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Boletín de la Sociedad Chilena de Química

versión impresa ISSN 0366-1644

Bol. Soc. Chil. Quím. v.45 n.3 Concepción set. 2000

http://dx.doi.org/10.4067/S0366-16442000000300017 

SEPARATION BY CAPILLARY ELECTROPHORESIS OF
C-GLYCOSYLFLAVONOIDS IN Passiflora sp. EXTRACTS

E. R. Pastene1, G. Bocaz 2a, I. Peric3, M. Montes1, V. Silva1,
and E. Riffo3

1 Fac. de Farmacia, Universidad de Concepción, P. O. Box 237, Concepción, Chile
2 Masaryk University, Faculty of Science, Department of Analytical Chemistry, Kotlárská 2,
61137 Brno, Czech Republic.
a On the leave from: Department of Laboratories, Legal Medical Service, Ministry of Justice
Av. La Paz 1012, Santiago, Chile
3 Depto. de Química Analítica e Inorgánica, Facultad de Ciencias Químicas, Universidad de
Concepción, P. O. Box 160-C, Concepción, Chile
(Received: May 11,2000 - Accepted: June 21, 2000)

In memorian of Dr. Guido S. Canessa C.

ABSTRACT

The potential of Capillary Electrophoresis (CE) for separation of C-Glycosylflavonoids has been examined. These compounds are found in Passiflora incarnata L. and Passiflora coerulea L. and their quantitative evaluation is the method for quality control of their extracts. Capillary zone electrophoresis (CZE) as well as micellar electrokinetic chromatography (MEKC) conditions were assayed. The optimum resolution of analytes was achieved within 12 min by using an electrolyte system of 50 mM sodium tetraborate, adjusted to pH 9.2 with NaOH and direct detection at 270 nm. Moreover, identification of several peaks was accomplished by repeating and comparing the migration times or electrophoretic mobilities and by spiking the sample solution with the pure compound prior to injection.

Key Words: Capillary zone electrophoresis, Micellar electrokinetic chromatography, C-Glycosylflavonoids, Passiflora sp, Separation.

RESUMEN

Se examina el potencial de la Electroforesis Capilar (EC) para la separación de C-Glicosilflavonoides. Estos compuestos se encuentran en Passiflora incarnata L. y Passiflora coerulea L. y su evaluación cuantitativa constituye el método para el control de calidad de sus extractos. En este trabajo se ensaya las modalidades de electroforesis capilar de zona (ECZ) y cromatografía micelar electrocinetica (CMEC). La resolución optima de los analitos, se alcanzó dentro 12 min, usando como sistema de electrolitos tetraborato de sodio 50 mM, ajustado a pH 9.2 con NaOH y detección directa a 270 nm. Adicionalmente, se realiza la identificación de algunos peaks, mediante comparación de los tiempos de migración o mobilidades electroforeticas y recarga previa a la inyección con sustancias patron puras.

Palabras claves: Electroforesis capilar de zona, Cromatografía micelar electrocinética, C-Glicosilflavonoides, Passiflora sp, Separación.

INTRODUCTION

Representative of Passifloraceae family are commonly used in phytotheraphy due to their tranquillising and sedative properties (1-3). Climatic conditions in Chile allow a good growth of Passiflora coerulea L. which is used as herbal tea. Passiflora incarnata L., a pharmacopoeia official drug, is found only within phytotherapheutic preparations (tinctures and capsules). Both species have C-Glycosylflavonoids derived from Luteolin and Apigenin, whose quantitative dosage is used as a quality control method. The techniques shown in DAB 10 and Ph. Helv. VII, consider the spectrophotometric determination of these complexed compounds with AlCl3 after acid hydrolysis. To the unsatisfactory extraction of the hydrolysis products, a clean-up problem of the lipophilic substances (mainly chlorophylls) is added. These lipophilic matters interfere with the UV absorption of the AlCl3 complex (4). Due to these deficiencies in the purification and extraction processes, some alternative methods avoiding acidic treatment and tedious purification processes of samples have been proposed. Modern HPLC methods allow a partial separation of C-Glycosylflavonoids. However, additionally solid - phase purification step is required (RP 18 cartridges), increasing the analysis time over 20 minutes per run (5,6). Other enhanced UV-VIS methods, which do not consider acidic hydrolysis have been developed. Nevertheless, UV-VIS analysis shows a good correlation with the HPLC studies, but it does not give information about the stability of the extracts (7,8).

On the other hand, HPTLC resulted to be a more versatile alternative, because the clean-up of the extracts is not required and in addition it allows high throughput of the samples. Despite this, partial resolution of C-Glycosyflavonoids remains (9).

Within the five last years, a series of papers related to the separation of flavonoids and other phenolic compounds, from different vegetable materials, by using capillary electrophoresis, have been published (10-13). Most of these studies consider the flavonoids as neutral compounds, feasible to be separated by MEKC (14-16). Nevertheless, there exist few information of the C-Glycosylflavonoids behaviour with this modern analytical tool, which has encouraged us to study the potential of CE in the control of Passiflora sp. extracts.

MATERIAL AND METHODS

High Performance Capillary Electrophoresis

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) modes for the separation of C-Glycosylflavonoids from Passiflora sp. extracts were investigated. Separations were conducted using a PrinCE 450 Capillary Electrophoresis System connected to a Lambda 1010 UV-Vis detector (Bishoff, Leonberg, Germany). System DAx software was used for data acquisition and analysis. Uncoated fused-silica capillaries were 75 mm i.d. and 375 mm o.d. (Polymicro Technologies Inc., Phoenix, AZ, USA ), with an end-to-end length of 70 cm and an end-to-detection window length of 55 cm.

Prior to the first use, a new capillary was activated for 20 min using 1 M NaOH and then washed for 10 min with Nanopure grade water (18 WW/cm), and finally equilibrated with the operating buffer for 10 min before any sample injection.

Samples and standards were injected hydrodynamically at 25 mbar for 12 s (5nl). The capillaries were sequentially washed for 2 min with 0.1 M NaOH, 2 min with Nanopure grade water and 5 min with the carrier solution, between injections. Sodium borate (Aldrich) buffer and buffers with different sodium borate/sodium dodecyl sulfate(SDS) ratios for CZE and MEKC modes were assayed respectively. Buffers were adjusted to pH 9.2 using NaOH (Merck), degassed for 20 min by sonication (ELMA Transsonic 820/H, Germany), and finally filtered through a 0.45mm Millex-HV membrane (Millipore, Milford, USA) prior to their use.

The experiments were performed at room temperature and detected by direct UV absorbance at 270 nm.

Materials

Aerial parts of Passiflora coerulea L. (Passifloraceae), were supplied by Knop Laboratories (Quilpué, Chile) and identified by Dr. Max Quezada (Department of Botany, School of Biological Sciences, Universidad de Concepción, Concepción, Chile). A voucher specimen (CONC146512), was deposited at the herbarium of the same Department. Dried extract of P. incarnata (3:1), was supplied by HECO Überseehandel Tietjen & Co. (Hamburg, Germany). Isoorientin, vitexin, orientin and saponarin flavonoids were supplied by Roth (Karlsruhe, Germany) and used as reference materials. Analytical grade sodium tetraborate decahydrate, sodium dodecyl sulphate and sodium hydroxide were purchased from Merck (Darmstadt, Germany) and were used without further purification. Methanol LiChrosolv‚ grade was obtained from Merck (Darmstadt, Germany). Water was deionized with a Barnstead/Thermolyne NANOpure Ultra Pure Water System (Dubuque, Iowa, USA).

Sample preparation

One Hundred milligrams of pulverised and sieved P. coerulea were extracted by refluxing three times during 15 min with 10 ml of 60% methanol. Extracts were filtered through a piece of cotton in a 25 ml volumetric flask and adjusted to volume with 60% methanol. No clean-up process was necessary before sample injection to HPCE device. Dried extract of P. incarnata was proportionally diluted with 60% methanol. Prior to capillary electrophoresis, both extracts were filtered through a 0,22 mm Millex-HV membrane (Millipore, Milford, USA).

RESULTS AND DISCUSSION

Optimisation of electrophoretic conditions

Extracts of P. coerulea and P. incarnata have a complex mixture of C-Glycosylflavonoids. These compounds are difficult to resolve satisfactorily through conventional chromatographic techniques. Separation of C-Glycosylflavonoids by using capillary electrophoresis in CZE and MEKC modes, was evaluated. The assayed conditions are summarised in Table I.

Separation of the P. coerulea flavonoids with the different systems assayed are presented in Fig. 1. Electropherograms obtained by using buffers with different sodium borate/SDS ratios show a poor resolution which is enhanced with decreasing SDS concentration until CZE condition is reached. The optimum resolution in CZE conditions is due to the formation of borate complexes with the phenolic OH groups, negatively charged at pH 9.2. Under these conditions, flavonoids with a lower negative charge and a higher molecular weight migrated together with the electroosmotic flow (EOF) being detected at the beginning. It is known that the EOF is decreased with increasing borate concentration, because the zeta potential is decreased (17). This means that the surface charge and the double layer thickness is decreased. However, with increasing borate concentration a current increase is produced. Consequently, the electroosmotic velocity and the theoretical plate number were increased to attain an improved selectivity (18).


Fig. 1 Efect of the borate/SDS ratio upon the separation pattern of P. coerulea C-Glycosyflavonoids. For MEKC used ratios were 10mM (a); 10mM/50mM (b) and 20mM/50mM (c). For CZE borate concentrations were 10mM (d) and 50mM (e). Fused-silica capillary: L = 70,0 cm (I=55,0 cm) x 75 mm I.D. Separation condition: +20 KV mA; 270 nm, 30 ºC, 12 s hydrodynamic injection.

It is known that the formed complexes between borate and flavonoids are type V. The boron influence on buffer pH and its interaction with the phenolic groups of flavonoids are not sufficient to explain the higher selectivity in the separation of the structurally very similar compounds. Although it is not clearly understood, it is believed that borate has also influence on sugar hydroxyl groups, which could contribute in an important way on the system selectivity (19).

To study the viscosity effect on separation, runs at different temperatures were carried out. The studied compounds are resolved efficiently by using standard conditions at 30°C. At 20 and 25°C separation was unsatisfactory (data not shown).

Study of extracts of P. coerulea and P. incarnata

Identification of flavonoids found in P. coerulea extract has been performed by comparing their migration times with those of the standard substances and samples spiking. Important amounts of isoorientin were found whereas vitexin and orientin were found at very low concentrations (Fig. 2). The latter two can be artefacts generated by Wessely-Mooser isomerization from isoorientin and isovitexin, or by partial hydrolysis of some O-glucoside derived from them (20). These results are in agreement with the HPTLC preceding studies, where these substances where detected only by fluorescence, using the Natural Products Reagent (diphenylboryloxyethylamine), for the derivatization procedure. None of the other available standards were present in this plant.


Fig. 2 Identification of isoorientin in P. coerulea extract by spiking with isoorientin standard. Borate concentration; 50 mM (pH = 9.2). Separation conditions: +20 KV, 200 mA; 270 nm, 30 ºC, 12 s hydrodynamic injection.

Using HPLC/MS, the presence of saponarin, schafttosid, isoshafttosid, luteoline 2´-glucoside and isovitexin in P. incarnata as principal components, has been informed elsewhere (21, 22). In our study, commercial extract showed the presence of vitexin, orientin and isovitexin whereas saponarin and isoorientin were detected in very low quantities (Fig. 3).


Fig. 3 Identification of flavonoids in P. incarnata extract by spiking with standars of saponarin, isoorientin and vitexin. Borate concentration; 50 mM (pH = 9,2). Separation conditions: +20 KV 200 mA; 270 nm, 30 ºC, 12 s hydrodynamic injection.

Capillary zone electrophoresis of both extracts allow to perform a "fingerprints" of them. This is very useful in quality control of the vegetal materials and the phytotherapeutic products. Both plants showed a "fingerprint" with substantial qualitative differences. In further investigations, which are still in progress, more emphasis will be paid to the quantitative aspects.

Preceding studies have shown the utility of MEKC in a high number of flavonol-3-O-Glycosides analysis usually found in medicinal plants. The use of CZE in separation of C-Glycosylflavonoids by using sodium borate buffer is confirmed in this work.

In summary, CE because of it high separation selectivity, small sample size requirement, high speed of analysis, efficiency, sensitivity (0,1 - 1mg/ml), low reagent and solvent consumption, automation feasibility in the CZE mode represent a powerful analytical tool alternative to HPLC and HPTLC for separation and identification of C-Glycosylflavonoids in P. coerulea and P. incarnata extracts. Generally, CE seems to be a valid alternative in natural product studies as well as a valuable complement to other techniques.

ACKNOWLEDGEMENTS

Financial support for this research was provided by the Dirección de Investigación of the Universidad de Concepción, DIUC, Project N° 98074023-11, Vicerrectoria de la Universidad de Concepción (Special Project for the development of the Capillary Electrophoresis Nº 96-001) and Facultad de Farmacia, Universidad de Concepción. Professor E. Pastene thanks to Reinaldo Knop from Knop Laboratories (Quilpué, Chile). Dr. I. Peric thanks to the Deutscher Akademischer Austauschdienst DAAD program of the Federal Republic of Germany for financing his stay at University of Tübingen and to the Deutsche Gesellschaft für Technische Zusammenarbeit (GTZ) for the provision of the capillary electrophoresis device.

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