A rapid and effective method for isocratic RP-HPLC separation of flavonol glycosides of the overground part of Bupleurum flavum is developed. The lipophilicity of the identified compounds is assessed by the partition coefficient logP calculated for n-octanol-water system. A good correlation between the capacity factors (log k') obtained in the RP-HPLC system and the calculated lipophilicity parameters is found. The results indicate that the RP-HPLC is useful method for rapid and accurate determination of the lipophilicity of the compounds investigated.

The Bupleurum genus, Apiaceae, includes about 150 species, which are spread exclusively in the subtropical and temperate zones of the Northern Hemisphere [1,2]. There are 14 species in Bulgaria [3]. A number of Asian species are widely used in the traditional Eastern medicine because of their biologically active compounds - flavonoids, polysaccharides, lignans and saponins [4,5]. Bupleurum flavum is a taxon spread on the Balkan Peninsula. We are carried out a phytochemical research on the overground part of the plant for the first time. By LC-MS technique are identified the following flavonol glycosides: quercetin-dirhamnoside, quercetin-3-rhamnosylglucoside, quercetin-3-glucoside, kaempferol-di-rhamnoside, kaempferol-3-rhamnosylglucoside, kaempferol-3-glucoside and isorhamnetin-3-rhamnosyl-glucoside [6]. It is isolated one lignan and five triterpenoids, two of them - new derivatives of lupane [7].

The present investigation concerns some of the flavonol glycosides isolated by solid-phase extraction (SPE) and separated by isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) system. Since the degree of the lipophilicity of the compounds varies in a large range, it is important to predict their partition behaviour for preliminary evaluation of the liquid chromatography system. The aim of the study is to assess a correlation between the calculated lipophilicity parameter logP for the n-octanol-water system and the retention of the compounds on RP-HPLC column.

Instrumentation

The analysis is performed on a VARIAN (USA) chromatographic system which includes tertiary pump Model 9012, Rheodyne injector with 10 ml sample loop, UV-VIS detector Model 9050 set at 360 nm according to the UV absorption maxima of the compounds determined. A reversed-phase Supershere RP18, 4 mm, 250 x 4 mm (Merck, Germany) column is used, maintained at room temperature.

Standards

The standards of the flavonol glycosides are provided by Extrasynthese (Genay, France). The solvents are HPLC-grade (Merck, Germany).

Herba Bupleuri

The overground part of Bupleurum flavum is collected in the region of South Black Sea during July 1997.

Sample preparation

The purification and preconcentration of the samples are accomplished by SPE on VARIAN Vac Elut10 vacuum manifold using cartridges Bond Elut C18, 500 mg, 3 ml. The flavonoid mixture is obtained from Herba Bupleuri by shaking in 50 % CH₃OH for 24 h, concentrated till dry in vacuo and the residue is dissolved in methanol. The plant extract is applied on the preliminary conditioned cartridges and eluted with 70% CH₃OH. Aliquot parts of 10 ml are injected in the liquid chromatographic system.

RESULTS AND DISCUSSION
Figure 1. RP-HPLC chromatogram of B. flavum methanol extract after SPE

A method for purification and isolation of flavonol glycosides from the overground part of Bupleurum flavum is developed by solid-phase extraction and the optimum elution of the samples is achieved with 70% CH₃OH.

The RP-HPLC behaviour of the compounds is tested on Supershere RP18, 4 mm, 250 x 4mm (Merck, Germany) column sequentially varying the composition of the aqueous-methanol mobile phase. The studied glycosides are separated isocratically: quercetin-dirhamnoside, quercetin-3-rhamnosylglucoside, quercetin-3-glucoside, isorhamnetin-3-rhamnosylglucoside, kaempferol-dirhamnoside, kaempferol-3-rhamnosylglucoside, kaemp-ferol-3-glucoside (Figure 1). The separation is improved by modifying the mobile phase with small amount of tert-butanol (figure 2).

Figure 2. RP-HPLC chromatogram of flavonol glycosides (standards)

The widespread flavonol glycosides have the same basic skeleton but differ in the degree and the position of hydroxyl, methoxy and sugar residues (Table 1). For this reason their partition behaviour is very different. Calculating the lipophilicity of the solutes is important for understanding how they might refer to the liquid chromatography separation system [8]. The obtained logP values by a computer programme Chem-X^R [9] and the experimentally derived logk' values in the applied RP-HPLC system (Figure 2) are shown in Table 2. It is established a good correlation between both the parameters (figure 3) generated the following equation:
 

logk' = -0.214 (0.046) logP + 0.837 (0.114) n = 6 R2 =0.844 s = 0.063 F1,4 = 21.624 Figure 3. Plot of capacity factors logk' vs. calculated lipophilicity logP of the flavonol glycosides studied The obtained equation shows that logk' is an appropriate parameter for the assessment of the lipophilicity of compounds by RP-HPLC as well.

The isocratic RP-HPLC procedure coupled with solid-phase extraction allows a good purification of the samples and rapid separation of the flavonol glycosides with some advantages in terms of the analysis time and reproducibility over the gradient elution mode and the classical methods of extraction. The methods developed are suitable for analysing a variety of plant species. The obtained results confirm the growing interest in the calculation of the lipophilicity parameter logP for predicting the retention of analyses (logk') in the RP-HPLC system.

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3. Assenov Iv. (1982): Bupleurum L., In: Kozuharov S. (Ed.), Flora republicae popularis bulgaricae Vol. 8, Sofia, In aedibus academiae scientiarum bulgaricae, 109 - 125.

4. Seto H. et al. (1986): Isolation of Triterpenoid Glycosides (Saikosaponins) from Bupleurum kunmingense and Their Chemical Structures. Agric. Biol. Chem. 4, 943 - 948.

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6. Gevrenova R., Dimitrova B., Assenov Iv. (1997): Flavonols from Bupleurum flavum Forsk. - fam. Apiaceae. Farmacia S. 3-4, 9-14.

7. Pistelli L. et al. (2000): Bupleurum flavum Forskal: Estrazione e caratterizzazione di composti fenolici e triterpenoidici. 10 Congresso nazionale, Societa' Italiana di fitochimica, Firenze.

8. Abraham M.H. et al. (1997): Determination of solute lipophilicity, as log P(octanol) and log P(alkane) using poly(styrene-divinylbenzene) and immobilised artificial membrane stationary phases in reverse-phase high-performance liquid chromatography. J. Chromatogr. A 766, 35-47.

9. Chem-X^R (1999): Chemical Design Ltd., Roundway Hous, Cromwell Park, Chipping Norton, Oxfordshire, UK.