Airway smooth muscle relaxant activity of Cordia

Journal of Ethnopharmacology 229 (2019) 280–287

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Airway smooth muscle relaxant activity of Cordia dodecandra A. DC. mainly by cAMP increase and calcium channel blockade

T

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Amanda Sánchez-Recillasa, , Laura Rivero-Medinaa, Rolffy Ortiz-Andradea, Jesus Alfredo Araujo-Leónb, J. Salvador Flores-Guidoc a

Laboratorio de Farmacología, Facultad de Química, Universidad Autónoma de Yucatán, Calle 43 No. 613 x calle 90, Colonia Inalámbrica, C.P. 97069 Mérida, Yucatán, Mexico b Laboratorio de Cromatografía, Facultad de Química, Universidad Autónoma de Yucatán, Calle 43 No. 613 x calle 90, Colonia Inalámbrica, C.P. 97069 Mérida, Yucatán, Mexico c Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Yucatán, Carretera Mérida-Xmatkuil Km. 15.5, Mérida, Yucatán, Mexico

A R T I C LE I N FO

A B S T R A C T

Keywords: cAMP Calcium channel blockade Chromatographic fingerprint Cordia dodecandra Relaxant activity Trachea relaxation Smooth muscle

Ethnopharmacological relevance: The fight against chronic respiratory diseases needs the exploration of new active compounds with properties that contribute to diminish the symptoms or resolve the disease alongside current therapy. Materials and methods: Eight extracts obtained from the bark and leaves of a Mayan medicinal plant used to treat asthma, Cordia dodecandra A. DC., were investigated for their relaxant effect on rat isolated tracheal rings precontracted with carbachol [1 µM]. The underlying functional mode of action of the most effective extract was assessed and the chromatographic fingerprints of more active extracts were analyzed. Results: The dichloromethane bark extract (DECd-b) was the most effective and potent (Emax= 87.57 ± 1.32 %; EC50 = 392.7 ± 5.18 µg/mL). DECd-b relaxant effect was maximized in presence of isoproterenol (β-adrenergic agonist, [10 µM]) and theophylline (phosphodiesterase unspecific inhibitor, [10 µM]). DECd-b also showed efficient relaxation of KCl [80 mM]-induced contraction and inhibition of CaCl2-induced contraction. Pre-incubation with propranolol (non-selective β-adrenergic antagonist, [10 µM]), SQ22536 (adenylyl cyclase inhibitor; [100 µM]), ODQ (guanylyl cyclase inhibitor; [10 µM]), L-NAME (nitric oxide synthase inhibitor; [10 µM]), indomethacin (a cyclooxygenase unspecific inhibitor; [10 µM]), glibenclamide (ATP-sensitive potassium channel blocker; [10 µM]) and 2-aminopyridine (voltage-gated potassium channel blocker [100 µM]) did not modify the DECd-b relaxant-effect curve. The chromatographic analysis of DECd-b suggests the cordiaquinones presence with double conjugated bounds such as menaquinone. Conclusions: Results suggest that DECd-b induces relaxation mainly by cAMP increase and Ca2+ channel blockade. The chromatographic profiles and UV spectrum of DECd-b and HECd-l suggest the presence of molecules with structure of meroterpenoid naphthoquinones. This work report scientific evidence of C. dodecandra medicinal specie, which contributes to the pharmacological and phytochemical background of C. dodecandra providing an added value to the traditional use of this specie.

1. Introduction Asthma is a chronic inflammatory airway disease that leads to expiratory airflow limitation and is associated with airway hyperresponsiveness (AHR). Its defined by the history of respiratory symptoms such as wheeze, shortness of breath, chest tightness and cough that vary over time and intensity (GINA, 2016; Kudo et al., 2013); according to WHO, asthma affects between 100 and 150 million people worldwide and

causes nearly 180,000 deaths each year (Organization, 2016). Current asthma therapy focuses on symptom control to prevent future risk of adverse outcomes (GINA, 2016), however, it doesn’t resolve the disease (Kudo et al., 2013; Sánchez-Recillas et al., 2014a). Due to the high level of therapy-tolerance that patients develop as the result of frequent drug use; in addition to the amount of side effects they present, the need to research new therapeutic strategies has increased (Hartley et al., 2014; Thirstrup, 2000).

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Corresponding author. E-mail addresses: [email protected] (A. Sánchez-Recillas), [email protected] (L. Rivero-Medina), rolff[email protected] (R. Ortiz-Andrade), [email protected] (J.A. Araujo-León), [email protected] (J.S. Flores-Guido). https://doi.org/10.1016/j.jep.2018.10.013 Received 1 May 2018; Received in revised form 17 September 2018; Accepted 9 October 2018 Available online 17 October 2018 0378-8741/ © 2018 Elsevier B.V. All rights reserved.


A. Sánchez-Recillas et al.

Plants remain today an important source of treatment for nearly 80% of the world population (León Jiménez, 2005). Drug discovery techniques have been applied to the standardization of herbal medicines (Butler, 2004), thus their use has led to the isolation and characterization of bioactive compounds that represent novel candidates for the development of phytomedicines or drugs which can be used to treat chronic, degenerative, and infectious diseases (Newman et al., 2003). Empirical use of medicinal plants by Mexican indigenous population takes account of approximately 80% of the 4000 species found in this country, some of them used to treat respiratory diseases such as asthma (Waizel Haiat and Waizel Bucay, 2009). Cordia (family Boraginaceae) is a genus of deciduous flowering trees or shrubs comprising more than 300 species distributed widely in the tropical region. Leaves, fruit, bark and seed of a majority of the species to possess abundant ethnomedicinal value, in has been found to be used most frequently to treat many ailments such as respiratory disorders, stomach pain, wound, inflammation, myalgia, cough, dysentery and diarrhea (Oza and Kulkarni, 2017). Cordia dodecandra A. DC. («Cordia dodecandra A.DC. — The Plant List», s. f.), commonly known as “Ciricote”, is utilized in Mayan traditional medicine. For the treatment of diarrhea and dysentery, a decoction of the bark is prepared, and for asthma, bronchitis and cough, a decoction of leaves or leaves plus bark is utilized (MéndezGonzález et al., 2012; Morales and Herrera, 2009; Waizel Haiat and Waizel Bucay, 2009). Nowadays, there are no systematic studies that support the therapeutic use of this vegetal species; but some studies report that other species from the Cordia genus could offer many pharmacological effects such as antioxidant (Al-musayeib et al., 2011; Nariya et al., 2013), anti-inflammatory (Al-musayeib et al., 2011; Dutra et al., 2016; Ranjbar et al., 2016), anti-parasitic (Saki et al., 2015), antibacterial and anti-micotic (De Sá De Sousa Nogueira et al., 2013; Matias Ferreira et al., 2013). In this context, current research was carried out in order to establish the ex vivo bronchorelaxant effect of eight organic extracts of C. dodecandra and to assess the underlying functional mode of action of the most effective one.

Table 1 Percentage yield obtained of extracts of Cordia dodecandra. Extract

Yield

HECd-l DECd-l MECd-l MESxCd-l HECd-b DECd-b MECd-b MESxCd-b

2.23% 0.92% 8.54% 12.09% 0.15% 0.43% 1.73% 2.5%

HECd-l: hexanic extract of C. dodecandra leaves (maceration), DECd-l: dichloromethanic extract of C. dodecandra leaves (maceration), MECd-l: methanolic extract of C. dodecandra leaves (maceration), MESxCd-l: methanolic extract of C. dodecandra leaves (soxhlet). HECd-b: hexanic extract of C. dodecandra bark (maceration), DECd-b: dichloromethanic extract of C. dodecandra bark (maceration), MECd-b: methanolic extract of C. dodecandra bark (maceration), MESxCd-b: methanolic extract of C. dodecandra bark (Soxhlet).

was deposited in the herbarium “Alfredo Barrera Marín” (voucher number: J. S. Flores 12576) at the Campus de Ciencias Biológicas y Agropecuarias, Facultad de Medicina Veterinaria y Zootecnia, UADY, Mexico. 2.2.1. Extraction of plant material Leaves and bark were separated, washed with water, dried at room temperature (25 °C), and crushed in an industrial blender. The powdered leaves (200 g) and bark (200 g) were separately submitted to successive maceration processes and exhaustively with hexane (2 Lt), dichloromethane (2 Lt), and methanol (2 Lt) with three solvent changes every 72 h. Soxhlet extraction was also carried out using three 10 g samples of plant material with methanol at 70 °C for 24 h. All extracts were filtrated and concentrated in vacuum at 50 °C using a Rotavapor (Buchi® R-200) and the percentages yields obtained were as shown in Table 1.

2. Materials and methods 2.1. Chemicals and drugs 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 2-aminopyridine (2-AP), calcium chloride (CaCl2), carbamylcholine chloride (carbachol), glibenclamide, indomethacin, isoproterenol, L-NGNitroArginine methyl ester (L-NAME), nifedipine, pentobarbital, propranolol, potassium chloride (KCl), SQ22536, theophylline, and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The methanol, acetonitrile (Fermont, México) and acetic acid (Sigma Adrich, USA) (chromatographic grade) were used in the chromatographic analysis. Deionized water was purified by an Epure water purification system (Thermo Scientific, USA). As internal standard to establish the relative retention time was used Antrhaquinone (Sigma Aldrich, USA, analytical grade). Dichloromethane (Fermont, Mexico) and n-hexane (High Purity, Mexico) (chromatographic grade) were used to dissolve each extract, respectively. All other reagents and solvents (analytical grade) were obtained from local sources. Stock solutions of extracts were prepared with distilled water and DMSO on the same day of experimentation.

2.3. Ex vivo pharmacological evaluations To carry out the experiments, all extracts were dissolved in DMSO maximum [1 %]. 2.3.1. Animals Healthy male Wistar rats (250–300 g bodyweight) were obtained from the Animal House of the Regional Investigation Center “Dr. Hideyo Noguchi”, UADY, Mexico. Animals were housed in polycarbonate cages and maintained under standard laboratory conditions (12 h light/dark cycle, 25 ± 2 °C temperature, and 45–65 % humidity), and were fed with standard rodent diet and water ad libitum. All animal procedures were conducted in accordance to our Federal Regulations for Animal Experimentation and Care of NOM-062-ZOO-1999 (SAGARPA, 2001) 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 five animals per group. All animals were sacrificed by cervical dislocation after deep anesthesia with pentobarbital (65 mg/kg, i.p).

2.2. Plant material Plant species was selected using an ethnomedical criteria (Abreu Guirado and Cuéllar Cuéllar, 2008) Aerial parts and bark were collected in Merida, Yucatan, Mexico (20.6552948, −89.6102314) by the work group of the Laboratorio de Farmacología, Facultad de Química, Universidad Autónoma de Yucatán (UADY), Mexico. José Salvador Flores Guido, Ph.D. carried out the identification. A sample of the specimen

2.3.2. General procedures Trachea was dissected, cleaned out of connective tissue and mucus, and cut into 4–5 mm long strips (two cartilage rings). Then, tissue 281



and presence of glibenclamide or 2-AP. f) To determine whether inhibition of extracellular Ca2+ influx was involved in the extract-induced relaxation, after a sensitization process the following two sets of experiments were carried out: ➣ Tracheal rings were contracted by depolarization with KCl [80 mM] and the relaxation CRC of DECd-b [3.03–1000 μg/mL] was built as described previously in Section 2.3.3. For this set of experiments, nifedipine (calcium channel blocker, [0.01–3.46 µg/mL]) was used as positive control. ➣ After a previous stabilization in Ca2+-free KHS (20 min), tracheal rings were washed with Ca2+-free KHS containing KCl [80 mM] and were allowed to stabilize for 15 min. Then, a CRC for the CaCl2-induced contraction [0.06 µM a 20 µM] was obtained in the absence of DECd-b (control group). Once the maximal contraction was reached, tissue was washed with Ca2+-free, KCl [80 mM] KHS and allowed to stabilize for 20 min. Finally, after a 15-min incubation with DECd-b EC50 [392.7 μg/mL], another CRC for the CaCl2-induced contraction was obtained [0.06–20 µM]. The contractile effect induced by CaCl2 was compared in the absence and presence of DECd-b. Positive control for this set of experiments was nifedipine [10 μM]

segments were assembled using stainless steel hooks, under optimal tension of 2.5 g in 10 mL organ baths containing 10 mL of warmed (37 °C) and oxygenated (O2/CO2, 95:5, v-v) Krebs-Henseleit solution (KHS; composition mM: NaCl 119, KCl 4.6, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.5, NaHCO3 20, glucose 11.4, EDTA 0.027, pH=7.4, in distilled water). Tension changes were recorded by Grass-FT03 force transducers (Astro Med, West Warwick, RI, USA) connected to an MP150 analyzer (BIOPAC 4.1 Instruments, Santa Barbara, CA, USA) as described previously by Sánchez-Recillas et al. (2014a). After the stabilization period (20 min), sensitization was carried out. The tissues were stimulated with Carbachol (CCh [1 μM]) during 15 min, washed with fresh KHS, and allowed to stabilize for 15 min. This procedure was repeated twice. 2.3.3. Airway smooth muscle relaxant activity of extracts, controls and vehicle on contraction induced by CCh After sensitization, tissues were allowed to stabilize for 20 min and then they were contracted with CCh [1 μM]. Extracts [3.03–1000 μg/ mL], vehicle (DMSO, [1% final concentration]) or positive control (theophylline [0.303–100 μg/mL]) were added to the chamber in cumulative concentrations (Concentration–Response Curves, CRC). The relaxant effect of the samples was determined by their ability to reduce the maximal tracheal contraction induced by CCh, comparing tissue tension before and after their addition.

2.4. Data analysis Results are expressed as the mean (n = 5) ± Standard Error of the Mean (S.E.M), considering "n" as experiments number in independent animals. Concentration–Response Curves (CRC) were plotted, and the experimental data from the CRC were adjusted by the nonlinear DoseResp equation with the curve-fitting program ORIGIN 8.0. Pharmacological parameters efficacy (Emax) and median effective concentration (EC50) values were calculated. The statistical significance of differences between means was assessed by a one-way analysis of variance (ANOVA) followed by the Tukey post hoc test; p values < 0.05 (*p < 0.05) were considered statistically significant (Bailey, 1995; Daniel, 2002).

2.3.4. Determination of the relaxant mode of action of DECd-b In order to establish the underlying mode of action of dichloromethanic extract of bark of Cordia dodecandra (DECd-b) the following ex vivo experiments were carried out: a) For the interaction with β2-adrenergic receptor, tissues were preincubated with isoproterenol (non-selective β-adrenergic agonist, [10 µM]) or propranolol (non-selective β-adrenergic antagonist, [10 µM]) for 15 min prior to the contraction with CCh [1 μM]. Relaxation CRC of DECd-b [3.03–1000 μg/mL] was then built as described in the smooth muscle relaxant set of experiments. The maximal relaxing effect of DECd-b was compared in the absence and presence of isoproterenol and propranolol respectively b) For direct activation of adenylyl cyclase enzyme (AC), tracheal rings were pre-incubated with SQ22536 (AC-inhibitor, [100 μM]) for 15 min prior to CCh [1 μM] induced contraction. DECd-b EC50 [392.7 μg/mL] was then added to the tissue. Maximum relaxation induced by the extract was compared with and without SQ22536. c) For the interaction with phosphodiesterases (PDE's) or prostacyclin (PGE2), and cAMP increase, tracheal rings were pre-incubated with theophylline (PDE's unspecific inhibitor, [10 µM]) or indomethacin (cyclooxygenase (COX) inhibitor [10 μM]), respectively for 15 min prior to the contraction with CCh [1 μM]. The relaxant effect induced by DECd-b was compared in absence and presence of theophylline or indomethacin. d) In order to know the role of participation of derivate of epithelium relaxing factor such as nitric oxide (NO) and to establish the possible inhibition of the soluble guanylyl cyclase enzyme (sGC), tracheal rings were pre-incubated with L-NAME (a nitric oxide synthase [NOS] inhibitor, [10 μM]) or ODQ (an sGC inhibitor, [10 μM]) for 15 min prior to the contraction with CCh [1 μM]. The relaxation CRC of DECd-b was built as described previously in Section 2.3.3. The maximal relaxing effect of DECd-b was compared in the absence and presence of ODQ or L-NAME. e) In order to know the role of ATP-sensitive potassium channel (KATP) or voltage-gated potassium channel in the extract-induced relaxant effect, tracheal rings were pre-incubated with glibenclamide (a KATP specific inhibitor, [10 μM]) or 2-aminopyridine (2-AP) [100 µM]) for 15 min prior to the contraction with CCh [1 μM]. The relaxation CRC of DECd-b was built as described previously in Section 2.3.3. The maximal relaxant effect of DECd-b was compared in the absence

2.5. Chromatographic fingerprint analysis by HPLC UV-DAD With the aim to correlate bioactive metabolites presence with pharmacological effect, chromatographic profiles of the most efficient extracts (HECd-l & DECd-b) were obtained. An Agilent Technologies 1200-series HPLC system (Agilent, San Jose, CA, USA) with a quaternary pump and a UV-DAD detector equipped with a Zorbax Poroshell 120 XDB-C18 column (150 mm × 4.6 mm i.d., 5 µm, Agilent, USA) was used. The chromatography was performed under gradient conditions with H2O (0.1 % with acetic acid v/v) and methanol: acetonitrile (1:1, v/v) with a flow rate of 1.0 mL/min, injecting 5 μL of sample. The column was purged with the mobile phase for 10 min, followed by equilibration for 10 min. The total time required for sample analysis was 25 min. Spectral data were collected and plotted at detection wavelengths around 190–900 nm. 3. Results and discussion Leaves and bark were subjected to extraction process by maceration with solvents of different polarity (low: hexane, medium: dichloromethane and high: methanol) with the aim to relate the pharmacological effect to the type of possible metabolites contained in the different extracts. Besides two extracts were obtained by soxhlet with methanol (leaves and bark) in aim to identify how the extraction temperature influences pharmacological effect. A total of 8 extracts were obtained and evaluated (4 for each organ of the plant). Details of their yields are shown in Table 1. The extracts obtained by soxhlet with methanol, followed by those obtained by maceration at room temperature with methanol showed, as expected, the highest yields for both leaves and bark. Metabolic content 282



Table 2 Pharmacological parameters obtained of concentration-response curves of airway smooth muscle relaxant activity of Cordia dodecandra extracts.

Relaxant effect (%)

0

*

20

*

40 60

*

*

Theophylline (control) HECd-l DECd-l MECd-l MESxCd-l

80 100

0.1

1

10

100

1000

(A)

0

Relaxant effect (%)

20

*

60

*

Theophylline (control) HECd-b DECd-b MECd-b MESxCd-b

80 100

0.1

1

10

* 100

Theophylline (positive control) HECd-l DECd-l MECd-l MESxCd-l HECd-b DECd-b MECd-b MESxCd-b

98.79 61.77 54.05 30.63 8.39 24.53 87.57 − 6.05 36.57

± ± ± ± ± ± ± ± ±

CE50 (µg/mL) 7.54 2.92 2.72 1.17 2.55 5.34 1.32* 1.42 2.28

53 ± 4.16 263.5 ± 5.98 721.7 ± 2.8 > 1000 > 1000 > 1000 392.7 ± 5.18 > 1000 > 1000

effect was discrete, since this experimental model does not evaluate the modulation of inflammatory process, airway hyperresponsiveness, hypersensitivity development or the inhibition of inflammatory mediators release during an asthmatic episode (Bazán-Perkins et al., 2009; Kudo et al., 2013; Sánchez-Recillas et al., 2014a; Wright et al., 2013). On the other hand, HECd-l (Emax= 61.77 ± 2.92 %; EC50 = 263.5 µg/mL) and DECd-b (Emax = 87.57 ± 1.32 %; EC50 = 392.7 µg/mL) were the extracts with the most significant effects. Statistical analysis showed that EDCd-b was significantly more efficient that EHCd-l but both were equipotent (one-way ANOVA and Tukey post hoc test, p < 0.05). Table 2 depicts the pharmacological parameters efficacy (Emax) and potency (EC50) obtained for the extracts and positive control employed. These results indicate that DECd-b was the most effective extract of the entire series evaluated, so we decided to assess its functional mode of action on tracheal rings. To determine the interaction with β2-adrenergic receptors we evaluated DECd-b induced relaxation in the presence of isoproterenol (non-selective β-adrenergic agonist) and propranolol (non-selective βadrenergic antagonist). Fig. 2A shows CRC of DECd-b relaxant effect in presence of isoproterenol and propranolol respectively. CRC with isoproterenol was significantly (p < 0.05) displaced to the left respect to control, which indicates a possible activation on β2-adrenergic receptor and/or accumulation of intracellular 3′-5′-cyclic adenosine monophosphate (cAMP) by adenylate cyclase enzyme stimulation or phosphodiesterase enzymes inhibition (Alemán-Pantitlán et al., 2016; Koike et al., 2004; Thirstrup, 2000). Likewise, CRC of DECd-b in presence of propranolol (non-selective β-adrenergic antagonist) was shifted to the left respect to control and was shifted to the right respect to CRC in presence of isoproterenol, discarding a possible antagonism of the β2 receptor. This behavior suggests intracellular cAMP increase by mechanisms independent from β2-adrenergic activation. The latter suggests that other relaxant effectors of the β2-adrenergic pathway such as cAMP rise directly by Adenylate Cyclase (AC) stimulation or indirectly by Phosphodiesterases (PDE) inhibition, or the triggering of cAMP-independent relaxant factors, could be responsible of the DECd-b relaxant effect (Alemán-Pantitlán et al., 2016; Koike et al., 2004; Thirstrup, 2000). To find out if DECd-b relaxant effect is was mediated by AC stimulation and subsequently increment of intracellular cAMP, we preincubated SQ22536, an enzyme AC inhibitor. Relaxant effect of DECd-b in presence of SQ22536 not was modified (supplementary data), discarding direct AC activation. On the other hand, to find out if DECd-b relaxant effect is was mediated by PDE inhibition; we pre-incubated

*

40

Emax (%)

Emax: Maximum effect, CE50: Median effective concentration. HECd-l: hexanic extract of C. dodecandra leaves (maceration), DECd-l: dichloromethanic extract of C. dodecandra leaves (maceration), MECd-l: methanolic extract of C. dodecandra leaves (maceration), MESxCd-l: methanolic extract of C. dodecandra leaves (soxhlet). HECd-b: hexanic extract of C. dodecandra bark (maceration), DECd-b: dichloromethanic extract of C. dodecandra bark (maceration), MECd-b: methanolic extract of C. dodecandra bark (maceration), MESxCd-b: methanolic extract of C. dodecandra bark (Soxhlet). (*) Indicate significative difference between HECd-l and DECd-b, one-way ANOVA (p > 0.05).

Concentration [μg/mL]

*

Sample

1000


(B) Fig. 1. (A) Concentration-response curve of the relaxant effect of the C. dodecandra leaves extracts and theophylline on trachea rat segments pre-contracted with carbachol [1 µM] and (B) Concentration-response curve of the relaxant effect of the C. dodecandra bark extracts and control (theophylline) on trachea rat segments pre-contracted with carbachol [1 µM]. Results are presented as mean ± S.E.M., n = 5 and (*) Values P < 0.05 represent significant difference respect to less active extracts.

of medicinal plants are mainly compounds with medium to high polarity (Jones and Kinghorn, 2012), and heat often increases solvent's capability to separate them from the plant material (Cechinel and Yunes, 1998; Kuklinski, 2000). Figs. 1A and 1B show concentration response curves (CRC) of the extracts evaluated. All test samples showed significant relaxant effect in a concentration-dependent manner on the contraction induced by CCh ([1 μM], acting as muscarinic cholinergic agonist), with exception of MECd-b that showed mild contractile effect (Emax = -6.05 ± 1.42 %). MECd-l (Emax = 30.63 ± 1.17 %; EC50 = > 1000 μg/mL), MESxCd-l (Emax = 8.39 ± 2.55 %; EC50 = > 1000 μg/mL), HECd-b (Emax = 24.53 ± 5.34 %; EC50 = > 1000 μg/mL) and MESxCd-b (Emax = 36.57 ± 2.28 %; EC50 = > 1000 μg/mL) showed moderate relaxant effect, less effective and less potent than theophylline (a non-selective phosphodiesterase inhibitor; Emax = 98.79 ± 7.54 %; EC50 = 53 µg/ mL) used as positive control. We cannot discard the anti-asthmatic properties of the latter fractions of C. dodecandra even though their 283



ψ

ψ

* *ψ

ψ

0

*ψ

20

*ψ

40 60

*ψ *ψ

80 100

Relaxant effect (%)

Relaxant effect (%)

0

*

Control Propranolol [10μM] Isoproterenol [10μM]

ψ

20 40 60 80 100

DECd-b Nifedipine (control)

120

120 10

100

0.1

1000

1

10

Concentration of DECd-b [μg/mL]

2.0 1.8

*

1.6

60

*

80 100

**

Control Theophylline [10μM]

1.2 1.0 0.8 0.6 0.4 0.2 0.0

120 10

Control Nifedipine [10μM] (positive control) DECd-b [392.7μg/mL]

1.4

*

Contraction (g)

Relaxant effect (%)

0

40

1000

(A)

(A)

20

100


100

-0.2 0.01

1000

Concentration of DECd-b [μg/mL]

*

* 0.1

*

* 1

*

* 10

*

*

*

100

Concentration of CaCl2 [μg/mL]

(B)

(B)

Fig. 2. (A) Effect of propranolol and isoproterenol and (B) Effect of theophylline pretreatment on DECd-b-induced relaxation in tracheal segments pre-contracted with carbachol [1 µM]. Results are presented as mean ± S.E.M., n = 5 and (*) or (Ψ) values P < 0.05 represent significant difference respect to control.

Fig. 3. (A) Inhibitory effect of the DECd-b on the contraction induced by KCl [80 mM] and (B) Inhibitory effects of the DECd-b on the cumulative-contraction curve dependent on extracellular Ca2+ influx induced by KCl [80 mM] in tracheal ring segments. Results are presented as mean ± S.E.M., n = 5 and (*) values P < 0.05 represent significant difference respect to control.

DECd-b [392.7 µg/mL] and a CRC in the presence of theophylline was carried out. Fig. 2B shows relaxant effect of DECd-b in the presence of theophylline. A parallel shift to left respect control with out modified significant maximum effect was observed, implying DECd-b potency was increased by theophylline and suggesting that rising levels of cAMP may play a role in DECd-b relaxant effect (Thirstrup, 2000). cAMP intracellular elevation in airway smooth muscle cells leads to cAMPdependent protein kinase A enzyme (PKA) stimulation. PKA is known to be the main effector of β2-adrenergic stimuli; nonetheless direct activation by DECd-b may also be taking place. PKA leads to inactivation (phosphorylation) of myosin light-chain kinase (MLCK). In addition, large-conductance Ca2+-activated K+ channels (KCa, BKCa, Maxi-K+) are markedly activated by PKA-induced phosphorylation. The opening of KCa channels also regulates airway smooth muscle tone mediated by membrane potential-dependent Ca2+ influx (Ca2+ dynamics), such as L-type voltage-dependent Ca2+ (VDC) channels (Kume, 2015). In this context CRC-DECd-b not was modify in presence of glibenclamide or 2-aminopyridine (an ATP-sensitive potassium channel blocker or voltage-gated potassium channel blocker)

(supplementary data), suggesting direct opening of potassium channels do not play part in the DECd-b induced relaxation. On the other hand PKA activation may stimulate calcium recapture towards the sarcoplasmic reticulum leading to hyperpolarization and airway smooth muscle relaxation, thus intracellular calcium concentration plays important role in ASMC contraction (Thirstrup, 2000). Airway smooth muscle cells (ASMC) contraction is determined by the balance between phosphorylation and de-phosphorylation of the regulatory light chain of myosin (rMLC) (Bai and Sanderson, 2006). Phosphorylation of rMLC is induced by Ca2+-calmodulin activated MLCK. rMLC-dephosphorylation is believed to be mediated by myosin light chain phosphatase (MLCP). As a result, ASMC relaxation may be the result of different cellular pathways that culminate in either or both a reduction in MLCK activity and an increase in MLCP activity (Bai y and Sanderson, 2006). Intracellular calcium concentration plays important role in ASMC contraction. In this context DECd-b was also capable to induce relaxation (Emax= 99.90 ± 2.97 %) on KCl [80 mM] pre-contracted tracheal rings, in a concentration-dependent manner (Fig. 3A) furthermore CaCl2-induced contraction was completely 284



ODQ and L-NAME did not modify the relaxant effect of DECd-b (supplementary data) discarding, respectively, prostacyclin's, cGMP and NO participation in the DECd-b-induced relaxation (Estrada-Soto et al., 2012). At the present time phytochemical studies reveal that meroterpenoids, phenilpropanoids, phenols, alkaloids, flavones, flavonoids, tanines and glycosides have been isolated from the genus Cordia (Almusayeib et al., 2011; De Sá De Sousa Nogueira et al., 2013; Matias Ferreira et al., 2013; Saki et al., 2015), The chromatogram of fingerprint analysis by HPLC with UV-DAD of hexanic extract of C. dodecandra leaves (HECd-l) shows 10 signals while the chromatogram of dichloromethanic extract of C. dodecandra bark (DECd-b) only five signals were found. Table 3 show results of retention time in fingerprint analyses of HECd-l and DECd-b extracts. The extraction process with middle-low polarity solvents like dichloromethane and hexane allow obtaining bioactive molecules such as meroterpenoid naphthoquinones. These molecules have been isolated of the Cordia genus and are known as cordiachromes or cordiaquinones (Ioset et al., 1999; da Silva et al., 2017). The UV-spectrum of the signals in the chromatogram (Fig. 4A-C) showed two maximum signals on 410 and 665 nm, these wavelengths correspond to cordiaquinones with double conjugated bounds such as menaquinone (vitamin K) or cordiaquinone A-D derivates (Bieber et al., 1994). The cordiaquinones have been reported with antifungal and larvicidal activities (Ioset et al., 2000) as well as cytotoxicity induced by oxidative stress (Marinho-Filho et al., 2010). On the other hand, menaquinone has been proved to significantly inhibit the degranulation of mesenteric mast cells of rats and to inhibit IgE mediated appearance of A form basophilic cells in patients with bronchial asthma (Kimura et al.,

Table 3 The results of relative retention time in fingerprint analyses (n = 3) of more efficient extracts of Cordia dodecandra. Relative retention time HECd-l

DECd-b

Signal

Retention time (Rt)

Averages

RSD

Averages

RSD

1 2 STDI 3 4 5 6 7 8 9 10

12.27 12.46 12.71 14.04 14.28 16.07 16.57 18.14 19.86 21.35 23.48

0.97 0.98 1.00 1.10 1.12 1.26 1.30 1.43 1.56 1.68 1.85

1.38 0.87 0 0.54 1.97 0.45 0.19 1.03 1.04 0.58 0.31

N/D N/D 1.00 1.10 N/D N/D 1.30 N/D 1.56 1.68 1.85

N/D N/D 0 1.72 N/D N/D 0.86 N/D 0.35 0.28 1.03

DECd-b: dichloromethanic extract of C. dodecandra bark, HECd-l: hexanic extract of C. dodecandra leaves, N/D: Not Detectable, RSD: Relative Standard deviation, STDI: Internal Standard (Anthraquinone).

abolished by DECd-b (Fig. 3B). The latter confirms a second mode of action related with possible blockade of Ca2+ channels (Gilani et al., 2005; Sánchez-Recillas et al., 2014b). In order to identify nitric oxide (NO)-3′-5′-cyclic guanosine monophosphate (cGMP) pathway activation, DECd-b relaxant effect was evaluated in presence of NO-cGMP pathways inhibitors. Indomethacin,

Fig. 4. Fingerprint analyses of Cordia dodecandra extracts (A) Anthraquinone as Internal Standard, (B) Hexanic extract from leaves (HECd-l) and (C) Dichloromethanic extract from bark (DECd-b) at detection wavelengths around 190–900 nm. 285



Appendix A. Supporting information

1975). Menaquinone has also been associated with a marked decrease in inflammatory markers such as C-reactive protein, and matrix metalloproteinase-3 (Ebina et al., 2013), which plays an important role on asthma mediated airway remodeling (Dahlen et al., 1999). Another study has shown that menaquinone produces its anti-inflammatory activity by suppressing the expression of inflammatory cytokines in cultured macrophage-like cells through the repression of IKKα/β phosphorylation and consequent inhibition of the activation of nuclear factor κB (Ohsaki et al., 2009). On the other hand, has been reported presence of rosmarinic acid, alantoine, e-siringine and salvianolic acid in the methanolic extract of C. dodecandra (Aguilar Vázquez and Quijano, 2016). Rosmarinic acid has numerous pharmacologic effects, including anti-oxidant, anti-inflammatory, and analgesic. Liang et al. (2016) report attenuation of airway inflammation and hyperresponsiveness in a murine model. In this context Al-Sereiti et al. suggest that rosmarinic acid has a therapeutic potential in treatment or prevention of bronchial asthma, spasmogenic disorders, peptic ulcer, inflammatory diseases, hepatotoxicity, atherosclerosis, ischaemic heart disease, cataract, cancer and poor sperm motility (Al-Sereiti et al., 1999). Based on the above, molecules with cordiaquinones structure as menaquinone maybe responsible of C. dodeandra pharmacologic effect, nevertheless it is necessary perform exhaustive phytochemical analysis to identify the bioactive metabolites.

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4. Conclusions For the first time, we reported the fingerprint analysis of dichloromethanic extract of bark and hexanic extract of leaves of C. dodecandra. The signals in the chromatographic profiles and UV spectrum suggest the presence of molecules with structure of meroterpenoid naphthoquinones. Results of mechanism of action suggest that dichloromethanic extract of C. dodecandra bark induces relaxation mainly by cAMP increase and calcium channel blockade. This work reports scientific evidence of C. dodecandra medicinal specie, which contributes their pharmacological and phytochemical background providing it an added value, and our result suggest that Cordia dodecandra A. DC bark could be considered as source of bioactive compounds with relaxant potential on airway smooth muscle cells. Acknowledgments This study was financed by Facultad de Química of Universidad Autónoma de Yucatán, Mexico through the project: Evaluación farmacológica ex vivo y estudio toxicológico de plantas medicinales de Yucatán usando musculatura lisa como modelo experimental, ID: SISTPROY-FQUI-2016-0007. Amanda Sánchez-Recillas, Rolffy OrtizAndrade and Jesus Alfredo Araujo-León belong to the Red Nacional de Investigación Preclínica de Productos Naturales. Declarations of interest None. Contribution of authors Amanda Sánchez-Recillas and Laura Rivero-Medina performed the ex vivo experiments, result discussion and analysis. Rolffy OrtizAndrade and Jesus Alfredo Araujo-León performed extracts preparation, chromatographic fingerprint and their analysis. J. Salvador FloresGuido performed collect and identification of vegetal specie. The writing and submission of the paper for the purpose of publication in the journal was an agreement reached by all authors. Rolffy OrtizAndrade performed design of the experiments and results analysis. 286



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Glossary µM: Micromolar. AC: Adenylyl cyclase. Ach: Acetilcholine. ADP: Adenosine diphosphate. AHR: Airway hyperresponsiveness. ANOVA: Analysis of variance. ASM: Airway smooth muscle. ASMC: Airway smooth muscle cells. ATP: Adenosine triphosphate. BKCa: Large-conductance Ca2+-activated K+ channel. CaCl2: Calcium chloride. cAMP: Cyclic adenosine monophosphate. CCh: Carbamoylcholine chloride (carbachol). cGMP: Cyclic guanosine monophosphate. CH2Cl2: Dicloromethane. CRC: Concentration-response curve. DECd-b: Dichloromethanic extract of C. dodecandra bark (maceration). DECd-l: Dichloromethanic extract of C. dodecandra leaves (maceration). DMSO: Dimethyl sulfoxide. EC50: Median effective concentration. Emax: Maximal effect (efficacy). EtOH: Ethanol. HECd-b: Hexanic extract of C. dodecandra bark (maceration). HECd-l: Hexanic extract of C. dodecandra leaves (maceration). Hx: Hexane. KCa: Large-conductance Ca2+-activated K+ channel. KCl: Potassium chloride. KHS: Krebs-Henseleit Solution. L-NAME: L-NG-NitroArginine methyl ester. Maxi-K+: Large-conductance Ca2+-activated K+ channel. MECd-b: Methanolic extract of C. dodecandra bark (maceration). MECd-l: Methanolic extract of C. dodecandra leaves (maceration). MeOH: Methanol. MESxCd-b: Methanolic extract of C. dodecandra bark (soxhlet). MESxCd-l: Methanolic extract of C. dodecandra leaves (soxhlet). mL: Milliliter. MLC: Myosin light chain. MLCK: Myosin light-chain kinase. MLCP: Myosin light chain phosphatase. mM: Milimolar. Nfd: Nifedipine. NO: Nitric oxide. nm: Nanometer NOS: Nitric oxide synthase. ODQ: 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. PDE: Phosphodiesterases. PGE: Prostacyclin. PKA: Protein kinase A enzyme. RKH: Ringer Krebs-Henseleit. rMLC: Regulatory light chain of myosin. sGC: Soluble guanylyl cyclase enzyme. VDC: Voltage-dependent Ca2+ channels. WHO: World Health Organization.

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