780 FARMACIA, 2009, Vol. 57, 6 HPLC ANALYSIS OF CAROTENOIDS FROM SENECIO VERNALIS AND S. JACOBAEA (ASTERACEAE) GEORGE DAN MOGOŞANU1*, ADELA PINTEA2, LUDOVIC EVERARD BEJENARU1, CORNELIA BEJENARU3, GABRIELA RĂU4, HONORIUS POPESCU1 1Department of Pharmacognosy & Phytotherapy 2Department of Biochemistry, Faculty of Zootechny and Biotechnologies, University of Agricultural Sciences and Veterinary Medicine, 3–5 Mănăştur Street, 400372 Cluj-Napoca, Romania 3Department of Vegetal & Animal Biology 4Department of Organic Chemistry & Medicinal Chemistry 1,3,4Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova, 2–4 Petru Rareş Street, 200349 Craiova, Romania *corresponding author: [email protected] Abstract Through HPLC analysis, in two Senecio species (S. vernalis Waldst. et Kit. and S. jacobaea L.), carotenoid fractions have been determined for the first time. Therefore, in increasing order of the retention times, seven compounds have been identified and quantitative determined, as follows: neoxanthin, violaxanthin, lutein-5,6-epoxide, lutein, β-cryptoxanthin, α- and β-carotene. High amounts of lutein have been found in all Senecio extracts. Rezumat Pentru prima dată au fost analizate, prin HPLC, fracţiunile carotenoidice provenite de la două specii de Senecio (S. vernalis Waldst. et Kit. şi S. jacobaea L.). Astfel, în ordinea crescătoare a timpilor de retenţie, au fost identificate şi determinate cantitativ şapte componente, după cum urmează: neoxantina, violaxantina, lutein-5,6-epoxid, luteina, β-criptoxantina, α- şi β-caroten. În extractele de Senecio, luteina a fost preponderentă din punct de vedere cantitativ. Keywords: Senecio vernalis; S. jacobaea; carotenoids; HPLC analysis. Introduction Senecio vernalis Waldst. et Kit., spring groundsel and S. jacobaea L., tansy ragwort, of Asteraceae family, are native in Eurasia, North America, Australia, New Zealand, Argentina, and Northern Africa. They are herbaceous, common in Romania’s flora, along roadsides, waste grounds, forests and crops, from the plain to the mountains’ zone. S. vernalis is annual or biennial and S. jacobaea is perennial [6]. Pyrrolizidine alkaloids, flavonosides, coumarins, volatile oils, sterols, triterpenes, heteroglycans, tannin, fatty acids, sugars, and vitamins FARMACIA, 2009, Vol. 57, 6 781 have been previously isolated from the aerial parts of Senecio species [4, 7, 10–12]. The aerial parts of Senecio species are used for their medicinal properties: astringent, antidiarrhoeal, diuretic, diaphoretic, emmenagogue, galactagogue, expectorant. A homeopathic remedy is used in the treatment of internal haemorrhages and dysmenorrhoea. An emollient poultice is made from the leaves. A decoction of the root is used for internal bruises and wounds. The fresh juice of the plants is cooling and has astringent effects for burns, sores, cancerous ulcers, eye inflammations, ulcerated mouth and throat [1, 4, 5, 7, 13]. Because of their cumulative effect, pyrrolizidine alkaloids have been reported by the World Health Organization to be toxic for animals and humans. If a sufficient quantity is consumed, it can cause irreversible liver cirrhosis. Senecio species posses a small risk for humans, the aerial parts being used under the generic name of Senecionis herba. However, some sensitive individuals can suffer from an allergic reaction, because of the sesquiterpene lactones’ content, which can cause dermatitis [4, 7, 10–12, 14]. In animals, carotenoids serve as a source of vitamin A and other retinoids, and as photoprotective and cancer prevention agents. Taking into account their importance, the qualitative and quantitative determination of carotenoids from S. vernalis and S. jacobaea have been established by HPLC analysis. Materials and Methods Plant material The raw material has been collected in the flowering period from two Senecio species, as follows: S. vernalis, from Craiova (Romanescu Park), in April 2004, and S. jacobaea, from Scăeşti village (Dolj County), in August 2003. Voucher specimens are deposited in the Herbarium of the Department of Pharmacognosy & Phytotherapy, Faculty of Pharmacy, University of Medicine and Pharmacy of Craiova. Reagents and solvents All of the analytical grade solvents and reagents were purchased from Merck (Darmstadt, Germany). Extraction Samples of accurately weighed, air-dried vegetal material of two Senecio species have been fine powdered in an Ultraturax device, and then 782 FARMACIA, 2009, Vol. 57, 6 repeatedly extracted with a mixture of ethyl acetate–methanol–petroleum ether (1:1:1, in volumes), under continuous agitation, in diminished light, in the presence of butylated hydroxytoluene (BHT), used as antioxidant, and sodium bicarbonate, added for the prevention of epoxidic rearrangements which can develop into an acid environment. After the filtering, the extracts have been dry-evaporated at 350C and low pressure. The residual samples have been added to a certain volume of ethylic ether, and then used for the quantitative spectrophotometric determination of carotenoids. Subsequently, the solvent has been evaporated and the raw extract has been preserved at low temperature (-200C), under nitrogen, and stored in a dark place. Saponification The raw extracts, which contain carotenoid pigments, are usually subdued to saponification (hydrolysis) for releasing carotenoids from the ester combinations and for the removal of the unsaponifiable fraction of the lipids. Thus, after the redissolving of the raw extracts in ethylic ether, an equal volume of 30% potassium hydroxide solution in methanol has been added. The saponification has been performed in a closed flask, in the dark and under continuous agitation, minimum eight hours. The extract has been transferred in a separation funnel with 1% sodium chloride solution and then washed until a neutral pH. The superior ether phase, which contains the carotenoid pigments, has been separated, dry-evaporated and preserved at -200C, under a nitrogen stream and stored in a dark place. Quantitative determination The UV–Vis absorption spectrum of the total carotenoids has been determined using a M40 spectrophotometer at a wavelength λmax 450 nm. The amount of carotenoids, in mg%, has been calculated based on the following equation [3]: A×V ×1000 mg% carotenoids = , 2500× l ×100 where: A – absorbance of the sample, at λmax 450 nm; V – sample volume 1% (mL); 2500 – specific absorbance for carotenoids (A 1 cm); l – optical path length (1 cm). HPLC experimental conditions The carotenoid fractions have been analysed using a HPLC system that included a Kontron 322 pump, a Kontron 465 injector, a Waters 990 photodiode array detector, and a 250 mm length × 4.6 mm diameter 5-µm C18 Discovery reversed phase column (Supelco) [8, 9]. FARMACIA, 2009, Vol. 57, 6 783 The mobile phase had a constant flow rate of 1 mL/min.: ▪ Solvent A: acetonitrile–water (9:1, in volumes) + 0.5% EPA (ethyl-isopropil-amine); ▪ Solvent B: ethyl acetate + 0.5% EPA. The following scheme has been used for the separation and identification of carotenoids: At the time (min.) 0 : 0% B in A; 16 : 60% B in A; 25 : 60% B in A; 27 : 0% B in A. Results and discussion The retention times of carotenoids from the analysed samples have been compared with those of the reference substances separated in the same conditions: lutein, zeaxanthin, canthaxanthin, β-cryptoxanthin, α- and β-carotene (Figure 1). In accordance with the references’ catalogue, within 5–10 min. neoxanthin (5.66 min.) and violaxanthin (7.22 min.) have been separated; it is supposed that the peak which appears ahead lutein, at 8.6 min., could be represented by the lutein-5,6-epoxide [2]. Other compounds have been identified starting from the correlation of their chromatographic behavior with UV–Vis absorption spectra specific for the carotenoids (Figures 2–5). The results of the quantitative UV–VIS spectrophotometric determination of the total carotenoids are shown below (Table I). Table I Quantitative determination of the total carotenoids Sample mg% total carotenoids, expressed in lutein Senecionis vernalis herba 7.74 Senecionis vernalis flos 4.24 Senecionis vernalis radix 0.37 Senecionis jacobeae herba 6.67 The aerial parts of Senecio species contain close values of the total carotenoids. In the flowers of S. vernalis, the content of carotenoids is lower then in the aerial parts. The lowest amount of total carotenoids has been determined in the roots of S. vernalis. The amount of carotenoids is relatively small, because the hydrocarbons are very susceptible to oxidation. That is why, in the analysed samples, high amounts of lutein and small quantities of β-carotene have been found. 784 FARMACIA, 2009, Vol. 57, 6 Figure 1 Figure 2 Figure 3 HPLC-chromatogram for HPLC-chromatogram for HPLC-chromatogram for the mixture of substances the saponified extract of the saponified extract of Senecionis vernalis herba Senecionis vernalis flos Figure 4 Figure 5 HPLC-chromatogram for the saponified HPLC-chromatogram for the saponified extract of Senecionis vernalis radix extract of Senecionis jacobeae herba FARMACIA, 2009, Vol. 57, 6 785 In the extracts from two Senecio species, some carotenoids with therapeutic implications like lutein, α- and β-carotene have been identified. Retention times and UV–Vis absorption maximums, obtained after the separation and analysis of the peaks, are shown in Table II. Table II HPLC analysis of carotenoids in Senecio extracts Retention time Absorption maximums Absorption maximums Carotenoids (HPLC) [min.] (HPLC) [nm] (literature) [nm]