Samane Arefani ¹, Seyyed Mohammad Mohseni Mehran ², Hassan Moladoust ³, Mohammad Reza Norasfard ¹, Ahmad Ghorbani ⁴, Mahmood Abedinzade ^{5 *}

Diseases of the respiratory system are one of the main causes of death and include situation such as chronic obstructive pulmonary disease, pneumonia or asthma. Medicinal plants have beneficial effects on multiple diseases include respiratory disorders like asthma and bronchitis. The aim of this study was to evaluate the effects of U. dioica and L. Album on tracheal smooth muscle contraction.

Hydroalcoholic extracts of L. Album and U. Dioica aerial parts were prepared by maceration method and standardized based on their total phenol content. The effect of the extracts on the KCl-induced contraction of rat trachea was examined in an organ bath chamber. Data was analyzed with spss software 22.

The extract of L. Album (5 mg/ml), similar to theophylline (20 mM), significantly reduced the KCl-induced tracheal contraction. On the other hand, U. Dioica (1 mg/ml and 5 mg/ml) augmented the KCl-induced contraction.

The relaxant effect of L. Album on the trachea makes it as a candidate for the managing patients with asthma and obstructive pulmonary diseases. But because of U. Dioica potential constrictive effect on the trachea it is suggested that patients avoid consuming it.

Asthma; herbal medicine; lung.

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Ischemic heart disease, stroke, chronic obstructive lung disease and lower respiratory infections have remained the top killers during the past decade. Chronic diseases cause increasing numbers of deaths worldwide. According to WHO estimates, 235 million people suffer from asthma. Asthma is the most common chronic disease among children. Asthma is not just a public health problem for high income countries: it occurs in all countries. Over 80% of asthma deaths happen in low and lower-middle income countries. Asthma is under-diagnosed and under-treated, creating a substantial load to individuals and families and possibly restricting individuals’ activities for a lifetime [1]. Airway smooth muscle is responsible for the acute constriction of the trachea and bronchi in asthma and chronic obstructive pulmonary disease (COPD). Therefore, it is the main target in the pharmacotherapy of these diseases. Agonists of β2-adrenoceptors and muscarinic antagonists are currently available drugs for rescue from airflow obstruction in asthma and COPD [2, 3]. Considering developing tolerance to these drugs and their potential adverse effects, the search for new bronchodilator agents is of great interest [4, 5]. In this context, the relaxant effect on airway smooth muscle has been reported for several medicinal plants and some natural polyphenols [6-8]. L. Album L. (White dead nettle) is a polyphenol-rich medicinal plant in the family of Lamiaceae [9]. Experimental studies showed that the Album has antioxidant, antimicrobial, hypoglycemic, cytoprotective, and anticancer activities [10-13]. Also, beneficial effects of L. Album have been reported on respiratory disorders including asthma and bronchitis [14]. U. Dioica, also known as stinging nettle, belonging to the family of Urticaceae. It has been shown that U. Dioica has hypoglycemic, antioxidant, antimicrobial, analgesic, and anticancer effects [15-17] Recently, Qayyum et al. Observed that U. Dioica extract induces a relaxant effect on the rabbit thoracic aorta. However, until now, there is no report on the effects of U. Dioica on airway muscle contraction [18]. Since there was no available data about the effects of L. Album and U. Dioica extracts on the trachea, the aim of this work was to test the potential relaxant effect of these extracts on the trachea in organ bath condition.

The plants L. Album and U. dioica (Fig. 1) were collected from Rasht region in Guilan Province, Iran. They were identified by Fatemeh Yousefbeyk, Department of Pharmacognosy, Guilan University of Medical Sciences, Iran (voucher specimen No. 202HGUM and 156HGUM for L. Album and U. Dioica, respectively). For each plant, the hydroalcoholic extract was prepared by macerating the powder of stems and leaves in 50% ethanol (10 ml/g powder) for 72 h at 40 °C [19]. Then, the extracts were filtered through mesh 250 μm, centrifuged 10 min at 2000 RPM, and their supernatants were dried on a water bath (40 °C).

The extracts were standardized based on their total phenol content by the Folin-Ciocalteu method. A sample of each extract (20 μL of 10 mg/ml) was mixed with the Folin-Ciocalteu reagent (100 μL) and sodium carbonate solution (300 μL, 1 M), and the volume was adjusted to 2 ml with deionized water [20]. After 2 h, the absorbance was measured by the spectrometer at 765 nm. The standard curve was prepared for Gallic acid and the total phenol content of the extracts was expressed as milligram of Gallic acid equivalents.

Male albino Wistar rats (200-250 g) were used in this study. They were kept in a standard room with controlled lighting (12 h light and 12 h darkness) at a temperature of 22 ± 2°C. The animals were having free access to food and tap water ad libitum. The protocol of this study complied with the guidelines of the animal care of Guilan University of Medical Sciences.

The animals were anesthetized with an intraperitoneal injection of thiopental (50 mg/kg). The chest was opened and a piece of the trachea (length 1 cm) was removed and placed in cool and oxygenated Krebs solution (37°C, pH = 7.4). Connective tissues were carefully removed and the trachea was split into two pieces. Each piece of the trachea (5 mm) was mounted between the two hooks, one fixed at the bottom of the organ bath and the other connected to an isometric transducer. The organ bath contained Krebs-Henseleit solution (NaCl 120 mM, KCl 4.72 mM, CaCl2 2.5 mM, MgSO4 0.5 mM, NaHCO3 25 mM, KH-2Po4 1.2 mM, glucose 11 mM, pH 7.4). An initial tension of 1.5 g was kept for all stages of the experiments and an acclimatization period of 60 min was used. Contraction of the trachea was induced by KCl (60 mM). When the contraction reached a plateau, 0.1 ml of L. Album extract (0.1, 1 and 5 mg/ml), U. Dioica extract (0.1, 1 and 5 mg/ml), theophylline 20 mM (Sigma, T1633), or saline was added to organ bath and the changes in contractile force were recorded.21 Each incubation was repeated six times.

Data were analyzed with Spss software 22 with One-way analysis of variance and Turkey’s tests. The results are presented as mean ± SEM and p < 0.05 was considered statistically significant.

Physio-chemical properties of L. Album and U. Dioica extracts are shown in (Table 1) The solid residue of macerated hydroalcoholic extracts of L. Album and U. Dioica was 16.5% and 12.5%, respectively. Total phenolic compound concentration was 70 mg and 60 mg Gallic acid equivalent per gram of the crude extract for L. Album and U. Dioica, respectively.

The tracheal smooth muscle contraction induced by KCl was set at 100%. As expected, theophylline as positive control significantly inhibited (26 ± 2.5%, p < 0.001) the KCl- induced contraction (Fig. 1) Similarly, the extract of L. Album at a concentration of 5 mg/ml exhibited a significant relaxant effect (16 ± 3.1 %, p < 0.05) on the trachea (Fig. 2)

Figure 4 shows the effect of U. Dioica extract on tracheal smooth muscle contraction. This extract at concentrations of 1 mg/ml and 5 mg/ml augmented the KCl-induced contraction of 11 ± 1% (p < 0.05) and 18 ± 1.6% (p < 0.05), respectively.

In the present work, the effects of L. Album and U. Dioica extracts on the contraction of the trachea were evaluated in organ bath condition. First, we standardized the hydroalcoholic extracts of these plants based on their total phenol content and found that they are polyphenol-rich medicinal plants. With regard to our findings, previous studies revealed the presence of various phenolic compounds such as chlorogenic acid, caffeic acid derivatives, rutin, and quercetin 3-O-glucoside in L. Album and U. Dioica [9, 10, 22]. The results of the present work for the first time showed that the extract of L. Album, similar to theophylline, reduced the KCl-induced tracheal contraction. The relaxing effects of phenolic compounds such as rutin, quercetin, and caffeic acid derivatives on human isolated bronchus have been previously reported [6, 23]. Therefore, the relaxant effect of L. Album extract on trachea might be induced by its phenolic compounds. The exact mechanisms by which L. Album relaxes trachea need to be determined by further studies. Previous studies revealed that the relaxant property of medicinal plants on tracheal smooth muscle could be achieved by calcium channel-blocking effect, potassium channel- opening activity, inhibitory effect on muscarinic receptors, histamine (the H1 receptor) antagonistic activity, stimulatory effect on β2-adrenoceptors, and methylxanthine activity [7]. But the present study showed that U. Dioica extract augmented the KCl-induced contraction of the trachea. In the literature, there are some conflicts about the effect of U. Dioica on the smooth muscle contraction. Dizaye et al. Reported that aqueous extract of the U. Dioica had no significant effects on the smooth muscle of the bladder and pulmonary artery in the rabbit [24]. Qayum et al. Showed that methanolic extract of the U. Dioica has a relaxant effect on phenylephrine-induced contraction in rat aorta [18]. where as Collier and Chesher revealed that U. Dioica string contains acetylcholine, histamine, and 5-hydroxytryptamine, and evokes contraction in the uterus of rats [25]. These controversies may be due to the differences in the method of extraction, parts of the plant used for extraction, animal species, and source of tissues for smooth muscle isolation. In conclusion, the results of the present study suggest that the album has a relaxant effect on the tracheal smooth muscle and might be a candidate for the management of patients with asthma and obstructive pulmonary diseases. Also, it is suggested that these patients, avoid consumption of the U. Dioica because of its potential constrictive effect on the trachea.

This work was financially supported by the Guilan University of Medical Sciences, Rasht, Iran and extracted from MSC student thesis. The authors declare that they have no competing interests

Research deputy of Guilan University of Medical Sciences.

All authors read the manuscript and declare that there is no conflict of interest.

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