Secondary Metabolites from Asclepias otarioides

Emma Maldonado,^1,* Sonia Amador,¹ and Verónica Juárez-Jaimes²

¹ Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, Coyoacán, 04510, D. F., México. emmaldon@unam.mx

² Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de México, Apartado Postal 70-367, 04510, D. F., México.

Received October 6^th, 2014 ]]> Accepted November 24^th, 2014

Abstract

Chemical study of the aerial parts of Asclepias otarioides led to the isolation of four pentacyclic triterpenes and one cardenolide glycoside. This is the first report on the occurrence of the triterpenes 1, 3, and 4 in the genus Asclepias.

Key words: Asclepias otarioides; Apocynaceae; Asclepiadoideae; Triterpenes; Cardenolide Glycoside.

Resumen

El estudio químico de las partes aéreas de Asclepias otarioides condujo al aislamiento de cuatro triterpenos pentacíclicos y de un glicósido de cardenólida. Este es el primer informe sobre la presencia de los triterpenos 1, 3 y 4 en el género Asclepias.

Palabras clave: Asclepias otarioides; Apocynaceae; Asclepiadoideae; triterpenos; glicósido de cardenólida.

The American genus Asclepias (fam. Apocynaceae, subfamily Asclepiadoideae) includes about 150 species, 68 of which grow in Mexico, and nearly half of them, are endemic [1-3]. Previous chemical investigations of Asclepias species have shown that different types of steroidal compounds such as cardenolides, pregnanes and androstanes, usually as glycosides, are the main metabolites of these plants [4-6]. However, flavonoid glycosides [7], megastigmane glycosides [8], triterpenes [4, 9], conduritols, and conduritol glycosides [10] have been also isolated from these plants. Asclepias species have ecological significance by their relationship with the monarch butterfly, Danaus plexippus; an insect that sequestrates cardenolides from Asclepias plants as a chemical defense mechanism against predators [11]. Although Asclepias species are considered toxic, some of them are used in folk medicine as anthelmintic, analgesic, cardiotonic, and for the treatment of dermatological problems [3], cancer [4], pleuresy, bronchitis [6], and asthma [9]. This paper describes the isolation and the structure elucidation of the major constituents of the aerial parts of Asclepias otarioides E. Fourn., an herbaceous plant, endemic to Mexico [12].

Results and Discussion

As result of the chemical study of the aerial parts of A. otarioides, three oleanane-type triterpenes (1-3), one lupane-type triterpene (4), one cardenolide glycoside (5), β-sitosterol glucoside and a mixture of β-sitosterol/stigmasterol were isolated. The oleanane-type triterpenes were identified as oleanonic acid (1) [13-15], oleanolic acid (2) [16, 17], and 3,4-seco-olean-12-en-3,28-dioic acid (3) [18], while the structure of the lupane-type triterpene corresponded to betulinic acid (4) [19, 20] (Fig. 1). Structures of compounds 1-4 were determined by analyses of their IR, MS and NMR spectra and comparison of these data with those reported in the literature. β-Sitosterol glucoside and the mixture of β-sitosterol/stigmasterol were identified by comparison of their ¹H NMR spectra and physical constants with those of authentic samples.

Compound 5 was part of a complex mixture from which it could not be isolated. So, the mixture was esterified (Ac₂O-pyridine) and compound 5 was isolated as the pentaacetyl derivative 6. The molecular formula of this derivative was assigned as C₃₉H₅₄O₁₅ by the pseudomolecular ion at m/z 785.3356 [M + Na] ⁺ observed in its HRESIMS (calcd. for, 785.3355). The ¹³C NMR spectrum of compound 6 showed 39 signals; 10 of them correspond to 5 acetyl groups, another 6 signals were assigned to a monosaccharide, and the remaining 23 signals were atributted to a cardenolide. The ¹H NMR spectrum showed the signals for the sugar moiety at δ 4.84 (d, H-1'), 4.79 (dd, H-2'), 5.60 (t, H-3'), 4.66 (dd, H-4'), 3.94 (dq, H-5'), and 1.21 (d, H-6'). The coupling constants of these signals (J_1-2 = 8.0 Hz, J_2-3 = J_3-4 = 3.0 Hz _, J_4-5 = 10.0 Hz) led to the identification of this sugar as β-allomethylose [21]. The presence of an α,β-unsaturated-γ-lactone in the aglycone was deduced from ¹H NMR signal at δ 5.85 for the vinyl proton H-22 and those for the γ-methylene protons (H₂-21) at δ 4.85 and 4.77; and confirmed by the ¹³C NMR signals at δ 173.0 (C-20), 73.2 (CH₂-21), 118.1 (CH-22), and 174.1 (C-23). The aglycone also presents signals for one oxymethylene (δ_H 4.32 d, J = 12.5 Hz, δ_H 4.12 d, J = 12.5 Hz; δ_C 61.7, CH₂-19), two oxymethines (δ_H 4.53 dd, J = 12.0, 4.0 Hz, δ_C 77.1, CH-12; δ_H 3.65 tt, J = 11.0, 5.0 Hz; δ_C 77.6, CH-3), a non protonated carbon bonded to oxygen (δ_C 85.6, C-14), and one methyl group (δ_H 0.88 s; δ_C 10.4, CH₃18). These assignments were based on analysis of the 2D NMR spectra, establishing the structure of the cardenolide as that of 12β-hydroxycoroglaucigenine. The HMBC correlation of H-1' to C-3 showed that the allomethylose was bonded to the oxygen at C-3. In the same manner was established that the acetyl groups were bonded to the oxygens at C-12, C-19, C-2', C-3', C-4' and C-6'. The derivative 6 was identified as 12-O,19-O,2'-O,3'-O,4'-O,6'-O-pentaacetyl-12β-hydroxycoroglaucigenine-3-O-β-Dallomethyloside, which has not been described previously. Thus, the cardenolide present in A. otariodes was identified as 12βhydroxycoroglaucigenine-3-O-β-D-allomethyloside (5) [21].

To our knowledge, this is the first report on the ocurrence of oleanonic acid (1), 3,4-seco-olean-12-en-3,28-dioic acid (3), and betulinic acid (4) in Asclepias genus. Oleanolic acid (2) and other pentacyclic triterpenes have been isolated from A. syriaca, A. linaria and A. speciosa [9,22,23]. 12β-Hydroxycoroglaucigenin-3-O-β-D-allomethyloside (5) and its aglycon were isolated from A. curassavica [21,24]. Thus, the chemical composition found in A. otarioides was consistent with those found in another species of Asclepias genus, in which the same type of compounds were present. This indicates that not only cardiac glycosides, but pentacyclic triterpenes are relevant constituents of Asclepias species.

General experimental procedures. Melting points (uncorrected) were determined on a Fisher Jones melting point apparatus. Optical rotations were measured on a Perkin Elmer 343 polarimeter. The IR spectra were recorded on a FTIR-Magna 750 spectrophotometer. NMR spectra were recorded on a Varian Unity Plus 500 or on Varian XR-300 spectrometers, using TMS as internal standard. EIMS were measured on a JEOL JMS-AX505HA mass spectrometer. HRESIMS was recorded on a Bruker microTOF II ESI mass spectrometer. Column chromatographies operated with vacuum (CC) were performed on silica gel 60 (Merck G). Thin layer chromatographies (TLC) were carried out on precoated Macherey-Nagel Sil G/UV₂₅₄ plates with thicknesses of 0.25 mm.

Vegetal material. The aerial parts of Asclepias otarioides E. Fourn. were collected in the Ajusco Mountain, Southwest Mexico City, in July 2006. V. Juárez-Jaimes authenticated the vegetal material. A voucher specimen (MEXU 1 248 428) was deposited at the National Herbarium.

Extraction and isolation. Fresh aerial parts of A. otarioides (446 g) were extracted with MeOH and then with EtOAc. Both extracts were combined (148.7 g) and fractioned by partition between EtOAc-H₂O and BuOH-H₂O, to obtain 44.7 and 8.1 g of extract, respectively. The EtOAc-soluble extract was fractioned by silica gel column chromatography (CC) with hexane-EtOAc mixtures to obtain five combined fractions (1A to 5A). Purification of fraction 2A (eluted with hexane-EtOAc 19:1) by silica gel CC eluted with hexane-EtOAc 19:1 and 9:1 gave fractions 1B-5B. A mixture of the ubiquitous β-sitosterol/stigmasterol (299 mg) was obtained from fraction 2B. Crystallization of fraction 3B led to the isolation of oleanonic acid (1) (mp 167-169 °C, [α]_D 93.5, c 0.23, CHCl_3, lit.: mp 170-176 °C, [α]_D 96.6, c 0.23, MeOH [13], [α]_D 73.6, c 0.26, CHCl₃ [15]) Mother liquors of 1 were combined with fraction 4B and subjected to silica gel CC eluted with CH₂Cl₂, followed by a second CC eluted with hexane-iPrOH 98:2 and crystallization to obtain 41.2 mg of betulinic acid (4) (mp 285-287 °C, [α]_D 7.0, c 0.20, CHCl_3, lit.: mp 290-292 °C, [α]_D 7.5, c 0.5, pyridine [19], [α]_D 8.0, c 0.37, CHCl₃ [20])_, together with an additional amount of 1, to make a total of 3.87 g. Fractions 3A (eluted with hexane-EtOAc 17:3 to 7:3) and 5B were combined, decoloured with activated charcoal and subjected to CC eluted with hexane-EtOAc gradient. Fractions eluted with hexane-EtOAc 9:1 afforded 568 mg of oleanolic acid (2) (mp 297-299 °C, [α]_D 70.8, c 0.226, CHCl_3, lit.: mp 301-302.5 °C, [α]_D 68.9, c 0.21, CHCl₃ [16,17]). Chromatography of fraction 5A (eluted with hexane-EtOAc 1:4 to 0:1) over a silica gel column eluted with CHCl₃-MeOH 19:1 to17:3 gave fractions 1C-4C. Fraction 1C was purified by CC eluted with mixtures of CHCl₃-MeOH of increasing polarity. Fractions eluted with CHCl₃-MeOH 98:2 gave 21 mg of 3,4-seco-olean-12-en-3,28-dioic acid (3) (mp 268-270 °C, [α]_D 60.0, c 0.21, CHCl_3, lit.: mp >250 °C, [α]_D 54.4, c 0.006, MeOH [18]. Crystallization of fraction 2C gave 53.2 mg of β-sitosterol glucoside.

Silica gel CC (CHCl₃-MeOH) of the BuOH-soluble extract gave fractions 1D-4D. Repeated CC of fraction 2D gave a mixture (77 mg) containing 12β-hydroxycoroglaucigenin-3-O-β-D-allomethyloside (5), which could not be purified. A portion (64.4 mg) of this mixture was acetylated in the usual manner (pyridine/Ac₂O, room temp., 24 h), and purified by silica gel CC eluted with hexane-Me₂CO 4:1, to obtain the pentaacetyl derivative of 5 (6, 71.7 mg).

Pentaacetyl-12β-hydroxycoroglaucigenin-3-O-β-D-allomethyloside (6). Amorphous solid; [α]_D²⁰ + 5.37 (c 0.28, CHCl₃); IR (CHCl₃, ν, cm^-1):1747, 1629, 1373, 1171, 1083, 1037; ¹H NMR (500 MHz, CDCl₃, δ, ppm, J/Hz): 2.18 (1H, dt, J = 14.0, 3.5 Hz, H-1a), 0.91 (1H, td, J = 14.0, 3.5 Hz, H-1b), 1.93 (1H, m, H-2a), 1.38 (1H, m, H-2b), 3.65 (1H, tt, J = 11.0, 5.0 Hz, H-3), 1.92 (1H, m, H-4a), 1.76 (1H, m, H-4b), 1.25 (1H, m, H-5), 1.43 (1H, m, H-6a), 1.24 (1H, m, H-6b), 2.02 (1H, m, H-7a), 1.13 (1H, m, H-7b), 1.67 (1H, td, J = 12.0, 3.5 Hz, H-8), 1.08 (1H, td, J = 12.0, 3.0 Hz, H-9), 1.87 (1H, m, H-11a), 1.33 (1H, m, H-11b), 4.53 (1H, dd, J = 12.0, 4.0 Hz, H-12), 1.73 (1H, m, H-15a), 1.36 (1H, m, H-15b), 2.15 (1H, m, H-16a), 1.92 (1H, m, H-16b), 2.87 (1H, td, J = 6.0, 4.0 Hz, H-17), 0.88 (3H, s, H-18), 4.32 (1H, d, J = 12.5 Hz, H-19a), 4.12 (1H, d, J = 12.5 Hz, H-19b), 4.85 (1H, br dd, J = 18.0, 1.5 Hz, H-21a), 4.77 (1H, dd, J = 18.0, 2.0 Hz, H-21b), 5.85 (1H, br s, H-22); 4.84 (1H, d, J = 8.0, Hz, H-1'), 4.79 (1H, dd, J = 8.0, 3.0 Hz, H-2'), 5.60 (1H, t, J = 3.0, Hz, H-3'), 4.66 (1H, dd, J = 10.0, 3.0 Hz, H-4'), 3.94 (1H, dq, J = 10.0, 6.5 Hz, H-5'), 1.21 (3H, d, J = 6.5, Hz, H-6'), 2.15, 2.10, 2.05, 2.02, and 2.01 (3H each, s, CH₃CO); ¹³C NMR (125 MHz, CDCl₃, δ, ppm): 31.9 (C-1), 29.2 (C-2), 77.6 (C-3), 32.9(C-4), 44.6 (C-5'), 27.9 (C-6), 27.4 (C-7), 41.7 (C-8), 45.9 (C-9), 38.1 (C-10), 27.4 (C-11), 77.1 (C-12), 53.9 (C-13), 85.6 (C-14), 34.4 (C-15), 27.1 (C-16), 45.9 (C-17), 10.4 (C-18), 61.7 (C-19), 173.0 (C-20), 73.2 (C-21), 118.1 (C-22), 174.1 (C-23), 96.9 (C-1'), 69.5 (C-2'), 68.8 (C-3'), 71.4 (C-4'), 68.1 (C-5'), 17.5 (C-6'), 21.2, 21.1, 20.74, 20.68, and 20.6 (CH₃CO), 170.9 170.8, 169.8, 169.3, and 169.0 (CH₃CO); FABMS m/z 763 [M + H] ⁺ (3), 703 (1), 473 (2), 413 (2), 353 (8), 335 (8), 273 [triacetylallomethylose] ⁺ (87), 231 (4), 213 (4), 153 (38), 111 (42), 69 (73), 57 (100), 43 (72). HRESI MS m/z 785.3356 [M + Na] ⁺ (calcd. for C₃₉H₅₄NaO₁₅, 785.3355).

Acknowledgments

We are very grateful to R. Patiño, H. Ríos, I. Chávez, B. Quiroz, E. Huerta, R. Gaviño, M. A. Peña, N. Zavala, L. Triana, L. Velasco, and J. Pérez for technical assistance.

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