key: cord-0049814-qhoi8st5
authors: Sakai, Hiroyasu; Misawa, Miwa
title: Effect of Sodium Azulene Sulfonate on Capsaicin‐Induced Pharyngitis in Rats
date: 2005-01-24
journal: Basic Clin Pharmacol Toxicol
DOI: 10.1111/j.1742-7843.2005.pto960108.x
sha: 87393fb1e93940a9cf6915c6122f3fea88cbd4ab
doc_id: 49814
cord_uid: qhoi8st5

Abstract: Sodium azulene sulfonate is a water‐soluble derivative of azulene which is an antiinflammatory component of chamomile of the family of Asteraceae. Sodium azulene sulfonate is clinically used as a therapeutic agent in the treatment of pharyngitis as well as other inflammatory diseases such as tonsillitis, stomatitis and conjunctivitis. There has been no documentation on the effect of sodium azulene sulfonate on pharyngitis in laboratory models, probably because of no availability of such models. We recently established a pharyngitis model using capsaicin application on pharyngeal mucosa in rats. The present study investigated the antipharyngitis activity of sodium azulene sulfonate comparing with those of ruthenium red (vanilloid receptor antagonist, 8.5 and 85 mg/ml), ascorbic acid (antioxidative compound, 100 μg/ml), povidone iodine (gargle as disinfectant, oxidative compound, 5 and 20 mg/ml) and diclofenac sodium (cyclooxygenase inhibitor, 0.1 and 1 mg/ml). As an antipharyngeal effect, the capsaicin‐induced plasma exudation in the pharyngeal mucosa of the rat was evaluated. The capsaicin‐induced plasma exudation in the pharyngeal mucosa was inhibited by sodium azulene sulfonate (100 and 200 μg/ml) as well as ruthenium red and ascorbic acid, but not by povidone iodine and dicrofenac sodium; povidone iodine rather promoted the plasma exudation. In conclusion, the antipharyngitis effect of sodium azulene sulfonate was demonstrated for the first time in a laboratory model. Although the mechanism by which sodium azulene sulfonate inhibited the capsaicin‐induced pharyngitis is not yet unraveled, antioxidative effect, but not inhibitory effect on cyclooxygenase pathway, might be involved.

The pharyngitis is a common inflammatory disease of the oropharynx presenting symptoms of sore throat, erythema and chapping of throat, which is attributed predominantly to infections of viruses and bacteria. Irritation and sore of throat accompanied by mild oedema and erythema of the pharynx are present in about 80% of patients with the common cold syndrome due to viruses such as rhinoviruses, coronaviruses, influenza viruses, parainfluenza viruses and adenoviruses, and due to bacteria including Streptococcus pyogenes (Lang & Singh 1990; Peter 1992) . Cigarette smoking also becomes a cause of sore throat. The complaint of pharyngitis is responsible for an estimated 40 million outpatient visits in the United States in a year (Vukmir 1992) .

Sodium azulene sulfonate is a water-soluble derivative of azulene that is an anti-inflammatory component of chamomile, Matricaria recutita, Asteraceae. Azulene and sodium azulene sulfonate are clinically used for the medical treatment of pharyngitis as well as gastric ulcer, gastritis, conjunctivitis, aenoiditis and stomatitis. It has been reported that guaiazulene, a lipophilic azulene derivative, shows not only an antiinflammatory effect (Yanagisawa et al. 1990) but also an antioxidative effect (Kourounakis et al. 1997 ). Thus, a possibility that sodium azulene sulfonate, which is a guaiazulene relative compound, also has an antioxididative Author for correspondence: Hiroyasu Sakai, Department of Pharmacology, School of Pharmacy, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan (fax 81 3 5498 5787, e-mail sakai/hoshi.ac.jp). effect can be presumed. An antioxidative effect may inhibit the tissue damage induced by the nimious oxygen radicals which leukocytes and macrophages produce in inflammatory process, and may bring about antiinflammatory effect (Nowak et al. 1991) . Recently, the microbes might be killed by proteases, activated by oxidase through the generation of a hypertonic, K π -rich and alkaline environment in the phagocytic vacuole (Reeves et al. 2002) . Furthermore it was showed that K π crosses the membrane through large-conductance Ca 2π activated K π channel in neutrophil (Ahluwalia et al. 2004) .

Capsaicin is known to be the prototype of neurogenic irritants. Topical application of capsaicin to rat skin leads to excitation of afferent neurons (Kinins 1982) , increase in skin blood flow (Inoue et al. 1993) , and vasodilation (Lynn et al. 1992) . Neuropeptides such as substance P and neurokinin A, which are released by capsaicin from peripheral endings of afferent neurones via vanilloid receptor (Caterina et al. 1997) , have been considered as chemical mediators of skin inflammation (Holzer 1988 (Holzer & 1991 Maggi & Meli 1988; Saria et al. 1988) . Substance P which is a tachykinin found in the C-fiber nerve endings of the airways of a variety of species including man, influences several airway functions; it increases mucus secretion, epithelial chloride secretion, and vascular permeability and stimulates airway smooth muscle contraction (Martling 1987) . Though suitable animal models for studying pharyngitis and for development of effective drugs for the disease have not hitherto been reported, we lately established a pharyngitis model 55 EFFECT OF SODIUM AZULENE SULFONATE ON PHARYNGITIS using capsaicin in rats. In the capsaicin-induced pharyngitis, tachykinins were mainly involved and a participation of NK 1 receptor was suggested (Yamabe et al. 1998) .

Prostaglandin E 2 , a prostanoid derived from arachidonic acid metabolism through the enzymatic action of cyclooxygenase and prostaglandin E 2 synthase, is released from a number of cells in the respiratory organs during various airway inflammatory reactions (Holtzman 1991) . The airway epithelium, which is the primary target of initial assault by the inhaled irritants, is also the major cellular source of prostaglandin E 2 (Holtzman 1991) . The prostaglandin E 2 activates sensory ending in the lungs; for example, inhalation of aerosolized prostaglandin E 2 elicits coughs and retrosternal soreness (Costello 1985; Taguchi 1992) . Furthermore, inhaled prostaglandin E 2 enhances the sensitivity of the cough reflex elicited by capsaicin in man (Choudry et al. 1989) , suggesting a prostaglandin E 2 -induced sensitization of pulmonary C-fiber afferents. Prostaglandin E 2 generated by cyclooxygenase may therefore participate in the capsaicin-induced pharyngitis.

In the present study, we investigated the effects of sodium azulene sulfonate on the capsaicin-induced pharyngeal plasma exudation in rats. Furthermore, in order to investigate the mechanism(s) of the capsaicin-induced plasma exudation, the effects of ruthenium red (vanilloid receptor antagonist), ascorbic acid (antioxidative compound), povidon iodine (oxidative compound) and diclofenac sodium (cyclooxygenase inhibitor) were also examined.

Animals. Animals were housed for appropriate time intervals in the animal center of Hoshi University after their arrival. Constant temperature and humidity (22∫1 ae, 55∫10%) were maintained with a fixed 12 hr light-dark cycle and free access to food and water. Experiments were performed under the guiding principles for the care and use of laboratory animals approved by the Animal Care Committee of Hoshi University (Tokyo, Japan).

Effects of sodium azulene sulfonate and several drugs on pharyngeal plasma exudation induced by capsaicin. Male Wistar rats, weighing 280-490 g (Tokyo Laboratory Animal Co., Japan), were used. Animals were anaesthetized with urethane (2 g/kg, intraperitoneally), placed in the supine position and given spontaneous respiration through a tracheal cannula after treatment with atropine sulfate (0.2 mg/kg, intraperitoneally). To study the effects of the drugs on the capsaicin-induced pharyngeal plasma exudation, rats were administered with a 0.5 ml drug solution into the oral cavity 30 min. prior to capsaicin treatment, after binding the upper part of the oesophagus and the trachea with thread. The treatment drugs were as follows: Azunol A (1:800, 1:400 and 1:200 dilute distilled water; 50, 100 and 200 mg/ml as sodium azulene sulfonate), vehicle of Azunol A , ruthenium red (8.5 and 85 mg/ml, vanilloid receptor antagonist), ascorbic acid (100 mg/ml), povidone iodine (5 and 20 mg/ ml), diclofenac sodium (0.1 and 1 mg/ml) and saline as a control. Experimental pharyngitis was induced by application of capsaicin solution with a cotton-tripped applicator on the surface of pharyngeal mucosa. Before capsaicin application, oral cavity was washed two times with 0.5 ml saline. When the capsaicin solution was applied, the tongue was slightly pulled out with a foreceps and the pharynx area was opened deep in the oral cavity with a small rib spreader. A 0.3 mM (0.25 ml) capsaicin-soaked cotton was swabbed each for about 3 sec. gently totally three times. Because capsaicin was dissolved in a mixture of 10% ethanol-10% Tween 80-80% distilled water, the rats in the control group were given vehicle alone. After capsaicin solution was applied, a period of 60 min. was allowed before evaluation of plasma exudation. For a quantitative evaluation of the capsaicin-induced plasma exudation in the rat pharyngeal mucosa, extravasation of Evans blue dye into the pharyngeal tissue was determined. Evans blue dye (30 mg/kg, intravenously) was injected into the femoral vein 10 min. prior to the application of capsaicin. Sixty min. after the capsaicin application, exsanguination was done from the abdominal aorta. Each animal's head portion was perfused with 180 ml of citric acid buffer (5% of paraformaldehyde in 0.05 M sodium citrate solution adjusted to the pH 3.5 with 0.05 M citric acid solution) at a rate of 15 ml/min. via the bilateral carotid arteries to expel the intravascular dye; the perfused buffer being eliminated from an incision of the right atrium. Then, the bilateral musculus masseter of the rat was incised and the lower jaw was removed to enable extirpation of the pharynx. The pharyngeal mucosa was isolated by separation from the oesophagus and trachea; the soft palate, tongue, larynx and nasal tissues were removed. The isolated pharynx contained the portion ranging from the caudal end of the soft palate to the epiglottis just at the beginning of the larynx, and weighed 60-90 mg. Evans blue dye in the tissue was extracted in formamide at 60 ae for 24 hr and determined spectrophotometrically at 620 nm. The tissue dye content was expressed as micrograms of dye per gram of wet weight of tissue. Statistical analyses. All data are expressed as mean∫S.E. Statistical significance was determined by one-way analysis of variance (ANO-VA). ANOVA was carried out with Bonferroni/Dunn's test.

The amount of Evans blue leakage increased significantly in the capsaicin-treated group (61.2∫5.4 mg/g) compared with that in the control group (vehicle of capsaisin, 25.4∫1.2 mg/g, PϽ0.001) ( fig. 1 ). Sodium azulene sulfonate in concentrations of 100 and 200 mg/ml significantly inhibited the capsaicin-induced pharyngeal plasma exudation (43.1∫3.3 mg/g and 45.7∫2.1 mg/g, ratio of inhibition: 50.6∫9.3 and 43.4∫6.0%, IC50Ω64.1 mg/ml, PϽ0.01 and PϽ0.05, respectively). Amount of the capsaicin-induced Evans blue leakage was significantly decreased by ruthenium red (antagonist of vanilloid receptor) in concentrations of 8.5 mg/ml and 85 mg/ml (42.1∫6.8 and 33.3∫3.4 mg/g, ratio of inhibition: 53.4∫19.1 and 77.9∫23.0%, PϽ0.05 and PϽ0.01, respectively) in a concentration-dependent manner ( fig. 2) . The finding suggestes that the increase in Evans blue leakage by capsaicin was attributed to the effects mostly via vanilloid receptors. Pretreatment with 100 mg/ml of ascorbic acid (antioxidative compound) significantly decreased the capsaicin-induced Evans blue extravasation to 36.9∫1.8 mg/ g (ratio of inhibition: 67.9∫5.0%, PϽ0.01) ( fig. 3 ). Fig. 4 shows the effect of povidon idoine (oxidative compound) on the pharyngeal plasma exudation. Povidon iodine (20 mg/ml) significantly increased the amount of Evans blue leakage induced by capsaicin to 77.6∫8.7 mg/g (ratio of inhibition: ª45.8∫24.3, P∞0.05). Diclofenac sodium (cyclooxygenase inhibitor) (0.1 and 1 mg/ml) was without effect Fig. 5 . Effect of diclofenac sodium on capsaicin-induced Evans blue exudation in the rat pharynx. Diclofenac sodium (0.1 and 1 mg/ ml) was administered into the oral cavity 30 min. before capsaicin application. Pretreatment with dicrofenac sodium (20 mg/ml) was without effect on the capsaicin-induced the Evans blue exudation. Values are means∫S.E. from 6-10 experiments. Figure 1 on the capsaicin-induced Evans blue leakage (ratio of inhibition: 29.4∫11.8 and 26.0∫18.8%) ( fig. 5 ).

Capsaicin is known to cause excessive activation of primary afferent sensory neurones and to cause a release of neuropeptides including substance P and neurokinin A from nerve endings (Buck & Burks 1986) . Substance P and neurokinin A have potent proinflammatory effects in the airway tissues. Substance P is localized in afferent nerve terminals and sensory neuronal cell bodies of the trigeminal ganglion (Saria et al. 1988; Barnes 1987; Barnes et al. 1991 Barnes et al. & 1998 . After released from the afferent nerve terminals, substance P acts on NK 1 -receptors and mediates neurogenic inflammation. Neurogenic inflammation can be described as increased vascular permeability, plasma extravasation, glandular secretion and pro-inflammatory cell influx which are mediated by substance P. Recently, it has been proposed that the phenomena found in neurogenic inflammation may be partly mediated via activation of capsaicin vanilloid receptor 1 known as a polymodal receptor (Caterina et al. 1997; Tominaga et al. 1998; Trevisani et al. 2002) . Ruthenium red, a functional vanilloid receptor antagonist, inhibits the capsaicin effect on sensory neurons by an action on the plasma membrane to prevent opening of capsaicincoupled ion channels (Dray et al. 1990; Amann & Maggi 1991 ). In the present study, the capsaicin-induced pharyngitis was inhibited by ruthenium red, suggesting that capsaicin caused pharyngeal plasma exudation via vanilloid receptors. Indeed, we priviously reported that the capsaicininduced pharyngitis was abolished by tachykinins antagonists (Yamabe et al. 1998 ). In a preliminary study, we evaluated the binding activity of sodium azulene sulfonate to NK 1 and NK 2 receptor using radioligand binding assay. Sodium azulene sulfonate did not significantly bind to NK 1 and NK 2 receptors. It is thus suggested that sodium azulene sulfonate did not inhibit the capsaicin-induced pharyngitis by directly blocking NK 1 and NK 2 receptors. Diclofenac sodium which is one of the non-steroidal antiinflammatory drugs (NSAIDs) did not inhibit the capsaicin-induced pharyngitis. The result shows that cyclooxygenase products such as prostaglandin E 2 are not participating in the pharyngitis that capsaicin induces. This finding was in agreement with our previous report using indomethacin (Yamabe et al. 1998) .

Preliminarily we demonstrated that sodium azulene sulfonate inhibited lipid peroxidation in rat hepatic microsome study. The finding suggests that sodium azulene sulfonate has an anti-oxidative activity. Radical mediated impairment of cellular functions has been known to associate with a number of disorders. Since biological membranes contain highly oxidizable polyunsaturated fatty acids, they are particularly vulnerable to radical attack, and the oxidation of biomolecules such as lipids, proteins and DNA is considered to be critical in the development of inflammation.

It was observed that povidon iodine (20 mg/ml) rather worsened the capsaicin-induced pharyngitis. Povidon iodine which is widely used for the purpose of the disinfection by oxidative action to various types of pharyngitis is therefore considered to have a paradoxical aspect: disinfectant effect as a beneficial one, and enhancing effect on microvascular exudation as a disadvantageous one. The capsaicin-induced pharyngitis was inhibited by ascorbic acid, antioxidative compound. Thus, oxidative radicals such as superoxide may be mediated in the capsaicin-induced pharyngitis in rats. Although mechanism of the therapeutic effect of sodium azulene sulfonate on the capsaicin-induced pharyngitis in the present study is not enough explainable, the antioxidative effect might be involve in mechanism(s) of inhibition of capsaicin-induced pharyngitis by sodium azulene sulfonate.

In conclusion, using the capsaicin-induced pharyngeal plasma exudation model in rats, the beneficial effect of sodium azulene sulfonate on pharyngitis was for the first time demonstrated experimentally. Moreover, our present pharyngitis model was shown to be useful to screen effective drugs.

The large-conductance Ca 2π -activated K π channel is essential for innate immunity

Ruthenium red as a capsaicin antagonist

Neuropeptides and nasal secretion

Substance P and neurokinin A in human nasal mucosa

Neuropetptides in human airways: function and clinical implications

Neuropeptides in the respiratory tract. Part I

Inflammatory mediators of asthma: an uptake

The neuropharmacology of capsaicin: review of some recent observation

The capsaicin receptor: a heat-activated ion channel in the pain pathway

Pride: Sensitivity of the human cough reflex: effect of inflammatory mediators prostaglandin E 2 , bradykinin and histamine

Characteristics of prostaglandin induced cough in man

Ruthenium red blocks the capsaicin-induced increase in intracellular calcium and activation of membrane currents in sensory neurons as well as the activation of peripheral nociceptors in vitro

Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons

Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides

Sources of inflammatory mediators in the lung: the role of epithelial and leukocyte pathway for arachidonic acid oxygenation

Lung biology in health and disease series

Profile of capsaicin-induced mouse ear oedema as neurogenic inflammatory model: comparison with arachidonic acid-induced ear oedema

Responses of single nerve fibers to capsaicin applied to the skin

Antioxidant activity of guaiazulene and protection against paracetamol hepatotoxicity in rats

The sore throat: when to investigate and when to prescribe

The actions of capsaicin applied topically to the skin of the rat on C-fiber afferents, antidromic vasodilation and substance P levels

The sensory-afferent function of capsaicinsensitive sensory neurons

Sensory nerves containing tachykinins and CGRP in the lower airways

Effect of ascorbic acid on hydroxyl radical generation by chemical, enzymatic and cellular systems. Importance for antioxidant prevention of pulmonary emphysema

Streptococcal pharyngitis: current therapy and criteria for evaluation of new agents

Killing activity of neutrophils is mediated through activation of proteases by K π flux

Release of multiple tachykinins from capsaicin-sensitive sensory nerves in the lung by bradykinin, histamine, dimethylphenyl piperazinium and vagal nerve stimulation

Prostaglandin E 2 inhalation increases the sensation of dyspnea during exercise

The cloned capsaicin receptor integrates multiple pain producing stimuli

Ethanol elicits and potentiates nociceptor response via the vanilloid receptor-1

Adult and pediatric pharyngitis: a review

A new pharyngitis model using capsaicin in rats

Synthesis and anti-ulcer activities of 6-isopropylazulene-I-sodium sulphonate derivatives