Cadillo Pata de Perro. II Parte.

Después de realizare el primer artículo referente al Cadillo Pata de Perro, he notado que es una de las secciones más visitadas de mi Blog, por lo que dedique un poco más de tiempo a realizar unas investigaciones al respecto y pude encontrar que el año anterior (2010) fue realizado un estudio exhaustivo para determinar las sustancias químicas presentes en el sumo de las hojas frescas del Cadillo Pata de Perro, cuyo nombre científico es Ureña Sinuata L, y que junto a la Ureña Lobata L conforman el género Ureña L (Malvaceae).

www.saber.ula.ve/avancesenquimica

Avances en Química, 5(2), 95-98 (2010)

Artículo científico

Flavonoids from Urena sinuata L.

Adakarleny Sosa & Carmelo Rosquete*

Laboratorio de Productos Naturales, Departamento de Química, Facultad de Ciencias.

Universidad de los Andes, Mérida 5101, Venezuela

(*) carmelor@ula.ve

Recibido: 12/02/2010 Revisado: 28/06/2010 Aceptado: 07/07/2010

                                                                           Resumen

En este manuscrito se reportan los resultados obtenidos del estudio fitoquímico de las hojas frescas de Urena sinuata L. (cadillo de perro). Los componentes mayoritarios identificados fueron los flavonoides: 3'-β-Dglucopiranosil-6,7-O-dimetilquercetagetina (I), 4'-β-D-glucopiranosil-6,7Odimetilquercetagetina (II) y 3-β-Dglucopiranosil-6,7-O-dimetil-quercetagetina (III). Los compuestos aislados fueron caracterizados mediante sus constantes físicas y el análisis de sus espectros ultravioleta, de masas y de resonancia magnética nuclear mono- y bi-dimensionales. También se muestran los resultados obtenidos para estos compuestos en el ensayo de citotoxicidad sobre Artemia salina.

Palabras claves: Urena sinuata; flavonoides; glicósidos de flavonoles; derivados de quercetagetina

                                                       Abstract 

In this work it is exposed the obtained results of the phytochemical study of the fresh leaves of Urena sinuata L.(dog wart). The major components are the flavonoids: quercetagetin-6,7-O-dimethylether-3'-β-D-gluco-pyranoside (I), quercetagetin-6,7-O-dimethylether-4'-β-D-glucopyranoside (II), and quercetagetin-6,7-O-dimethylether-3-β-D-glucopyranoside (III). These products were characterized through their physical constants, UV, MS, and oneand two-dimensional NMR studies. By other hand, the obtained results of the Artemia salina cytotoxicity bioassay carried out to the isolated products are exposed.

Keywords: Urena sinuata; Flavonoids; Flavonol glycosides; Quercetagetin derivatives

  Introduction

Urena L. (Malvaceae) is a genus composed by two species:Urena lobata L. and Urena sinuata L. although some Botanist suggest that U. sinuata is subspecie of U. lobata. This plant (U. lobata) has been phytochemically studied by some authors, and steroids (stigmasterol and βsitosterol) 1, xanthones (mangiferin) 2, flavonoids (quercetina2-4, kaempferol, hypolaetin, gossypetin, luteolin, apigenin and chrysoeriol3), sugars (glucose, mannose, xylose and fructose) 5, and vitamins (ascorbic acid) 5 has been reported.

For U. sinuata only has been reported fatty acids: sterculic and malvalic acids6.

 In Venezuela, the infusion of foliage from Urena sinuata L.is used as anti-inflammatory, analgesic, and against kidneypain and gall stone7. For U.lobata antiparasitic8,9, antibacterial10-12, antidiarrheal13, and immunomodulatory

activity 14 have been mentioned.

Introducción

Ureña L. (Malvaceae) es un género compuesto por dos especies: Urena lobata L. y L. Ureña sinuata aunque algunos botánicos sugieren que la U. sinuata es una subespecie de U. lobata. Esta planta (U. lobata) ha sido estudiada fitoquímicamente por algunos autores que han  reportado  los esteroides (estigmasterol y βsitosterol) 1, xantonas (mangiferina) 2, flavonoides (quercetina2-4, kaempferol, hypolaetin, gossypetin, luteolina, apigenina y chrysoeriol3), azúcares (glucosa, xilosa manosa, y fructosa) 5, y vitaminas (ácido ascórbico) 5 

Para U. sinuata sólo se ha reportado ácidos grasos: acids6 estercúlico y malvalic.

En Venezuela, la infusión de las hojas de Ureña sinuata L.es utilizada como anti-inflamatorio, analgésico, y contra el dolor de riñón y de la vesícula calculos7. Para antiparasitario U.lobata 8,9, antibacterial10-12, antidiarreico13, y se han mencionado actividad de inmunomoduladora 14

.

Experimental

General Experimental Procedures

IR spectra were recorded as KBr disc on a Perkin ElmerFT-IR Spectrometer 1725X. UV spectra were recorded on an UV Varian Scan 3 using methanol as solvent. NMR spectra were run on Bruker Avance DRX 400 using CDCl3

as solvent and TMS as internal standard. Mass spectra were recorded on a Hewlett-Packard Mass Spectrometer model 5930a (70 eV). Si gel 60 (Merck, 70-230 mesh) with dry assembly was used in CC and Si gel Merck HF 254

(10 – 40 μ) on glass sheet (0.25 and 0.5 mm thickness, respectively) was used as adsorbent for TLC and PTLC.

Experimental

Procedimientos Experimentales Generales

Los espectros IR se registraron como disco de KBr en un espectrómetro Perkin-IR 1725X ElmerFT. Espectros UV se registraron en un UV Varian scanner 3 utilizando metanol como disolvente. Los espectros de RMN se realizaron en Bruker Avance DRX 400 con CDCl3

como disolvente y TMS como patrón interno. Los espectros de masas se registraron en un Hewlett-Packard modelo 5930a espectrómetro de masa (70 eV). Si gel 60 (Merck, malla 70-230) con montaje en seco se utilizó en CC y Si gel de Merck IC 254

(10 - 40 μ) en la hoja de vidrio (0,25 y 0,5 mm de espesor, respectivamente) fue utilizado como adsorbente para TLC y PTLC.

Plant Material

Urena sinuata L. (Malvaceae) was collected at SanCristóbal suburbs (Táchira State-Venezuela). Voucherspecimens were stored at MERC Herbarium, SciencesFaculty, Universidad de los Andes-Venezuela.

Material Vegetal

Ureña sinuata L. (Malvaceae) fue colectada en los suburbios San Cristóbal (Estado Táchira-Venezuela).Varios especímenes se almacenaron a MERC Herbario, Facultad de Ciencias, Universidad de los Andes-Venezuela.

Extraction and Isolation

1 Kg of fresh plant was extracted in a Soxhlet with nhexane, dichloromethane, acetone and methanol successively. Hexane extract was percolated on Sephadex LH-20® column for eliminate chlorophylls and analyzed

by GC/MS. None additional to previously reported interesting compounds was detected6. Dichloromethane extract (10.0 g) afforded before Si gel CC hexadecyl 4-monoitaconate (500 mg) as mainly product. Acetone extract (8.9 g) was also percolated on Sephadex LH-20® column for eliminate chlorophylls using firstly a mixture of n-hexane/chloroform/methanol (1:1:1) and then methanol, as solvents. From methanol eluate (525 mg) were obtained, before PTLC using n-hexane/acetone (1:8) x 8 as eluent, quercetagetin-6,7-O-dimethylether-3'-β-Dglucopyranoside, I (63 mg), quercetagetin-6,7-O-dimethylether-4'-β-D-glucopyrano-side, II (72 mg), and quercetagetin- 6,7-O-dimethylether-3-β-D-glucopyranoside, III (45mg).

Extracción y aislamiento

1 Kg de planta fresca se extrajo en un Soxhlet con nhexano, diclorometano, acetona y metanol sucesivamente. Se filtró extracto de Hexano sobre Sephadex LH-20 ® en la columna para eliminar clorofilas y se analizaron

por GC / MS. Se detectó compuestos interesantes6  ninguno adicional a informes anteriores. Extracto de diclorometano (10,0 g) proporcionó antes de Si gel de CC hexadecilo 4-monoitaconate (500 mg) como principal producto. Extracto de acetona (8,9 g) se percola también sobre Sephadex LH-20 ® en la columna para eliminar clorofilas utilizando en primer lugar una mezcla de n-hexano/chloroformo/methanol (1:1:1) y luego metanol, como disolventes. De eluato metanol (525 mg) se obtuvieron, antes PTLC utilizando n-hexano/acetona  (1:8) como eluyente x 8, quercetagetin-6 ,7-O-dimetil-éter-3 '-β-Dglucopyranoside, I (63 mg), quercetagetin-6 ,7-O-dimetil-éter-4' -β-D-glucopyrano-lado, II (72 mg), y quercetagetin-6,7-O-dimetil-éter-3-β-D-glucopiranósido, III (45 mg).

Biological Essay

Artemia salina cytotoxicity test was performed following the procedure described by Meyer et al.16

Ensayo biológico

Artemia salina la prueba de citotoxicidad se realizó siguiendo el procedimiento descrito por Meyer et al16

Quercetagetin-6,7-O-dimethylether-3'-β-D-glucopyranoside, I: dark yellow prisms, mp > 300 ºC. IR (KBr) υmax: 3377, 2923, 1654, 1554, 1130, 812 cm-1. UV (MeOH): see Table 2. 1H- and 13C-NMR (CDCl3): see table 1. MS m/z: [M]+. 508 (< 1), [M – Glu + H]+. 346 (100), [M – Glu –

H2O + H]+. 328 (31), [M – Glu – H2C=C=O + H]+. 303 (66), [M – Glu – H2C=C=O – H2O + H]+. 285 (16), [M – Glu – H2C=C=O – H2O – CH3 + H]+ 260 (9), [A1 – OCH3 – CO]+ 137 (19).

Quercetagetin-6,7-O-dimethylether-4'-β-D-glucopyranoside, II: dark yellow prisms, mp > 300 ºC. IR (KBr) υmax: 3378, 2929, 1654, 1547, 1131, 811 cm-1. UV (MeOH): see Table 2. 1H- and 13C-NMR (CDCl3): see table 1. MS m/z: [M]+. 508 (< 1), [M – Glu + H]+. 346 (100), [M – Glu –

H2O + H]+. 328 (34), [M – Glu – H2C=C=O + H]+. 303 (70), [M – Glu – H2C=C=O – H2O + H]+. 285 (18), [M – Glu – H2C=C=O – H2O – CH3 + H]+ 260 (9), [A1 – OCH3 – CO]+ 137 (19).

Quercetagetin-6,7-O-dimethylether-3-β-D-glucopyranoside, III: pale yellow prisms, mp > 300 ºC. IR (KBr) υmax: 3413, 2945, 1658, 1556, 1134, 806 cm-1. UV (MeOH): see Table 2. 1H- and 13C-NMR (CDCl3): see table 1. MS m/z: [M]+. 508 (< 1), [M – Glu + H]+. 346 (100), [M – Glu – H2O

+ H]+. 328 (31), [M – Glu – H2C=C=O + H]+. 303 (68), [M – Glu – H2C=C=O – H2O + H]+. 285 (16), [M – Glu – H2C=C=O – H2O – CH3 + H]+ 260 (9), [A1 – OCH3 – CO]+ 137 (19).

Results and Discussion

All compounds, I to III, showed very similar IR, mass and 13C-NMR spectra and only slight differences at 1H-NMR spectra (see table 1). The whole analysis of 1D spectroscopic data allowed us to determine that I to III correspond to flavone-type compounds, O-substituted at 3, 5, 6, 7, 3', and 4' positions, with one β-D-glucopyranosyl (mainly identified by 13C-NMR chemical shift), twomethoxyl,  and three hydroxyl groups as substituents. HMBC experiments confirmed us that methoxyl groups were placed at 6 and 7 positions for all three compounds, remain therefore to insert t he β-D-glucopyranosyl group between 3, 5, 3' and 4' positions, which was made using

shift reagent for UV spectra.

During UV spectra analysis of the compounds (see table 2) were observed: For compound I, the bathochromic shift for band I (+ 42 nm) showing a low intensity decrease with in time (hypochromic effect) observed when UV spectrum was recorded in methanol + sodium methoxyde can let us to place hydroxyl group on positions 3 and 4'. When UV spectrum was recorded in methanol + AlCl3, a + 53 nm bathochromic shift was observed; that shift remain unchanged after hydrochloric acid addition, which place hydroxyl group on positions 3 and 5. In consequence, the β-D-glucopyranosyl moiety should be located on 3' carbon, and compound I should be quercetagetin-6,7-Odimethylether-3'-β-D-glucopyranoside.

Similar to I, compound II showed for band I, abathochromic shift (+ 54 nm) in presence of AlCl3 and AlCl3 + HCl, which place hydroxyl groups on positions 3 and 5 too. The bathochromic displacement (+ 42 nm) of band I observed after sodium methoxyde addition without in time intensity decrease, together with the existence of C3 hydroxyl group previously established, place in this case, the β-D-glucopyranosyl moiety on C4', and compound II

should be quercetagetin-6,7-O-dimethylether-4'-β-Dglucopyranoside.

Different to previously described compounds, band I of UV spectrum in methanol + AlCl3 of compound III was affected after HCl addition, underwent a hypsochromic shift of -37 nm (respect to methanol + AlCl3) which indicated the existence of an orto-dihydroxyl moiety on Bring

(3' and 4' positions), which was corroborated by band I hypsochromic displacement (-32 nm) observed on methanol + sodium acetate UV spectrum after boric acid addition. The stability in time of the methanol + sodium methoxyde, together with the 12 nm bathochromic shift remanent respect to methanol UV spectrum observed on band I of methanol + AlCl3 + HCl, place the third hydroxyl group on C5 carbon, and therefore compound III should

be quercetagetin-6,7-O-dimethylether-3-β-D-glucopyranoside. Although this compound has been reported from Brickellia dentata15, none spectroscopic data has been published in reference 15 and references cited therein.

The UV β-D-glucopyranosyl moieties locations for I-III were confirmed by HMBC interactions observed between the hydrogen on anomeric carbon from each saccharide moiety and it flavonoid moiety carbon (3’, 4’, and 3, for I, II, and III respectively)

Table 1: NMR data for compounds I – III.

_______________________________________________________________________________________

                                    I                                                II                                                    III_______________                              

           δC                 δH, m (J Hz)             δC               δH, m (J Hz)                  δC                  δH, m (J Hz)

2                 156.9                                            156.8                                                 156.6

3                 133.5                                            133.5                                                 133.3

4                 177.8                                            177.9                                                 177.7

5                 151.9                                            151.9                                                 151.7

6                 131.9                                            131.8                                                 131.6

7                 158.8                                            158.8                                                 158.3

8                   91.4                 6.85, s                   91.3               6.82, s                         91.2                  6.83, s

9                 151.9                                             151.8                                                151.6

10               105.5                                             105.5                                                105.3

1'                 121.3                                            121.3                                                121.1

2'                116.5                 7.62, d (2.0)       116.5              7.60, d (2.0)               116.3              7.61, d (2.0)

3'                144.9                                             150.0                                                144.8

4'                148.7                                             148.8                                                148.5

5'               115.4               6.84, d (8.0)           115.4              6.83, d (8.0)              115.1            6.84, d (8.0)

6'               121.8        7.61, dd (8.0, 2.0)        121.8           7.57, dd (8.0, 2.0)        121.6       7.58, dd (8.0, 2.0)

1''              100.9        5.46, d (7.3)                  100.9          5.44, d (7.3)                  100.7          5.48, d (7.3)

2''                76.6      3.23, dd (8.0, 7.3)            76.6        3.24, dd (8.0, 7.3)            76.5       3.19, dd (8.0, 7.3)

3''                70.1      3.26, dd (8.0, 8.0)            70.0       3.25, dd (8.0, 8.0)            69.9       3.27, dd (8.0, 8.0)

4''               74.3        3.08, dd (8.0, 8.0)           74.2        3.08, dd (8.0, 8.0)           74.1       3.08, dd (8.0, 8.0)

5''               77.6     3.08, ddd (8.0, 5.5, 1.5)    77.6    3.07, ddd (8.0, 5.5, 1.5)      77.5     3.07, ddd (8.0, 5.5, 1.5)

6''a             61.1       3.37, dd (11.5, 1.5)          61.1       3.34, dd (11.5, 1.5)          61.8        3.55, dd (11.5, 1.5)

6''b             61.1       3.30, dd (11.5, 5.5)          61.1       3.31, dd (11.5, 5.5)          61.8         3.29, dd (11.5, 5.5)

6-OMe      60.3                  3.71, s                   60.3               3.73, s                        60.3                     3.71, s

7-OMe       56.6                 3.90, s                   56.6                 3.97, s                      56.5                     3.90, s

5-OH                             12.58, s                                  12.55, s                                            12.60, s

Table 2: UV data for compounds I – III.

                                   ___________________________UV λmax (nm)_______________________________

                                   ___________I_______________________ II ____________________III___________

                                           Band I            Band II             Band I            Band II            Band I             Band II

MeOH                                 352                 256                    352                 259                  353                    258

MeOH+NaOMe                  394↓      272↓         405                270                  393                   270

MeOH+NaOAc                  404                 258                      376                266                  407                   256

NaOAc+H3BO3                404                  258                     376                266                  375                   258

MeOH+AlCl3                    405                 271                      406                276                  402                   273

MeOH+AlCl3+HCl           408                 272                      406                274                  365                   266

↓ Intensities decrease in time.

Due to the frequently ingestion of U. sinuata leaves infusion by Andean peoples and, by other hand, by the possibility of use of the three flavonoids in

pharmacological essays, the cytotoxicity of these compounds was tested. Compounds I to III showed similar values for DL50≈ 1000 ppm, which point out the low cytotoxicity showed for the three compounds to Artemia salina16.

Hexadecyl 4-monoitaconate isolated from dichloromethane extract probably arises from fungus of the Aspergillus genus which colonized the plant material

during the storage.

Conclusions

From Urena sinuata leaves, three quercetagetin glucosides were isolated and identified; two of them are new natural products. The presence of I-III in U. sinuata leaves difference chemically to this plant from U. lobata, from the which only flavonoid aglycones were isolated; this sentence support the location of these taxa in different species.

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Una profusa gama de ilustraciones se puede encontrar en: https://picasaweb.google.com/118369454231235428280/CadilloPataDePerro

Pido disculpas por la mala traducción que he podido hacer y por los errores ortográficos que haya cometido al realizar la traducción, si alguno puede asistirme en este sentido le estaré muy agradecido.  William Piña