Phytochemical Investigation of Senecio vulgaris L . Growing in Egypt

Senecio vulgaris Coumarin Cytotoxicity Antioxidant activity Abstract Senecio vulgaris L. is a well known toxic plant, phytochemical investigation of the whole plant afforded the isolation of a new coumarin; 3,7-dihydroxy-5,6 dimethoxycoumarin7-O-β-D-glucoside (1), in addition to the isolation of isorhamnetin-3-O-β-D-glucoside (2) and two known alkaloids of common occurrence in genus Senecio; senecionine (3) and seneciphylline (4). The isolated compounds were identified by physical and chemical analyses and by intensive investigation of their spectral data. EtOH extract, light petrol and CHCl3 fractions of S.vulgaris were found to have cytotoxic activity on colorectal cell line HCT-116 cell line, with IC50 values of 19.9, 27.3 and 17.8 μg mL -1, respectively. That indicates the most active cytotoxic fraction is the alkaloid-bearing chloroform fraction. These findings promote the future perspective of S. vulgaris as an anticancer agent. The antioxidant activity of EtOH extract, light petrol fraction and CHCl3 fraction was evaluated by DPPH assay, %inhibition was 23.31, 4.677 and 17.598, respectively. Also the ratio between senecionine and seneciophylline in their mixture was determined using 1H-NMR spectrum.


Introduction
Genus Senecio can be considered one of the largest genera of family Asteraceae (subfamily Asteroideae, tribe Senecionae) as it comprises about 1250 species of annual or perennial herbs, shrubs or small trees which are distributed all over the world, especially in South Africa, Mediterranean floristic region and in temperate areas of Asia and America (Teles et al., 2008;Lotfi et al., 2010).

Senecio vulgaris L. is one of the common Egyptian
Senecio species.It is known by the common English names: groundsel and common groundsel; and by the vernacular Arabic names: Morrar, Murrar and eshbet salema (Umberto, 2000).It can be considered as a problematic weed as it is rich in toxic pyrrolizidine alkaloids.Many compounds were isolated from it, such as; alkaloids, flavonoids and terpenes (El-Shazly et al., 2002, 2014;Gharbo et al., 1969;Habib, 1981).
Pyrrolizidine alkaloids show either no or low acute toxicity; but they can undergo a 3-step metabolic toxication process leading to alkylating agents which affect the liver and other organs.Metabolic toxication process of pyrrolizidine alkaloids starts first by conversion of PAs into unstable hydroxyl-PAs by cytochrome p-450 monoxygenase enzyme complex in the liver.Then, instability of these hydroxyl PAs leads to dehydration and formation of dehydro-PAs.After that, rearrangement and formation of stabilized carbonium ions by loss of hydroxyl groups or ester functions as hydroxyl or acid anions these ions react with electrophilic parts of proteins, DNA and RNA leading to genotoxicity (Wiedenfeld et al., 2008;Mattocks et al., 1986).
In this context, our present work has focused on the measured using Sturat SMP heating stage microscope, United Kingdom, UV measurements was done using Pye Unicam SP8-100 UV/VIS spectrophotometer and TLC plates were examined under Duo-UV lamp, Desage, Heidelberg, Germany. 1 H-NMR (500 MHz), 13 C-NMR (125MHz), DEPT-135, and 2D-NMR spectra were recorded using the residual solvent signal as an internal standard on a Varian AS 400 NMR spectrometer in National Center for Natural Products Research, School of Pharmacy, University of Mississippi.

Extraction and isolation
The aerial parts of S.vulgaris (4 kg) were extracted with 95% MeOH at room temperature and filtered.The filtrate was evaporated under reduced pressure to give a MeOH extract (300 g), which was divided into two equal portions, one of them (150 g) was suspended in water (300 mL) and then successively partitioned with light petrol, CH 2 Cl 2 , EtOAc and n-BuOH, respectively.The other portion (150 g) was suspected to acid-base fractionation.

Column chromatography of ethyl acetate fraction
Isolation of compounds 1 and 2 from EtOAc fraction: the EtOAc soluble fraction (7 g) was chromatographed phytochemical study of Senecio vulgaris L., which includes isolation of four compounds (1-4) their structures are shown in Figure 1; as well as evaluation of the cytotoxic activity of different fractions against colorectal cell line HCT-116 and their free radical scavenging activity using DPPH assay.

Plant materials
Senecio vulgaris L. was collected in March 2013 (flowering stage) from El-Shalalat garden in Alexandria, Egypt and identified at Plant Taxonomy Department, Faculty of Science, Alexandria University, Egypt.A voucher sample (No Sv169) has been kept in the herbarium of Faculty of Science, Alexandria University.

Determination of the ratio between senecionine and seneciphylline using 1 H NMR data
Senecionine and seneciphylline are ubiquitous coexisting alkaloid-pair among many Senecio species (Habib., 1980;Segall et al., 1983).In the present work, determination of the ratio of these two alkaloids in the studied plant was attempted by 1 H-NMR evidence; referring to the integration of their H-13' signal(s) in 1 H-NMR spectrum of their mixture.

Early investigation of Senecio toxicity in rats
Early investigation of the toxicity with senecio plant was done by detecting its characteristic key elements in the stool of the rat that was fed with powdered senecio plant at a dose lower than the lethal dose.
Compound 1 was isolated as a white amorphous powder; the mass spectrometry suggested the molecular formula to be C 17 H 20 O 11 (m/z 400.12).In thin-layer chromatography, it was detected using UV lamp at 366 nm as a sky blue fluorescing spot which intensified upon exposure to ammonia vapor. 1 H-NMR spectrum ( Observation of broad singlet peaks for both H-4 and H-8 was due to long range coupling between both protons and indicated unsubstitution of the corresponding carbons (Hammoda et al., 2008).The absence of the typical AB system for H-3 and H-4 indicated a 3-substituted coumarin, this substitution was proved to be with oxygenated substituents due to downfield shift of C-3 (δ C 141.0) (Cussans et al., 1975).
The two signals at δ C 61.9 and 62.3 along with their proton signals at δ H 3.59 (3H, s) and 3.64 (3H, s) indicated the presence of 2 methoxy groups (Hammoda on a silica gel column, (300 g, 4 cm X 70 cm).The elution was performed using CHCl 3 : MeOH mixtures with gradual increase in polarity.Fractions (35% MeOH in CHCl 3 ) showed a major violet spot at R f 0.52 (CHCl 3 : MeOH, 8:2).Twentyfive mg of the residue left after removal of the solvent was subjected to pTLC on fluorescent silica gel plates using (CHCl 3 : MeOH, 8:2) as a developing system.The plate was visualized by UV light and ammonia vapor showing a major sky blue florescent zone.The zone was scraped off and eluted using a mixture of CHCl 3 : MeOH (1:2) to yield 16 mg of white amorphous powder (compound 1).

Column chromatography of chloroform fraction
Isolation of compounds 3 and 4 from CHCl 3 fraction: the residue left after evaporation of CHCl 3 fraction obtained by the acid-base treatment (8 g) was fractionated over a silica gel column (350 g, 4 cm X 70 cm).The elution was performed using CHCl 3 : MeOH mixtures with gradual increase in polarity.
Fractions (30% MeOH in CHCl 3 ) showed one major violet spot when sprayed with anisaldehyde /H 2 SO 4 followed by heating in the oven (150 ο C) for 2 min and orange color when sprayed by Dragendorff's reagent.After NMR analysis of the residue obtained by crystallization, it was found to be a mixture of two alkaloids, so 40 mg of the residue was subjected to pTLC on fluorescent silica gel plates using (CHCl 3 :MeOH:NH 3 , 8:2:0.2) as a developing system.The plate was visualized by UV showing two zones with R f 0.65 and 0.64.Both zones was scraped off and eluted with a mixture of (CHCl 3 : MeOH, 1:2) to yield 6 mg of 3 and 11 mg of 4, respectively.

Cytotoxicity evaluation using viability test
The cytotoxic activity of EtOH extract, light petrol and CHCl 3 fractions of S.vulgaris were evaluated by using viability assay (Skekhan et al.,1990) using vinblastin as a reference compound against colorectal carcinoma cell line (HCT-116 cell line).

Free radical scavenging activity screening using DPPH assay
The free radical scavenging activity of ethanol extract, petroleum ether and chloroform fractions et al., 2008).The location of the two methoxy groups at C-5 and C-6 was deduced from 13 C NMR spectrum as their chemical shifts above 60 ppm indicating that they are both ortho-flanked (Kaysar et al., 1995).
NMR spectra showed the sugar moiety to be one glucose unit with anomeric proton at δ H 5.20 (1H, d) along with its anomeric carbon at δ C 103.1 (Godecke et al., 2005).
The site of glycosylation was deduced from UV spectrum, as it was not strongly modified in alkaline medium (NaOAc) indicating the absence of a free hydroxylated C-7 (Alqasomy et al., 2013;Girard et al., 2005), thus the location of the sugar moiety should be at C-7.The β configuration of glucose was indicated from the chemical shift and J-value of its anomeric proton (She et al., 2009).The structure was confirmed as 3,7-dihydroxy-5,6 dimethoxycoumarin-7-O-β-Dglucoside by 2D-NMR analyses.
Compound 2 was isolated as yellow crystals with MP 215 ο C. In thin-layer chromatography, its spot gave a yellow color upon exposure to ammonia vapor and identified as isorhamnetin-3-O-β-D-glucoside.
Compound 3 and 4 were isolated as white powder.
In thin-layer chromatography, both compounds gave orange spot upon spraying with Dragendorrf's reagent indicating its alkaloidal nature, identified as senecionine and seneciphylline.
The cytotoxic activity of EtOH extract, light petrol and CHCl 3 fractions of S.vulgaris indicated that the most active cytotoxic fraction is the alkaloidbearing chloroform fraction.These findings promote the future perspective of pyrrolizidine alkaloids as anticancer agent.
In senecionine C-13' is a methyl group, giving a three protons which observed at δ H 0.90 (3H, d, J=5 Hz) with integration value of 3.05 ; whereas in seneciphylline C-13' is terminal methylene group that gives two well separated signals at δ H 5.34 and δ H 5.00 with integration values of 2.82 and 2.39, respectively (Roeder et al., 1992).That means in case of senecionine the one proton (CH 3 ) is approximately equivalent to one, but in case of seneciphylline each proton (CH) is approximately equivalent to two, so the ratio of senecionine to seneciphylline is 1:2.
Early investigation of the toxicity with Senecio plant was done by detecting its characteristic key elements in the stool of the rat that was fed with powdered Senecio plant at a dose lower than the lethal dose.

Conclusion
A flavonoid and a new coumarin were isolated from ethyl acetate fraction and two pyrrolizidine alkaloids were isolated from chloroform fraction of the aerial parts of Senecio vulgaris L. Cytotoxic and free radical scavenging activities of different fractions were evaluated.

Conflict of interest
The authors report no declaration of conflict of interest.