The composition of phenolic acids in Scleranthus perennis, Caryophyllaceae is analyzed for the first time. The ether extract from the overground parts is purified and preconcentrated using a solid-phase extraction procedure. The subsequent high-performance liquid chromatography separation is achieved on Spherisorb ODS-2, 5mm (250 x 4.6 mm) column by linear gradient elution mode. The obtained results reveal the presence of protocatechuic, 4-hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, o-coumaric, salicylic and benzoic acids in the studied samples.

The Scleranthus genus, Caryophyllaceae includes seven species widely spread in Bulgaria [1]. S. annuus and S. perennis are commonly used in the traditional Eastern medicine and Bulgarian folk veterinary medicine due to the biological properties of flavonoid, coumarin and saponin constituents [2]. The naturally occurring phenolic acids (derivatives of benzoic and cinnamic acids) are proved to be pharmacologically active as antioxydants, antimutagenic and anticarcinogenic agents [3].

In this study we attempt to assay the phenolic acids in the overground part of Scleranthus perennis for the first time. A solid-phase extraction (SPE) procedure is developed for addititional fractionation of the investigated acids from the plant extract. The reversed-phase high-performance liquid chromatography (RP-HPLC) method of Guillen et al. [4] is suitably modified for the subsequent determination of the isolated derivatives of benzoic and cinnamic acids.

Instrumentation

The assays are performed on VARIAN (USA) chromatographic system which includes tertiary pump Model 9012, Rheodyne injector with 20 ml sample loop, UV-VIS detector Model 9050 set at 220, 280 and 310 nm according to the UV absorption maxima of the compounds determined. Varian Star Chromatography workstation and computer software (version 4.5) are used for controlling the system and collecting the data. The reversed-phase column Spherisorb ODS-2, 5 mm (250 x 4.6mm) is used, fitted with Spherisorb ODS-2, 30 mm precolumn 30 x 4.6 mm (Interchim, Montlucon, France), maintained at room temperature.

Standards and chemicals

The standards of phenolic acids are obtained from Extrasynthese (Genay, France) and Fluka (Buchs, Switzerland). Methanol (HPLC-gradient grade) and all other reagents are supplied from Merck (Germany).

Herba Scleranthi

The overground part of Scleranthus perennis is collected in the region of Lulin mountain during July 1996.

Sample preparation

The plant extract of Herba Scleranthi is obtained by refluxing the powdered samples sequentially with ethanol and ethanol-water mixture (70:30 v/v) and the pooled solutions are concentrated in rotary vacuum evaporator. The aqueous residue (adjusted to pH=2.0 by concentrated hydrochloric acid) is thoroughly extracted with ether and evaporated to dryness in vacuo (0.4282 g sample extract). For additional purification and preconcentration of the acids in the obtained dry fraction a solid-phase extraction is performed using VARIAN Vac Elut 10 vacuum manifold and Bond Elut C18, 500 mg, 3 ml cartridges. The retained analytes are eluted by tetrahydrofuran that is thoroughly evaporated under nitrogen stream. The residue is dissolved in 150 ml of water-methanol (1:1 v/v) mixture and aliquot parts of 20 ml are injected for the subsequent HPLC determination.

Optimization of SPE and HPLC procedures

The fractionating of the plant extract by SPE is tested on several types of reversed-phase C18 supports with different physical characteristics as carbon load, pore and particle size. Series of experiments for conditioning and loading of cartridges are carried out varying the volume, composition, pH of washing and eluting solvent systems, pH-values of the applied samples, drying time, etc. Finally, the cartridge is conditioned sequentially with 10 ml MeOH and 10 ml 0.1M NaOOCCH₃. The sample dissolved in 5 ml 0.1M NaOOCCH₃ (pH 8.0) is loaded onto a cartridge and washed with 5 ml 0.1M NaOOCCH₃ solution. The effluent portion (adjusted to pH 2.0 with glacial acetic acid) is passed through the cartridge conditioned with 10 ml MeOH, 10 ml 0.07M CH₃COOH (pH 2.0) and washed with 5 ml 0.07M CH₃COOH. After drying of the cartridge with nitrogen stream an optimum elution of the adsorbed phenolic acids is achieved with 1ml tetrahydrofuran.

The accuracy of the solid-phase extraction method and the effect of plant matrix are measured by recovery percentages. Certain amounts of phenolic acids, which are as close as possible to those found in the real plant extract, are added to the sample and the spiked solution is subjected to the entire analytical procedure. The recovery study is done in duplicate. Good results are obtained for 4-hydroxybenzoic acid with recovery 103.53±5.26% (Table 1). In the case of protocatechuic acid the recovery is slightly lower most probably due to the higher polarity of the compound. In general, the recovery characteristics of the proposed SPE method are adequate.

The reversed-phase liquid chromatography method applied by Guillen et al. [4] is suitably modified for our study to achieve optimum separation of the main derivatives of benzoic and cinnamic acids in the plant samples. The gradient elution profile is made up from 100% solvent A (4% methanol-2% acetic acid in water) to 100% solvent B (45% methanol-2% acetic acid) in a period of 55 min at flow rate 1.2 ml/min. Under these conditions the obtained resolution of the solutes is acceptable and no interference is observed from other compounds in the plant matrix (Figures 1 and 2).

Figure 1. RP-HPLC chromatogram of S. perennis ether extract after SPE (l=280 nm)

Figure 2. RP-HPLC chromatogram of S. perennis ether extract after SPE (l=220 nm)

Quantitative analyses

A standard curve for each studied acid is obtained by plotting the peak area of the determined compound against the concentration in the corresponding standard solution. The stock solution of acid mixture is suitably diluted with methanol. Aliquots of five or eight different concentrations (mg/ml) are injected into the HPLC column and the peak areas are determined at 220 nm for the both acids - benzoic and salicylic - and 280 nm for the others. The data obtained are subjected to statistical analysis utilizing the Statistikprogram STL, Kalibrierung und Validierung in der Analytischen Chemie. The quantitative data of the assayed acids are expressed in ppm (Table 2).

The composition of phenolic acids from the overground part of Scleranthusperennis is analyzed for the first time. A method for purification and isolation by solid-phase extraction with good recovery is developed. The phenolic acids of the plant extract are separated and identified by RP-HPLC applying properly modified gradient elution mode. The results obtained by these techniques revealed the presence of protocatechuic, 4-hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, o-coumaric, salicylic and benzoic acids. The proposed SPE coupled with HPLC methods are suitable for analyzing a variety of plant species.

1. Kuzmanov B. and Kozuharov S. (1966): Scleranthus L., In: Kozuharov S. (Ed.), Flora republicae popularis bulgaricae, Vol. 3, Sofia, In aedibus academiae scientiarum bulgaricae, 291 - 298.

2. Zdraveva P. and Assenov Iv. (1997): Phytochemical study of Scleranthus perennis L. (Caryophyllaceae). Pharmacia (Sofia). 2, 2-10.

3. Newmark, H.L. (1992): Phenolic Compounds in Food and Their Effects on Health. In: Huang M.T., Ho C.T. and Lee C.Y. (Eds.), ACS Symposium Series Nr.507, American Chemical Society, Washington, DC, 48-76.

4. Guillen D.A., Barroso C.G. and Perez-Bustamante J.A. (1996): Selection of column and gradient for the separation of polyphenols in sherry wine by high-performance liquid chromatography incorporating internal standards. J. Chromatogr. A 724, 117-124.