Investigation of antitussive and toxicological activity of Ballota limbata in mice

Abstract

Context: Ballota limbata Benth. (Lamiaceae) (syn, Otostegia limbata Hook.f.) is a species grown in the North West Frontier Province and the lower hills of West Punjab, Pakistan. Ballota species are renowned for their antispasmodic, antiulcer, diuretic, vermifuge, and especially sedative effects. However, little is known about the biological activity of B. limbata.

Objective: Evaluation of antitussive activity and safety profile of dried B. limbata extract.

Materials and methods: Whole air-dried plants were partitioned with various solvents and the butanol fraction was subjected to antitussive evaluation using a sulfur dioxide (SO₂)-induced cough model in mice. Codeine and dextromethorphan were used as positive control. Safety profile of the testing material was established using standard toxicity tests.

Results: B. limbata extract inhibited cough provoked by SO₂ gas in mice in a dose-dependent manner. The extract exhibited maximum protection against SO₂-induced cough after 60 min of administration. B. limbata offered maximum cough suppressive effects, that is, number of coughs during 60 min was 11.66 ± 1.2 (mean ± SEM), after s.c. administration of 800 mg/kg, as compared with codeine 10 mg/kg, s.c., dextromethorphan 10 mg/kg, s.c., and saline showing a frequency of cough of 11.75 ± 1.18, 12.25 ± 0.83, and 46.25 ± 1.52, respectively. LD₅₀ value of B. limbata was greater than 5000 mg/kg. No sign of neural impairment was observed at antitussive doses and the extract has been well-tolerated at higher doses.

Discussion and conclusion: This study demonstrates that the extract of B. limbata has shown strong cough suppressive effect in mice without yielding any notable toxicity.

Keywords::

• Cough
• antitussive
• Ballota limbata
• Otostegia limbata
• codeine
• dextromethorphan
• sulfur dioxide

Introduction

Cough is a universal phenomenon that has been experienced by every human being. It is an essential protective and defensive action that secures the removal of mucus, noxious substances, and infections from the larynx, trachea, and larger bronchi (Chung, 2003). On the other hand, a number of patients have a non-productive cough, which is not associated with mucus clearance and may be the first overt sign of disease of the airways or lungs, may significantly contribute to the spread of airborne infections, and in some instances may result in severe functional and structural damage to the organism (for review, see Irwin et al., 2006; Brignall et al., 2008; Young & Smith, 2010).

Drugs currently used to treat cough are among the most widely used over-the-counter drugs in the world, despite a recent analysis indicating that there is little evidence to suggest that such drugs produce any meaningful efficacy (Schroeder & Fahey, 2002). The primary action of currently available cough suppressants (opiates, dextromethorphan, etc.) is on the central cough pathway. The significant side effects of these agents such as constipation, respiratory depression, dependence, drowsiness, and death limit their use in humans (Rang et al., 1999; Chung & Chang, 2002). Currently, there is a huge unmet need for the development of safe, effective antitussive therapeutic options in the treatment of persistent cough as an alternative to existing medications (Dicpinigaitis, 2010; Birring, 2011).

The genus Ballota (Lamiaceae) consists of about 33 species growing mainly in Asia in the Mediterranean region and adjoining Asia Minor. Ballota species are famous for their antispasmodic, antiulcer, diuretics, vermifuge, and especially sedative effects. In folk medicine, decoctions or infusions of whole plant or leaves of Ballota africana Benth. and Ballota nigra L. are applied in respiratory ailments such as asthma, cough, bronchitis, and pulmonary troubles (Watt & Breyer-Brandwijk, 1962; Duke, 2002). Ballota limbata Benth. (syn. Otostegia limbata Hook f.) is found in the North West Frontier Province and lower hills of West Punjab, Pakistan. The juice of aerial parts of this plant (locally called “Bui” or “Phutkanda”) is used for treatment of children’s gums and ophthalmia in man (Chopra et al., 1956; Nasir & Ali, 1972). A literature search reveals that apart from few phytochemical investigation reports and in vitro bioassays, no pharmacological study has been carried out in vivo on this plant. Phytochemical investigations indicate that a rare class of tetracycline diterpenoid with novel structures has been isolated from this plant (Ahmad et al., 2004a,b; Khan et al., 2009).

Cough can be induced in experimental animals by chemical, mechanical, or electrical stimulation of sensory nerve afferents in the larynx, trachea, or bronchial mucosa or by stimulation of central nervous system (CNS). However, it is considered that cough obtained by chemical stimulation is more comparable with that in humans than is obtained with other tussigenic stimuli (Braga, 1989a). In present studies, the sulfur dioxide (SO₂) gas-induced murine cough model was used to investigate antitussive efficacy of testing materials (Miyagoshi et al., 1986).

Materials and methods

Plant material and extraction

The plant B. limbata was collected from Abbottabad, Pakistan in June 2001, and identified by Dr. Manzoor Ahmad (Taxonomist) at the Department of Botany, Post Graduate College, Abbottabad, Pakistan. A voucher specimen (#6872) has been deposited in the herbarium of Botany Department of Post Graduate College, Abbottabad, Pakistan for future references. Air-dried whole plant (35 kg) was exhaustively extracted with methanol (40 L) at room temperature. The extract was evaporated to yield residue (315 g), which was partitioned between hexane (47 g), chloroform (95 g), ethyl acetate (69 g), butanol (33 g), and water (59 g). The butanol fraction was subjected to antitussive evaluation and used for the safety profile.

Experimental animals

Studies were carried out on Naval Medical Research Institute (NMRI, USA) mice weighing 25 ± 5 g, obtained from the animal house facility of Section of Pharmacology, H.E.J. Research Institute of Chemical Sciences, University of Karachi. Animals of either sex were used. Animals were kept in groups of 5–6 in transparent plastic cages and provided standard 12 h light/dark cycle beginning at 8 a.m. Standard food and water were available ad libitum. With some exception, all experiments were carried out between 8 a.m. and 6 p.m. Animal procedures were performed in accordance with the guidelines specified by the Animal Care and Use Committee of our Institution. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Chemicals

Sulfuric acid and Tween 80 were purchased from Sigma Chemical Company, St. Louis, MO. Sodium hydrogen sulfite was purchased from Merck, Darmstadt, Germany. Codeine phosphate and dextromethorphan hydrobromide were received as gifts from Wilson Pharmaceutical Co., Islamabad Pakistan and Abbott Laboratories (Pakistan) Limited, Karachi, Pakistan, respectively.

Testing material was suspended in aqueous Tween 80 solution (1% v/v). All chemicals were dissolved in normal saline. All solutions were freshly made at the day of testing and administered to a final volume of 0.1 mL/10 g body weight of mice. Testing material and all chemicals were administered subcutaneously (s.c.). All doses (of testing material and chemicals) were expressed in mg/kg of body weight of animals, excluding the weight of their salts.

Antitussive evaluation

Antitussive identification and quantification of testing material was determined with the SO₂ gas-induced murine cough model described by Miyagoshi et al. (1986). The experimental model is shown in Figure 1, where “A” is a 500-mL three-necked round bottom flask containing aqueous saturated (39%) sodium hydrogen sulfite solution. By opening the stopcock “a” of dropping funnel “B,” concentrated sulfuric acid (98%) was introduced to generate SO₂ gas. SO₂ gas filled previously in “A,” and by opening stopcocks “b” and “c” the pressure in the gas reservoir “C” was elevated, which was recorded by the water manometer “D.” The stopcock “b” was then closed and stopcock “d” was opened slightly until the pressure in “D” reached 6 mm Hg (constant throughout the experiment), then stopcock “d” was closed. These procedures were conducted in a draught. Cough response of an animal was observed by placing the animal in desiccator “E.” Stopcocks “c,” “e,” and “f” were opened, respectively, and when the pressure in “D” became 0 mm Hg, all the stopcocks were closed immediately. A certain amount of SO₂ (which was fixed throughout the experiment) was introduced into the desiccator this way. One minute after introduction of the gas, the animal was taken out of the desiccator and the number of coughs produced during the initial 5 min of exposure were counted by an up-ended filter funnel, with a stethoscope at the tip in which the mouse was confined.

Figure 1.  Apparatus used in sulfur dioxide gas-induced cough model. (A) Three-necked round bottom flask, containing 39% NaHSO₃ solution, (B) dropping funnel having concentrated H₂SO₄, (C) gas reservoir, (D) water manometer, and (E) desiccator. The procedure used to produce sulfur dioxide gas is described in the text.

Animals were divided into seven groups, containing eight mice each. One served as control group (saline, 0.1 mL/10 g, s.c.), three groups for butanol fraction of B. limbata (BFBl 200, 400 and 800 mg/kg, s.c.), two groups for the standard drugs codeine phosphate (10 mg/kg, s.c.) and dextromethorphan (10 mg/kg, s.c.) and the remaining group was used for vehicle (Tween 80, 0.1 mL/10 g, s.c.). These doses of testing material were selected on the basis of the toxicity profile and preliminary antitussive screening (data not shown). Initially, frequency of coughs for each animal was determined at 0 min, before administration of any chemical or testing material. It has been illustrated that cough response to a given stimulus varies from animal to animal but that repeated assessments within the same animals are fairly reproducible (Belvisi and Hele, 2003). Thus, animals having low or high cough threshold were not entertained for further studies. Number of coughs was observed for all animal groups at 30, 60, and 90 min (after drug administration) intervals using the same procedure. The time interval at which the greatest antitussive action was observed was taken as time of the peak effect.

Toxicity profile

Acute neurotoxicity

The primary safety test used to determine BFBl-induced neural impairment was the inverted screen acute neurotoxicity test (Coughenour et al., 1977). The apparatus consists of six 12.6 cm² platforms of 0.6 cm wire mesh supported by metal bars that in turn are mounted on a metal rod. Mice were pretested on the apparatus the day preceding the experiment, and those failing the task were not used for the subsequent drug test. For the test procedure, mice were dosed with incremental doses of BFBl (250, 500, 1000, and 2000 mg/kg, s.c.), placed 30, 60, and 120 min on individual platforms while the rod rotated through an arc of 180°. Mice unable to climb to an upright position for the duration of 1 min were considered to be impaired (Coughenour et al., 1977). Eight mice were used for each dose to determine neural impairment in mice. Mice were then monitored for mortality throughout 24 h.

LD₅₀

The dose of testing material that caused death in 50% of the animals within 24 h (LD₅₀) was calculated using the method described by Lorke (1983). In brief, BFBl at the dose of 10, 100, and 1000 mg/kg was administered s.c. to groups of three mice each. Upon the results of mortality in each group after 24 h, four more mice were administered different doses of EAFTc in order to obtain the least and most toxic value. LD₅₀ was calculated by geometric mean of these values.

Statistical analysis

Data were analyzed using statistical analyzing software “Microcal Origin version 6.” The number of coughs was expressed as means ± standard error of the mean (SEM). Student’s t-test was performed for each experiment to determine the difference between control and experimental groups. Difference was considered significant if P ≤ 0.05.

Results

Antitussive effects

BFBl (200 mg/kg, s.c.) produced a significant inhibition of SO₂ gas-induced cough in mice (Figure 2, P < 0.01; P < 0.001), that is, number of coughs was 36.9 ± 1.4, 33.9 ± 1.4, and 35.7 ± 1.2 (mean ± SEM) after 30, 60, and 90 min of dosing, respectively, as compared with saline having a frequency of cough 46.6 ± 1.3, 46.2 ± 1.5, and 46.7 ± 1.5 in the same time frame. BFBl 200 exhibited maximum cough inhibition after 60 min of dosing, that is, cough efforts = 25.5 ± 2.5, representing 26.75% reduction in cough efforts, which was approximately one-third as active as codeine (10 mg/kg, s.c.) and dextromethorphan (10 mg/kg, s.c.), that is, cough efforts = 11.75 ± 1.18 and 12.25 ± 0.83 reflecting 75.70 and 74.74%, respectively. At moderate dose, such as 400 mg/kg, the extract of B. limbata resulted in a marked reduction of frequency of cough induced by SO₂ gas, as illustrated in Figure 3. Number of cough efforts was reduced from 48.1 ± 1.6 (number of coughs before dosing) to 33.5 ± 2.07, 25.5 ± 2.5, and 31.4 ± 1.8 (means ± SEM) after 30, 60, and 90 min of extract administration, respectively. Cough suppressive effects of BFBl were obviously increased at elevated doses. At a higher dose (800 mg/kg, s.c.), extracts of B. limbata resulted in more significant reduction of frequency of cough induced by SO₂ gas. Number of cough efforts was decreased from 48.2 ± 2.1 (coughs before dosing) to 24.6 ± 1.7, 18 ± 1.7, and 24.7 ± 1.6 (means ± SEM) after 30, 60, and 90 min of dosing, respectively. The antitussive activity of BFBl (800 mg/kg) was comparable with codeine (10 mg/kg, s.c.) and dextromethorphan (10 mg/kg, s.c.), as illustrated in Figure 4. BFBl showed maximum protection against SO₂ elicited cough after 60 min of s.c. administration, which was taken as time of peak effect (TPE). The inhibitory potential of different doses of BFBl and standard drugs at TPE are shown in Figure 5. The cough suppressant activity of BFBl tended to reduce after 60 min. However, there was a significant reduction in frequency of cough at 30 and 90 min, which revealed that this extract has a long duration of action. Vehicle (Tween 80, 0.1 mL/10 g, s.c.) alone was without influence on the incidence of cough after SO₂ gas challenge (data not shown).

Figure 2.  Antitussive effect of s.c. administered butanol fraction of Ballota limbata (BFBl 200 mg/kg) in sulfur dioxide gas-induced cough model in mice. Codeine (10 mg/kg) and dextromethorphan (10 mg/kg) were used as positive control. Each column represents mean ± SEM. Number of coughs was recorded for 5 min for each condition. n = 8 mice in each group. Significant differences from saline control were indicated as *P < 0.01, **P < 0.001 by Student’s t-test.

Figure 3.  Antitussive effect of s.c. administered butanol fraction of Ballota limbata (BFBl) 400 mg/kg in mice. Each column represents mean ± SEM. Number of coughs was recorded for 5 min. n = 8 mice in each group. Significant differences from saline control were indicated as *P < 0.01, **P < 0.001 by Student’s t-test.

Figure 4.  Antitussive effect of s.c. administered butanol fraction of Ballota limbata (BFBl) 800 mg/kg in mice. Each column represents mean ± SEM. Number of coughs was recorded for 5 min. n = 8 mice in each group. Significant differences from saline control were indicated as *P < 0.001 by Student’s t-test.

Figure 5.  Comparison of antitussive efficacy of butanol fraction of Ballota limbata (BFBl 200, 400, and 800 mg/kg), codeine 10 mg/kg, and dextromethorphan 10 mg/kg in mice.

Toxicity profile

Acute neurotoxicity

The effects of BFBl on neural impairment and motor function were determined by inverted screen acute neurotoxicity test. BFBl was observed at different doses (250, 500, 1000, and 2000 mg/kg, s.c.) and time intervals (30, 60, 90, and 120 min) in mice. B. limbata did not express any sign of acute neurotoxicity at any specified time, when tested up to 2000 mg/kg.

LD₅₀

The LD₅₀ of BFBl after s.c. dosing was calculated to be greater than 5000 mg/kg per 24 h.

Discussion

Plants have provided and continue to provide essential material for treatment of numerous diseases, shelter, food, furniture, clothing, writing, weapons, cosmetics, and numerous other purposes (Balick & Cox, 1996). Certainly, the great civilizations of ancient Chinese, Indian, and North African provided written evidence of Man’s ingenuity in utilizing plants for treatment of a wide variety of diseases (Fabricant & Farnsworth, 2001; Houghton, 2001; Phillipson, 2001; Schmidt et al., 2008). Most popular cough medicines throughout the world are based on herbal derivatives (Ziment, 2002). Numerous compounds such as codeine, morphine, noscapine, bromhexine, guaifenesin, ephedrine, cromolyn, and so on and their derivatives, which are isolated from different plant species, are well-established western medicines for treating cough or underlying pathologies (März & Matthys, 1997; Ziment, 2002).

The results of our present study reveal that the extract of whole plant B. limbata significantly inhibited SO₂ gas-provoked cough in mice. It was found that the extract of B. limbata, administered at a high dose, caused a significant inhibition of cough reflex comparable with positive controls (codeine 10 mg/kg and dextromethorphan 10 mg/kg).

The successful treatment of cough is inextricably linked to the establishment of its etiology. In many patients, there is no apparent cause associated with persistent cough; therefore, it is necessary to suppress irritable coughing by antitussive agents (Dicpinigaitis, 2010). Currently, the most commonly used antitussives are centrally acting narcotic antitussives (also called codeine group). They produce obvious antitussive action at doses below those required for pain relief. However, at their effective doses these antitussives cause a high rate of unwanted effects, like drowsiness, nausea and vomiting, constipation, depression of respiratory center, decreased secretion in the bronchioles, inhibition of ciliary activity, and often cause physical dependence. Their administration can lead to increased sputum viscosity, decreased expectoration, and hypotension. All these disadvantages usher the development of new antitussive agents, especially non-narcotic ones, which might prevent the pathological cough (Rang et al., 1999). Plants extracts have the ability to exhibit a multiplicity of pharmacological actions such as antiallergic, anti-inflammatory, analgesic, antimicrobial, and so on, which are triggered by several different types of chemical compounds present in the extract.

Airway inflammatory conditions such as asthma, cough variant asthma, and eosinophilic bronchitis trigger numerous mechanical events in the wall of airways including smooth muscle contraction, vasodilatation and edema, mucus secretion, and decrease in lung compliance by release of different inflammatory mediators. All of these changes in the airways wall trigger coughing. Ahmad and colleagues (2004a) have isolated new diterpenoids from B. limbata, which have inhibitory potential against lipoxygenase enzymes. These enzymes play a vital role in the biosynthesis of a variety of chemical mediators such as hydroxyeicosatetraenoic acids (HETEs), leukotrienes, lipoxins, and hepoxilins in mammalian cells. These mediators play an important role in bronchial asthma and airway inflammation (Barnes et al., 1998).

Phytochemical studies have reported that B. limbata also contains flavonoids (Ikram et al., 1979). Many studies have reported the antiallergic properties of flavonoids and their derivatives like disodium cromoglycate, a chromone known for its clinical use in the therapy of cough and asthma (Gábor, 1986). The presence of flavonoids in B. limbata may partially explain its antitussive effect.

The possible mechanism of the antitussive effect of B. limbata, on the basis of ethnopharmacological reports and our observations, may be characterized as peripheral, that is, acting on afferent or efferent limb of the cough reflex (antiallergic and spasmolytic). However, the possibility of central action by suppressing the cough motor pattern generator (mainly due to mild sedation) could not be excluded. Although a sedative property of agents is not essential for an antitussive effect, it may illustrate that these agents have the capacity to provoke CNS activity.

Our study, using different tests for acute toxicity/neural impairment, demonstrated a relative lack of unwanted side effects for BFBl at antitussive doses. The test for acute toxicity, the neural impairment test, indicated that after s.c. administration BFBl possessed a more favorable safety profile, therapeutic range, and tolerability in mice when compared with findings for most of the current available antitussives, namely codeine and dextromethorphan. For example, subcutaneously administered BFBl to mice yielded a drastically better LD₅₀ value, that is, greater than 5000 mg/kg, than codeine and dextromethorphan, representing LD₅₀ values of 140 and 275 mg/kg by same route of drug administration in mice (Braga, 1989b).

Conclusion

In conclusion, the present data indicates that B. limbata possesses antitussive potential against chemically induced cough in mice. The antitussive activity of this plant correlates with various pharmacological properties, which may justify its widespread use in various respiratory conditions in folk medicine. Further studies aimed at isolation of the active compounds responsible for antitussive activity and the related mechanisms of action are ongoing in our laboratory.

Acknowledgements

This work was accomplished by an institutional grant from Department of Pharmacology, University of Karachi, Pakistan and an exceptional assistance from Mr. Muhammad Liaquat Raza, Mr. Shamsher Ali, and Mr. Afsar Khan. We are grateful to Marlene L. Anderson for helpful comments on the manuscript.

Declaration of interest

We declare that we have no conflict of interest with any organization or person.