Homoisoflavonoids and Chalcones Isolated from Haematoxylum campechianum L., with Spasmolytic Activity

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.


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][4][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 these compounds showed activity on the inhibition of tyrosine kinase, being hematoxylin, hematoxylol A (3) and epihematoxylol B the most active compounds [13][14][15].The flavonoid sapanchalcone which also was isolated from Caesalpinia sappan L., has an anti-inflammatory effect in a model of rheumatoid arthritis in mice [16].However, the biological studies reported so far have only been focused on isolated compounds, but not on extracts and fractions for the heartwood of H. campechianum.Our interest is focused on compounds of the flavonoid type because they have been shown to have important biological activity on disorders of the central nervous system [17,18].The aim of this study was to demonstrate the spasmolytic activity of the methanolic extract.Five fractions (HcF5-HcF9) obtained from the chromatographic separation of this extract exhibited five isolated compounds: two chalcones (compounds 1 and 2) and three homoisoflavonoids structurally derived from hematoxylin (compounds 3, 4 and 5), all of them have been reported previously for this plant [6,11].The biological model employed was the in vitro electrical stimulation of the isolated ileum of guinea pig.Additionally, all five compounds were characterized by one and two-dimensional NMR.

Evaluation of the Ethanolic Extract and Fractions
The results presented in Figure 1 demonstrate that the methanolic extract and the fractions of H. campechianum induced a concentration-dependent inhibition of the electrical stimulation induced contractions in guinea pig ileum, using papaverine as a positive control.The values of EC 50 and E max are presented in Table 1.
sappan L., has an anti-inflammatory effect in a model of rheumatoid arthritis in mice [16].However, the biological studies reported so far have only been focused on isolated compounds, but not on extracts and fractions for the heartwood of H. campechianum.Our interest is focused on compounds of the flavonoid type because they have been shown to have important biological activity on disorders of the central nervous system [17,18].The aim of this study was to demonstrate the spasmolytic activity of the methanolic extract.Five fractions (HcF5-HcF9) obtained from the chromatographic separation of this extract exhibited five isolated compounds: two chalcones (compounds 1 and 2) and three homoisoflavonoids structurally derived from hematoxylin (compounds 3, 4 and 5), all of them have been reported previously for this plant [6,11].The biological model employed was the in vitro electrical stimulation of the isolated ileum of guinea pig.Additionally, all five compounds were characterized by one and two-dimensional NMR.

Evaluation of the Ethanolic Extract and Fractions
The results presented in Figure 1 demonstrate that the methanolic extract and the fractions of H. campechianum induced a concentration-dependent inhibition of the electrical stimulation induced contractions in guinea pig ileum, using papaverine as a positive control.The values of EC50 and Emax are presented in Table 1.The methanol extract obtained by the maceration of the heartwood of H. campechianum showed inhibition of the electrically induced smooth muscle contraction (EC 50 = 62.11 ± 3.23), which is similar to the HcF7 fraction (EC 50 = 61.75 ± 3.55) obtained from the chromatographic separation; whereas, the HcF9 fraction (EC 50 = 125.5 ± 10.65) presented the lower activity when compared with papaverine (EC 50 = 20.08 ± 2.0).It is noteworthy that both the methanolic extract and the HcF7 fraction showed a similar effect to the one exerted by papaverine, as shown by the comparison of the Emax (85.16 ± 6.34 and 84.6 ± 1.7 versus 85.72 ± 2.8).The HcF5, HcF6, and HcF8 fractions showed no significant spasmolytic activity., n = 3 independent experiments performed in triplicates, and were determined by linear regression analysis using GraphPad Prism 6.0 Software; a-f Values are statistically significant at p < 0.05.ND = not determined.

Evaluation of Compounds 1, 2 and 3a-5a
The results presented in Figure 2 demonstrate that compounds 1 and 2 isolated from the heartwood of H. campechianum induced a concentration-dependent inhibition of the electrical stimulation-induced contractions in the guinea-pig ileum, as did the papaverine positive control.The methanol extract obtained by the maceration of the heartwood of H. campechianum showed inhibition of the electrically induced smooth muscle contraction (EC50 = 62.11 ± 3.23), which is similar to the HcF7 fraction (EC50 = 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 (EC50 = 20.08 ± 2.0).It is noteworthy that both the methanolic extract and the HcF7 fraction showed a similar effect to the one exerted by papaverine, as shown by the comparison of the Emax (85.16 ± 6.34 and 84.6 ± 1.7 versus 85.72 ± 2.8).The HcF5, HcF6, and HcF8 fractions showed no significant spasmolytic activity.

Evaluation of Compounds 1, 2 and 3a-5a
The results presented in Figure 2 demonstrate that compounds 1 and 2 isolated from the heartwood of H. campechianum induced a concentration-dependent inhibition of the electrical stimulation-induced contractions in the guinea-pig ileum, as did the papaverine positive control.Chromatographic separation allowed us to isolate five homoisoflavonoids-type compounds; two chalcones known as sapachalcone (1, EC50 = 16.06 ± 2.15) and 3-deoxysappachalcone (2, EC50 = 25.37 ± 3.47), that presented similar relaxing activity as papaverine (EC50 = 20.08 ± 2.0) and hematoxylol A tetraacetate (3a, EC50 = 204.5 ± 2.75), 4-O-methylhematoxylol tetracetate (4a, EC50 = 285 ± 11.0), and hematoxin diacetate (5a, EC50 = 203.1 ± 7.2).It must be noted that compounds 3a, 4a and 5a had no significant relaxing activity (Table 1).These results suggest that the effect produced by the methanolic extract is mainly caused by the chalcones.With this finding, both the biological activity of H. campechianum, as well as its use in traditional medicine were demonstrated.However, further studies are required in order to determine the biological activity of this plant for specific diseases, in order to develop treatments focused on solving particular illness.

Structural Elucidation of Homoisoflavoids
Chromatographic fractionation of HcF6 and HcF7 allowed for the purification of 1 which was Chromatographic separation allowed us to isolate five homoisoflavonoids-type compounds; two chalcones known as sapachalcone (1, EC 50 = 16.06 ± 2.15) and 3-deoxysappachalcone (2, EC 50 = 25.37 ± 3.47), that presented similar relaxing activity as papaverine (EC 50 = 20.08 ± 2.0) and hematoxylol A tetraacetate (3a, EC 50 = 204.5 ± 2.75), 4-O-methylhematoxylol tetracetate (4a, EC 50 = 285 ± 11.0), and hematoxin diacetate (5a, EC 50 = 203.1 ± 7.2).It must be noted that compounds 3a, 4a and 5a had no significant relaxing activity (Table 1).These results suggest that the effect produced by the methanolic extract is mainly caused by the chalcones.With this finding, both the biological activity of H. campechianum, as well as its use in traditional medicine were demonstrated.However, further studies are required in order to determine the biological activity of this plant for specific diseases, in order to develop treatments focused on solving particular illness.
The carbon-hydrogen assignment was performed with HSQC and the correlations at two and three bonds were established using the HMBC spectra (Figure 3).Compound (2) was obtained as an orange powder with a melting point of 242 °C.The NMR spectra of 1 H and 13 C, showed the same signals as compound (1) (see Tables 1 and 2), except for one of the ABX systems of aromatic rings, which is now AB in 7.49 (dd, 1.8, 8.4 Hz, H-2, H-6), and 6.81 (dd, 1.8, 8.4 Hz, H-3, H-5).Direct comparison of spectroscopic data with those described indicate that this compound corresponds to 2′-methoxy-4,4′-dihydroxychalcone, also known as 3-deoxysappanchalcone (2) [11].
The carbon-hydrogen assignment was performed with HSQC and the correlations at two and three bonds were established using the HMBC spectra (Figure 3).

Hematoxylol A Tetraacetate (3a)
Compound (3) was isolated as a white powder with a melting point of 239 • C. According to the analysis of NMR spectroscopic data (see Tables 2 and 3) and the comparison with data described in the literature, this compound was identified as 3,4,10,11-tetrahydroxy-7,8-dihidroxy-6H-dibenz[b,d]oxocin-7-one (see Figure 1) on the basis of the DEPT, COSY HSQC, and HMBC spectra of its peracetate derivative (3a), also known as hematoxylol A(3), which had been previously isolated from Haematoxylum campechianium and tested as a protein tyrosine kinase inhibitor [6,13].3) was isolated as a white powder with a melting point of 239 °C.According to the analysis of NMR spectroscopic data (see Tables 2 and 3) and the comparison with data described in the literature, this compound was identified as 3,4,10,11-tetrahydroxy-7,8-dihidroxy-6H-dibenz[b,d] oxocin-7-one (see Figure 1) on the basis of the DEPT, COSY HSQC, and HMBC spectra of its peracetate derivative (3a), also known as hematoxylol A(3), which had been previously isolated from Haematoxylum campechianium and tested as a protein tyrosine kinase inhibitor [6,13].4) was isolated as a yellow powder with a melting point of 98.2 °C.On the basis of the DEPT, COSY HSQC, and HMBC spectra (see Tables 1 and 2) of its peracetate derivative (4a) and the comparison with data described in the literature this compound was identified as 4-O-methylhematoxylol tetraacetate (4a).The natural compound (4) was isolated from Haematoxylum campechianium and tested as a protein tyrosine kinase inhibitor [6,13].5) was isolated as a yellow powder with a melting point of 136.9 °C.On the basis of the DEPT, COSY, HSQC, and HMBC spectra (see Tables 1 and 2) of its peracetate derivative (5a), and the comparison with data described in the literature this compound was identified as hematoxin diacetate (5a).The natural compound (5) was isolated from Haematoxylum campechianium and tested as a protein tyrosine kinase inhibitor [6,13].

4-O-Methylhematoxylol Tetracetate (4a)
Compound (4) was isolated as a yellow powder with a melting point of 98.2 • C. On the basis of the DEPT, COSY HSQC, and HMBC spectra (see Tables 1 and 2) of its peracetate derivative (4a) and the comparison with data described in the literature this compound was identified as 4-O-methylhematoxylol tetraacetate (4a).The natural compound (4) was isolated from Haematoxylum campechianium and tested as a protein tyrosine kinase inhibitor [6,13].3) was isolated as a white powder with a melting point of 239 °C.According to the analysis of NMR spectroscopic data (see Tables 2 and 3) and the comparison with data described in the literature, this compound was identified as 3,4,10,11-tetrahydroxy-7,8-dihidroxy-6H-dibenz[b,d] oxocin-7-one (see Figure 1) on the basis of the DEPT, COSY HSQC, and HMBC spectra of its peracetate derivative (3a), also known as hematoxylol A(3), which had been previously isolated from Haematoxylum campechianium and tested as a protein tyrosine kinase inhibitor [6,13].4) was isolated as a yellow powder with a melting point of 98.2 °C.On the basis of the DEPT, COSY HSQC, and HMBC spectra (see Tables 1 and 2) of its peracetate derivative (4a) and the comparison with data described in the literature this compound was identified as 4-O-methylhematoxylol tetraacetate (4a).The natural compound (4) was isolated from Haematoxylum campechianium and tested as a protein tyrosine kinase inhibitor [6,13].5) was isolated as a yellow powder with a melting point of 136.9 °C.On the basis of the DEPT, COSY, HSQC, and HMBC spectra (see Tables 1 and 2) of its peracetate derivative (5a), and the comparison with data described in the literature this compound was identified as hematoxin diacetate (5a).The natural compound ( 5) was isolated from Haematoxylum campechianium and tested as a protein tyrosine kinase inhibitor [6,13].

Hematoxin Diacetate (5a)
Compound ( 5) was isolated as a yellow powder with a melting point of 136.9 • C. On the basis of the DEPT, COSY, HSQC, and HMBC spectra (see Tables 1 and 2) of its peracetate derivative (5a), and the comparison with data described in the literature this compound was identified as hematoxin diacetate (5a).The natural compound (5) was isolated from Haematoxylum campechianium and tested as a protein tyrosine kinase inhibitor [6,13].

General Experimental Procedures
Melting points were obtained on a Termo Scientific IA9000 series melting point apparatus (Electrothermal, Essex, UK) and were left uncorrected.All NMR spectra were recorded on a Bruker Advance III HD-600 at 600 MHz for 1 H-NMR, NOESY, 1 H-1 H COSY, HSQC, and HMBC, and 150 MHz for 13 C-NMR and DEPT in CDCl3, CD3OD, CD3COCD3 and DMSO.Chemical displacements are reported in ppm relative to TMS.
For quantification of the isolated compounds, 5 mg in one milliliter of methanol were dissolved in dilution series of 25, 50, 100 and 200 g/mL.

Plant Material
The heartwoods of Haematoxilumn campechanium L. (7.5 kg), were collected in Cunduacán Tabasco, Mexico in February 2016.The voucher specimen of this material (No. 35455) was deposited in the herbarium of Juarez Autonomous University of Tabasco at Villahermosa, Tabasco, México.

Extraction and Chromatographic Fractionation of Heartwood
The dried and powdered heartwood was successively extracted three times with methanol (14 L, 3 times) for 24 h, at a room temperature.The obtained methanolic extract was evaporated to dryness with a rotary evaporator under reduced pressure (Heidolph G3, Schwabach, Germany), producing a residue of 300 g.

General Experimental Procedures
Melting points were obtained on a Termo Scientific IA9000 series melting point apparatus (Electrothermal, Essex, UK) and were left uncorrected.All NMR spectra were recorded on a Bruker Advance III HD-600 at 600 MHz for 1 H-NMR, NOESY, 1 H-1 H COSY, HSQC, and HMBC, and 150 MHz for 13 C-NMR and DEPT in CDCl 3 , CD 3 OD, CD 3 COCD 3 and DMSO.Chemical displacements are reported in ppm relative to TMS.
For quantification of the isolated compounds, 5 mg in one milliliter of methanol were dissolved in dilution series of 25, 50, 100 and 200 g/mL.

Plant Material
The heartwoods of Haematoxilumn campechanium L. (7.5 kg), were collected in Cunduacán Tabasco, Mexico in February 2016.The voucher specimen of this material (No. 35455) was deposited in the herbarium of Juarez Autonomous University of Tabasco at Villahermosa, Tabasco, México.

Extraction and Chromatographic Fractionation of Heartwood
The dried and powdered heartwood was successively extracted three times with methanol (14 L, 3 times) for 24 h, at a room temperature.The obtained methanolic extract was evaporated to dryness with a rotary evaporator under reduced pressure (Heidolph G3, Schwabach, Germany), producing a residue of 300 g.
The fraction HcF8(14 g) was separated using a chromatographic column (10 × 50 cm) with normal phase silica gel (170 g, 70-230 mesh, Merck) and eluted with a gradient system with dichloromethane/methanol with an increase in polarity of 5%, obtaining 105 fractions (HcF8-F1 to HcF8-F18) of 100 mL each.The 18 fractions were grouped according to their similarity in TLC.

Model of Ileum Isolated Guinea Pigs
To evaluate the spasmolytic activity, the experimental model of electrically induced guinea pig isolated ileum was performed.Guinea pigs of either sex (250-500 g) were used and subjected to cervical dislocation.Ethical guidelines for the handling and slaughter of experimental animals indicated by the American Veterinary Medical Association (AVMA, 2001) were followed.
(Official Mexican Standard NOM-062-ZOO-1999).Their abdomens were opened.Their ileum were removed and maintained in Petri dishes containing Tyrode's solution, and constantly aerated with carbogen gas (O 2 95%, CO 2 5%).Portions of about 1.5 cm length of the tissue were mounted in a set of 3 mL chambers.One end of the tissue was attached to the bottom of the chamber to an electrode while the other was attached with a silk thread to a force transducer, which was connected to an acquisition system (PanLab, BIOPAC Systems, Goleta, CA, USA).After 30 min of an adaptation period, the tissue was electrically stimulated (25 V, 5 mS, 1 Hz, 5 S, every 2 min; with a Grass stimulator) by isolated tungsten electrodes connected to the end of the tissue.Induced contractions were recorded and after homogeneous response, different concentrations of drugs under study were added into the chamber, and the ability for inhibiting the electrically induced contraction was evaluated [20].The positive control used was papaverine.

Preparation of Extracts, Fractions and Compounds for Evaluations
The methanolic extract, fractions, and compounds were diluted in Tyrode's solution and PVP (polyvinylpyrrilodine, 1:2) for the less polar.

Statistical Analysis
The effect in this model was expressed as the mean ± standard deviation (SD).From n = 3 independent experiments were performed in triplicates, and were determined by linear regression analysis using GraphPad Prism 6.0 Software(GraphPad Software, San Diego, CA, USA).The values of the mean effective concentrations (EC 50 ) and maximum effects (E max ) were obtained from the concentration-response curves.The data were analyzed by a one-way ANOVA and the Tukey test.Differences between the means were considered to be significant when p < 0.05.

Conclusions
Five compounds of H. campechianum methanolic extract were isolated and characterized: two chalcones known as sappanchalcone (1), 3 -deoxy-sappanchalcone (2), three homoisoflavonoids derivatized known as hematoxylol A (3a), 4-O-methylhematoxylol (4a), and hematoxin (5a).The correct structures were assigned by detailed spectroscopy analysis of NMR in one and two dimensions.The most bioactive chalcones 1 and 2 were isolated from the spasmolytic fraction (Hc-F7).In the biological test in vitro, extract, fractions, and compounds 1 and 2. These results validate the traditional use of this plant.

Figure 1 .
Figure 1.Concentration-response curves of the methanolic extract, HcF5 to HcF9 fractions of Haematoxilum campechianum induced contractions in isolated guinea pig ileum compared with papaverine as control (+).

Figure 1 .
Figure 1.Concentration-response curves of the methanolic extract, HcF5 to HcF9 fractions of Haematoxilum campechianum induced contractions in isolated guinea pig ileum compared with papaverine as control (+).
* Values are mean ± SD.