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Tracheal relaxation through calcium channel blockade of Achillea millefolium hexanic extract and its main bioactive compounds
Journal Pre-proof Tracheal relaxation through calcium channel blockade of Achillea millefolium hexanic extract and its main bioactive compounds Luis Arias-Durán, Samuel Estrada-Soto, Monserrat Hernández-Morales, Fabiola Chávez-Silva, Gabriel Navarrete-Vázquez, Ismael León-Rivera, Irene Perea-Arango, Rafael Villalobos-Molina, Maximiliano Ibarra-Barajas PII:
S0378-8741(19)34431-9
DOI:
https://doi.org/10.1016/j.jep.2020.112643
Reference:
JEP 112643
To appear in:
Journal of Ethnopharmacology
Received Date: 5 November 2019 Revised Date:
26 January 2020
Accepted Date: 1 February 2020
Please cite this article as: Arias-Durán, L., Estrada-Soto, S., Hernández-Morales, M., Chávez-Silva, F., Navarrete-Vázquez, G., León-Rivera, I., Perea-Arango, I., Villalobos-Molina, R., Ibarra-Barajas, M., Tracheal relaxation through calcium channel blockade of Achillea millefolium hexanic extract and its main bioactive compounds, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/ j.jep.2020.112643. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Tracheal relaxation through calcium channel blockade of Achillea millefolium hexanic extract and its main bioactive compounds
Luis Arias-Durána,+, Samuel Estrada-Sotoa,*, Monserrat Hernández-Moralesa,+, Fabiola Chávez-Silvaa, Gabriel Navarrete-Vázqueza, Ismael León-Riverab, Irene Perea-Arangoc, Rafael Villalobos-Molinad, Maximiliano Ibarra-Barajasd.
a
Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca,
Morelos 62209, México b
Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos,
Cuernavaca, Morelos 62209, México. c
Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos,
Cuernavaca, Morelos 62209, México. d
Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional
Autónoma de México, Tlalnepantla, Estado de México 54090, México
*Corresponding author. Tel.: (+52) 777 329 7089; E-mail address: [email protected] (S. Estrada-Soto). +
Taken in part from the B. Sc. Pharm. Thesis of M. Hernández-Morales and M. Pharm
Thesis of L. Arias-Durán.
1
ABSTRACT Ethnopharmacological importance Achillea millefolium L. (Asteraceae) is used for the treatment of respiratory diseases, diabetes, and hypertension. Aim to explore its tracheal relaxant properties and clarify its functional mechanism of action on smooth muscle cells, which allow us to propose it as a potential anti-asthmatic drug. Material and methods organic and hydro-alcoholic extracts from A. millefolium were obtained by macerations, then their relaxing effect on ex vivo isolated rat trachea rings was determined. Most active extract (hexanic extract, EHAm) was studied to determine its functional mechanism of action using synergic, antagonist and inhibitor agents related with the contraction/relaxation process of the smooth muscle. Also, EHAm was subjected to bioguided fractionation by open-column chromatography (on silica gel) using cyclohexaneEtOAc (80:20) in an isocratic way to isolate main bioactive compounds. Results: organic and hydro-alcoholic extracts showed relaxant effect in a concentrationresponse dependent manner, being EHAm the most active. Functional mechanism of action indicates that a non-competitive antagonism to the muscarinic receptors by EHAm was observed; in addition, the NO/cGMP pathway is involved in the relaxation process of the tracheal smooth muscle. However, the most important mechanism of action showed by EHAm was related with the calcium channel blockade influx into the smooth muscle cells. On the other hand, epimeric sesquiterpene lactones leucodin (1) and achillin (2) were isolated and purified, which are responsible for the observed smooth muscle relaxant activity of the extract. Conclusion: hexanic extract of A. millefollium induced a significant relaxant effect on tracheal rat rings by calcium channel blockade and NO release. 2
Keywords: Achillea millefolium; achillin; calcium channel blockade; leucodin; trachea.
Abbreviations: 3',5'-cyclic adenosine monophosphate, cAMP; 3',5'-cyclic guanosine monophosphate, cGMP; centro de investigación en biotecnología, CEIB; ethyl acetate, EtOAc; N-nitro-L-arginine methyl ester hydrochloride, L-NAME; 1-H-[1,2,4]-oxadiazolo[4,3a]- quinoxalin-1-one, ODQ; dimethylsulphoxide, DMSO; hexanic extract of Achillea millefollium, EHAm; dichloromethanic extract of Achillea millefollium, EDAm; methanolic extract of Achillea millefolium, EMAm; hydro-alcoholic extract of Achillea millefolium, EEA Am; concentration-response curves, CRC; maximum effect, Emax; half maximal effective concentration, EC50; Nitric oxide, NO.
Highlights Achillea millefolium hexanic extract showed a significant relaxant effect on isolated rat trachea rings. Relaxant effect mode of action is mainly mediated by calcium channel blockade and NO release. Bio-guided fractionation allowed the isolation of leucodin and achillin.
3
1. Introduction Asthma is a common, complex and heterogeneous respiratory disease associated with increased response of the airways to a variety of stimuli and leads to recurrent episodes of respiratory symptoms, such as wheezing, dyspnea, chest tightness and cough. These symptoms are associated with the generalized, but variable, obstruction of air flow regulated by the airways of the respiratory system, consisting mainly of epithelium and bronchial smooth muscle mediating contraction and relaxation stimuli (GINA, 2019). There are different groups of drugs for the treatment of these conditions. First group corresponds to drugs that block the effect of contractile agonists, such as anti-cholinergic, antileukotriene and 5-lipoxygenase inhibitors; second group includes drugs that act directly as smooth muscle relaxants through the release of cAMP, cGMP, inhibition of the degradation of cyclic nucleotides and/or modulating the activity of the ion channels of the cell membrane; and third group is associated with anti-inflammatory drugs. In addition, complementary and alternative medicine is frequently used in the treatment of these illnesses, and is historically important. Since some classes of drugs used for their treatment originated in herbal medicine, traditional medicines and practices are currently being reexamined, leading potentially to the discovery of new bioactive compounds (Kim et al., 2010; Shin et al., 2009; Ying et al., 2006). Thus, Mexican traditional medicine has a wide use of plants for the treatment of respiratory diseases, and some of these plants have already been studied pharmacologically, such as Argemone ochroleuca, Gnaphalium liebmannii, Bougainvillea spectabilis, Bursera simaruba, Croton glabellus and Thymus vulgaris (Rodríguez-Ramos et al., 2011; SánchezMendoza and Navarrete, 2008; Waizel and Waizel, 2009). Therefore, Achillea millefolium L. (Asteraceae), known as yarrow (milenrama), is commonly used in folk medicine for the 4
treatment of inflammatory and spasmodic gastrointestinal disorders, hepatobiliary complaints, and overactive cardiovascular and respiratory ailments (Dall’Acqua et al., 2011). In Mexico, and in different parts of the world, A. millefollium is also used for the treatment of diabetes and related diseases. On the other hand, the main components of the plant are: flavonoids, phenolic acids, alkaloids, terpenes, tannins, among others. The observed pharmacological effects for A. millefolium are antioxidant and antimicrobial activities, anti-inflammatory, antihypertensive and bronchodilatory, gastrointestinal, antispasmodic, diuretic, urinary antiseptic, astringent, antidiabetic and antihemorrhagic effects
(Ali
et
al.,
2017).
The
antitussive
and
antispasmodic
effects
of A.
millefolium extracts on smooth muscle derived from isolated guinea pig trachea, ileum and pulmonary artery have also been described (Ali et al., 2017; Chávez-Silva et al., 2018; Petlevski et al., 2001). Moreover, A. millefollium is a part of the official medicine in several countries. This plant is included in the Pharmacopoeia of countries such as Germany, Czech Republic, France and Switzerland; and in Brazil, it is included in the 16 medicinal plants of “Verde Saúce”, which includes the anti-inflammatory, antispasmodic and antiseptic properties. Also, the Russian Federation Pharmacopoeia mentions that an infusion of the aerial parts of A. millefolium (15 g in 200 mL), about 65-100 mL can be used 2 to 3 times daily as a hemostatic, anti-inflammatory and sedative agent (Shikov et al., 2014; 2017). In this framework, the aim of current research was to establish the relaxant effect of organic and hydro-alcoholic extracts of A. millefollium on tracheal rat rings, to determine the underlying mechanism of action, and to develop the bio-guided fractionation of the most active extract in order to isolate the active compounds responsible of the relaxant action. 5
2. Materials and Methods 2.1 Chemical and Drugs Carbamylcholine chloride ≥98% (carbachol), theophylline, N-nitro-L-arginine methyl ester hydrochloride ≥98% (L-NAME), 1-H-[1,2,4]-oxadiazolo-[4,3a]- quinoxalin-1one (ODQ), dimethylsulphoxide (DMSO) and Nifedipine were purchased from Sigma– Aldrich Co. (St. Louis, MO, USA). All other reagents were analytical grade from local sources.
2.2 Plant material, Preparation of the Extract and Isolation. Aerial parts of A. millefolium (flowers, leaves and stem) used in our experiments were collected in June 2014 by Dr. Guillermo Ramirez and identified by Dr. Irene PereaArango (CEIB, UAEM) in Tres Marias, Huitzilac, in the State of Morelos, Mexico. Voucher specimen was deposited at the Herbarium (HUMO Herbarium, UAEM) under number 34332. The A. millefolium material was subjected to a cleaning process, and dried at room temperature in the shade. Then, dried plant material (500 g) was powdered and extracted by successive macerations with hexane, dichloromethane and methanol for 72 hours (3 times). A plant hydro-alcoholic extract (EEA Am) was prepared by macerating the powdered plant material (50 g) in 1000 mL ethanol (70%) for 72 hours (3 times). Solvent was then removed under reduced pressure using a rotary evaporator (Buchi® R-200). Yields for extracts obtained were: hexanic 1.96% (9.8g, EHAm), dichloromethanic 1.76% (8.8 g, EDAm), methanolic 4.28 % (21.42 g, EMAm) and hydro-alcoholic 35% (17.5 g, EEA Am).
6
Hexane soluble-extract (EHAm, 7 g) was subjected to bio-guided fractionation, separated by column chromatography over silica gel (0.063–0.200 mm, Merck, Germany), using cyclohexane-EtOAc (80:20) in an isocratic way, TLC Silica gel plates 60 F254 (Merck) were used for monitoring purification. A total of 299 fractions (30 mL each), grouped into six pools according to similar chromatographic profiles was obtained. Yields for fractions were: F1 (0.789 g, 11.27%), F2 (0.985 g, 14.02 %), F3 (0.180 g, 2.57%), F4 (0.841 g, 12.01%), F5 (0.250 g, 3.57%), and F6 [2.156 g, 30.8 % obtained with EtOAc (100%)]. For ex vivo pharmacological study, the dry extracts were solubilized in DMSO 1% (in water).
2.3 Pharmacological evaluations 2.3.1 Animals Male Wistar rats (250-350 g) were used. They were maintained under standard laboratory conditions with free access to food and water. All animal procedures were conducted in accordance with the Official Mexican Rule for Animal Experimentation and Care (SAGARPA, NOM-062-ZOO-1999), and approved by the Institutional Animal Care and Use Committee based on US National Institute of Health publication (No. 85-23, revised 1985). All experiments were carried out using six animals per group.
2.3.2 Solutions
7
The composition of Krebs solution was (in mM): NaCl, 118.0; KCl, 4.6; CaCl2.H2O, 2.5; MgSO4.H2O, 5.7; NaHCO3, 25.0; KH2PO4.H2O, 1.1; EDTA, 0.026; and glucose, 11.0, pH 7.4. Also, KCl 80 mM Krebs solutions were prepared by an equimolar replacement of Na+ for K+. In nominally Ca2+-free solution, CaCl2 was omitted.
2.3.3 Preparation of tracheal rat rings Male Wistar rats were killed by cervical dislocation. The trachea was dissected and cleaned of connective tissue and mucus, and immediately sectioned into rings 4-5 mm in length (each containing 2-3 cartilaginous rings). Then, tissue segments were mounted on stainless steel hooks under an optimal tension of 2 g, in 10-ml organ baths containing warmed (37°C) and oxygenated (O2/CO2, 95:5) Krebs solution. Changes in tension were recorded by Grass-FT03 force transducers (Astromed, West Warwick, RI, USA) connected to an MP100 Analyzer (Biopac Instruments, Santa Barbara, CA, USA) as described. (Sánches-Recillas et al., 2014). After equilibration, the rings were contracted by carbachol [1 µM] and washed every 40 min for 2 h. After contraction with carbachol, the test samples (Extracts, pure compounds and positive controls) were added at the bath in a volume of 100 µL; then, cumulative Concentration-Response Curves (CRC) were obtained for each ring. The relaxant effect of the test samples was determined by comparing the muscular tone of the contraction before and after addition of the test materials. Muscular tone was calculated from the tracings using Acknowledge software (BiopacTM). The vehicle for the test samples was dimethyl sulfoxide 1% (DMSO diluted with water).
2.3.4 Determination of the possible functional mode of action of the EHAm
8
a) To determine the involvement of cGMP, cAMP and/or NO in the EHAm relaxant effect, EHAm was evaluated in absence and presence of the following pharmacological agents: isoproterenol [(10 µM) a β-adrenoceptor agonist], LNAME [(10 µM) a nonspecific nitric oxide synthase inhibitor], and ODQ [(10 µM) a soluble guanylate cyclase inhibitor] (El-Yazbi et al., 2008; Vlkovicova et al., 2008) which were incubated on tracheal rat rings for 15 minutes. Subsequently, the tissues were contracted with carbachol and the test samples concentrations were added. On the other hand, cumulative CRC for theophylline were measured in absence and presence of EHAm (412 µg/mL). b) To verify that the relaxation induced by EHAm involved Ca2+ influx blockade, two kinds of experiments were performed. Firstly, after the stabilization period, isolated rat trachea rings were washed with Krebs solution, then rinsed with a solution containing KCl (80 mM). Once a plateau was attained, CRC of EHAm-induced relaxation were obtained by adding cumulative concentrations of the extract to the bath. Secondly, the experiments were carried out in Ca2+-free Krebs solution. Isolated rat trachea rings were washed with Ca2+-free Krebs solution containing KCl (80 mM) (15 min), and cumulative CRC for CaCl2 were obtained in the absence of EHAm (control group) or after a 15-min incubation with EHAm (131.8 and 412 µg/mL). Finally, the contractile effect induced by CaCl2 was compared in the absence and presence of EHAm.
2.4 Statistical analysis
9
All experimental results are expressed as mean ± S.E.M. Statistical analysis was done by two-way analysis of variance (ANOVA) with Bonferroni test using the software Origin 7.0. P value less than 0.05 was considered to be statistically significant.
3. Results and Discussion As described previously, A. millefollium is widely used for the treatment of several diseases, and for those there are pharmacological and phytochemical studies to corroborate its folkloric medicinal uses (Ali et al., 2017; Chávez-Silva et al., 2018). Thus, in order to support and to give additional evidence about its anti-asthmatic effect, the organic and hydro-alcoholic extracts were evaluated in an ex vivo model, using isolated rat trachea to observe the relaxing effect of the airway smooth muscle. As shown in Fig. 1, the hexanic (EHAm), dichloromethanic (EDAm), methanolic (EMAm), and aqueous-ethanolic (EEA Am) extracts of A. millefolium induced relaxing effect on carbachol (1 µM)-contraction, which depends on the concentration. It should be noted that extracts EDAm, EMAm and EEA Am are less effective (Emax) and potent than the positive control theophylline (Emax= 94.1±1.8% and EC50 of 51.0±4.2 µg/mL). However, EHAm was the most active and efficient extract evaluated (Emax= 80.4±6.8 2% and EC50 of 412±5.8 µg/mL). Based on these results, it can be established EHAm as a perfect candidate for to determine its functional mode of action, and to develop the bio-guided fractionation study to obtain pure compounds responsible for the relaxant action. In this sense, relaxing effect of A. millefolium extracts on different types of smooth muscle, including tracheal rat rings, has been determined in previous studies (Feizpour et al., 2013; Raju et al., 2009; Koushyar et al., 2013). Relaxant effect observed for EHAm could be due to different mechanisms,
10
including muscarinic receptor antagonism (Loenders et al., 1992). EHAm inhibitory effect was examined in muscarinic receptors, Fig. 2a shows the concentration-response curves of the contraction induced by carbachol (muscarinic agonist), in the presence and absence (control) of EC50 of EHAm (412.0 µg/mL), which shows a significant shift to the right of the curve in a non-parallel manner; in addition, it induces a significant decrease in efficacy, that clearly indicates an opposite effect to the contraction induced by carbachol, i.e., it is a possible non-competitive antagonist of muscarinic receptors, caused by allosteric binding to the active site that prevents the agonist-receptor binding, or by a functional antagonism that interferes with the signaling process for the development of the contraction, such as the production and/or accumulation of NO, cAMP or cGMP, intra- and extra-Ca2+ channel blockade or K+ channel opening, among others. Similarly, it may lead to the signaling by IP3/DAG being interrupted, as well as previous studies carried out on this plant species, where Feizpour et al. established that A. millefolium aqueous-ethanolic extract acts through a competitive antagonism on guinea pig trachea muscarinic receptors (Feizpour et al., 2013). These data are important since an ideal asthma antimuscarinic drug should diminish bronchoconstriction and mucus secretion by antagonizing M1 and M3 receptors. On the other hand, EHAm may also be acting on other signaling pathways involved in the relaxant effect, counteracting the effect produced by carbachol (Billigton and Penn, 2003; FloresSoto and Segura-Torres, 2005; Gosens et al., 2006). Thus, we decided to evaluate the ß-adrenergic receptors stimulation as a possible mechanism responsible for the EHAm relaxant effect, which is well-known that it leads to increased intracellular cAMP levels, resulting in muscle relaxation, the sense of airways; in this meaning, the stimulatory effect of EHAm on adrenergic receptors was examined by
11
producing relaxing concentration-response curves. Fig. 2b shows the relaxing effect of EHAm in absence (control) and presence of isoproterenol, and the curve did not show a significant change in the relaxing effect compared to control, which indicates that ßadrenergic receptors are not involved in the relaxing effect by EHAm, as suggested by Koushyar and Cols., who demonstrated that A. millefollium aqueous-ethanolic extract is not involved in the relaxing effect through ß-adrenergic receptors participation (Koushyar et al., 2013), nonetheless, it is important to mention that the difference in the mode of action between extracts is directly related with the chemical composition, because we are studying a hexanic extract, and previous studies were on hydro-alcoholic extract. Instead, the effect could be due to inhibition of phosphodiesterases (PDEs), a superfamily of proteins whose members are responsible for cAMP and cGMP hydrolysis to their corresponding nucleoside 5'-monophosphates AMP and GMP, respectively, and consequently their inhibition results in a bronchodilation (Bornouf and Pruniaux, 2002; Rodríguez-Ramos et al., 2011). Relaxing concentration-response curves were performed using theophylline as a positive control (inhibitor of PDEs). Fig. 2c displays no significant changes in the curve compared to the positive control; this result suggests that EHAm does not participate through a synergism with theophylline increasing intracellular cyclic messengers’ accumulation, by inhibiting PDEs. Latter evidence allowed us to discard the production and/or accumulation of cAMP in the relaxant effect of the extract; however, the relaxing activity could be due to increase in cGMP, through NO production by the ciliated epithelial cells, alveolar type II cells and nerve fibers that innervate the smooth muscle of the airway cells (Ricciardolo et al., 2004) by the nitric oxide synthases (NOS). Once NO is produced, its main molecular target is soluble guanylate cyclase (GC), which contains in its
12
structure a specific recognition site for NO, called heme group and it can pass from an inactive to an active state according to intracellular conditions. GC interaction with NO forms cGMP from guanosine triphosphate that results in smooth muscle relaxation (Strijdom et al., 2009). In this sense, Fig. 2d shows a significant shift to the right of the concentration-response curve when NOS inhibitor L-NAME is present, then efficacy and potency of EHAm decreased compared to the control curve, this result suggests that in NO absence there is a partial blockade of the relaxing effect, which indicates that EHAm contains compounds capable of triggering NOS activity to produce nitric oxide. Likewise, ODQ a GC inhibitor, slightly shifted the curve to the right, which corroborates the potential activation of NOS. This latest evidence is important because in human airways nerves, considered inhibitory bronchodilators of the non-adrenergic or non-cholinergic type (iNANC) do exist. NO neural release modulates cholinergic neural response in human airways in vitro, apparently through a functional antagonism with the released ACh, which reduces resistance of the airway, and that NO released by nerve fibers is a major neurotransmitter responsible for respiratory tract relaxation (Lammers et al., 1992). On the other hand, there are several possible mechanisms involved in the relaxing effect of the smooth muscle of the airways, some of these are directly involved in smooth muscle cells and could be acting at different levels, such as: Ca2+ influx decrease, cell membrane Ca2+ channels blockade, inhibition of Ca2+ release from intracellular stores; increase in K+ flow through channels opening and contractile apparatus inhibition. Increasing K+ external concentration to 80 mM induces depolarization and voltage-gated Ca2+ channels opening that induces smooth muscle to contract (Chen et al., 2003; Gao et al., 2010; Gilani et al., 2005). Therefore, as seen in Fig. 3a, EHAm induced relaxation on
13
isolated rat trachea rings using a depolarizing Krebs solution (80 mM KCl), which suggest that EHAm could block voltage-gated Ca2+ channels. Nifedipine was used as a control (selective blocker of L-type Ca2+ channels). To corroborate the effect, it was found that EHAm (412 µg/mL) completely opposes the contraction induced by CaCl2 (Fig. 3b); however, at lower dose (131 µg/mL) a partial concentration-dependent blockade is observed, which suggests that one of the main mechanisms of action of EHAm is the Ca2+ influx blockade (Cribbs, 2006; Mustafa et al., 2011). These results are related with those described by an A. millefollium hydroalcoholic extract (Khan and Gilani, 2011) in tracheal strips, which induced inhibition of high K+ and CCh-induced contractions with a similar potency, which suggest a non-specific tracheal relaxation effect perhaps by Ca2+ blockade mode of action. Calcium channel blockers are known to be beneficial in airway hypersensitivity, and the presence of these kind of compounds in A. millefollium, could explain its medicinal use in the respiratory diseases. Subsequently, EHAm was subjected to a bio-guided fractionation study; a primary fractionation was carried out in order to isolate the responsible compounds for the pharmacological activity. A total of 299 fractions (30 mL each), grouped into six pools according to similar chromatographic profiles were obtained. Fractions 1 (0.789 g), 2 (0.985 g), 3 (0.180 g), 4 (0.841 g), 5 (0.250 g), and 6 [2.156 g. obtained with EtOAc (100%)] were evaluated using the EC50 calculated for EHAm (412 µg/mL). Ultraviolet light lamp UV254 (Fig. 4a), and 10% ammonium ceric sulfate (Fig. 4b) were used as developers for the monitoring of the purification process. For this, TLC silica gel plates were used; as observed in Fig. 5, F-5 is the fraction that shows the highest pharmacological activity, which has the capacity to reduce the contractile state produced by carbachol at 86.2± 2.4%,
14
which suggests that in fraction F-5 one of the components that provide the greatest pharmacological effect is present. Also, the fraction F-3 showed an interesting and significant effect of 68.4±4.6%, which suggests that another major compound responsible for the activity is present. Moreover, in fraction F-4, composed for a mixture of compounds located in F-3 and F-5, showed 71.9±2.2% effect, which suggests that in mixture or by separate the compounds have significant effect. The fractions F-1, F-2 and F-6 show less activity than the others; however, it is not ruled out that in these fractions there are compounds that intervene in the observed pharmacological effect. From Fraction F-3 a crystalline solid with melting point of 203-205 °C was obtained and identified as γ-lactone leucodin (1, Fig. 6). Also, from F-5 a yellowish white powder with a melting point of 133-135 ºC was described as achillin (2, Fig. 6). Both 1 and 2 were characterized by 1H NMR and 13C NMR data compared with those reported in the literature (Glasl et al., 2002; Martinez-Vazquez et al., 1988). Finally, the relaxant effect of 1 and 2 was determined. Both compounds are responsible for the pharmacological activity observed for EHAm. In Fig. 7 it is observed that 1 presented an Emax of 74.0±6.4% and EC50 of 258±2.7 µM, meanwhile, 2 showed an Emax of 87.0±3.9% and EC50 of 270.5±4.5 µM. Compound 1 is less effective than 2; however, both have similar potency. The pattern of the relaxant curves is analogous in both compounds, suggesting that the mode of action could be generated in a similar way, possibly through a Ca2+ channels blockade as shown by the EHAm extract, further functional and molecular experiments are necessary to corroborate this hypothesis. 4. Conclusion
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Hexanic extract of A. millefollium induced a significant relaxant effect on tracheal rat rings by calcium channel blockade and NO release. Leucodin and achillin are the main bioactive compounds responsible of the relaxant action.
Conflict of interest The authors declare no conflict of interest. Author contributions to the paper were as follows: Extracts preparation, and leucodin and achillin isolation: L. A-D., M. H-M, F. ChS. Structural elucidation: G. N-V., I. L-R. Pharmacological evaluation: R. V-M., M. I-B., L. A-D., S. E-S. Identification and recollection of plant material: I. P-A. Study design: S. E-S. Manuscript preparation: all authors. Notes: The authors declare no competing financial interest.
Acknowledgment This work was supported by SEP-CONACYT (Proyecto de Ciencia Básica A1-S13540). L. Arias-Durán acknowledges the fellowship awarded by CONACyT (431596) to carry out graduate studies.
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Kim, H.Y., DeKruyff, R.H., Umetsu, D.T., 2010. The many paths to asthma: phenotype shaped by innate and adaptive immunity. Nat. Immunol. 11, 577-584. http:// doi: 10.1038/ni.1892. Koushyar, H., Koushyar, M.M., Byrami, G., Feizpour, A., Golamnezhad, Z., Boskabady, M.H. 2013. The effect of hydroethanol extract of Achillea Millefolium on βadrenoceptors of guinea pig tracheal smooth muscle. Indian J. Pharm. Sci. 75, 400-405. http:// doi: 10.4103/0250-474X.119810. Lammers, J.W., Barnes, P.J, Chung, K.F., 1992. Nonadrenergic, noncholinergic airway inhibitory nerves. Eur Respir J. 5, 239-246. Loenders, B., Rampart, M., Herman, AG. 1992. Selective M3 muscarinic receptor inhibits smooth muscle contraction in rabbit trachea without increasing the release of acetylcholine. J. Pharmacol. Exp. Ther. 263, 773-779. Martinez-Vazquez, M., Muñoz-Zamora, A., Joseph-Nathan, P., 1988. Conformational analysis
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Raju, D., Chitra, V., Das, K., Janiki, P., Shankari, M. 2009. Evaluation of antiasthmatic activity of aqueous extract of Achillea mellifolium Linn flowers. Arch. Appl. Sci. Res. 1, 287-93. Ricciardolo, F.L., Sterk, P.J., Gaston, B., Folkerts, G., 2004. Nitric oxide in health and disease
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List of figures. Figure 1. Relaxing concentration–response curves of extracts obtained from Achillea millefolium on isolated rat trachea rings contracted with carbachol. Data are expressed as the mean ± S.E.M. of six experiments (*p < 0.05). Figure 2. (a) Concentration–response curves of the contraction induced by carbachol on isolated rat trachea rings in absence and presence of EHAm (EC50) (b) Concentrationresponse curves of the relaxant effect induced by EHAm on isolated rat trachea rings pretreated with isoproterenol, (c) Concentration–response curves of the relaxant effect induced by theophylline in absence and presence of EHAm (EC50) (d) Concentration–response curves of the relaxant effect induced by EHAm on isolated rat trachea rings pre-treated with L-NAME, and ODQ and pre-contracted with carbachol. Data are expressed as the mean ± S.E.M. of six experiments (*p < 0.05). Figure 3. (a) Concentration-response curves of the relaxant effect induced by EHAm and nifedipine on isolated rat trachea rings contracted with KCl (80 mM); (b) Inhibitory effects of EHAm on the cumulative-contraction curve dependent on extracellular Ca2+ influx in Ca2 + -free solution. Data are expressed as mean ± S.E.M. of six experiments (*p < 0.05). Figure 4. TLC of fractions obtained from the bio-guided primary fractionation of the EHAm, F-1 to F-6. Stationary phase: silica gel 60 Merck; mobile phase: ciclohexane, EtOAc (65:35). Detection: a) UV254 b) 10% of ammoniacal ceric sulfate. Figure 5. Relaxing percentage of primary fractions obtained from bio-guided fractionation of EHAm. Results are presented as mean ± SEM, of six experiments *p<0.05 compared with control 22
Figure 6. Chemical structure of leucodin (1) and achillin (2). Figure 7. Relaxing effects of 1 and 2 on contractions produced by Carbachol (0.1 µM). Results are presented as mean ± SEM, of six experiments *p<0.05 compared with control.
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Figure 1.
Figure 2.
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c)
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Figure 3.
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Figure 4. a)
b) Front
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Figure 5.
Figure 6. 1)
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Figure 7.
S0378-8741(19)34431-9
DOI:
https://doi.org/10.1016/j.jep.2020.112643
Reference:
JEP 112643
To appear in:
Journal of Ethnopharmacology
Received Date: 5 November 2019 Revised Date:
26 January 2020
Accepted Date: 1 February 2020
Please cite this article as: Arias-Durán, L., Estrada-Soto, S., Hernández-Morales, M., Chávez-Silva, F., Navarrete-Vázquez, G., León-Rivera, I., Perea-Arango, I., Villalobos-Molina, R., Ibarra-Barajas, M., Tracheal relaxation through calcium channel blockade of Achillea millefolium hexanic extract and its main bioactive compounds, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/ j.jep.2020.112643. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Tracheal relaxation through calcium channel blockade of Achillea millefolium hexanic extract and its main bioactive compounds
Luis Arias-Durána,+, Samuel Estrada-Sotoa,*, Monserrat Hernández-Moralesa,+, Fabiola Chávez-Silvaa, Gabriel Navarrete-Vázqueza, Ismael León-Riverab, Irene Perea-Arangoc, Rafael Villalobos-Molinad, Maximiliano Ibarra-Barajasd.
a
Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca,
Morelos 62209, México b
Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos,
Cuernavaca, Morelos 62209, México. c
Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos,
Cuernavaca, Morelos 62209, México. d
Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional
Autónoma de México, Tlalnepantla, Estado de México 54090, México
*Corresponding author. Tel.: (+52) 777 329 7089; E-mail address: [email protected] (S. Estrada-Soto). +
Taken in part from the B. Sc. Pharm. Thesis of M. Hernández-Morales and M. Pharm
Thesis of L. Arias-Durán.
1
ABSTRACT Ethnopharmacological importance Achillea millefolium L. (Asteraceae) is used for the treatment of respiratory diseases, diabetes, and hypertension. Aim to explore its tracheal relaxant properties and clarify its functional mechanism of action on smooth muscle cells, which allow us to propose it as a potential anti-asthmatic drug. Material and methods organic and hydro-alcoholic extracts from A. millefolium were obtained by macerations, then their relaxing effect on ex vivo isolated rat trachea rings was determined. Most active extract (hexanic extract, EHAm) was studied to determine its functional mechanism of action using synergic, antagonist and inhibitor agents related with the contraction/relaxation process of the smooth muscle. Also, EHAm was subjected to bioguided fractionation by open-column chromatography (on silica gel) using cyclohexaneEtOAc (80:20) in an isocratic way to isolate main bioactive compounds. Results: organic and hydro-alcoholic extracts showed relaxant effect in a concentrationresponse dependent manner, being EHAm the most active. Functional mechanism of action indicates that a non-competitive antagonism to the muscarinic receptors by EHAm was observed; in addition, the NO/cGMP pathway is involved in the relaxation process of the tracheal smooth muscle. However, the most important mechanism of action showed by EHAm was related with the calcium channel blockade influx into the smooth muscle cells. On the other hand, epimeric sesquiterpene lactones leucodin (1) and achillin (2) were isolated and purified, which are responsible for the observed smooth muscle relaxant activity of the extract. Conclusion: hexanic extract of A. millefollium induced a significant relaxant effect on tracheal rat rings by calcium channel blockade and NO release. 2
Keywords: Achillea millefolium; achillin; calcium channel blockade; leucodin; trachea.
Abbreviations: 3',5'-cyclic adenosine monophosphate, cAMP; 3',5'-cyclic guanosine monophosphate, cGMP; centro de investigación en biotecnología, CEIB; ethyl acetate, EtOAc; N-nitro-L-arginine methyl ester hydrochloride, L-NAME; 1-H-[1,2,4]-oxadiazolo[4,3a]- quinoxalin-1-one, ODQ; dimethylsulphoxide, DMSO; hexanic extract of Achillea millefollium, EHAm; dichloromethanic extract of Achillea millefollium, EDAm; methanolic extract of Achillea millefolium, EMAm; hydro-alcoholic extract of Achillea millefolium, EEA Am; concentration-response curves, CRC; maximum effect, Emax; half maximal effective concentration, EC50; Nitric oxide, NO.
Highlights Achillea millefolium hexanic extract showed a significant relaxant effect on isolated rat trachea rings. Relaxant effect mode of action is mainly mediated by calcium channel blockade and NO release. Bio-guided fractionation allowed the isolation of leucodin and achillin.
3
1. Introduction Asthma is a common, complex and heterogeneous respiratory disease associated with increased response of the airways to a variety of stimuli and leads to recurrent episodes of respiratory symptoms, such as wheezing, dyspnea, chest tightness and cough. These symptoms are associated with the generalized, but variable, obstruction of air flow regulated by the airways of the respiratory system, consisting mainly of epithelium and bronchial smooth muscle mediating contraction and relaxation stimuli (GINA, 2019). There are different groups of drugs for the treatment of these conditions. First group corresponds to drugs that block the effect of contractile agonists, such as anti-cholinergic, antileukotriene and 5-lipoxygenase inhibitors; second group includes drugs that act directly as smooth muscle relaxants through the release of cAMP, cGMP, inhibition of the degradation of cyclic nucleotides and/or modulating the activity of the ion channels of the cell membrane; and third group is associated with anti-inflammatory drugs. In addition, complementary and alternative medicine is frequently used in the treatment of these illnesses, and is historically important. Since some classes of drugs used for their treatment originated in herbal medicine, traditional medicines and practices are currently being reexamined, leading potentially to the discovery of new bioactive compounds (Kim et al., 2010; Shin et al., 2009; Ying et al., 2006). Thus, Mexican traditional medicine has a wide use of plants for the treatment of respiratory diseases, and some of these plants have already been studied pharmacologically, such as Argemone ochroleuca, Gnaphalium liebmannii, Bougainvillea spectabilis, Bursera simaruba, Croton glabellus and Thymus vulgaris (Rodríguez-Ramos et al., 2011; SánchezMendoza and Navarrete, 2008; Waizel and Waizel, 2009). Therefore, Achillea millefolium L. (Asteraceae), known as yarrow (milenrama), is commonly used in folk medicine for the 4
treatment of inflammatory and spasmodic gastrointestinal disorders, hepatobiliary complaints, and overactive cardiovascular and respiratory ailments (Dall’Acqua et al., 2011). In Mexico, and in different parts of the world, A. millefollium is also used for the treatment of diabetes and related diseases. On the other hand, the main components of the plant are: flavonoids, phenolic acids, alkaloids, terpenes, tannins, among others. The observed pharmacological effects for A. millefolium are antioxidant and antimicrobial activities, anti-inflammatory, antihypertensive and bronchodilatory, gastrointestinal, antispasmodic, diuretic, urinary antiseptic, astringent, antidiabetic and antihemorrhagic effects
(Ali
et
al.,
2017).
The
antitussive
and
antispasmodic
effects
of A.
millefolium extracts on smooth muscle derived from isolated guinea pig trachea, ileum and pulmonary artery have also been described (Ali et al., 2017; Chávez-Silva et al., 2018; Petlevski et al., 2001). Moreover, A. millefollium is a part of the official medicine in several countries. This plant is included in the Pharmacopoeia of countries such as Germany, Czech Republic, France and Switzerland; and in Brazil, it is included in the 16 medicinal plants of “Verde Saúce”, which includes the anti-inflammatory, antispasmodic and antiseptic properties. Also, the Russian Federation Pharmacopoeia mentions that an infusion of the aerial parts of A. millefolium (15 g in 200 mL), about 65-100 mL can be used 2 to 3 times daily as a hemostatic, anti-inflammatory and sedative agent (Shikov et al., 2014; 2017). In this framework, the aim of current research was to establish the relaxant effect of organic and hydro-alcoholic extracts of A. millefollium on tracheal rat rings, to determine the underlying mechanism of action, and to develop the bio-guided fractionation of the most active extract in order to isolate the active compounds responsible of the relaxant action. 5
2. Materials and Methods 2.1 Chemical and Drugs Carbamylcholine chloride ≥98% (carbachol), theophylline, N-nitro-L-arginine methyl ester hydrochloride ≥98% (L-NAME), 1-H-[1,2,4]-oxadiazolo-[4,3a]- quinoxalin-1one (ODQ), dimethylsulphoxide (DMSO) and Nifedipine were purchased from Sigma– Aldrich Co. (St. Louis, MO, USA). All other reagents were analytical grade from local sources.
2.2 Plant material, Preparation of the Extract and Isolation. Aerial parts of A. millefolium (flowers, leaves and stem) used in our experiments were collected in June 2014 by Dr. Guillermo Ramirez and identified by Dr. Irene PereaArango (CEIB, UAEM) in Tres Marias, Huitzilac, in the State of Morelos, Mexico. Voucher specimen was deposited at the Herbarium (HUMO Herbarium, UAEM) under number 34332. The A. millefolium material was subjected to a cleaning process, and dried at room temperature in the shade. Then, dried plant material (500 g) was powdered and extracted by successive macerations with hexane, dichloromethane and methanol for 72 hours (3 times). A plant hydro-alcoholic extract (EEA Am) was prepared by macerating the powdered plant material (50 g) in 1000 mL ethanol (70%) for 72 hours (3 times). Solvent was then removed under reduced pressure using a rotary evaporator (Buchi® R-200). Yields for extracts obtained were: hexanic 1.96% (9.8g, EHAm), dichloromethanic 1.76% (8.8 g, EDAm), methanolic 4.28 % (21.42 g, EMAm) and hydro-alcoholic 35% (17.5 g, EEA Am).
6
Hexane soluble-extract (EHAm, 7 g) was subjected to bio-guided fractionation, separated by column chromatography over silica gel (0.063–0.200 mm, Merck, Germany), using cyclohexane-EtOAc (80:20) in an isocratic way, TLC Silica gel plates 60 F254 (Merck) were used for monitoring purification. A total of 299 fractions (30 mL each), grouped into six pools according to similar chromatographic profiles was obtained. Yields for fractions were: F1 (0.789 g, 11.27%), F2 (0.985 g, 14.02 %), F3 (0.180 g, 2.57%), F4 (0.841 g, 12.01%), F5 (0.250 g, 3.57%), and F6 [2.156 g, 30.8 % obtained with EtOAc (100%)]. For ex vivo pharmacological study, the dry extracts were solubilized in DMSO 1% (in water).
2.3 Pharmacological evaluations 2.3.1 Animals Male Wistar rats (250-350 g) were used. They were maintained under standard laboratory conditions with free access to food and water. All animal procedures were conducted in accordance with the Official Mexican Rule for Animal Experimentation and Care (SAGARPA, NOM-062-ZOO-1999), and approved by the Institutional Animal Care and Use Committee based on US National Institute of Health publication (No. 85-23, revised 1985). All experiments were carried out using six animals per group.
2.3.2 Solutions
7
The composition of Krebs solution was (in mM): NaCl, 118.0; KCl, 4.6; CaCl2.H2O, 2.5; MgSO4.H2O, 5.7; NaHCO3, 25.0; KH2PO4.H2O, 1.1; EDTA, 0.026; and glucose, 11.0, pH 7.4. Also, KCl 80 mM Krebs solutions were prepared by an equimolar replacement of Na+ for K+. In nominally Ca2+-free solution, CaCl2 was omitted.
2.3.3 Preparation of tracheal rat rings Male Wistar rats were killed by cervical dislocation. The trachea was dissected and cleaned of connective tissue and mucus, and immediately sectioned into rings 4-5 mm in length (each containing 2-3 cartilaginous rings). Then, tissue segments were mounted on stainless steel hooks under an optimal tension of 2 g, in 10-ml organ baths containing warmed (37°C) and oxygenated (O2/CO2, 95:5) Krebs solution. Changes in tension were recorded by Grass-FT03 force transducers (Astromed, West Warwick, RI, USA) connected to an MP100 Analyzer (Biopac Instruments, Santa Barbara, CA, USA) as described. (Sánches-Recillas et al., 2014). After equilibration, the rings were contracted by carbachol [1 µM] and washed every 40 min for 2 h. After contraction with carbachol, the test samples (Extracts, pure compounds and positive controls) were added at the bath in a volume of 100 µL; then, cumulative Concentration-Response Curves (CRC) were obtained for each ring. The relaxant effect of the test samples was determined by comparing the muscular tone of the contraction before and after addition of the test materials. Muscular tone was calculated from the tracings using Acknowledge software (BiopacTM). The vehicle for the test samples was dimethyl sulfoxide 1% (DMSO diluted with water).
2.3.4 Determination of the possible functional mode of action of the EHAm
8
a) To determine the involvement of cGMP, cAMP and/or NO in the EHAm relaxant effect, EHAm was evaluated in absence and presence of the following pharmacological agents: isoproterenol [(10 µM) a β-adrenoceptor agonist], LNAME [(10 µM) a nonspecific nitric oxide synthase inhibitor], and ODQ [(10 µM) a soluble guanylate cyclase inhibitor] (El-Yazbi et al., 2008; Vlkovicova et al., 2008) which were incubated on tracheal rat rings for 15 minutes. Subsequently, the tissues were contracted with carbachol and the test samples concentrations were added. On the other hand, cumulative CRC for theophylline were measured in absence and presence of EHAm (412 µg/mL). b) To verify that the relaxation induced by EHAm involved Ca2+ influx blockade, two kinds of experiments were performed. Firstly, after the stabilization period, isolated rat trachea rings were washed with Krebs solution, then rinsed with a solution containing KCl (80 mM). Once a plateau was attained, CRC of EHAm-induced relaxation were obtained by adding cumulative concentrations of the extract to the bath. Secondly, the experiments were carried out in Ca2+-free Krebs solution. Isolated rat trachea rings were washed with Ca2+-free Krebs solution containing KCl (80 mM) (15 min), and cumulative CRC for CaCl2 were obtained in the absence of EHAm (control group) or after a 15-min incubation with EHAm (131.8 and 412 µg/mL). Finally, the contractile effect induced by CaCl2 was compared in the absence and presence of EHAm.
2.4 Statistical analysis
9
All experimental results are expressed as mean ± S.E.M. Statistical analysis was done by two-way analysis of variance (ANOVA) with Bonferroni test using the software Origin 7.0. P value less than 0.05 was considered to be statistically significant.
3. Results and Discussion As described previously, A. millefollium is widely used for the treatment of several diseases, and for those there are pharmacological and phytochemical studies to corroborate its folkloric medicinal uses (Ali et al., 2017; Chávez-Silva et al., 2018). Thus, in order to support and to give additional evidence about its anti-asthmatic effect, the organic and hydro-alcoholic extracts were evaluated in an ex vivo model, using isolated rat trachea to observe the relaxing effect of the airway smooth muscle. As shown in Fig. 1, the hexanic (EHAm), dichloromethanic (EDAm), methanolic (EMAm), and aqueous-ethanolic (EEA Am) extracts of A. millefolium induced relaxing effect on carbachol (1 µM)-contraction, which depends on the concentration. It should be noted that extracts EDAm, EMAm and EEA Am are less effective (Emax) and potent than the positive control theophylline (Emax= 94.1±1.8% and EC50 of 51.0±4.2 µg/mL). However, EHAm was the most active and efficient extract evaluated (Emax= 80.4±6.8 2% and EC50 of 412±5.8 µg/mL). Based on these results, it can be established EHAm as a perfect candidate for to determine its functional mode of action, and to develop the bio-guided fractionation study to obtain pure compounds responsible for the relaxant action. In this sense, relaxing effect of A. millefolium extracts on different types of smooth muscle, including tracheal rat rings, has been determined in previous studies (Feizpour et al., 2013; Raju et al., 2009; Koushyar et al., 2013). Relaxant effect observed for EHAm could be due to different mechanisms,
10
including muscarinic receptor antagonism (Loenders et al., 1992). EHAm inhibitory effect was examined in muscarinic receptors, Fig. 2a shows the concentration-response curves of the contraction induced by carbachol (muscarinic agonist), in the presence and absence (control) of EC50 of EHAm (412.0 µg/mL), which shows a significant shift to the right of the curve in a non-parallel manner; in addition, it induces a significant decrease in efficacy, that clearly indicates an opposite effect to the contraction induced by carbachol, i.e., it is a possible non-competitive antagonist of muscarinic receptors, caused by allosteric binding to the active site that prevents the agonist-receptor binding, or by a functional antagonism that interferes with the signaling process for the development of the contraction, such as the production and/or accumulation of NO, cAMP or cGMP, intra- and extra-Ca2+ channel blockade or K+ channel opening, among others. Similarly, it may lead to the signaling by IP3/DAG being interrupted, as well as previous studies carried out on this plant species, where Feizpour et al. established that A. millefolium aqueous-ethanolic extract acts through a competitive antagonism on guinea pig trachea muscarinic receptors (Feizpour et al., 2013). These data are important since an ideal asthma antimuscarinic drug should diminish bronchoconstriction and mucus secretion by antagonizing M1 and M3 receptors. On the other hand, EHAm may also be acting on other signaling pathways involved in the relaxant effect, counteracting the effect produced by carbachol (Billigton and Penn, 2003; FloresSoto and Segura-Torres, 2005; Gosens et al., 2006). Thus, we decided to evaluate the ß-adrenergic receptors stimulation as a possible mechanism responsible for the EHAm relaxant effect, which is well-known that it leads to increased intracellular cAMP levels, resulting in muscle relaxation, the sense of airways; in this meaning, the stimulatory effect of EHAm on adrenergic receptors was examined by
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producing relaxing concentration-response curves. Fig. 2b shows the relaxing effect of EHAm in absence (control) and presence of isoproterenol, and the curve did not show a significant change in the relaxing effect compared to control, which indicates that ßadrenergic receptors are not involved in the relaxing effect by EHAm, as suggested by Koushyar and Cols., who demonstrated that A. millefollium aqueous-ethanolic extract is not involved in the relaxing effect through ß-adrenergic receptors participation (Koushyar et al., 2013), nonetheless, it is important to mention that the difference in the mode of action between extracts is directly related with the chemical composition, because we are studying a hexanic extract, and previous studies were on hydro-alcoholic extract. Instead, the effect could be due to inhibition of phosphodiesterases (PDEs), a superfamily of proteins whose members are responsible for cAMP and cGMP hydrolysis to their corresponding nucleoside 5'-monophosphates AMP and GMP, respectively, and consequently their inhibition results in a bronchodilation (Bornouf and Pruniaux, 2002; Rodríguez-Ramos et al., 2011). Relaxing concentration-response curves were performed using theophylline as a positive control (inhibitor of PDEs). Fig. 2c displays no significant changes in the curve compared to the positive control; this result suggests that EHAm does not participate through a synergism with theophylline increasing intracellular cyclic messengers’ accumulation, by inhibiting PDEs. Latter evidence allowed us to discard the production and/or accumulation of cAMP in the relaxant effect of the extract; however, the relaxing activity could be due to increase in cGMP, through NO production by the ciliated epithelial cells, alveolar type II cells and nerve fibers that innervate the smooth muscle of the airway cells (Ricciardolo et al., 2004) by the nitric oxide synthases (NOS). Once NO is produced, its main molecular target is soluble guanylate cyclase (GC), which contains in its
12
structure a specific recognition site for NO, called heme group and it can pass from an inactive to an active state according to intracellular conditions. GC interaction with NO forms cGMP from guanosine triphosphate that results in smooth muscle relaxation (Strijdom et al., 2009). In this sense, Fig. 2d shows a significant shift to the right of the concentration-response curve when NOS inhibitor L-NAME is present, then efficacy and potency of EHAm decreased compared to the control curve, this result suggests that in NO absence there is a partial blockade of the relaxing effect, which indicates that EHAm contains compounds capable of triggering NOS activity to produce nitric oxide. Likewise, ODQ a GC inhibitor, slightly shifted the curve to the right, which corroborates the potential activation of NOS. This latest evidence is important because in human airways nerves, considered inhibitory bronchodilators of the non-adrenergic or non-cholinergic type (iNANC) do exist. NO neural release modulates cholinergic neural response in human airways in vitro, apparently through a functional antagonism with the released ACh, which reduces resistance of the airway, and that NO released by nerve fibers is a major neurotransmitter responsible for respiratory tract relaxation (Lammers et al., 1992). On the other hand, there are several possible mechanisms involved in the relaxing effect of the smooth muscle of the airways, some of these are directly involved in smooth muscle cells and could be acting at different levels, such as: Ca2+ influx decrease, cell membrane Ca2+ channels blockade, inhibition of Ca2+ release from intracellular stores; increase in K+ flow through channels opening and contractile apparatus inhibition. Increasing K+ external concentration to 80 mM induces depolarization and voltage-gated Ca2+ channels opening that induces smooth muscle to contract (Chen et al., 2003; Gao et al., 2010; Gilani et al., 2005). Therefore, as seen in Fig. 3a, EHAm induced relaxation on
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isolated rat trachea rings using a depolarizing Krebs solution (80 mM KCl), which suggest that EHAm could block voltage-gated Ca2+ channels. Nifedipine was used as a control (selective blocker of L-type Ca2+ channels). To corroborate the effect, it was found that EHAm (412 µg/mL) completely opposes the contraction induced by CaCl2 (Fig. 3b); however, at lower dose (131 µg/mL) a partial concentration-dependent blockade is observed, which suggests that one of the main mechanisms of action of EHAm is the Ca2+ influx blockade (Cribbs, 2006; Mustafa et al., 2011). These results are related with those described by an A. millefollium hydroalcoholic extract (Khan and Gilani, 2011) in tracheal strips, which induced inhibition of high K+ and CCh-induced contractions with a similar potency, which suggest a non-specific tracheal relaxation effect perhaps by Ca2+ blockade mode of action. Calcium channel blockers are known to be beneficial in airway hypersensitivity, and the presence of these kind of compounds in A. millefollium, could explain its medicinal use in the respiratory diseases. Subsequently, EHAm was subjected to a bio-guided fractionation study; a primary fractionation was carried out in order to isolate the responsible compounds for the pharmacological activity. A total of 299 fractions (30 mL each), grouped into six pools according to similar chromatographic profiles were obtained. Fractions 1 (0.789 g), 2 (0.985 g), 3 (0.180 g), 4 (0.841 g), 5 (0.250 g), and 6 [2.156 g. obtained with EtOAc (100%)] were evaluated using the EC50 calculated for EHAm (412 µg/mL). Ultraviolet light lamp UV254 (Fig. 4a), and 10% ammonium ceric sulfate (Fig. 4b) were used as developers for the monitoring of the purification process. For this, TLC silica gel plates were used; as observed in Fig. 5, F-5 is the fraction that shows the highest pharmacological activity, which has the capacity to reduce the contractile state produced by carbachol at 86.2± 2.4%,
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which suggests that in fraction F-5 one of the components that provide the greatest pharmacological effect is present. Also, the fraction F-3 showed an interesting and significant effect of 68.4±4.6%, which suggests that another major compound responsible for the activity is present. Moreover, in fraction F-4, composed for a mixture of compounds located in F-3 and F-5, showed 71.9±2.2% effect, which suggests that in mixture or by separate the compounds have significant effect. The fractions F-1, F-2 and F-6 show less activity than the others; however, it is not ruled out that in these fractions there are compounds that intervene in the observed pharmacological effect. From Fraction F-3 a crystalline solid with melting point of 203-205 °C was obtained and identified as γ-lactone leucodin (1, Fig. 6). Also, from F-5 a yellowish white powder with a melting point of 133-135 ºC was described as achillin (2, Fig. 6). Both 1 and 2 were characterized by 1H NMR and 13C NMR data compared with those reported in the literature (Glasl et al., 2002; Martinez-Vazquez et al., 1988). Finally, the relaxant effect of 1 and 2 was determined. Both compounds are responsible for the pharmacological activity observed for EHAm. In Fig. 7 it is observed that 1 presented an Emax of 74.0±6.4% and EC50 of 258±2.7 µM, meanwhile, 2 showed an Emax of 87.0±3.9% and EC50 of 270.5±4.5 µM. Compound 1 is less effective than 2; however, both have similar potency. The pattern of the relaxant curves is analogous in both compounds, suggesting that the mode of action could be generated in a similar way, possibly through a Ca2+ channels blockade as shown by the EHAm extract, further functional and molecular experiments are necessary to corroborate this hypothesis. 4. Conclusion
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Hexanic extract of A. millefollium induced a significant relaxant effect on tracheal rat rings by calcium channel blockade and NO release. Leucodin and achillin are the main bioactive compounds responsible of the relaxant action.
Conflict of interest The authors declare no conflict of interest. Author contributions to the paper were as follows: Extracts preparation, and leucodin and achillin isolation: L. A-D., M. H-M, F. ChS. Structural elucidation: G. N-V., I. L-R. Pharmacological evaluation: R. V-M., M. I-B., L. A-D., S. E-S. Identification and recollection of plant material: I. P-A. Study design: S. E-S. Manuscript preparation: all authors. Notes: The authors declare no competing financial interest.
Acknowledgment This work was supported by SEP-CONACYT (Proyecto de Ciencia Básica A1-S13540). L. Arias-Durán acknowledges the fellowship awarded by CONACyT (431596) to carry out graduate studies.
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List of figures. Figure 1. Relaxing concentration–response curves of extracts obtained from Achillea millefolium on isolated rat trachea rings contracted with carbachol. Data are expressed as the mean ± S.E.M. of six experiments (*p < 0.05). Figure 2. (a) Concentration–response curves of the contraction induced by carbachol on isolated rat trachea rings in absence and presence of EHAm (EC50) (b) Concentrationresponse curves of the relaxant effect induced by EHAm on isolated rat trachea rings pretreated with isoproterenol, (c) Concentration–response curves of the relaxant effect induced by theophylline in absence and presence of EHAm (EC50) (d) Concentration–response curves of the relaxant effect induced by EHAm on isolated rat trachea rings pre-treated with L-NAME, and ODQ and pre-contracted with carbachol. Data are expressed as the mean ± S.E.M. of six experiments (*p < 0.05). Figure 3. (a) Concentration-response curves of the relaxant effect induced by EHAm and nifedipine on isolated rat trachea rings contracted with KCl (80 mM); (b) Inhibitory effects of EHAm on the cumulative-contraction curve dependent on extracellular Ca2+ influx in Ca2 + -free solution. Data are expressed as mean ± S.E.M. of six experiments (*p < 0.05). Figure 4. TLC of fractions obtained from the bio-guided primary fractionation of the EHAm, F-1 to F-6. Stationary phase: silica gel 60 Merck; mobile phase: ciclohexane, EtOAc (65:35). Detection: a) UV254 b) 10% of ammoniacal ceric sulfate. Figure 5. Relaxing percentage of primary fractions obtained from bio-guided fractionation of EHAm. Results are presented as mean ± SEM, of six experiments *p<0.05 compared with control 22
Figure 6. Chemical structure of leucodin (1) and achillin (2). Figure 7. Relaxing effects of 1 and 2 on contractions produced by Carbachol (0.1 µM). Results are presented as mean ± SEM, of six experiments *p<0.05 compared with control.
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Figure 1.
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