Acylated flavonol glycosides and terpenoids from the leaves of Alibertia sessilis

Abstracts

Two novel acylated flavonol glycosides, along with iridoids, triterpenes, steroids and a-tocopherolquinone, were isolated from the leaves of Alibertia sessilis (Rubiaceae). The determination of the structures of the new compounds was based mainly on ¹H- and 13C-NMR.

Alibertia sessilis; Rubiaceae; acylated flavonol glycosides; iridoids; terpenoids; α-tocopherolquinone

Dois novos flavonóis glicosilados, juntamente com iridóides, triterpenóides, esteróides e tocoferolquinona, foram isolados das folhas de Alibertia sessilis (Rubiaceae). As estruturas dos flavonóides foram determinadas através de métodos espectroscópicos, principalmente da RMN de 13C e de ¹H.

Article

Acylated flavonol glycosides and terpenoids from the leaves of Alibertia sessilis

Roberto S. Gallegos Olea^a, Nídia F. Roque^a*, and Vanderlan da S. Bolzani^b

^a Instituto de Química, Universidade de São Paulo, CP 26077, 05599-970 - SP, Brazil

^bInstituto de Química, UNESP, SP, Brazil

Received: June 17, 1996

Dois novos flavonóis glicosilados, juntamente com iridóides, triterpenóides, esteróides e tocoferolquinona, foram isolados das folhas de Alibertia sessilis (Rubiaceae). As estruturas dos flavonóides foram determinadas através de métodos espectroscópicos, principalmente da RMN de ¹³C e de ¹H.

Two novel acylated flavonol glycosides, along with iridoids, triterpenes, steroids and a-tocopherolquinone, were isolated from the leaves of Alibertia sessilis (Rubiaceae). The determination of the structures of the new compounds was based mainly on ¹H- and ¹³C-NMR.

Keywords: Alibertia sessilis, Rubiaceae, acylated flavonol glycosides, iridoids, terpenoids, α-tocopherolquinone

Introduction

In the course of our continuing search for new active antifungal compounds from Alibertia (Rubiaceae) found in "Cerrado" region of S. Paulo, we studied Alibertia sessilis (Vell.) K. Schum. collected in Itirapina, in the state of São Paulo, Brazil. Previous investigations of the leaves of A. macrophylla resulted in the isolation of fungitoxic non-glycosidic iridoids 1b and 1a-hydroxydihydrocornin aglycones, and the caffeic acid esters: 2-phenylethyl caffeoate and 2-methyl-4-hydroxybutyl caffeoate¹. A. edulis, an other species collected in the same region, was also investigated, and no active compound was detected². Relatively few chemical studies of the Alibertia genus have been reported, despite evidence of its rich terpenoidic constitution³.

In the present study, from the leaves of A. sessilis we obtained large amounts of the triterpenes oleanolic, ursolic, and epi-betulinic acid, and small amounts of the iridoids gardenoside, deacetylasperuloside, and 10-dehydrogardenoside, together with the fungitoxic a- and b-gardiol. Besides iridoids and triterpenes, the flavonols quercetin-3-O-rutinoside, quercetin-3-O-b-D-(2"-O-trans-p-coumaroyl)-rutinoside (1) and kaempferol-3-O-b-D-(2"-O-trans-p-coumaroyl)-rutinoside (2) were isolated. These last two glycosides are being reported for the first time from a natural source.

Results and Discussion

The hexanic extract of the leaves of Alibertia sessilis was submitted to a chromatographic process to afforded sitosterol and a-tocopherolquinone, identified by comparison with authentic samples. After methylation, the CH₂Cl₂ extract of the leaves gave ursolic, oleanolic, and epi-betulinic acid methyl esters^2,4. After chromatographic separations, the butanolic soluble part of the hydroalcoholic extract afforded a mixture of the isomeric iridoids a and b gardiol⁵, in addition to gardenoside and deacetylasperulosidic acid methyl ester^6,7. These compounds were identified by comparing the spectroscopic data with the literature^5-7.

Gel permeation chromatography of the EtOAc soluble part of the hydroalcoholic extract gave quercetin-3-O-rutinoside⁸ (rutin) and a mixture of the new acylflavonol glycosides 1 and 2. These were purified by subsequent preparative TLC. The ¹H-NMR of 1 and 2 showed signals that suggested the structure of quercetin and kaempferol, respectively, for the aglycones of the compounds. In both spectra signals corresponding to the trans p-coumaroyl group and a rhamnose sugar portion were also found (Table 1). The anomeric proton of the rhamnose appeared as a singlet (d 4.36) in accordance with the a configuration. Another signal, which could be assigned to an anomeric proton of another sugar appeared at d ~ 5.5 (d, J = 8 Hz) in both spectra.

The ¹³C-NMR spectra (Table 2) confirmed the structure of quercetin and kaempferol aglycones to 1 and 2, respectively. The presence of rhamnose and coumaroyl moieties was also confirmed. The localization of the carbohydrates in the flavonols at C-3 was deduced by the chemical shifts of C-3, C-2, and C-4, the first being shielded and the latter two deshielded in relation to the free aglycones⁸. The rhamnose signals are unchanged, so the acyl group should be located on the other sugar moiety. The methylenic carbon of that sugar is deshielded by 6 ppm when compared with that of glucose. A COSY ¹H^-1H spectrum of 1 showed correlations between the anomeric proton signal at d 5.56 and a signal at d 4.87 (t, J = 8.4 Hz). These values suggested an acylation of the hydroxyl at C-2 and a diaxial hydrogen coupling between H-2/H-1 and H-2/H-3. These data are in agreement with an acylated C-2 glucose.

Comparative analyses of the ¹³C-NMR carbohydrate data of 1 and 2 with those of the glucose in rutin^8,9,10 revealed that the C-2 of the glucose in 1 and 2 was deshielded by +2 ppm, and that C-1 and C-3 are shielded by -2 ppm. These observations are in agreement with the location of the coumaroyl group at C-2 of the glucose moiety. Thus, the structures of the two new acylated flavonol glycoside isolated from Alibertia sessilis were established as quercetin-3-O-b-D-(2"-O-trans-p-coumaroyl)-rutinoside (1) and kaempherol-3-O-b-D-(2"-O-trans-p-coumaroyl)-rutinoside (2).

The occurrence of iridoids and triterpenes in Alibertia is in agreement with chemosystematic correlations and botanical positioning of this genus in Gardenieae³. However, the isolation of this type of flavonol is unusual if we consider the chemical composition of other Alibertia already described in the literature.

Experimental

General experimental procedures

NMR spectra were measured at 200 MHz for ¹H, with TMS as the internal standard and 50 MHz for ¹³C, using the solvent signal as reference.

Plant material

Alibertia sessilis (Vell.) K. Shum. (Rubiaceae) was collected in the Itirapina Botanic Reserve, in the Cerrado region of São Paulo, Brazil. A voucher specimen is deposited at the Botanical Institute of São Paulo (number S.P. 110683).

Extraction and isolation of the constituents

Dried and powdered leaves (515 g) of A. Sessilis were successively extracted with hexane, methylene chloride and ethanol/water (6:4). The crude hexane extract (10.4 g) was fractionated on silica gel column chromatography. Further purification of the fractions by preparative TLC, eluted with C₆H₆:EtOAc (9:1) yielded sitosterol (86 mg), a-tocopherolquinone (29 mg), and fatty material.

The CHCl₃ insoluble fraction (1.2 g) of the crude dichloromethanic extract (9.5 g) was methylated with CH₂N₂. Preparative TLC eluted with CHCl₃:MeOH (97:3) of this material gave three fractions: the first was a mixture of ursolic acid and oleanolic acid methyl esters (300 mg), the second was the methyl ester of ursolic acid (800 mg), and the third was the methyl ester of epi-betulinic acid (30 mg).

The crude ethanol/water (6:4) extract (48 g) was partitioned into ethyl acetate and then into n-butanol. The soluble part of n-Butanol (2.5 g) was submitted to column chromatography on silica gel, and eluted with CHCl₃ with increasing amounts of MeOH. After analysis by TLC some fractions were combined. Further preparative TLC eluted with CHCl₃:MeOH (85:15) of the less polar fractions gave a mixture (5 mg) of the isomeric iridoids -gardiol and b-gardiol. Another fraction of that column was submitted to reversed-phase HPLC [column C-8 (25 x 0.4 cm) and elution with a H₂O - MeOH gradient, at a flow rate of 8 mL/min, and detector UV (240 nm)], affording the iridoids gardenoside (17 mg) and deacetylasperulosidic acid methyl ester (4 mg).

The soluble part of ethyl acetate (200 mg) was precipitated with CHCl₃. The precipitate was dissolved in MeOH and submitted to column chromatography on Sephadex LH-20, and eluted with MeOH. After TLC, some fractions were combined and one of them was identified as quercetin-3-O-rutinoside (50 mg). Another fraction was submitted to preparative TLC, and eluted with CHCl₃:MeOH: n-BuOH:H₂O (25:5:10:1) to afford 12 mg of quercetin-3-O-b-D-(2"-trans-p-coumaroyl)-rutinoside (1) and 25 mg of kaempferol-3-O-b-D-(2"-trans-p-coumaroyl)-rutinoside (2).

Acknowledgements

The authors are grateful to CNPq for financial support and schalarships.

FAPESP helped in meeting the publication costs of this article

Present address: Departamento de Química, UFMa.

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Publication Dates

• Publication in this collection
  31 Jan 2011
• Date of issue
  June 1997

History

• Received
  June 1996