Isolation of lignans glycosides from Alibertia sessilis ( Vell . ) K . Schum . ( Rubiaceae ) by preparative high-performance liquid chromatography

Enantiomeric aglycone lignans contained in a mixture were separated from a fraction of the extract of the stems of Alibertia sessilis(Vell.) K. Schum. (Rubiaceae) by preparative high-performance liquid chromatography. An efficient and fast separation can be achieved with methanol-water (30:70, v/v). Their structures were identified as (+)-lyoniresinol 3 α-O-β-glucopyranoside and (-)lyoniresinol 3α-O-β-glucopyranoside, being reported for the first time in Rubiaceae.


Introduction
Rubiaceae is widely distributed in Brazilian main ecosystems (Amazon, Cerrado and Atlantic Forest).This family is well known due to economic and therapeutic importance of these species, specially Coffea arabica and Cinchona ledgeriana [1][2].Reported chemical constituents of Rubiaceae revealed a great diversity of secondary metabolites such as iridoids, alkaloids, anthraquinones, flavonoids, phenolics derivatives, triterpenes and diterpenes [1].
From the stems of A. sessilis we report the isolation of a mixture of lignan glycosides.The separation of (+)-lyoniresinol 3α-O-β-glucopyranoside (1) and (-)-lyoniresinol 3α-O-βglucopyranoside (2) by conventional methods such as silica gel and RP18 low pressure column chromatography was unsuccessful.However, it was quite easy and fast to purify each compound by preparative high-performance liquid chromatography (HPLC).Reversed-phase HPLC is commonly used for the separation of compounds present in complex mixtures of plant extracts [8][9].
Nuclear magnetic resonance (NMR) analysis was used to identify the compounds.Compound 1 exhibit antioxidant [10] and antitumor-promoting activities [11].These compounds, whose chemical structures are given in Figure 1, are being reported for the first time in Rubiaceae.

Extraction, fractionation and preparation of sample
Dried and powered stems (50.0 g) of A. sessilis were successively extracted with ethanol at room temperature.The solvent was removed under reduced pressure yielding 3.7 g of crude extract.This extract was dissolved in methanolwater (80:20, v/v) and partitioned with hexane, ethyl acetate and n-butanol to yield 0.5 g of hexane extract, 1.4 g of ethyl acetate extract and 1.3 g of n-butanol extract after evaporated to dryness under reduced pressure.
The ethyl acetate extract was submitted to column chromatography using Sephadex LH-20.
The column was eluted with methanol, to give 15 fractions reunited after thin layer chromatography (TLC) analysis.Fractions 2-3 (146.4 mg) were subjected to chromatography on a RP18 column using a water-methanol gradient system affording iridoid geniposidic acid (11.8 mg), a derivative fenolic glycoside (6.7 mg) not yet identified and the mixture of lignans glycosides (36.6 mg).

Separation procedure
The separation of lignans was performed using a Phenomenex Luna RP18 (2) column (250 x 21.20 mm I.D. x 10 µm, Torrance, CA, USA) in a Rheodyne 7125 sample injector with a 2.0 mL sample loop (Rheodyne, Cotati, CA, USA).The mobile phase composed of methanol-water (30:70, v/v, isocratic system) was eluted using a flow-rate of 10.0 mL min -1 , the injection volume was 1.0 mL.Pure lignans were examined by HPLC on an analytical column.The analysis was performed with a Phenomenex Luna RP18 (2) column (250 x 4.6 mm I.D. x 5 µm, Torrance, CA, USA) in a Rheodyne 7125 sample injector with a 20 µL sample loop (Rheodyne, Cotati, CA, USA).The mobile phase composed of methanol-water (30:70, v/v, isocratic system) was eluted at a flowrate of 1.0 mL min -1 , the injection volume was 20 µL.For both preparative and analytical HPLC, the detector was set as 238 nm.

Structural identification of the compounds
The NMR spectra in CD 3 OD of all compounds were obtained using a Varian, INOVA 500 spectrometer (Varian, Palo Alto, CA, USA), operating at 500 MHz for 1 H and 125 MHz for 13 C, two-dimensional technique such as gCOSY (gradient chemical shift correlation spectroscopy), gHMQC (gradient heteronuclear multiple quantum coherence) and gHMBC (gradient heteronuclear

Results and Discussion
The crude extract from stems of A. sessilis was partitioned using hexane, ethyl acetate and n-butanol.The ethyl acetate extract afforded geniposidic acid, a derivative fenolic glycoside and a third compound.The 1 H-and 13 C-NMR data for this last showed overlapping signals, suggesting that third compound could be a diastereomeric mixture of lignans containing enantiomeric aglycone parts that were not separated.Conventional methods such as silica gel and RP18 low pressure column chromatography were not successful to separate this lignans.
So, this partially purified sample was analysed by HPLC and a series of experiments was performed to determine the optimal solvent system for the HPLC separation.The following systems were tested: methanol-water (35:65, 30:70, 25:75, v/v), and the separation time were: 18 min, 25 min and 42 min, respectively, for an analytical separation run.But, despite of first system has faster separation time, the peaks concerning to lignans didn't have good separation, and the third system had a long separation time.A good and fast separation can be achieved with the system methanol-water (30:70, v/v).
Another evidence that the mixture was composed of a enantiomeric aglycone parts is the identical UV spectras with absorption maximum at 215 and 270 nm and one spot when analysed by TLC.

Conclusions
Reversed-phase HPLC was successful in the preparative separation of a mixture containing enantiomeric aglycone parts, it was easy and fast to purify each compound of the mixture.So, this study contributed significantly to improve the knowledge about secondary metabolites of more one species of Brazilian Cerrado, when these compounds are being reported for the first time in Rubiaceae.