Physiology of the respiratory system and asthma

The respiratory system is composed of the lungs and the air passages, the muscles of the thorax, of pleural sacs and nerves. The air passage consists of the paired nasal cavities, pharynx, larynx, trachea and bronchial tree. The trachea bifurcates to form the primary bronchi which further divide into secondary bronchi leading to smaller respiratory bronchioles terminating in alveoli, through which oxygen passes from air into blood and carbon dioxide passes from blood into air.

On the lungs and air passages, receptors such as adrenoceptor, histamine and muscarinic are present, which are responsible for regulation of different physiological functions. Stimulation of β-adrenergic receptors decreases smooth tone of the airways and inhibits the release of inflammatory mediators from mast cells. Muscarinic receptors in airways belong to M₃ subtypes, which occur almost exclusively in proximal airways, and mediate contractile responses and increase the mucous secretion. Histamine receptors of H1-type are present on bronchial muscle, causing contraction of smooth muscles, but have little physiological role; thus antihistaminic drugs have limited therapeutic contribution.

The hyperactivity of respiratory smooth muscles results in airway constriction leading to asthma. Most of the disorders of the respiratory system, other than infectious diseases, results from the hyperactivity of airways. Asthma is a major congestive respiratory disorder, characterised by episodic wheezing, cough and chest tightness associated with airflow obstruction. The worldwide prevalence of asthma has been increasing, particularly in children. According to the World Health Organization (WHO), it affects about 5-10% of adults and 10% of children globally. The mortality rates from asthma have been increasing steadily over recent decades. According to the National Center for Health Statistics, the death rate from asthma in the United States increased from 0.8 per 100 000 in 1971 to 2 per 100 000 in 1991.

The pathogenesis of asthma is multifactorial and multicellular since macrophages, mast cells, eosinophils, neutrophils and platelets are involved in its pathogenesis. The cells produce an arsenal of mediators such as bradykinin, histamine, leukotrienes, platelet-activating factor, prostaglandins and thromboxane which interact in a complex way to produce numerous pathological effects. These include constriction of airway smooth muscle, increased microvascular leakage, mucus secretion and recruitment of inflammatory cells into airways. Histopathological studies of patients with asthma have shown inflammation in the airways with infiltration of inflammatory cells, particularly eosinophils, disruption of airway epithelium and mucus hypersecretion, thus indicating that airway inflammation may underlie bronchial hyperresponsiveness.

Asthma is classified into extrinsic and intrinsic types. The extrinsic type generally appears in early stages of life in individuals with a family history of either asthma or various allergies including hay fever, eczema and dermatitis. The intrinsic type, on the other hand, develops at around 40 years of age and occurs because of non-specific factors (common cold, exercise or emotion) that may trigger the asthmatic attack. Many stimuli including viral infection, environmental allergens, animal dander, stress, air pollutants, emotion (fear, anger, frustration), cold air and changes in weather enhance symptoms of asthma and alter airway physiology.

In many instances asthma has been found to run in families and multiple genes are involved in its expression. In the traditional Greco-Arab Unani system of medicine, the human race is divided genetically into four classes based on their susceptibility to develop different diseases, i.e, choleric, sanguine, phlegmatic and melancholic. Those who have the tendency to develop asthmatic disorders belong to the phlegmatic category. It has been observed that such individuals with sensitive airways respond adversely and develop bronchoconstriction and/or cough when taking allopathic medicines, such as angiotensin-converting enzyme inhibitors.

Drugs used to treat asthma

The common classes of drugs with proven efficacy in asthma are bronchodilators such as β₂-agonists, anticholinergics, phosphodiesterase inhibitors, while glucocorticosteroids, mass cell stabilisers and leukotriene modifiers are used usually as preventive therapy in chronic cases. More recently, Ca²⁺ antagonists and potassium channel openers have been added to the list of potential bronchodilators. All bronchodilators currently in use are known to manifest cardiac stimulation as a serious side-effect, particularly when given orally. Inhalers are used to avoid cardiac side-effects, but are very expensive and beyond the reach of a large part of the population in developing countries, so alternate measures are being explored for safe and cost-effective treatment.

Herbs in this regard have potential not only as a source of new clinical drugs but are also gaining popularity in the form of crude herbal products or botanicals. Interestingly, a constituent of Aspalathus linearis (a popular herbal tea in South Africa, commonly known as rooibos) is chrysoeriol, a flavonoid, and was found to exhibit high selectivity for airways compared with other smooth muscles, so placing itself amongst the candidates to be developed for congestive airways disorders.

Pathology of coughing

Cough is a spasmodic contraction of the thoracic cavity that results in abrupt release of air from the lungs. It is usually very sudden in onset and very often repetitive. The cough reflex is complex, involving the central and peripheral nervous system as well as the smooth muscle of the bronchial tree. It has been suggested that irritation of the bronchial mucosa causes bronchoconstriction, which in turn stimulates cough receptors (which probably represent a specialised type of stretch receptor) located in tracheobronchial passages. The cough reflex probably includes several mechanisms or centres that are distinct from the mechanisms involved in the regulation of respiration. Excessive cough is one of the most common symptoms for which the patient seeks medical care and may represent up to one-third of a pulmonologist’s outpatient referrals. Persistent severe cough, seen in interstitial lung disease or bronchiectasis, may impair respiration as well as disrupt sleep and social functioning. Bronchospasm, syncope, rib fractures and urinary incontinence are all potential complications. On the basis of duration, cough has been divided into acute (less than 3 weeks’ duration), subacute (3-8 weeks) and chronic (more than 8 weeks) types.

The causes of acute cough are viral or bacterial infection, pneumonia, pulmonary embolism and pulmonary oedema. The most common causes of subacute and chronic cough are asthma, weather changes, smoking, inflammation of larynx or pharynx and allergies.

The drugs that directly or indirectly can affect the cough are diverse. Cough may be the first, or the only, symptom of asthma or allergy and in such cases bronchodilators and antihistaminergics have been shown to reduce cough without having significant central effects. The drugs acting primarily on central or peripheral nervous system components of the cough reflex are opioid agents, i.e. codeine and dextromethorphan, which are structurally related to morphine and act on the cough centre of the medulla, increasing the cough threshold and thus depressing the cough.

Models for respiratory studies

ln-vivo studies

Pulmonary function test

The pulmonary functions are assessed using a spirometer, just before and 2 h after administration of the test and control drugs to the patients with asthma. The subjects are asked to take a deep inspiration followed by forcible expiration into the spirometer. The various parameters such as forced vital capacity, forced expiratory volume in first second, peak expiratory flow rate and forced expiratory flow rate between 25% and 75% of forced vital capacity are recorded. Because a significant improvement is observed after 2 h, this schedule is fixed for the measurements throughout the study.

Bronchodilatory activity

Rats are anaesthetised with sodium thiopental, then intubated with a tracheal tube and ventilated with a volume ventilator (Miniature ideal pump, Bioscience, UK) adjusted at a rate of 70-80 strokes/min (to deliver 7-10 mL/kg of room air) in the supine position. A polythene catheter is inserted into the jugular vein for drug administration. Changes in airway resistance are measured by connecting a side arm of the tracheal cannula to a pressure transducer (MLT 1199). Bronchoconstriction is induced with carbachol or histamine, which is reversed within 7-10 min. The test drugs are given to the animals 5-8 min prior to administration of carbachol.

Aerosol inhalation method

Guinea pigs that reacted positively on the preliminary test of the histamine aerosol are selected and used during the in-vivo test. Four groups of the selected animals are prepared. The first group served as controls. For the three other groups, different doses of the test drug are administered by oral route 2 h before the histamine aerosol test. Then animals are placed into a 10-L transparent plastic bell jar. They are aerosolised with 5 mg/mL histamine solution during 3 min. The reaction of each animal is noted. The animal which did not present any suffocation sign during 3 min is considered as protected.

Histamine and antigen-induced bronchospasm

The animals are anaesthetised by ethyl urethane (1.25 g/kg intraperitoneally). After tracheotomy a tracheal cannula is introduced and connected to a ventilation pump and a pressure transducer. The ventilation pressure is registered with a Gemini recorder. The animals are ventilated artificially at a frequency of 50 breaths/min and the respiratory volume is adjusted to 10 mL/breath. Maximal changes in pulmonary ventilation pressure (PVP) are expressed as the percentage of the basal pulmonary ventilation pressure. For histamine-induced bronchospasm, histamine (20 µg/kg) is injected intravenously through a short polyethylene catheter inserted into the jugular vein. For antigen-induced bronchospasm, the animals are first sensitised by two successive inhalations (50 µL each) of a nebulised Oleaceae allergen. After 48 h, the animals are anaesthetised and placed under the assisted respiration and are administered with the Oleaceae allergen (100 µL) by intratracheal instillation.

In-vitro studies

Isolated tracheal strips

The trachea is dissected from a guinea pig or rabbit killed by cervical dislocation and kept in Krebs solution. The tracheal tube is cut into rings, 2-3 mm wide. Each ring is opened by a longitudinal cut on the ventral side opposite to the smooth muscle layer, forming a tracheal strip with a central part of smooth muscle in between the cartilaginous portions on the edges. The preparation is then mounted in a 20 mL tissue bath containing Krebs solution, at 37°C and aerated with carbogen (5% CO₂ in 95% O₂). A tension of 1 g is applied to each of the tracheal strips and is kept constant throughout the experiment. The tissue is equilibrated for 1 h before the addition of any drug. The tracheal preparations are then constricted with carbachol, histamine and potassium and the relaxant effect of a drug is assessed by adding in a cumulative fashion.

Lung parenchyma slicing

Male guinea pigs weighing 250-350 g are killed by cervical dislocation. The thoracic cavity is opened and the lungs are removed. A 1.5 mm x 20 mm strip of subpleural parenchyma is cut from an area of grossly normal lung and prepared for recording of contractile responses. The tissues are placed in organ baths containing physiological salt solution and aerated with carbogen. One end of each tissue is tied with a silk thread to a glass hook located at the bottom of the organ bath and the other end is connected by a silk thread to a force transducer. An initial load of 1 g is applied to each of the lung parenchyma strips and equilibrated for 1 h before the addition of any drug, then the preparations are constricted with different spasmogens, such as carbachol, histamine or potassium, to assess the bronchodilator effect.

Sensitisation procedure

Guinea pigs are sensitised by intraperitoneal injection of 5 mL of 0.9% saline containing 10 µg of oval-bumin dispersed with 1 mg of aluminium hydroxide. The injection is repeated after 14 days and the animals are killed 7-10 days after the second injection. After removing the trachea from the adjacent tissues, preparations are mounted for isometric recording. Following the equilibration period the tissues are constricted with carbachol. After 30 min tissues are exposed to ovalbumin (1-3 g/mL) and a contraction of the trachea confirm that the guinea pigs are successfully sensitised. The test drug is preincubated with the preparation 20 min before ovalbumin addition.

Mast cell stabilisation assay to assess prevention potential

The rats are sensitised by subcutaneous injection of horse serum along with 0.5 mL of triple antigen containing Bordetella pertussis organisms. The rats are divided into eight groups of six and treated with either saline, positive control (prednisolone or ketotifen) or the different doses of the test drug or plant extract. On the 14th day, 3 h after the last dose treatment, the rats are killed, and intestinal mesentery is taken for study of mast cells. Mesenteric and intestinal pieces are kept in a Ringer Locke’s solution at 37°C. Mesenteric pieces are then challenged with 5% horse serum in vitro for 10 min. Pieces of mesentery are stained supravitally with toluidine blue. Tissue is first immersed in 0.1% toluidine blue in 4% aqueous formal saline for 10 min. The tissue is then transferred to xylene for 5-10 min and finally rinsed two or three times with acetone and examined under a microscope. The numbers of intact and disrupted mast cells per high field are counted.

According to the WHO, about three-quarters of the world population relies on traditional remedies (mainly herbs) to fulfil their healthcare needs (WHO, 2003). In developing countries, remedies prepared by traditional healers from plants of the local flora are the only drugs available for a large number of people. It is not uncommon that a single plant possesses a wide range of medicinal applications. Several scientific studies, in parallel to this, have also shown the presence of synergistic and/or side-effect-neutralising combinations in plants, which is the result of the presence of multiple constituents in a single plant. Herbs have always played an important role in the treatment of respiratory diseases and Atropa belladonna is one such example, with folkloric repute to treat asthma. This species is the source of atropine, a prototype antimuscarinic drug, which has a wide range of clinical applications including asthma.

Chinese traditional medicine has also claimed to treat many diseases, especially bronchial asthma. The number of plants considered useful for respiratory disorders is surprisingly large. Some of the species, which have been shown to be effective in the disorders of airway hyperactivity in experimental animals or clinical trials, confirming their traditional use in respiratory disorders, are listed in Table: Studies on medicinal plants useful in respiratory disorders.

Table: Studies on medicinal plants useful in respiratory disorders

Plants mentioned for their effect against potassium-induced contraction are considered to be calcium-channel blockers (CCBs) as high K⁺ (>30 mmol/L) is known to cause smooth-muscle contraction via Ca²⁺ influx through voltage-dependent calcium channels. Interestingly, we found some novel combinations and observed that CCB-like constituents are abundantly present in plants and usually coexist with other active constituents, such as phosphodiesterase inhibitors and anticholinergics.

The drugs used in conventional medicine as bronchodilators in asthmatic conditions belong to the catagories of β₂-adrenoceptor stimulants (salbutamol), phosphodiesterase inhibitors (theophylline) and anticholinergics (ipratropium), all of which cause cardiac stimulation as a side-effect. Calcium antagonists (which are devoid of cardiac stimulant effect) have been shown to possess therapeutic usefulness in asthma in recent years. These have an inhibitory effect, opposite to what is seen with currently used anti-asthmatic drugs. Hence, it is logical to combine Ca²⁺ antagonists with any of the above mentioned bronchodilators to achieve an enhanced bronchodilator effect with neutralisation of cardiac side-effects.

Based on the assumption that medicinal plants do contain such combinations of activities, a few plants with folkloric use in asthma were screened. Interestingly, two popular plants, commonly used in asthma and cough, i.e. turmeric (Curcuma longa) and St John’s wort (Hypericum perforatum) were found to possess bronchodilatory effects through the combination of CCB and phosphodiesterase inhibitory activities. Similarly, the essential oil of Nepeta cataria also causes bronchodilation through a combination of the same inhibitory mechanisms (unpublished data). We observed in several patients that turmeric, when mixed with honey, caused cough suppression (unpublished data). In a clinical trial, garlic (Allium sativum) was found to be very effective in patients with asthma, which also results from its in-vitro tracheorelaxant effect.

Henbane (Hyocyamus niger) is smoked to relieve asthmatic attack. Carefully planned experiments revealed that in addition to the anticholinergic activity, it contains Ca²⁺ antagonist constituent(s), responsible for its bronchodilator and antispasmodic actions, thus offering a novel combination of activities, which explain its medicinal use in asthma. In a clinical study, Terminalia bellerica was found to possess antiasthmatic and antitussive effects. More recent studies showed that the underlying mechanism(s) for effectiveness in airway hyperactivity disorders is the dual blockade of muscarinic receptors and Ca²⁺ channels. Carum copticum, a popular herbal remedy in airway hyperactivity disorders, has been shown to possess a combination of anticholinergic, antihistaminic and calcium-channel blocker activities.

As mentioned above, the side-effect profile of anticholinergics and Ca²⁺ antagonists in the heart oppose each other and it would not be surprising to see if the net effect of this combination is more than the sum of the individual components, with lesser side-effects when studied in a clinical set up. The boiled extract of Carum copticum caused a significant increase in pulmonary function test values in patients with asthma. The aqueous and macerated extracts of Carum copticum reduced the number of coughs in guinea pigs produced by citric acid aerosol, being greater than that of the standard drug, codeine. Aspalatbus linearis (rooibos or bush tea) was found to exhibit bronchodilatory effect through the opening of potassium channels. Interestingly enough, one of its known constituents, chrysoeriol, was found to exhibit high selectivity for its inhibitory effect on airways compared with other smooth muscles, thus showing the potential to be developed for airway disorders.

Nigella sativa (black seeds) are used in folk medicine for its usefulness in airway disorders, along with multiple other medicinal uses. The volatile oil of N. sativa induced a dose-dependent increase in the respiratory rate and intratracheal pressure of guinea pigs. It has been reported that Nigella sativa exhibits mast-cell stabilising (inhibiting the release of histamine) and Ca²⁺ antagonist effects. The results of a Phase I study, involving 29 adults with asthma, generally suggest a prophylactic effect of boiled extract of Nigella sativa on asthma disease.

Ginger (Zingiber officinale) exhibited antiasthmatic and antitussive effects in patients with airways hyperactivity. It was reported to possess a Ca²⁺ antagonist mechanism, which might be responsible for the bronchodilatory action, though additional mechanism(s) cannot be ruled out. Sarcococca saligna and Borago officinalis were also found to exhibit CCB effect, which may account for their antiasthmatic potential.

Randomised trials with ginkgo (Ginkgo biloba) liquor and dried extract of ivy (Hedera helix) produced clinically relevant improvements in lung function, significantly better than placebo or control treatment. Four trials with Tylophora indica alcoholic extract reported significant improvement in asthma symptoms. One clinical trial with Boswellia serrata resulted in a significant improvement in forced expiratory volume of patients with asthma. In a double-blind trial, 32 people with steroid-dependent asthma were given either placebo or essential oil of eucalyptus for 12 weeks. The results showed that people using eucalyptus were more able to reduce their steroid dosage gradually than those taking placebo. In a clinical study, the powder of Lepidium sativum dried seeds was found to increase the forced vital capacity, forced expired volume and peak expiratory flow rate in patients with mild-to-moderate bronchial asthma, indicating its usefulness in reducing asthma severity.

We also observed the bronchodilatory potential of some of the well-known naturally occurring compounds, such as curcumin, catechin and thymoquinone, with common characteristics being a calcium-channel blocker effect along with phosphodiesterase inhibitory activity for catechin (unpublished data). Theophylline, a plant-derived compound is well known to exhibit a bronchodilator effect through inhibition of phosphodiesterase and adenosine receptors.

Knowing that the aetiology of respiratory disorders is complex, where multiple sites are targeted for better control, medicinal plants in singular or poly-herbal formulation (contain multiple active components acting on different sites) have the potential for preventive and curative therapeutic success. A limited number of clinical trials have been conducted to date. Further studies in this direction are likely to result in novel additions to modern therapeutics.