|Year : 2016 | Volume
| Issue : 3 | Page : 256-263
Evaluation of antitussive and anti-asthmatic activity of Tabernaemontana divaricata(L.) R. Br. Ex Roem. and Schult
Amit Kumar Srivastava1, Hemant Nagar1, Rajnish Srivastava2, Varsha Ahirwar1, Harinarayan Singh Chandel1
1 Department of Pharmacology, Truba Institute of Pharmacy, Bhopal, Madhya Pradesh, India
2 Department of Pharmacology, Moradabad Educational Trust Group of Institutions, Faculty of Pharmacy, Moradabad, Uttar Pradesh, India
|Date of Web Publication||30-Jan-2018|
Dr. Amit Kumar Srivastava
Department of Pharmacology, Truba Institute of Pharmacy, Karond Gandhi Nagar, Bhopal - 462 038, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: The study was aimed to investigate the antitussive and anti-asthmatic activities of ethanolic extract of Tabernaemontana divaricata (TDEE) leaves byin vivo andin vitro models. Recently, indole alkaloids (monoterpenoid indole alkaloids) have been approved as investigational new drug for clinical trial in respiratory diseases, and T. divaricata has already proven its potential for the presence of indole alkaloids. Materials and Methods: Acute toxicity studies of TDEE were performed in accordance with the Organization for Economic Cooperation and Development guidelines no. 425. The sensitized guinea pigs were screened out and divided into control, standard, and TDEE-treated groups. Anti-asthmatic activity of TDEE was assessed byin vitro guinea pig tracheal chain method andin vivo bronchoprotective test method using aminophylline as a standard drug. Taken codeine as standard, antitussive activity was evaluated by in vivo citric acid-induced tussive response. Results: TDEE was found to be safe up to 2000 mg/kg, body weight. TDEE exhibits maximum bronchi relaxation of 91.66% and 92.83% against acetylcholine and histamine-induced contraction, respectively. TDEE exhibited maximum and significant (P < 0.001) bronchoprotection of 42.28% at the dose level of 200 mg/kg, body weight. TDEE at aerosolic dose of 6% (w/v) exhibited decreased average cough frequency (4.83 ± 0.30) which is quite significant (P < 0.001) and effective as compared to standard drug codeine. Based on the histopathological evidences, TDEE-treated groups showed reduced inflammatory cell infiltration and had restored epithelial damage. Conclusion: The results of the study revealed the potent antitussive and anti-asthmatic activities of T. divaricata, which support its further implication for the treatment of cough-associated complications such as cough variant asthma.
Keywords: Acetylcholine, aminophylline, bronchoprotection, codeine, histamine, Tabernaemontana divaricata
|How to cite this article:|
Srivastava AK, Nagar H, Srivastava R, Ahirwar V, Chandel HS. Evaluation of antitussive and anti-asthmatic activity of Tabernaemontana divaricata(L.) R. Br. Ex Roem. and Schult. AYU 2016;37:256-63
|How to cite this URL:|
Srivastava AK, Nagar H, Srivastava R, Ahirwar V, Chandel HS. Evaluation of antitussive and anti-asthmatic activity of Tabernaemontana divaricata(L.) R. Br. Ex Roem. and Schult. AYU [serial online] 2016 [cited 2020 Dec 1];37:256-63. Available from: https://www.ayujournal.org/text.asp?2016/37/3/256/224178
| Introduction|| |
Asthma is a chronic inflammatory disease characterized by the association of bronchial hyperresponsiveness, inflammation, and remodeling.,, Current medications are effective in treating acute airway narrowing and decreasing inflammation but are relatively less effective in preventing chronic structural changes. Asthma affects as many as 334 million people in the world. Further, avoidable asthma deaths are still occurring due to inappropriate management. The etiological basis for effective management of asthma needs bronchodilator action as muscle relaxant medication, anti-inflammatory as preventive medication, and long-acting β2-agonist for symptomatic control.,
Alkaloids are the leading pharmacological active phytoconstituents which have a nitrogen atom in a heterocyclic ring structure. About 12,000 different types of alkaloids are produced by the diverse species of plants, and a quarter from this large group of natural product accounts for indole alkaloids. Complex structures and diverse pharmacological activities have made the research on indole alkaloids more interesting and attractive including the investigation of biosynthetic pathways for many decades.
The ethnopharmacological relevance of Tabernaemontana divaricata reveals its antioxidant, anti-bacterial, anti-carcinogenic, analgesic and the enhancement of cholinergic activities in both peripheral and central nervous systems.T. divaricata also possess immunomodulatory effect as it decreases the cytokine expression of interleukin-1 beta and tumor necrosis factor alpha-α (TNF-α) in human, in vivo and in vitro murine models, respectively., This supports a novel approach for the treatment of allergic asthma via inhibition of the T-helper 2 derived cytokine expression, resulting in downstream effects on immunoglobulin E and eosinophils. Studies showed that expression of TNF-α is increased at both protein and mRNA levels in different inflammatory cells such as T-cells, eosinophils, mast cells, and macrophages in asthmatic airways. It has been also demonstrated that TNF-α is also responsible for the release of inflammatory mediators from human lung mast cells via the activation of transcription factors, such as nuclear factor-κB and activator protein 1., Increased expression of various inflammatory system genes leads to increased synthesis of inflammatory cytokines such as interleukin 10, interleukin 12, interferon γ, inflammatory receptors and adhesion molecules. The cough is considered to be the most common complication associated with the respiratory diseases such as asthma, bronchitis, and pneumonia, and the treatments available bring some inevitable side effects that synergize complications. A number of alkaloids from the different plant sources have been found to possess antitussive activity.,,
As a diverse source of alkaloids, the main objective of the study was to elucidate the pharmacological potential of T. divaricata for antitussive and anti-asthmatic activities so that it can be further implicated as the drug of choice for cough-associated respiratory complications such as cough variant asthma.
| Materials and Methods|| |
Drugs and chemicals
Gallic acid, quercetin, folin-ciocalteu reagent, and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) were purchased from Sigma-Aldrich, St. Louis, USA. Acetylcholine chloride (CAS number 60-31-1), histamine dihydrochloride (CAS number 56-92-8), aminophylline (CAS no. 317-34-0), and citric acid (CAS number. 77-92-9) were procured from HiMedia Pvt. Ltd. Mumbai, India.
Fresh leaves of T. divaricata for the present study were collected in the month of January from Bhopal, Madhya Pradesh, India. Plant material was identified and authenticated by the Head of Department, Department of Botany, Saifia Science College, Bhopal, and a specimen voucher (443/Bot/Saifia/13) was deposited at the Department of Pharmacognosy, Truba Institute of Pharmacy, Bhopal, for future reference.
The leaves were shade dried for 2 weeks and then pulverized to coarse powder and passed through sieve no. 20. Coarsely dried powder was first defatted with petroleum ether (60°C–80°C) for 72 h to remove fatty materials and then extracted with ethanol (95%) using Soxhlet apparatus for 36 h; the extract was collected and concentrated in vacuum under reduced pressure and the dried crude extract was stored at 4°C for further study.
Preliminary phytochemical screening
Ethanolic extract of T. divaricata leaves (TDEE) was subjected to various phytochemical tests for the identification of the phytoconstituents (carbohydrates, tannins, alkaloids, glycosides, flavonoids, steroids, proteins, and amino acids) present in the extract using standard procedures.,
In vitro free radical scavenging activity using 1-diphenyl-2-picryl-hydrazyl method
The free radical scavenging activity (FRSA) of TDEE was measured by DPPH method. 0.1 mM solution of DPPH was prepared in methanol, and 1 ml of it was added to different concentrations of TDEE (50, 100, 200, 400, and 500 μg/ml) and the final volume of 3 ml was made with methanol. The mixture was shaken vigorously and incubated at room temperature for 30 min. Absorbance of the resulting mixture was measured at 517 nm against methanol as blank, using a ultraviolet-visible spectrophotometer (Systronics, 2203, Japan). Each sample was evaluated in triplicate and results were represented as mean. The ascorbic acid was used as a standard antioxidant in this method. Percentage of DPPH FRSA was determined as follows:
The experiment was carried out on healthy guinea pigs (400–600 g). The animals were acclimatized to the standard laboratory conditions, fed with standard pellet diet and water ad libitum during the study. The study protocol was approved by the Institutional Animal Ethics Committee as per Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines, India (Approval number 1196/a/08/CPCSEA).
Acute oral toxicity study
The acute oral toxicity study was evaluated as per the Organization for Economic Cooperation and Development (OECD) guidelines no. 425, on guinea pigs (400–600 g) of either sex. Before the experiment, animals were fasted overnight with water ad libitum. Six animals were selected which received a dose of 2000 mg/kg, body weight. All six animals were given a single dose of 2000 mg/kg, body weight of TDEE by oral gavage. Animals were observed individually for any sign of toxicity, behavioral changes, and mortality after dosing, with special attention given during the first 4 h, and thereafter for 24 h, for a total period of 7 days.
In vitro method
Effect of Tabernaemontana divaricata on isolated guinea pig tracheal chain preparation
The male guinea pig was sacrificed by the overdose of sodium pentobarbital (100 mg/kg, body weight, i.p.) and was confirmed by cervical dislocation to isolate tracheal rings and at least 5–6 rings were tied together by their cartilage portion to form a tracheal chain and 12–14 such preparations were transferred to a dish containing Krebs's solution. The chain was mounted in a 20 ml organ bath containing Krebs–Henseleit (K–H) solution ([mM]: NaCl, 118.4; KCl, 4.7; KH2 PO4, 1.2; NaHCO3, 25.0; CaCl2, 2.5; MgSO4, 1.2; glucose, 11.1; pH 7.4 ± 0.05), at 35°C–37°C under 0.5 g tension and allowed to equilibrate at least for 1 h before commencing the experiment. During the experiment, the K–H solution was replaced after every 10 min. After the equilibrium period, contraction was induced by adding acetylcholine or histamine. Thereafter, standard aminophylline (1 μg/ml) and the TDEE at 2 mg/ml and 4 mg/ml were added serially as 0.2, 0.4, 0.6, 0.8, and 1 ml in cumulative doses and observed for bronchodilation using a Biopac MP 45, data acquisition system, respiratory transducers, and expressed as percent bronchorelaxation.,
In vivo method
Effect of Tabernaemontana divaricata on histamine and acetylcholine-induced respiratory distress: Bronchoprotective test
The sensitivity and suitability of the guinea pig for the study were screened by challenging each animal by an equal volume mixture of 0.1% histamine and 2% acetylcholine chloride in a histamine chamber under pressure of 450 ± 50 mmHg for 15 s. The onset of respiratory distress in seconds (preconvulsive) was carefully measured for each animal during an aerosol challenge. Animals having a pre-convulsive time >120 s were considered insensitive and unsuitable and not included in the study. The selected 24 male guinea pigs were divided into control, standard, and two test groups (n = 6). The control group animals received 5 ml/kg of 0.1% carboxy methyl cellulose (CMC), standard group animals received aminophylline (10 mg/kg, body weight) suspended in 0.1% CMC, and test group animals received 100 and 200 mg/kg, body weight of TDEE suspended in 0.1% CMC. All the treatments were given by the oral gavage route. Animals of all groups except control were pretreated with a single dose of their respective treatment (standard or test drug), daily for 3 days prior to the bronchial challenge while the last dose of the extract or standard drug was administered 1 h before the bronchial challenge. The delitescence/subsidence of convulsion and tumble numbers for each animal were recorded within a 6-min interval of exposure. Bronchial challenge by aerosol provoked a bronchospastic reaction in all the sensitive animals within 3 min. The delay in the appearance of the bronchospastic reaction was considered as bronchoprotective effect and expressed as percentage protection relative to the control group.
Where T1 is the preconvulsive breathing time (s) in the control group; T2 is the preconvulsive breathing time (s) in the treatment or standard group.
On the last day of the bronchoprotective test, lungs of guinea pig from each group were isolated; embedded in paraffin and tissue sections were stained using hematoxylin and eosin dye for histopathological studies.
Effect of Tabernaemontana divaricata on in vivo citric acid-induced cough response
Non anesthelized and unrestrained 24 male guinea pigs were selected for the study and were divided into four groups (n = 6). Each animal was individually placed in a transparent chamber of 30 cm × 20 cm × 20 cm and exposed to nebulized aqueous solution of 0.1 g/ml of citric acid (nebulizing rate: 0.7 ± 0.04 ml/min) continuously for 7 min. During the last 5 min of exposure, each animal was observed continuously and closely to determine the number of cough responses and there was a clear difference between cough and sneezing in sound and behavior of the animal as well. The above protocol was performed for every animal from each group for 10 min after exposing animals to the aerosol solutions of saline (for a baseline measurement), codeine solution (0.03 g/ml, standard), TDEE (3% w/v), and TDEE (6% w/v) for at least 7 min and was observed for cough response.,
All the experimental results were represented as mean ± standard error of mean. Data was analyzed using one-way analysis of variance followed by Tukey–Kramer multiple comparisons test. Statistical calculations were carried out using GraphPad InStat 3 software, San Diego, CA, USA. P < 0.05 was considered statistically significant in all the cases.
| Results|| |
Preliminary phytochemical investigation of TDEE revealed the presence of alkaloids, glycosides, flavonoids, tannins, triterpenoids, polyphenols, carbohydrates, and proteins [Table 1].
|Table 1: Phytochemical screening of ethanolic extract of Tabernaemontana divaricata leaves|
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In vitro antioxidant activity of Tabernaemontana divaricata by 1-diphenyl-2-picryl-hydrazyl method
DPPH is a well-known stable free radical used for the evaluation of FRSA. Reduction of DPPH by antioxidant compounds present in the plant extract produces a color change reaction from purple to pale yellow. The reduction of DPPH-free radicals could be assessed by determining its absorbance at 517 nm using a spectrophotometer. Although TDEE shows less antioxidant activity as compared to ascorbic acid, it was found to show a gradual increase in percentage inhibition with increasing concentration at 517 nm [Table 2] as an antioxidant with maximum inhibition of 64.85% at 500 μg/ml as compared to ascorbic acid (82.78%) at 500 μg/ml. TDEE exhibits significant difference (P < 0.0001) in antioxidant activity with corresponding concentrations in a dose-dependent manner as compared with ascorbic acid. Findings of in vitro FRSA study revealed the role of TDEE as a primary antioxidant and free radical inhibitor.
|Table 2: Antioxidant activity of ethanolic extract of Tabernaemontana divaricata leaves by 1,1-diphenyl-2-picrylhydrazyl assay|
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Acute oral toxicity study
Acute toxicity studies of TDEE were performed in accordance with the OECD 425, and the extract was found to exhibit a great margin of safety up to dose of 2000 mg/kg, body weight; there was no change in the behavioral pattern and no sign of toxicity and mortality was observed during the overall toxicity studies. Accordingly, 1/10th of this dose was considered to be the experimental safe dose.
Effect of Tabernaemontana divaricata on isolated guinea pig tracheal chain preparation
TDEE when added cumulatively to the organ bath containing either acetylcholine or histamine precontracted tracheal chain exhibited varying degrees of bronchorelaxation. TDEE exhibited maximum bronchorelaxation of 91.66% (P < 0.0001) and 92.83% (P < 0.1) against acetylcholine- induced and histamine-induced bronchocontraction, respectively, when compared with standard [Table 3] and [Table 4].
Effect of Tabernaemontana divaricata on histamine- induced and acetylcholine-induced respiratory distress: Bronchoprotective test
For the bronchoprotective test, first the sensitive guinea pigs were screened for the study by challenging them through an equal volume aerosolic mixture of 0.1% histamine and 2% acetylecholine chloride in a histamine chamber for 15 s. For this screening, 36 guinea pigs were randomly selected for aerosolic challenge and 33 animals were found sensitive and suitable for the study and the remaining three animals were found unsuitable for the study. After sensitivity screening, all the animals were re-randomized before treatment to reduce the error in-between the groups. In bronchoprotective test model, TDEE at the dose level of 100 and 200 mg/kg, body weight, showed significant bronchoprotection (P < 0.0001) with increased latency when compared with before treatment values [Figure 1]a. TDEE was also found to exhibit a potential bronchoprotection with reduced tumble numbers (P < 0.001) compared with control group [Figure 1]b. The percentage bronchoprotection exhibited by TDEE at 200 mg/kg, body weight, was quite significant as compared to the standard drug aminophylline (42.73%) so that we had obtained the order of potency, i.e., aminophylline ≥ TDEE at 200 mg/kg, body weight > TDEE at 100 mg/kg, body weight [Figure 1]c.
|Figure 1: Bronchoprotective effect of Tabernaemontana divaricata against histamine and acetylcholine induced bronchospasm. (a) Results are represented as mean α standard error of mean, n = 6 animals in each group, ****P < 0.001. (b) Results are represented as mean α standard error of mean, n = 6 animals in each group, a- significant difference versus control group and b- significant difference versus standard group, ***P < 0.001, ****P < 0.001, (c) Tabernaemontana divaricata at 200 mg/kg, possess significant percentage of bronchoprotection as compared to aminophylline|
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Effect of test drug on in vivo citric acid-induced cough response
TDEE at 3% and 6% w/v aerosolic dose against citric acid-induced tussive reaction was found to exhibit a significant (P < 0.001) reduction in cough response as compared to control [Figure 2].
|Figure 2: Antitussive effect of Tabernaemontana divaricata against citric acid-induced tussive response, all values are represented as mean ± standard error of mean, n = 6 animals in each group, data were analyzed by one-way analysis of variance using Turkey-Kramer multiple comparisons test, a- significant difference vs. control group and b- significant difference versus standard group, ***P < 0.001, ****P < 0.001|
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Histological examination reveals that the lungs of the control (bronchial challenged with no treatment) group animals showed a marked population of inflammatory cells surrounded by the damaged epithelium in alveolar walls, perivascular and peribronchial spaces [Figure 3]a. TDEE treatment markedly reduced the inflammatory cell infiltration around the damaged epithelium [Figure 3]c and d] as compared to control [Figure 3]a and standard [Figure 3]b.
|Figure 3: Histopathological sections of guinea pig lungs showing the effect of Tabernaemontana divaricata, (a) control group; epithelium (E); surrounded by peribronchial and perivascular inflammatory cells and blood vessels, (b) standard group; reduced epithelial damage with minimal inflammatory cells, (c) bronchial challenged group treated with Tabernaemontana divaricata (100 mg/kg); reduced inflammatory cells; improved airway with no epithelial repair, (d) Bronchial challenged plus treatment with Tabernaemontana divaricata (200 mg/kg) significant reduction in inflammatory cells, minute damaged epithelium was observed as compared to untreated group|
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| Discussion|| |
Various cellular elements such as T-lymphocytes, mast cells, neutrophils, and epithelial cells play a pivotal role in the pathophysiology of asthma. Due to the vast variety of the associated side effects of the currently available treatment options (bronchodilators and anti-inflammatory agents), herbal medicines proved their potential as safe and efficacious alternatives, for the treatment of asthma which is also supported by some of the clinical studies.
The present study was aimed to investigate the antitussive and anti-asthmatic activities of TDEE. Preliminary phytochemical screening of TDEE showed the presence of alkaloids, glycosides, flavonoids, tannins, triterpenoids, polyphenols, carbohydrates, and proteins while acute oral toxicity studies revealed the non-toxic nature of the extract for subsequent studies. We have also investigated the in vitro FRSA of TDEE by DPPH method and found that TDEE could act as a key antioxidant and free radical inhibitor which could be associated with the polyphenolics present in the extract.
Histamine is the most dominant factor for asthma  and an important mediator of bronchial muscle contraction and the obstruction of these may occur via H1 receptors. In addition, acetylcholine released from efferent nerve endings of the inner bronchus results in the excessive formation of inositol 1, 4, 5-triphosphate (IP-3) in bronchial muscles which leads to the intracellular release of calcium and initiate bronchoconstriction. It has been reported that bronchial acetylcholine and histamine H1 receptor blockade results in bronchodilation, which is considered as vital in the treatment of asthma. A prominent effect caused by both leads to a varying degree of bronchoconstriction that causes asphyxia and death. Bronchodilators can delay the occurrence of these symptoms.
The results of histamine induced and acetylcholine-induced tracheal chain contraction as well as bronchospasm depict the significant bronchorelaxant and bronchoprotective effects of TDEE, which suggest that the extract might have some histamine as well as cholinergic receptor antagonistic properties. Histological evidences also revealed the anti-inflammatory efficacy of the TDEE, namely, reduction in the inflammatory cell infiltration and repaired epithelium damage.
Cough is a very common complication that is mostly related to a number of pulmonary diseases. It is a normal physiological response to an irritation of the laryngo-tracheo-bronchial system caused by mechanical or chemical stimulation. It may be painful and fatiguing and require suppression by antitussive drugs. In animals, coughing has been elicited by various methods and mechanisms such as mechanically induced cough reflex in unanesthetized dogs, chemically induced cough response (sulfur dioxide-induced cough response) in guinea pigs and mice, ammonia-induced cough response in mice, and citric acid-induced coughing in guinea pigs.,,,,
In the present study, the antitussive activity of TDEE has been compared with that of standard drug codeine against coughing induced by chemical stimulation (citric acid) in guinea pigs. Guinea pigs were selected for in vivo citric acid-induced cough due to their afferent nerve reaction and similarity in cough production in response to citric acid like in humans. Tussive response induced by the citric acid was a result of the stimulation of the histaminergic and acetylcholinergic receptors followed by the bronchial hyperresponsiveness and hypersensitivity, tracheobronchial contraction and cough induction. The extract was found to show significant inhibition of cough as compared with standard treatment (codeine) in a dose-dependent manner. Alkaloids are widely reported for their mast cell stabilizing and α and β-adrenergic receptor stimulating property., Phytochemical analysis of the TDEE confirmed the presence of alkaloid and polyphenolics. The antitussive mechanism of TDEE in citric acid induced cough response was probably due to the mast cell stabilizing property via the inhibition of histaminergic and acetylcholinergic receptors located in the bronchial tract or by the activation of α and β-adrenergic receptors.
Excluding alkaloids, other important pharmacologically active secondary metabolites like polyphenolics, flavonoids and tannins are also thought to be key modulators of the histamine and acetylcholine-induced respiratory distress and tussive response by their well-known antioxidant, anti-inflammatory, expectorant and antimicrobial properties.,, On the basis of findings of the present study, observed anti-asthmatic and antitussive activity may be related to the presence of alkaloids and other secondary metabolites present in TDEE.
| Conclusion|| |
The results take the lead to provide the preliminary and pre-clinical experimental evidences demonstrating that the TDEE has anti-asthmatic activities in both in vivo and in vitro animal models and anti tussive activity in in vivo animal model. Even though the results obtained are not sufficient to prove the mechanism of the TDEE in the inhibition of allergic, inflammatory and tussive response. Further investigations are needed to clarify the exact bioactive phytoconstituents and the precise mechanism of the TDEE.
Authors are thankful to Truba Institute of Pharmacy, Bhopal, Madhya Pradesh, India for providing the necessary research facilities for the successful completion of study.
Financial support and sponsorship
Truba Institute of Pharmacy Bhopal.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]