and half an hour before the study. The following schedule of treatment was administered: MATERIALS AND METHODS Group I: 0.5% CMC (control)Group II: Ketotifen (1 mg/kg) (standard) Group III: Alcoholic extract of M. oleifera (100 mg/kg) Seed kernels of M. oleifera were purchased from the Group IV: Alcoholic extract of M. oleifera (200 mg/kg) local market of Ahmedabad and were identifiedand authenticated by Dept. of pharmacognosy, Later the animals were exposed to an aerosol of L.M. College of Pharmacy, Ahmedabad, India. A 0.25% histamine and time for preconvulsion state voucher specimen was deposited at the Dept. of was noted for each animal (Sheth et al., 1972). After pharmacognosy, Ahmedabad. The coarse powder about 15 days of wash out period, the same (500 g) of the dried seed kernels was defatted using animals were given the above treatments and time petrol ether and then it was exhaustively extracted for preconvulsion state was noted for 0.5% using 95% ethanol (2,000 ml) in a soxhlet extractor.
acetylcholine bromide aerosol spray.
Cold aqueous extract of M. oleifera was prepared byextracting 1 part of seed kernels with 10 parts of Studies on isolated Guinea pig ileum water for 2 h without heating, while hot aqueous Overnight fasted guinea pigs of either sex weighing extract was prepared by heating the seed kernels 400 - 600 g were sacrificed using cervical dislocation with water. The extracts were concentrated under method. Ileum was quickly dissected out and reduced pressure to yield a syrupy mass and mounted in an organ bath maintained at 37 ± 1°C stored in air tight container in cool place and used and containing 20 ml Tyrode's solution under throughout the project.
basal tension of 500 mg. The composition of solutionin mM was NaCl, 137; CaCl2, 1.8; KCl, 2.7; glucose, 5.55; NaHCO3, 11.9; MgCl2, 1; NaH2PO4, 0.4. The All animals were housed at ambient temperature solution was continuously bubbled with air. The (22 ± 1°C), relative humidity (55 ± 5%) and 12/12 h responses to drug were recorded on a Student 2008 Oriental Pharmacy and Experimental Medicine 8(1), 24-31 Anita Mehta and Babita Agrawal physiograph (BioDevices) using isotonic transducer, number of degranulated mast cells from total of which exerted a basal tension equivalent to 500 mg atleast 100 mast cells counted. load on tissue. The issue was allowed to equilibrate In another study, rats were sensitized by for 30 min, during which, the bathing solution was administering three doses of 350 μg of egg albumin changed at every 10 min. The contractile responses adsorbed on 60 mg of aluminum hydroxide gel, of ileum to various agonists (Acetylcholine, histamine, the doses being given on the first, third and fifth 5-HT and BaCl2) were recorded in presence and day subcutaneously. The mast cells were collected absence of alcoholic extract of M. oleifera.
on the tenth day of sensitization. The study wasconducted in the same manner as above and the Studies on compound 48/80 and egg albumin sensitized cells were degranulated using egg induced rat peritoneal mast cell degranulation albumin (1 mg/ml). Percent protection of the mast Normal saline containing 5 units/ml of heparin cells in the control group and the treated groups was injected in the peritoneal cavity of male rats were calculated by counting the number of lightly anaesthetized with ether. After a gentle degranulated mast cells from total of atleast 100 abdominal message, the peritoneal fluid containing mast cells counted. Control group is consisted of mast cells was collected in centrifuge tubes placed positive control group in which egg albumin was over ice. Peritoneal fluid of 4 - 5 rats was collected added without addition of test agent and a and pooled and centrifuged at 2,000 rpm for 5 min.
negative control group in which neither egg Supernatant solution was discarded and the cells albumin nor the test agent was added to correct for were washed twice with saline and resuspended in spontaneous degranulation of mast cells without any degranulating agent.
0.1 ml of the peritoneal cell suspension was transferred to 6 test tubes and was treated as follows.
Anti inflammatory studyCarrageenan induced rat paw edema Test tube no. 1 & 2 - Saline Albino rats of either sex weighing 200 - 250 g were Test tube no. 3 - 0.1 ml of 0.5 mg/ml alcoholic divided in 4 groups of 6 animals each. The following extract of M. oleifera in Saline schedule of treatment was administered: Test tube no. 4 - 0.1ml of 1.0 mg/ml alcoholic Group I: 0.5% CMC extract of M. oleifera in Saline Group II: Diclofenac sodium (20 mg/kg p.o.) Test tube no. 5 - 0.1 ml of 2.0 mg/ml alcoholic Group III: Alcoholic extract of M. oleifera (200 mg/kg extract of M. oleifera in Saline p.o.) in 0.5% CMC Test tube no. 6 - 0.1 ml of 10 μg/ml of Ketotifen Group IV: Alcoholic extract of M. oleifera (400 mg/ kg p.o.) in 0.5% CMC Each test tube was incubated for 15 min at 37°C Animals were treated with drugs and subsequently and then Compound 48/80 (0.1 ml, 10 μg/ml) was 1 h after treatment; 0.1 ml of 1% carrageenan was added to each test tube except test tube no. 1. After injected subcutaneously into the planter region of further incubation for 10 min. at 37°C, the cells right hind paw to induce edema. The paw volume were stained with 0.1% toluidine blue solution was measured initially and at 1, 3 and 5 h after made in distilled water and examined under the carrageenan injection using plethysmographic high power of light microscope. Percent protection method of Harris and Spencer (1962). Percentage of the mast cells in the control group and the increase in paw volume from baseline was calculated treated groups were calculated by counting the and compared with control.
2008 Oriental Pharmacy and Experimental Medicine 8(1), 24-31 Investigation into the mechanism of action of Moringa oleifera for its anti-asthmatic activity Anti microbial studies observed due to pretreatment with M. oleifera (100 The in vitro antimicrobial activity of the M. oleifera mg/kg and 200 mg/kg) when the guinea pigs were was studied by broth dilution method and exposed to either acetylcholine (0.5%) or histamine minimum inhibitory concentration was found out.
(0.25%) aerosol. The increase in preconvulsion Cold aqueous extract, hot aqueous extract and the time was comparable to that of Ketotifen (1 mg/ alcoholic extract were prepared from the seeds of M. oleifera. These extracts at different concentrations(5 - 100 mg/ml) were tested against the organisms Effect of M. oleifera on agonists induced contractions Escherichia coli, Staphylococus aureus and pseudomonas of guinea pig ileum Alcoholic extract of M. oleifera (50 - 150 μg/ml) dosedependently inhibited ileal contractions induced by Statistical analysis: All the results were tested for histamine (3.84×10-4 mM), Ach (4.12×10-5 mM), significance using Student's t-test at the probability 5HT (5.67×10-5 mM) and BaCl2 (2.4×10-3 mM) level of 95%.
Compound 48/80 induced rat mast cell degranulationCompound 48/80 (10 μg/ml) produced significant Effect of M. oleifera on Ach and Histamine induced disruption of mast cells which was significantly bronchospasm in guinea pigs inhibited in a dose-dependent manner by Significant increase in preconvulsion time was pretreatment with the alcoholic extract of M. oleifera Table 1. Effect of M. oleifera on Ach and Histamine induced bronchospasm in guinea pigs % Increase in preconvulsion time M. oleifera (100 mg/kg p.o.) M. oleifera (200 mg/kg p.o.) 56.31 ± 3.11*** Ketotifen fumarate (1 mg/kg p.o.) *Significantly different from baseline (Student's paired t test) (n = 6). *P < 0.05, ***P < 0.001.
Table 2. Effect of M. oleifera on agonists induced contractions of guinea pig ileum Conc. of M.oleifera % Inhibition of Hista- % Inhibition of 5HT % Inhibition of BaCl2 mine contractions of Ach contractions Table 3. Effect of M. oleifera extract on compound 48/80 induced rat peritoneal mast cell degranulation Concentration (mg/ml) % Mast cells degranulation ± S.E.M. % Inhibition of degranulation M. oleifera extract M. oleifera extract M. oleifera extract Ketotifen fumarate N=6 in each group. *Significantly different from control. **P < 0.01 (Student's t test).
2008 Oriental Pharmacy and Experimental Medicine 8(1), 24-31 Anita Mehta and Babita Agrawal Table 4. Effect of M. oleifera extract on Egg Albumin induced rat peritoneal mast cell degranulation degranulation ± S.E.M.
M. oleifera Extract M. oleifera Extract M. oleifera Extract Ketotifen fumarate N=6 in each group. *Significantly different from control. **P<0.01 (Student's t test).
Table 5. Effect of M. oleifera on Carageenan induced rat paw edema % Increase volume of paw (% anti-inflammatory effect) 117.96 ± 0.787***(12.48) 16.87 ± 0.96**(39.01) 19.214 ± 1.328*(55.1) 16.40 ± 0.429**(76.87) Diclofenac sodium 18.175 ± 1.17*(60.16) 14.58 ± 0.819**(83.43) Significantly different from control. *P<0.05, **P<0.01, ***P<0.001 (Student's t test).
Table 6. Anti microbial study of M. oleifera Minimum Inhibitory concentration (mg/ml) Cold water extract Hot water extract Alcoholic extract E-coli ATCC-25922 P. aeruginosa ATCC-27853 S. aureus ATCC-25923 in concentrations of 0.5 - 2.0 mg/ml (Table 3). Egg Anti microbial study Albumin (1 mg/ml) induced rat mast cell Minimum Inhibitory Concentration for alcoholic degranulation was significantly inhibited by extract was low as compared to cold-water extract pretreatment of the animals with the alcoholic and hot water extract of M. oleifera. Hot water extract of M. oleifera in concentrations of 0.5 - 2.0 extract and alcoholic extract were ineffective against mg/ml. The protection was comparable to the P. aeruginosa. Cold-water extract of M. oleifera was reference standard Ketotifen (10 μg/ml) (Table 4).
found to be more active against Gram-positivebacteria, while alcoholic extract was found to be Effect of M. oleifera on Carrageenan induced rat active against Gram-negative bacteria (Table 6). paw edemaAlcoholic extract of M. oleifera at the dose of 200 mg/kg and 400 mg/kg decreased rat paw edema(76.87% reduction in edema volume), which was The results from our earlier clinical study on M.
comparable to that of standard Diclofenac Sodium oleifera suggest that, there was appreciable decrease 20 mg/kg (83.43% reduction in edema volume) in severity of symptoms of asthma and also simultaneously improvement in lung function 2008 Oriental Pharmacy and Experimental Medicine 8(1), 24-31 Investigation into the mechanism of action of Moringa oleifera for its anti-asthmatic activity parameters. Also, none of the patients showed oleifera produced dose dependent inhibition of ileal change in any general parameters or any adverse contractions induced by histamine, Ach, 5-HT and effect suggest safety of drug in dose used.
BaCl2. These indicate that M. oleifera has a non- Considering the availability along with convenience specific spasmolytic activity on smooth muscle.
and efficacy in oral administration, the drug offers Tylophora asthmatica has also been shown to possess a good future in treatment of asthma. Since non-specific spasmolytic activity (Harnath and bronchodilators, mediator release inhibitors, anti- Shyamalakumari, 1975). These effects of M. oleifera inflammatory drugs and anti-microbials are the correlate with our earlier results of improvement in different classes of drugs used conventionally in the symptoms and lung function parameters of the treatment of bronchial asthma; various animal asthmatic subjects.
models and experimental protocols were used in In addition to bronchodilating activity, a significant the present study to determine the mechanisms of number of therapeutic approaches for bronchial anti-asthmatic activity of M. oleifera.
asthma have been designed based on the antagonism Bronchial asthma is characterized by increased of specific mediators released from mast cells. Mast airway reactivity to spasmogens (Cockcroft, 1983).
cell degranulation is important in the initiation of An initial event in asthma appears to be the release immediate responses following exposure to allergens.
of inflammatory mediators (e.g. Histamine, Tryptase, Degranulated cells liberate mediators of inflammation Leukotrienes and prostaglandins). Some of these such as histamine, leukotrienes, platelet activating mediators directly cause acute bronchoconstriction, factors and chemotactic factors for eosinophils, airway hyperresponsiveness and bronchial airway neutrophils etc. from mast cells (Cushing, 1957; inflammation. Spasmolytic drugs like beta adrenergic Bellanti, 1971). They play a significant role in agonists, xanthine derivatives and anticholinergics airway inflammatory response such as airway relax the airway smooth muscles and are used as eosinophilia, late asthmatic response and airway quick relief medications in acute asthmatic attacks.
hyperresponsiveness as well as in immediate Beta adrenergic agonists promote bronchodilation hypersensitivity reaction like bronchial contraction.
by direct stimulation of beta adrenergic receptors Degranulation of mast cells has been taken as the in the airway smooth muscle, that lead to relaxation criteria of positive anaphylaxis. Ketotifen fumarate, of bronchial smooth muscle by rapid decrease in a well-known mast cell stabilizer, reduces synthesis airway resistance in vivo. Specific β2 agonists like of prostaglandins E2, thromboxane A2, leukotriene salbutamol, salmeterol etc. are used since long for C4 and B4. It also inhibits release of histamine, symptomatic relief in asthma. In present study, serotonin and other inflammatory mediators from significant increase in preconvulsion time was mast cells. Simultaneously it blocks H1 receptors.
observed due to pretreatment with M. oleifera, Khellin is a compound isolated from Ammi visnaga when the guinea pigs were exposed to either Ach and its structural analogue furanochromone or histamine aerosol. This bronchodilating effect of khellin. Cromolyn sodium, which is developed M. oleifera was comparable to ketotifen. It has been from the structural modification of Khellin (Cox et reported that Albizzia lebbeck (Tripathi and Das, al., 1970) is the mast cell stabilizer used in the 1977) and Ocimum sanctum (Singh and Agarwal, treatment of mild to moderate asthma. Adhatoda 1991), which are well known anti-asthmatic herbal vasica, Albizzia lebbeck, Coleus forskohlii, Tylophora drugs have similar mechanism of action. Spasmolytic asthmatica etc. are several well known drugs from effect of M. oleifera was also evaluated by observing indigenous plant sources used in asthma and have the effect of its alcoholic extract on histamine, Ach, been reported to have mast cell stabilizing activity 5HT and BaCl2 induced ileal contractions. M.
(Tripathi et al., 1979; Atal, 1980; Geetha et al., 1981; 2008 Oriental Pharmacy and Experimental Medicine 8(1), 24-31 Anita Mehta and Babita Agrawal Marone et al., 1987). A significant protection of rat nonpathogenic bacteria accumulate due to the peritoneal mast cells from disruption by antigen bronchial obstruction and plugging, causing and compound 48/80 by alcoholic extract of M.
serious infection. Plants produce a range of chemical oleifera points towards its ability to interfere the substance to protect themselves from the attack of release and/or synthesis of mediators of various pathogenic microorganisms. The substances inflammation, indicating its mast cell stabilizing that can either inhibit the growth of microorganisms or kill them are considered for developing new Further, airway inflammation has been demonstrated drugs for various infectious diseases. Use of these in all forms of asthma. Even in mild asthma, there medicinal plants can substitute antibiotics to treat is an inflammatory response involving infiltration, associated infection. In the present study, M.
particularly with activated eosinophils and oleifera possess good Antimicrobial activity when lymphocytes, with neutrophils and mast cells. The tested against various respiratory pathogens that degree of bronchial hyperresponsiveness and can be used to control respiratory complications.
airway obstruction is closely linked to the extent of In conclusion our data suggests that M. oleifera inflammation (Bousquet et al., 2000). Anti-inflammatory seed kernels have potential anti-asthmatic activity drugs suppress the inflammatory response by that may be due to its bronchodilator, mast cell inhibiting infiltration and activation of inflammatory stabilization, anti- inflammatory and anti microbial cells as well as their synthesis, or release of mediators property. Further study is ongoing to characterize and the effects of inflammatory mediators. The the active principles of the ethanolic extract which carrageenan induced paw edema model in rats is are responsible for antiasthmatic activity.
known to be sensitive to cycloxygenase inhibitors.
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