Investigación

 

Sesqui- and Tri- Terpenoids from Esenbeckia species (Rutaceae)

 

Arturo Cano,* José Luis Bucio, Marina Espinoza and Alejandro Ruíz-Cancino^†

 

Facultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma de México. Av. Guelatao No. 66 (Eje 7 Oriente). Col. Ejército de Oriente. Iztapalapa 09230, México D. F. Teléfono: 5623-0758. Email: cafa6205@prodigy.net.mx.

 

Recibido el 18 de marzo del 2000.
Aceptado el 16 de junio del 2000.

 

Abstract

Three Esenbeckia species (Rutaceae) were chemically analyzed by means of conventional chromatographic, spectroscopic and spectrometric techniques. From the aerial parts of E. berlandieri ssp berlandieri were obtained friedelin, β-sitosterol, and β-sitosteryl-β-D-glucoside. Limonin was characterized as the main constituent from the seeds of Esenbeckia ovata. From the aerial parts of E. velutinosa friedelin, caryophyllene β-oxide, lupeol, lupenone, cryptomeridiol and β-sitosterol were isolated and characterized.

Key Words: Esenbeckia berlandieri ssp. berlandieri, E. ovata, E. velutinosa, Rutaceae, sesquiterpenes, triterpenes, limonin.

 

Resumen

Fueron analizadas químicamente tres especies del género Esenbeckia (Rutaceae) por medio de técnicas cromatográficas, espectroscópicas y espectrométricas convencionales. De las partes aéreas de E. berlandieri ssp berlandieri se aisló fridelina, β-sitosterol y el β-D-glucósido de β-sitosterilo. La limonina fue caracterizada como el principal constituyente de las semillas de E. ovata. De las partes aéreas de E. velutinosa se aisló e identificó a la fridelina, β-óxido de cariofileno, lupeol, lupenona, criptomeridiol y β-sitosterol.

Palabras clave: Esenbeckia berlandieri ssp. berlandieri, E. ovata, E. velutinosa, Rutaceae, sesquiterpenos, triterpenos, limonina.

 

In memoriam to Dr. Jacobo Gómez-Lara

 

Introduction

Esenbeckia is an American genus of ca. 30 species, 17 of which are located mainly in the tropical regions of the states of Veracruz, Guerrero, Michoacán, Oaxaca and Chiapas in Mexico [1-2]. Previous chemical studies from the aerial parts of some species indicate that limonoids, acridone and furoquinoline alkaloids, and furanocoumarines are the main secondary metabolites [3-8]. In addition, phloroglucinols and polyprenols have been characterized from E. nesiotica [9], alkaloids from E. almawilla [10], indole alkaloids, triterpenes, lignanes and amides from the roots of E. leiocarpa [11-12], furanocoumarines and quinoline alkaloids from E. glandiflora [13], and terpenes and coumarins from E. stephani and E. ovata [14].

 

Results and Discussion

From the aerial parts of E. berlandieri ssp berlandieri were isolated and identified friedelin [15], β-sitosterol and β-sitosteryl-β-D-glucoside. From the seeds of E. ovata was isolated a white solid that exhibited positive reaction in the Ehrlich test for furanes [16], whose presence was according to the signals at δ 7.42 (2H, s, H-21, H-23) in the 1H NMR spectrum, and was confirmed by the fragment at m/z 95 in the EIMS. The presence of an AB system centered at δ 4.92 (1H, J = 13 Hz) and δ 4.48 (1H, J = 13 Hz) in the 1H NMR spectrum is characteristic for an oxygenated function at C-19 of polycyclic modified triterpenes, according to the molecular formula C₂₆H₃₀O₈ determined by EIMS. The presence of an α, β-epoxy-δ-lactone in the D-ring was established by the signals at δ 5.47 (H-17) y δ 4.04 (H-15), suggesting a limonoid-type substance. COSY experiments allowed the assignments of the signals at δ 2.68 (dd, J = 16.8, 2.1) and δ 2.98 (dd, J = 16.8, 3.9 Hz) for the hydrogens α to the carbonyl group in the A-ring, while those at δ 2.86 (t, J = 15) and δ 2.47 (dd, J = 15, 3.5 Hz) corresponded to the hydrogens at C(6). The methyl signals at δ 1.30, 1.18, 1.17 and 1.08 were assigned to CH₃-18, CH₃-25, CH₃-26 and CH₃-24, respectively, confirming the structure of limonin A (1) for this secondary metabolite [17]. Friedelin, β-sitosterol, lupeol [18], cryptomeridiol [19], lupenone and caryophyllene epoxide [20] were isolated from the aerial parts of E. velutinosa and identified by direct comparison with authentic samples.

These results along with those previously reported [3-14] indicate that Esenbeckia species biosynthesize different structural types of secondary metabolites. Additional chemical studies of other species are necessary in order to establish the chemotaxonomic significance of the isolated substances.

 

Experimental

Esenbeckia berlandieri ssp berlandieri ex Hemsley was collected in the state of Veracruz. A voucher specimen is deposited at the National Herbarium, (MEXU, CHR-30). The organic extract was obtained by maceration with ethanol of the air-dried aerial plant material (1.9 kg). This extract was chromatographed at reduced pressure (VLC) [21] using n-hexane and mixtures of n-hexane-ethyl acetate as elution system. This procedure allowed to isolate friedelin (240 mg), β-sitosterol (100 mg), and β-sitosteryl-β-D-glucoside (390 mg). Friedelin. White crystals, mp 245-247°C [15]; IR (CHCl₃) ν_max 2943, 2867, 1703, 1455, 1389, 1361, 1192, 1139, 1110 cm⁻¹; 1H NMR (CDCl₃, 300 MHz): δ 0.72 (3H, s, CH₃-27), 0.85 (3H, d, CH₃-23), 0.87 (3H, s, CH₃-29), 0.95 (3H, s, CH₃-24), 1.0, 1.18 y 1.25 (9H, s, CH₃-25, CH₃-26, CH₃-28), 2.25 (1H, c, J = 7 Hz, H-4), 2.42 (1H, dd, J = 5 Hz, H-2), 2.38 (1H, dd, J = 5, 2 Hz, H-2); EMIE m/z (int. rel.): 426 [M+] (29), 302 8169, 274 (22), 273 8379, 246 (25), 218 8299, 205 824), 191 830), 179 (32), 164 (36), 125 (59), 123 (60), 121 (40), 109 (77), 96 852), 95 (100), 81 (60), 69 (62), 55(93).

The seeds of E. ovata Brandegee (940 g; collected in the state of Veracruz, provided by Prof. C.H. Ramos, key: CHR 32) were extracted with n-hexane and then with CHCl₃ at room temp. affording 63.4 g of residue. The crude CHCl₃ was adsorbed onto silica gel and analyzed via vacuum liquid chromatography (VLC) [21], the cromatographic fractions were monitored using the Ehrlich´s test. Limonin (1 [17], 123 mg) was isolated after purification by recristallization from EtOH-iPrOH. White crystals, mp: 232-233°C, orange with Ehrlich's test; IR (CHCl₃) ν_max 3064, 2958, 2876, 1753, 1284, 1028 cm⁻¹; NMR 1H (CDCl₃, 300 MHz): δ1.30, 1.18, 1.78 and 1.08 (s, 12H), 2.23 (dd, J = 15.9 0, 3 Hz, 1H, H-5), 2.47 (dd, J = 14.7, 3.5 Hz, 1H, H-6a), 2.56(dd, J = 12.2, 3 Hz, 1H, H-9), 2.68 (dd, J = 16.7, 2.1 Hz, 1H, H-2a), 2.86 (t, J = 15.5 Hz, 1H, H-6b), 2.98 (dd, J = 16.8, 3.9 Hz, 1H, H-2b), 4.04 (s, 2H, H-1 and H-15), 4.47 (d, J = 13.2 Hz, 1H, H-19a), 4.77 (d, J = 13.2 Hz, 1H, H-19b), 5.47 (s, 1H, H-17), 6.34 (dd, J = 1.35, 0.9, 1H, H-22), 7.41 (s, 2H, H-21 and H-23). 1H NMR (DMSO-d₆, 500 MHz): δ 1.18, 1.11, 1.03 and 1.00 (s, 12H), 2.28 (dd, J = 15, 3 Hz, 1H, H-5), 2.46 (dd, 15.8, 3.5 Hz, 1H, H-6a), 2.58 (dd, J = 19.5, 3 Hz, 1H, H-9), 2.62 (dd, J = 16.5 Hz, 1H, H-2a), 2.77 (t, J = 16 Hz, 1H, H-2b), 3.12 (dd, J = 15 Hz, 1H, H-6a), 4.11 (d, J = 15 Hz, 2H, H-1 and H-15), 4.92 (d, J = 13.2 Hz, 1H, H-19a), 4.48 (d, J = 13 Hz, 1H, H-19b), 5.48 (s, 1H, H-17), 6.51 (dd, J = 1.5, 1.0, 1H, H-22), 7.68 (t, J = 2 Hz, 2H, H-23), 7.72 (d, J=0.5 Hz, 1H. H-21). 13C NMR (CDCl₃, 75 MHz, assignments by APT): δ77.8 (t, C-2), 36.4 (C-2), 166.6 (C-3), 80.3 (C-4), 53.9 (C-5), 35.7 (C-6), 206.1 (C-7), 45.9 (C-8), 48.1 (C-9), 51.3 (C-10), 18.9 (C-11), 30.8 (C-12), 37.9 (C-13), 65.7 (C-14), 60.6 (C-15), 169.1 (C-16), 79.2 (C-17), 21.4 (C-18), 65.3 (C-19), 119.9 (C-20), 141.1 (C-21), 109.6 (C-22), 143.2 (C-23), 17.6 (C-24), 20.7 (C-25), 30.2 (C-26). EMIE m/z (rel. int.). 348 (100), 347 (28.2), 329 (12.8), 147 (20.5), 121 (28.2), 119 (20.5), 97 (33.3), 95 (100), 93 (35.9), 91 (56.4), 69 (59), 43 (100), 41 (61).

After drying, the aerial parts of E. velutinosa C. H. Ramos (sp. nov.) (1.8 kg, provided by C. H. Ramos and deposited at the National Herbarium) were macerated with acetone. The crude extract (62 g) was adsorbed onto silica gel and carefully chromatographed on a silica gel column at reduced pressure (VLC) [21] eluting with n-hexane and n-hexane containing increasing proportions of EtOAc. This procedure allowed the isolation of friedelin (250 mg) [15], β-sitosterol (220 mg), lupeol (22 mg) [18]. Elution with n-hexane-EtOAc (3:2), afforded 3.45 g of a residue, which was rechromatographed at reduced pressure (VCC), and some fractions (TLC control) were further purified by prep. TLC (silica gel, n-hexane-AcOEt, 5:9), to give cryptomeridiol (19 mg) [19]. The mother liquors from the isolation of friedelin, were rechromatographed via VLC using n-hexane as eluent, to give lupenone (20 mg) and caryophyllene β-oxide (40 mg) [20]. All the substances were identifed by direct comparison with authentic samples.

 

Acknowledgements

The authors are grateful to M. Sc. Clara H. Ramos (Instituto de Biología, UNAM) for the supply and identification of plant material, to M. Sc. María Isabel Chávez, Beatriz Quiróz, Rocío Patiño, Luis Velasco and Francisco J. Pérez Flores from Instituto de Química de la UNAM for technical assistance, and Dr. Guillermo Delgado for stimulating interest and support to this work. We also thank to Carrera de Biología and Coordinación de Investigación de la FES-Zaragoza (UNAM) for partial financial support.

 

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