Smooth Muscle Relaxant Action of Benzyl Benzoates

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Ileum segments (2 cm each) were isolated from male Hartley gui- nea-pigs (600 ± 800 g) .... with an excess of ethereal CH2N2 to yield 15 (17 mg) as a colour- less oil. Isolates .... spectrum showed a resonance for a phenolic group at δH = 10.0 .... effects, mode of action and structure-activity relationships of stilbe- noids from ...
Smooth Muscle Relaxant Action of Benzyl Benzoates and Salicylic Acid Derivatives from Brickellia veronicaefolia on Isolated Guinea-Pig Ileum Original Paper

Abstract A dichloromethane-MeOH extract prepared from the aerial parts of Brickellia veronicaefolia inhibited the spontaneous contractions (IC50 = 39.22 g/mL) of the guinea-pig ileum. Bioassayguided fractionation of the extract led to the isolation of three new benzoic acid derivatives, 1,2-bis-O-(2-methoxybenzoyl)-bD-glucopyranoside (1), 3-(b-glucopyranosyloxy)benzyl 2,6-dimethoxybenzoate (2) and 3-hydroxybenzyl 2,6-dimethoxybenzoate (3), together with the known compounds taraxasteryl acetate (4), 4-allyl-2-methyloxyphenyl-b-glucopyranoside (5), 2-hydroxy-6-methoxybenzoic acid (6), 2-methoxybenzoic acid (7), chamazulene (8), 2-methoxybenzyl 2-hydroxybenzoate (9), benzyl 2,6-dimethoxybenzoate (10), 3-methoxybenzyl 2-hydro-

Blanca Rivero-Cruz1 María Alejandra Rojas2 Rogelio Rodríguez-Sotres1 Carlos M. Cerda-García-Rojas3 Rachel Mata1

xy-6-methoxybenzoate (11), benzyl 2-hydroxy-6-methoxybenzoate (12), benzyl 2,3,6-trimethoxybenzoate (13), benzyl 2hydroxy-3,6-dimethoxybenzoate (14) and 3-methoxybenzyl 2,6-dimethoxybenzoate (15). The isolates were characterized by spectral means. Compounds 2 ± 6, 8 ± 11, 14 and 15 induced a concentration-dependent inhibition of the spontaneous contractions of the guinea-pig ileum with IC50 values ranging from 1.49 to 4.96 M. Their activity was comparable to that of papaverine (IC50 = 4.23 M). Key words Smooth muscle relaxant ´ benzyl benzoates ´ Brickellia veronicaefolia ´ Asteraceae ´ chamazulene

320

Introduction Brickellia veronicaefolia (Kunth) Gray (Asteraceae) is a bittertasting shrub growing in the oak and pine woodlands of Mexico where it is commonly known as ªpextoº and ªpeistonº. Previous phytochemical studies of the aerial parts of this species resulted in the isolation and characterization of several 6-methoxyflavonols and labdane-type diterpenes [1], [2], [3], [4], [5]. B. veronicaefolia is one of the species most widely commercialized in Mexico for the treatment of gastrointestinal discomforts including stomachache biliary colic and dyspepsia [1]. In addition,

the plant is highly valued for the treatment of arthritis, local inflammations and infectious diseases. The traditional uses of B. veronicaefolia suggested the presence of smooth muscle relaxant and/or anti-inflammatory agents. Furthermore, a preliminary pharmacological test revealed that a CH2Cl2-MeOH (1 : 1) extract prepared from the aerial parts of the plant inhibited the spontaneous contractions of the guinea-pig ileum. Accordingly, in our continuing efforts to investigate Mexican plants widely used to treat gastrointestinal disorders ([6], [7], inter alia), the aim of the present investigation was to isolate and characterize the antispasmodic principles of B. veronicaefolia.

Affiliation 1 Facultad de Química, Universidad Nacional Autónoma de MØxico, Ciudad Universitaria, MØxico DF, MØxico 2 Facultad de Química, Universidad Autónoma de QuerØtaro, Centro Universitario, QuerØtaro, MØxico 3 Departamento de Química, Centro de Investigación y de Estudios Avanzados del Instituto PolitØcnico Nacional, MØxico DF, MØxico Correspondence Dr. Rachel Mata ´ Facultad de Química ´ Universidad Nacional Autónoma de MØxico ´ Coyoacµn 04510 ´ MØxico City ´ MØxico ´ Phone: +525-55-622 5289 ´ Fax: +525-55-622 5329 ´ E-mail: [email protected] History Taken in part from the PhD thesis of B. Rivero-Cruz submitted to Posgrado en Ciencias Químicas, UNAM 2005 Received June 14, 2004 ´ Accepted October 30, 2004 Bibliography Planta Med 2005; 71: 320±325 ´  Georg Thieme Verlag KG Stuttgart ´ New York DOI 10.1055/s-2005-864097 ISSN 0032-0943

Materials and Methods

Plant material The aerial parts of B. veronicaefolia were collected in Municipio de la Soledad, Aculco, State of Mexico, Mexico on December 10, 2000. An authenticated voucher specimen (BRC-1M) is preserved in the National Herbarium (MEXU), Instituto de Biologia, UNAM. The plant material was identified by Dr. Fernando Calzada. Isolated guinea-pig ileum test Ileum segments (2 cm each) were isolated from male Hartley guinea-pigs (600 ± 800 g) and prepared for recording the contractions using force transducers (Grass FTO3) connected to a polygraph (Grass 7D) as previously described [7], [8]. After a stabilization time of 30 min, a 10 min control period was recorded. The test substance (extract, fraction or compound) dissolved in DMSO was added to the bath (10 mL) in a volume of 50 L at different concentrations (one concentration was used per ileum segment). All the responses were recorded during 10 min. The effect of the extract, fractions and isolated compounds was determined by comparing the areas under the curve (AUC) inscribed by the frequency and the amplitude of the ileum contractions before and after the application of the test materials. Areas were calculated from polygraph tracings using an analog-digital tablet (CPLAB-10). Statistical analysis All the results are expressed as the mean of at least six experiments  SEM. CRC for the extract and pure compounds were plotted and the experimental data from the CRC were adjusted by non-linear, least squares, curve fitting program (PRISMA). The statistical significance (p < 0.05) of differences between means was assessed by an analysis of variance (ANOVA) followed by a Tukey's test [8], [9]. Extraction and isolation The air-dried plant material (2.5 kg) was ground into powder and extracted exhaustively by maceration at room temperature with a mixture of CH2Cl2-MeOH (1 : 1). After filtration, the extract was

The hexane fraction FI [60 g] was subjected to column chromatography on silica gel (1.3 kg) eluting with a gradient of n-hexane-CH2Cl2-MeOH [n-hexane (1.8 L); n-hexane-CH2Cl2 95 : 5 (7.2 L); n-hexane-CH2Cl2 9 : 1 (6.6 L); n-hexane-CH2Cl2 8 : 2 (3.3 L); nhexane-CH2Cl2 7: 3 (3.6 L); n-hexane-CH2Cl2 6 : 4 (2.4 L); n-hexane-CH2Cl2 1 : 1 (3.0 L); n-hexane-CH2Cl2 4 : 6 (3.3 L); n-hexaneCH2Cl2 3 : 7 (2.1 L); n-hexane-CH2Cl2 2 : 8 (2.7 L); n-hexaneCH2Cl2 1 : 9 (1.8 L); CH2Cl2 (1.2 L); CH2Cl2-MeOH 9 : 1 (1.5 L)]. This process furnished fifteen secondary fractions. HPLC separation [6 mL/min, n-hexane] of the fraction FI-1 [eluted with n-hexane-CH2Cl2 (95 : 5)] led to the isolation of 8 (50 mg, tR = 13.8 min). From fraction FI-10 [eluted with n-hexane-CH2Cl2 (80 : 20)] crystallized 200 mg of 4. Preparative HPLC [6 mL/min; n-hexane-EtOAc (8 : 2)] of the mother liquors from fraction FI-10 yielded 9 (15 mg, tR = 13.95 min). Fraction FI-12 [eluted with nhexane-CH2Cl2 (70 : 30)] was also separated by HPLC [6 mL/min, n-hexane-EtOAc (75 : 25)] to render 11 (10 mg) and 10 (30 mg), tR's = 13.4 and 15.1 min, respectively. Finally, preparative HPLC [6 mL/min, n-hexane-EtOAc (88 : 12)] of fraction FI-15 [(2.8 g), eluted with CH2Cl2] afforded 10 mg of 12 (tR = 14.7 min), 6 mg of 14 (tR = 22.9 min), 3.5 mg of 13 (tR = 24.0 min) and 98 mg of 15 (tR = 27.2 min). Acid hydrolysis of compound 1 Compound 1 (8 mg) was refluxed with 5 mL of HCl (1 N) for 3 h. The reaction mixture was extracted with CH2Cl2 ( ” 2). Upon evaporation of the solvent 2 mg of compound identical (IR, NMR) to 7 was obtained. The glycone was identified as glucose (TLC) by direct comparison with an authentic sample. Basic hydrolysis of compound 2 Compound 2 (2 mg) dissolved in 2 mL of KOH (10 %) in MeOH was refluxed for 1 h. The reaction mixture was neutralized with Rivero-Cruz B et al. Smooth Muscle Relaxant ¼ Planta Med 2005; 71: 320 ± 325

Original Paper

General experimental procedures Melting points were determined on a Fisher-Johns apparatus and are uncorrected. IR spectra were recorded as KBr pellets or films on a Perkin-Elmer 59 913 spectrophotometer. Optical rotations were registered on a Perkin-Elmer 241 digital polarimeter. NMR spectra including COSY, NOESY, HMQC and HMBC experiments were recorded on a Varian VXR-300S or a Varian Mercury 300 spectrometer in CDCl3 or CD3OD, either at 300 (1H) or 75.4 (13C) MHz, using tetramethylsilane (TMS) as an internal standard. EIMS were obtained on a JMS-AX505HA mass spectrometer. Positive HR-FAB-MS data were obtained in a JEOL SX 102 mass spectrometer using an NBA matrix. HPLC was carried out with a Waters HPLC instrument equipped with Waters 996 UV photodiode array detector (900) set at 270 ± 400 nm, using a Porasil column (19 mm i. d. ” 300 mm). Control of the equipment, data acquisition, processing and management of chromatographic information were performed by the Millennium 32 software program (Waters). Column chromatography: silica gel 60 (70 ± 230 mesh, Merck). TLC analyses were performed on silica gel 60 F254 plates (Merck), and visualization of plates was carried out using a ceric sulfate (10 %) solution in H2SO4.

concentrated under vacuum to yield 342 g of a greenish residue. Part of the active extract (230 g) was suspended in aqueous MeOH (10 % water), and sequentially partitioned with n-hexane (FI), CH2Cl2 (FII) and EtOAc (FIII). FII (75.4 g) was chromatographed on a silica gel column (1 kg) using a gradient of CH2Cl2-MeOH [CH2Cl2 (6.3 L); CH2Cl2-MeOH 99 : 1 (3.6 L); CH2Cl2-MeOH 98 : 2 (5.1 L); CH2Cl2-MeOH 97: 3 (3.6 L); CH2Cl2-MeOH 96 : 4 (3.0 L); CH2Cl2-MeOH 95 : 5 (1.8 L); CH2Cl2-MeOH 93 : 7 (1.5 L); CH2Cl2MeOH 9 : 1 (2.1 L); CH2Cl2-MeOH 8 : 2 (1.5 L)] to yield six secondary fractions (FII-1 to FII-6). Pharmacological testing indicated three active fractions (FII-2, FII-3 and FII-5). The most active fraction FII-5 [3 g, eluted with CH2Cl2-MeOH (9.8 : 0.2); 79 % of inhibition when tested at the IC50 of the original extract] was subjected to column chromatography on silica gel (150 g) using CH2Cl2 with increasing volumes of MeOH [CH2Cl2 (600 mL); CH2Cl2MeOH 99 : 1 (750 mL); CH2Cl2-MeOH 98 : 2 (600 mL) and CH2Cl2-MeOH 95 : 5 (500 mL)] as mobile phase to furnish six tertiary fractions (FII-5-I to FII-5-VI). HPLC separation [3 mL/min; CH2Cl2-MeOH (98 : 2)] of fraction FII-5-III afforded 1 (20 mg, tR = 6.7 min). Fraction FII-5-VI was subjected to HPLC separation [0.6 mL/min, CH2Cl2-MeOH (95 : 5)] to yield 5 (10 mg, tR = 18.7 min) and 2 (13 mg, tR = 27.8 min). Active fraction FII-2 [1 g, eluted with CH2Cl2-MeOH (95 : 5), 70 % of inhibition when tested at the IC50 of the original extract] was further chromatographed on a silica gel column (30 g) using CH2Cl2 to furnish 20 mg of 6, 200 mg of 3 and 10 mg of 7.

321

HCl 1 N and extracted with CH2Cl2 to obtain aglycone 3. The water-soluble residue was analyzed by TLC; glucose was identified by direct comparison with an authentic sample. Methylation of compound 3 A solution of 3 (20 mg) in diethyl ether (0.5 mL) was treated with an excess of ethereal CH2N2 to yield 15 (17 mg) as a colourless oil.

Original Paper

Isolates 1,2-Bis-O-(2-methoxybenzoyl)-b- glucopyranoside (1): glassy yellow solid; [a]D: + 2648 (c 0.01, MeOH); UV (EtOH): lmax (log e) = 210 (3.2), 234 (2.8), 303 (4.3) nm; IR (KBr): nmax = 3400, 2500, 1732, 1615 and 1487 cm±1; 1H- and 13C-NMR data, see Table 1; HR-FAB-MS: m/z = 449.4264 [M + H]+ (calcd. for C22H25O10 : 449.4266). 3-(b-Glucopyranosyloxy)benzyl 2,6-dimethoxybenzoate (2): yellow oil; [a]D: ±448 (c 0.09, MeOH); UV (EtOH): lmax (log e) = 270 (1.9); 300 (3.2) 360 (4.0) nm; IR (KBr): nmax = 3400, 1700, 1629, 1487, 1244, 1172, 1062 cm±1; 1H- and 13C-NMR data, see Table 2; HR-FAB-MS: m/z = 451.4419 [M + H]+ (calcd. for C22H27O10 : 451.4424). 3-Hydroxybenzyl 2,6-dimethoxybenzoate (3): colorless crystals; UV (EtOH): lmax (log e) = 270 (1.8); 278 (4.46), 300 (2.4) 360 (4.2) nm; IR (KBr): nmax = 3271, 1693, 1590, 1473, 1257, 1113 cm±1; 1H- and 13C-NMR see Table 2; EI-MS: m/z (rel. int) = 288 [M+] (65), 165 [ M+ ± C7H7O] (base peak), 150 (27), 138 (50), 107 (77), 77 (36); HR-EI-MS: m/z = 288.30 (calcd. for C16H16O5 : 288.3001).

Results and Discussion A CH2Cl2-MeOH (1 : 1) extract prepared from the aerial parts of Brickellia veronicaefolia showed a concentration dependent inhibition of the tone and amplitude of the spontaneous contraction of the guinea-pig ileum with an IC50 = 39.22 g/mL (Fig. 1). In order to identify the active principles, the crude extract was initially fractionationated by solvent partition to yield four primary fractions (FI - FIV). FI and FII, tested at the IC50 of the original extract, showed the highest inhibitory effect on the spontaneous contraction of the guinea-pig ileum (79 and 50 % inhibition, respectively). Repeated chromatography of the active fractions allowed the isolation of compounds 1 ± 15. Compounds 1 ± 3 are new natural products and were characterized by spectral means as 1,2-bis-O-(2-methoxybenzoyl)-b-glucopyranoside (1), 3-(bglucopyranosyloxy)benzyl 2,6-dimethoxybenzoate (2) and 3-hydroxybenzyl-2,6-dimethoxybenzoate (3). Secondary metabolites 4 ± 15 were identified by comparison of their NMR and MS data with those previously described in the literature as taraxasteryl acetate (4) [10], 4-allyl-2-methyloxyphenyl-b-D-glucopyranoside (5) [11], 2-hydroxy-6-methoxybenzoic acid (6) [12], 2-methoxybenzoic acid (7) [12], chamazulene (8) [13], 2-methoxybenzyl 2hydroxybenzoate (9) [14], benzyl 2,6-dimethoxybenzoate (10) [15], 3-methoxybenzyl 2-hydroxy-6-methoxybenzoate (11) [16], benzyl 2-hydroxy-6-methoxybenzoate (12) [14], benzyl 2,3,6trimethoxybenzoate (13) [15], benzyl 2-hydroxy-3,6-dimethoxybenzoate (14) [16] and 3-methoxybenzyl 2,6-dimethoxybenzoate (15) [15]. Compound 1 (C22H24O10) showed IR absorptions at 3400 ± 2500, 1732 and 1615, 1487 cm±1 consistent with the presence of hydro-

322

Table 1

1

H- (300 MHz) and 13 C-NMR (75.4 MHz) data for 1 (in CD3OD) dC

HMBC

1

122.0

3

2

161.1

4, 6

Position

dH

3

7.02 br d (J = 9.0)

114.0

1, 5

4

7.47 ddd (J = 9.0, 8.4, 1.8)

135.7

2, 6

5

6.92 br dd (J = 8.4, 7.8)

121.9

3

6

7.70 dd (J = 7.8, 1.8)

133.0

2

7

166.4

2¢ , 6



5.97 d (J = 8.4)



5.24 dd (J = 9.9, 8.4)

75.3





3.78 m

76.8





3.56 complex m

72.1

3¢, 6¢a



3.56 complex m

80.0

3¢, 4¢, 6¢b



3.92 br d (J = 12.0); 3.78 m

63.1



1¢¢

120.1

3¢¢

2¢¢

162.0

4¢¢, 6¢¢

94.7



3¢¢

7.05 br d (J = 9.0)

114.1

5¢¢, 1¢¢

4¢¢

7.50 ddd (J = 9.0, 8.4, 1.8)

136.6

2¢¢, 6¢¢

5¢¢

6.95 br dd (J = 8.4, 7.8)

121.9

3¢¢

6¢¢

7.76 dd (J = 7.8, 1.8)

133.8

2¢¢

168.1

1¢, 6¢¢

7¢¢ OMe-2

3.63 s

57.1

OMe-2¢

3.70s

57.1

Values in parentheses are coupling constants in Hz.

Rivero-Cruz B et al. Smooth Muscle Relaxant ¼ Planta Med 2005; 71: 320 ± 325

Table 2

1

H- (300 MHz) and 13C-NMR (75.4 MHz) spectral data for compounds 2 (in CD3OD), 3 and 15 (in CDCl3)

Position

2

dH

3

dC

15

dH

dC

1

114.1

112.7

2

158.5

157.3

dH

dC 113.5 157.5

3

6.65 d (J = 8.4)

105.0

6.55 d ( J = 8.4)

103.9

6.55 d (J = 8.4)

4

7.33 t (J = 8.4)

132.4

7.28 t (J = 8.4)

131.1

7.28 t (J = 8.4)

103.9 131.1

5

6.65 d (J = 8.4)

105.0

6.55 d (J = 8.4)

103.9

6.55 d (J = 8.4)

103.9

6

158.5

157.3

157.5

7

168.1

166.5

166.4



138.8

137.7

7.17 br t (J = 2.5)



117.3

6.93 br t (J = 2.5)

158.9

114.8

137.7 6.83 br t (J = 2.5)

155.6

113.5 159.7



7.06 br dd (J = 8.1, 2.5)

117.0

6.76 br dd (J = 8.1, 2.5)

114.9

6.86 br dd (J = 8.1, 2.5)

113.4



7.28 dd (J = 7.9, 8.1)

130.2

7.21 dd (J = 7.9, 8.1)

129.5

7.27 dd (J = 7.9, 8.1)

129.3

122.6

6.98 br d (J = 7.9)

120.1

7.01 br d (J = 7.9)

120.2



7.07 br d (J = 7.9)



5.28 br s

1¢¢

4.92 d (J = 7.5)

2¢¢

3.43 m

74.8

3¢¢

3.40 complex m

78.0*

4¢¢

3.40 complex m

71.1

5¢¢

3.40 complex m

77.9*

6¢¢

3.86 dd (J = 11.7, 1.5) 3.69 dd (J = 11.7, 3.9)

62.3

OCH3 (2,6)

3.80 s

56.5

67.4

5.32 br s

66.6

5.36 br s

66.6

3.80 s

56.0

3.80 s

55.9

3.81 s

55.2

Original Paper



102.1

OCH3 (3¢) * May be interchanged. Values in parentheses are coupling constants in Hz.

Fig. 1 Structures of compounds 1 ± 3 and 9 ± 15.

xy, ester and aromatic functionalities in the molecule. The UV maximum at l = 303 nm supported a conjugated aromatic function. The NMR (Table 1) spectra of 1 were well dispersed in MeOD and showed the presence of two disubstituted aromatic rings and one hexose unit [17]. Furthermore, the 13C-NMR chemical shifts were consistent with a b-D-glucopyranose unit esterified with two 2-methoxybenzoic acid moieties at C-1¢ and C-2¢. Basic hydrolysis (KOH) yielded glucose (TLC) and salicylic acid; the latter compound was identified by comparison with an authentic sample. Two dimensional NMR experiments, in-

cluding NOESY and HMBC, were used to establish the position of the salicylic acid units along the sugar core. The HMBC correlations C-7¢¢/H-6¢¢, H-1¢ linked the first moiety at C-1¢, while the correlations C-7/H-2¢, H-6 connected the second to C-2¢. Finally, the coupling pattern observed for H-2¢ (dd, J = 9.2, 8.4 Hz) and H-1¢ (d, J = 8.4 Hz) in the 1H-NMR spectrum of 1 revealed their trans-diaxial relationship and thus, the equatorial orientation of both salicylic acid units. Therefore, compound 1 was identified as 1,2-bis-O-(2-methoxybenzoyl)-b-glucopyranoside. Rivero-Cruz B et al. Smooth Muscle Relaxant ¼ Planta Med 2005; 71: 320 ± 325

323

Table 3

Inhibition of the spontaneous contraction of isolated guinea-pig ileum induced by the extract and compounds from B. veronicaefolia

Substance

Emax

IC50 (mM)

Potency

Original Paper

Papaverine

91.76  2.65

4.23  0.68

Extract

84.07  1.47

39.22  12.88

1

2

80.34  4.91

1.49  0.17

2.8

3

78.88  1.57

2.61  0.24

1.6

4

90.18  4.05

2.47  0.15

1.7

5

87.77  1.18

1.90  0.12

2.2

6

58.11  7.66

3.51  0.04

1.2

8

83.11  1.95

2.88  0.08

1.5

9

78.88  4.80

2.07  0.16

2.0

10

93.14  1.68

1.84  0.11

2.3

11

76.50  6.96

2.29  0.28

1.8

14

76.64  4.69

4.96  0.39

0.8

15

83.62  3.15

1.52  0.11

2.8

The IC50 of the extract is expressed in g/mL. Values as means  SEM; n = 6; p < 0.05. Potency was obtained by the formula: IC50 (M) papaverine/IC50 (M) compound, assuming a value of 1.00 for papaverine. Emax indicates the percentage of maximum inhibition.

324

Fig. 2 Concentration-response curves for the inhibitory effect of the extract and isolated compounds from Brickellia veronicaefolia on the spontaneous contractions of isolated guinea-pig ileum. Values are expressed as the percentages of inhibition of contractile responses calculated as the mean six data  S.E.M.

Compound 3 had the composition C16H16O5 as determined by HR-EI-MS and 13C-NMR, differing from 15 by 14 mass units. The NMR spectra of 3 (Table 2) and 15 were almost identical, the most obvious difference being the absence of the signals for the methoxy group at C-3 in the spectra of 3. Instead, the 1H NMR spectrum showed a resonance for a phenolic group at dH = 10.0 Rivero-Cruz B et al. Smooth Muscle Relaxant ¼ Planta Med 2005; 71: 320 ± 325

which disappeared upon equilibration with D2O. Altogether, these observations suggested that 15 was the methyl derivative of 3. Indeed, treatment of compound 3 with an ethereal solution of CH2N2 yielded 15. Thus, compound 3 was characterized as 3hydroxybenzyl 2,6-dimethoxybenzoate.

The spasmolytic activity of the isolates was determined for compounds 2 ± 15. Fig. 2 shows the concentration-response curves (CRC) for the phytochemicals and Table 3 lists the corresponding IC50 and Emax values. In general, all the compounds were more potent than the crude extract. Compounds 2, 9 ± 11, and 15 induced a significant concentration-dependent inhibition of the spontaneous contractions of the guinea-pig ileum. These compounds were more potent than papaverine, used as a positive control. The data presented in the Fig. 2 show that the presence of a glucosyl or methoxy residue at C-3¢ in the benzyl unit as well as the 2,6-dimethoxybenzoyloxy moiety, as in the case of compounds 2, 10 and 15, are important structural requirements for the biological activity. Compound 3 has good activity, although lower than that of 2, indicating how the presence of a free hydroxy group at C-3¢ provoked a significant decrease in the pharmacological action. Compounds 1 and 7 were not pharmacologically tested due to the scarcity of the samples. The spasmolytic properties of 4 were determined by Kirimer et al. [18]. These authors found that the ethanolic extract of the root bark of Scolymus hispanicus L. and 4, identified as the major active principle, evoked strong biphasic responses (i. e., antispasmodic and spasmogenic effects) on isolated rat ileum preparations. On the other hand, chamazulene, also a component of the essential oil of German chamomile (Matricaria recutita L.), has demonstrated anti-inflammatory, antioxidant and antiseptic activities [19], [20], [21]. Its anti-inflammatory activity stems from its ability to inhibit the biosynthesis of leukotrienes. Interestingly, B. veronicaefolia is combined with M. recutita in many overthe-counter (OTC) Mexican herbal remedies. In conclusion, B. veronicaefolia's phytochemicals have a spasmolytic effect on intestinal smooth muscle; this pharmacological activity is consistent with the popular use of this plant for the treatment of gastrointestinal disorders such as flatulence, dyspepsia and colitis. From the chemotaxonomic point of view this is the first report of benzyl benzoates in a species of the genus Brickellia. Also the spasmolytic property of this type of compounds is newly described. Further work is in progress to determine the mechanism of action of their antispasmodic effects.

Acknowledgements This work was partially supported by the ICBG ªBioactive Agents from Dry Land Biodiversity of Latin Americaº, grant 2U01 TW 00 316 from the National Institutes of Health (NIH), the National Science Foundation (NSF), and the U.S. Department of Agriculture (USDA) and by Consejo Nacional de Ciencia y Tecnología (CONACyT, Proyecto C01 ± 018. We thank Rosa del Villar, Marisela GutiØrrez and Georgina Duarte-Lisci for recording IR, UV, NMR, and mass spectra. The technical assistance of Isabel Rivero-Cruz, Laura Acevedo and Juana Isela Rojas is also acknowledgment. B. Rivero acknowledges fellowships from CONACyT and Dirección General de Estudios de Posgrado, UNAM, to carry out graduate studies. References 1

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Rivero-Cruz B et al. Smooth Muscle Relaxant ¼ Planta Med 2005; 71: 320 ± 325

Original Paper

Compound 2 analyzed for C22H26O10 (HR-FAB-MS). The 1D and 2D NMR data (Table 2) suggested a strong resemblance between 2 and compounds 3 and 15, the common features of the three structures being the 2,6-dimethoxybenzoyloxy unit. However, they differed in the nature of the substituent in the second aromatic ring; thus, the resonances for the m-hydroxy or m-methoxy group of the benzyl unit in 3 and 15, respectively, were replaced in the spectra of 2 by those of a b-D-glucopyranosyloxy moiety (Table 2). The HMBC correlations C-2¢/H-2¢, H-7¢ and C3¢/H-1¢¢ confirmed the placement of the b-D-glucopyranosyloxy unit at C-3¢. The b stereochemistry of the O-glycoside linkage was deduced using the same criteria as for compound 1 [i. e., the coupling pattern of the anomeric proton (d, J = 7.5 Hz) and the chemical shift of the anomeric carbon (dC = 102.1)]. Therefore, compound 2 was characterized as 3-(b- glucopyranosyloxy) benzyl 2,6-dimethoxybenzoate.

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