molecules Article Homoisoflavonoids and Chalcones Isolated from Haematoxylum campechianum L., with Spasmolytic Activity Armando Escobar-Ramos 1, Carlos Ernesto Lobato-García 1,*, Alejandro Zamilpa 2 ID , Abraham Gómez-Rivera 1, Jaime Tortoriello 2 and Manasés González-Cortazar 2,* ID 1 Academics Division of Basic Sciences, University Juárez Autonomous of Tabasco, Highway Cunduacán-Jalpa Km. 0.5, Cunduacán Tabasco 86690, Mexico; [email protected] (A.E.-R.); [email protected] (A.G.-R.) 2 Southern Biomedical Research, Mexican Institute of Social Security, Argentina No. 1, Col. Centro, Xochitepec 62790, Mexico; [email protected] (A.Z.); [email protected] (J.T.) * Correspondence: [email protected] (C.E.L.-G.); [email protected] (M.G.-C.); Tel.: +52-(777)-3-612-155 (M.G.-C.) Received: 13 July 2017; Accepted: 18 August 2017; Published: 24 August 2017 Abstract: Haematoxylum campechianum is a medicinal plant employed as an astringent to purify the blood and to treat stomach problems such as diarrhea and dysentery. A bio-guided chemical fractionation of the methanolic extract obtained from this plant allowed for the isolation of five compounds: two chalcones known as sappanchalcone (1); 3-deoxysappanchalcone (2); three homoisoflavonoids known as hematoxylol A (3); 4-O-methylhematoxylol (4); and, hematoxin (5). The spasmolytic activity was determined in an in vitro model (electrically induced contractions of guinea pig ileum), and allowed to demonstrate that the methanolic extract (EC50 = 62.11 ± 3.23) fractions HcF7 (EC50 = 61.75 ± 3.55) and HcF9 (EC50 = 125.5 ± 10.65) and compounds 1 (EC50 = 16.06 ± 2.15) and 2 (EC50 = 25.37 ± 3.47) of Haematoxylum campechianum present significant relaxing activity as compared to papaverine (EC50 = 20.08 ± 2.0) as a positive control. Keywords: homoisoflavonoids; Haematoxylum campechianum; chalcone; spasmolytic 1. Introduction The Haematoxilon genus (Leguminosae) has two species: Haematoxylum campechianum and Haematoxylum brassileto. Both are native trees of Central America and belong to the family of the Fabaceae and the subfamily Caesalpinioideae [1]. H. campechianum in Mexico is known as the “red wood”, “black wood”, and “Campeche wood” because it originally was found on the shores of the Gulf of Mexico, in the state of Campeche [2]. The heartwood of this tree is an important source for the well-known dye hematoxylin which is a staining agent employed in histology [3–5]; also, the extract from the wood has been used as a sweetener [6]. Traditional medicine refers to the use of the heartwood of this plant for the treatment of depression, kidney disorders, heart problems, toothache, fever, diarrhea, and hemorrhoids [7,8]; and the branches are mainly used to remove toxins [9]. Chemical studies of this plant lead to identifying: hematoxylin, which by oxidation produces hematein [6]; brazilin [10,11], which was also isolated from Caesalpinia echinata (Leguminosae); hematoxylol A (3)[6]; protossapanin A and B; sappanchalcona (1); 3-deoxisappanchalcone (2), isolated from Caesalpinia sappan L., tetra-O-methylhematoxylol B [6,12]; and, trimethylether of protosappanin B [6], as well as: hematoxylol, epihematoxylol, 4-O-methylhematoxylol, 4-O-methylepihematoxylol, sappanene, hematoxylene, hematoxylone, hematoxin, epihematoxin, hematoxilol B, and isohematoxylin. It is noteworthy that Molecules 2017, 22, 1405; doi:10.3390/molecules22091405 www.mdpi.com/journal/molecules Molecules 2017, 22, 1405 2 of 10 Molecules 2017, 22, 1405 2 of 10 sappanthese compounds L., has an anti-inflammatory showed activity on effect the inhibition in a model of tyrosineof rheumatoid kinase, arthritis being hematoxylin, in mice [16]. hematoxylol However, theA(3 biological) and epihematoxylol studies reported B the mostso far active have compoundsonly been focused[13–15] .on The isolated flavonoid compounds, sapanchalcone but not which on extractsalso was and isolated fractions from forCaesalpinia the heartwood sappan L.,of hasH. campechianum an anti-inflammatory. Our interest effect is in focused a model on of compounds rheumatoid ofarthritis the flavonoid in mice [type16]. because However, they the have biological been sh studiesown to reported have important so far have biological only been activity focused on disorderson isolated of compounds,the central nervous but not onsystem extracts [17,18]. and fractionsThe aim forof this the heartwoodstudy was ofto H.demonstrate campechianum the. spasmolyticOur interest activity is focused of onthe compoundsmethanolic ofextract. the flavonoid Five fractions type because (HcF5-HcF9) they have obtained been shownfrom the to chromatographichave important biological separation activity of this on extract disorders exhi ofbited the centralfive isolated nervous compounds: system [17, 18two]. Thechalcones aim of (compoundsthis study was 1 toand demonstrate 2) and three the spasmolytichomoisoflavonoids activity ofstructurally the methanolic derived extract. from Fivehematoxylin fractions (compounds(HcF5-HcF9) 3 obtained, 4 and from5), all the of chromatographicthem have been separationreported previously of this extract for exhibitedthis plant five [6,11]. isolated The biologicalcompounds: model two employed chalcones was (compounds the in vitro1 and electrical2) and stimulation three homoisoflavonoids of the isolated ileum structurally of guinea derived pig. Additionally,from hematoxylin all five (compounds compounds 3were, 4 and char5acterized), all of themby one have and two-dimensional been reported previously NMR. for this plant [6,11]. The biological model employed was the in vitro electrical stimulation of the isolated ileum 2.of Results guinea pig.and Additionally,Discussion all five compounds were characterized by one and two-dimensional NMR. 2.1.2. Results Evaluation and of Discussion the Ethanolic Extract and Fractions 2.1. EvaluationThe results of presented the Ethanolic in Figure Extract 1 and demonstrate Fractions that the methanolic extract and the fractions of H. campechianumThe results induced presented a concentration-dependent in Figure1 demonstrate thatinhibition the methanolic of the electrical extract stimulation and the fractions induced of contractionsH. campechianum in guineainduced pig aileum, concentration-dependent using papaverine as inhibitiona positive ofcontrol. the electrical The values stimulation of EC50 and induced Emax are presented in Table 1. contractions in guinea pig ileum, using papaverine as a positive control. The values of EC50 and Emax are presented in Table1. Figure 1. Figure 1. Concentration-responseConcentration-response curves curves of of the the methanolic methanolic extract extract,, HcF5 HcF5 to to HcF9 HcF9 fractions of Haematoxilum campechianum induced contractions in isolated guinea pig ileum compared with Haematoxilum campechianum induced contractions in isolated guinea pig ileum compared with papaverine as control (+). papaverine as control (+). TableThe methanol 1. Inhibitory extract effect of obtained the methanolic by the extract maceration, fractions of (HcF5 the heartwood to HcF9) and of compoundsH. campechianum (1–2, 3a–showed5a) inhibitionof Haematoxilum of the electrically campechianum induced induced smooth contractions muscle in contraction isolated guinea (EC pig50 =ileum 62.11 (Effective± 3.23), concentration which is similar to thefifty HcF7 EC50 fraction, relaxation (EC Emax50). = 61.75 ± 3.55) obtained from the chromatographic separation; whereas, the HcF9 fraction (EC50 = 125.5 ± 10.65) presented the lower activity when compared with papaverine Extract, Fraction and Compounds Effective Concentration Fifty (CE50 = µg/mL) Relaxation (%) Emax ± (ECExtract50 = 20.08 MeOH 2.0). It is noteworthy that both the methanolic62.11 ± 3.23 b extract and the HcF7 fraction85.16 ± 6.34 showed d a similarFraction effect to the one exerted by papaverine, as shown by the comparison of the Emax (85.16 ± 6.34 and 84.6HcF5± 1.7 versus 85.72 ± 2.8). The HcF5, HcF6,346.7± 19.32 and f HcF8 fractions showed56.7 no ± 1.8 significant c spasmolyticHcF6 activity. 324.9 ± 10.15 f 80.9 ± 2.5 d HcF7 61.75 ± 3.55 b 84.6 ± 1.7 d HcF8 343.7 ± 10.04 f 95.1 ± 1.2 e HcF9 125.5 ± 10.65 c 24.45 ±1.6 a Compound 1 16.06 ± 2.15 a 50.9 ± 2.2 b,c 2 25.37 ± 3.47 a 48.5 ± 2.0 b Molecules 2017, 22, 1405 3 of 10 Table 1. Inhibitory effect of the methanolic extract, fractions (HcF5 to HcF9) and compounds (1–2, 3a–5a) of Haematoxilum campechianum induced contractions in isolated guinea pig ileum (Effective concentration fifty EC50, relaxation Emax). Molecules 2017, 22, 1405 3 of 10 Effective Concentration Fifty Extract, Fraction and Compounds Relaxation (%) Emax (CE50 = µg/mL) 3a 204.5 ± 2.75 d 85.78 ± 2.7 d b d 4aExtract MeOH 62.11285± ±3.23 11.0 e 85.16 ± 6.34ND 5aFraction 203.1 ±7.2 d 100 ±0.3 e PapaverineHcF5 (+) 346.720.08± 19.32 ± 2.0 fa 56.7 ±85.721.8 c± 2.8 d f d * ValuesHcF6 are mean ± SD., n = 3 independent experiments324.9 ± performed10.15 in triplicates, and80.9 were± 2.5determined ± b ± d by linearHcF7 regression analysis using GraphPad Prism61.75 6.0 Software;3.55 a–f Values are statistically84.6 1.7 significant HcF8 343.7 ± 10.04 f 95.1 ± 1.2 e at p ˂ 0.05. ND = not determined. HcF9 125.5 ± 10.65 c 24.45 ±1.6 a TheCompound methanol extract obtained by the maceration of the heartwood of H. campechianum showed a b,c inhibition1 of the electrically induced smooth muscle16.06 contraction± 2.15 (EC50 = 62.11 ±50.9 3.23),± 2.2which is similar a b to the HcF72 fraction (EC50 = 61.75 ± 3.55) obtained25.37 from± the3.47 chromatographic separation;48.5 ± 2.0 whereas, the 3a 204.5 ± 2.75 d 85.78 ± 2.7 d HcF9 fraction (EC50 = 125.5 ± 10.65) presented the lower activity when compared with papaverine 4a 285 ± 11.0 e ND (EC50 = 20.08 ± 2.0). It is noteworthy that both the methanolic extract and the HcF7 fraction showed a 5a 203.1 ±7.2 d 100 ±0.3 e similarPapaverine effect to the (+) one exerted by papaverine, as 20.08 shown± 2.0 bya the comparison of 85.72the Emax± 2.8 (85.16d ± 6.34 and 84.6 ± 1.7 versus 85.72 ± 2.8).