The genus Senecio (family Asteraceae) is one of the largest in the world. It comprises about 1100 species which are the rich source of pyrrolizidine alkaloids. Plants containing pyrrolizidine alkaloids are among the most important sources of human and animal exposure to plant toxins and carcinogens. The pyrrolizidine alkaloids of seven Senecio species (S. erucifolius, S. othonnae, S. wagneri, S. subalpinus, S. carpathicus, S. paludosus and S. rupestris) were studied. Fourteen alkaloids were isolated and their structures determined from spectroscopic data (1H- and 13C-NMR, IR and MS). Five of them were identified in S. erucifolius, four in S. othonnae, two in S. wagneri, four in S. subalpinus, two in S. carpathicus, three in S. paludosus and three in S. rupestris. Seven pyrrolizidine alkaloids were found for the first time in particular species. The results have chemotaxonomic importance. The cytotoxic activity and antimicrobial activity of some alkaloids were also studied

BORIS M. MANDIC

DEJAN N. GODJEVAC

MILENA R. SIMIC

SLOBODAN M. MILOSAVLJEVIC

SNEZANA S. TRIFUNOVIC

VELE V. TESEVIC

VLADIMIR P. BESKOSKI

VLATKA V. VAJS

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 J. Serb. Chem. Soc. 74 (1) 27–34 (2009)  UDC 582.475:547.77+547.94: JSCS–3805 :615.28(497.11)(497.16)    Original  scientific  paper doi: 10.2298/JSC0901027M 27 Pyrrolizidine alkaloids from seven wild-growing Senecio species in Serbia and Montenegro BORIS M. MANDIĆ1, DEJAN N. GOĐEVAC2, VLADIMIR P. BEŠKOSKI2, MILENA R. SIMIĆ3, SNEŽANA S. TRIFUNOVIĆ2, VELE V. TEŠEVIĆ1, VLATKA V. VAJS2# and SLOBODAN M. MILOSAVLJEVIĆ1*# 1Faculty of Chemistry, Studentski trg 16, P.O. Box 158, 11000 Belgrade, 2Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Njegoševa 12, 11000 Belgrade, 3Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia (Received 17 June 2008) Abstract: The genus Senecio (family Asteraceae) is one of the largest in the world. It comprises about 1100 species which are the rich source of pyrro-lizidine alkaloids. Plants containing pyrrolizidine alkaloids are among the most important sources of human and animal exposure to plant toxins and carcino-gens. The pyrrolizidine alkaloids of seven Senecio species (S. erucifolius, S. othonnae, S. wagneri, S. subalpinus, S. carpathicus, S. paludosus and S. rupes-tris) were studied. Fourteen alkaloids were isolated and their structures deter-mined from spectroscopic data (1H- and 13C-NMR, IR and MS). Five of them were identified in S. erucifolius, four in S. othonnae, two in S. wagneri, four in S. subalpinus, two in S. carpathicus, three in S. paludosus and three in S. ru-pestris. Seven pyrrolizidine alkaloids were found for the first time in particular species. The results have chemotaxonomic importance. The cytotoxic activity and antimicrobial activity of some alkaloids were also studied. Keywords: Senecio; pyrrolizidine alkaloids; antitumor and antimicrobial activity. INTRODUCTION The toxic pyrrolizidine alkaloids (PA) are a large group of related com-pounds that occur in plants, mainly in species of Crotalaria (Leguminosae), Se-necio and related genera (Compositae), Heliotropium, Trichodesma, Symphytum, Echium and other genera of the Boraginaceae. The worldwide distributed genus Senecio (family Asteraceae) is a rich source of pyrrolizidine alkaloids. It was shown that the alkaloid pattern differs between some subspecies.1–4 Previous investigations of alkaloids from seven Senecio spe-                                                                                                                    * Corresponding author. E-mail: smilo@chem.bg.ac.rs # Serbian Chemical Society member. 28 MANDIĆ et al. cies (S. erucifolius, S. othonnae, S. wagneri, S. subalpinus, S. carpathicus, S. pa-ludosus and S. rupestris) are presented in short in Table I.5–27 TABLE I. Alkaloids (1–37) isolated from species of genus Senecio (Columns: I – S. eruci-folius; II – S. othonnae; III – S. wagneri; IV – S. subalpinus; V – S. carpathicus; VI – S. palu-dosus; VII – S. rupestris). The Arabic numbers in the columns correspond to the reference numbers of the papers Alkaloid I  II  III  IV  V  VI  VII Senecionine (1) 5–11  12  –  5,  13,14 – –  5,  15, 16 Seneciphylline (2)  5, 6, 8, 9,10, 11 17 –  5,  13,14 5 18–20 – O-Acetylseneciphylline (3)  10  – – – – – – Seneciphylline N-oxide (4)  21  – – – – – – O-Acetylseneciophylline N-oxide (5) 21  – – – – – – Senecivernine (6)  6,  10 – – 5 – – 5 Integerimmine (7)  5, 6, 9, 10 –  –  5, 13 –  –  5, 15, 16 21-Hydroxyintegerimmine (8)  5  – – – – – 5 Integerimmine N-oxide (9)  21  – – – – – – Senkirkine (10)  5,  9  – 5 – – – – O-Acetylsenkirkine (11)  9  – – – – – – Neosenkirkine (12)  –  – 5 – – – – Otosenine (13) –  17,  22, 23 – – – – – Doronine (14)  –  22,  23 – – – – – Platyphylline (15)  –  12  – – – – – Neoplatyphylline (16)  –  – – 5 – – – 7-Angeloylplatynecine (17) –  –  –  –  –  24  – 9-Angelylplatynecine (18) –  –  –  –  –  25  – Retronecine (19)  –  12  – – – – – Onetine (20)  –  17,  22 – – – – – Othonnine (21)  –  12  – – – – – Retrorsine (22)  8  12  – – – –  16 Dihydroretrorsine (23) –  –  –  –  –  24  – Erucifoline (24)  6,  10 – – – – – – O-Acetylerucifoline (25)  10  – – – – – – Erucifoline N-oxide (26)  21  – – – – – – Eruciflorine (27)  6  – – – – – – Procerine (28)  –  – 5 – – – – Racemonine (29) –  –  –  –  –  26,  27  – Racemocine (30) –  –  –  –  –  26,  27  – Racemodine (31) –  –  –  –  –  24  – Floridanine (32)  –  22,  23 – – – – – Senecioracenine (33)  –  – – –   25  – Sarracine (34) –  –  –  –  –  25  – Riddeline (35)  –  12  – – – – – 7-Angelylheliotridine (36)  –  – 5 – – – – Spartioidine (37)  6  – – – – – –   PYRROLIZIDINE ALKALOIDS FROM Senecio  29 Despite the fact that secondary metabolites (especially PAs) of Senecio spe-cies have been the subject of investigations for many years, interest in them re-mains. The investigations of the activities of plants PAs indicated their neuro-toxic, mutagenic, carcinogenic, but also antitumor effects.28,29 PAs are readily absorbed from the digestive tract and cause harmful effects only after undergoing activation in the liver to toxic metabolites. The effects include a variety of chan-ges leading to permanent damage to genes and chromosomes, the ability of cells to divide, the development of cancer and even cell death. Some of them are strong toxins for humans and domestic animals.30,31 The acute toxicity of PAs varies widely. The rat LD50 of most alkaloids known to be significant for human health are in the range of 34–300 mg/kg, although some approach 1000 mg/kg. On the other hand, many Senecio species are used in traditional medicine in Asia and Africa, which makes them a very interesting for phytochemical invest-tigation. In this study, the pyrrolizidine alkaloids from seven Senecio species (S. eru-cifolius, S. othonnae, S. wagneri, S. subalpinus, S. carpathicus, S. paludosus and S. rupestris) were isolated and their structure elucidated. Also, the cytotoxicity and antimicrobial activity of some of PAs were investigated. EXPERIMENTAL General The IR spectra were measured in the form of KBr pellets on a Perkin-Elmer FT-IR spec-trometer 1725X. The 1H- (200 MHz) and 13C-NMR (50 MHz) spectra were recorded on a Varian Gemini 2000 spectrometer. The mass spectra were obtained on a Finnigan MAT 8230, BE DCI (150 eV, iso-butane). Silica gel, 0.008 mm (Merck, Darmstadt, Germany), was used for preparative column chromatography (CC) and silica gel F-254 (Merck, Darmstadt, Germany) for analytical and preparative thin layer chromatography (TLC). Plant material The studied species and collection data are listed in Table II. TABLE II. Studied species and collection data Species Collection  data S. erucifolius  Deliblatska peščara, Serbia, July 2003 S. othonnae  Sinjajevina, Montenegro, August 2003 S. wagneri Majа Rusalijа, Montenegro, July 2004 S. subalpinus  Hajla, Montenegro, July 2003 S. carpathicus  Stara planina, Serbia, July 2004 S. paludosus  Suva planina, Serbia, July 2005 S. rupestris  Lisa, Montenegro, July 2005 Extraction and isolation The dried and powdered plant material of each sample was extracted with methanol. After solvent removal, the residue was dissolved in 1.0 M sulfuric acid, washed with CH2Cl2, the pH adjusted to 9.0 with NH4OH, extracted with CH2Cl2 and purified by silica gel CC and 30 MANDIĆ et al. prep. TLC to yield pure alkaloids. The elution was commenced with CH2Cl2:metha-nol:NH4OH (9:1:0.1) and the polarity was gradually increased. The spots were detected under UV254, by Dragendorff reagent or by spraying with 50 % H2SO4. Investigation of antiproliferative and antimicrobial activity Stock solutions of the isolated alkaloids were prepared in DMSO at a concentration of 10 mM and afterwards diluted with nutrient medium (RPMI 1640), supplemented with L-gluta-mine (3 mM), streptomycin (100 μg/ml), penicillin (100 IU/ml), 10 % heat inactivated (56 °C) fetal bovine serum and 25 mM Hepes, adjusted to pH 7.2 with bicarbonate solution and ap-plied to the target cells (human cervix carcinoma HeLa cells, human myelogenous leukemia K562 cells, human melanoma Fem-X cells and normal human peripheral blood mononuclear cells) at various final concentrations ranging from 0 to 100 μM. HeLa and Fem-X cell survival was determined indirectly by measuring the total cellular protein by the Kenacid Blue R dye binding method.32 Inhibition of the growth of PBMC and K562 cells was determined by the MTT test.33 The antibacterial activity of alkaloids (otosenine, seneciphylline and a mixture of sene-cionine and seneciphylline) was tested against the microorganisms Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Candida albicans and Aspergillus niger. The concentration of the tested samples was 1.0 mg/ml. The surface of an agar plate was inoculated by streaking the bacterial suspension (ca. 105 cfu/ml). After incubation at 37 °C for 24 h, the inhibitory effect was determined as the prevention of visible growth. RESULTS AND DISCUSSION The isolation procedure for alkaloids from the crude extract (of seven Sene-cio species: S. erucifolius, S. othonnae, S. wagneri, S. subalpinus, S. carpathicus, S. paludosus and S. rupestris) yielded fourteen alkaloids (1, 2, 4, 6, 7, 10, 13, 14, 17, 33, 36, 38–40) (Table III, Scheme 1). The structural assignments of alkaloids were based on comparison of their spectral data with those published in lite-rature.34,35 TABLE III. Alkaloids isolated from species of genus Senecio (Columns: I – S. erucifolius; II – – S. othonnae; III – S. wagneri; IV – S. subalpinus; V – S. carpathicus; VI – S. paludosus; VII – – S. rupestris). Mark PA indicates alkaloids isolated for the first time from the Senecio species Alkaloid  I  II III IV V VI  VII Senecionine (1)  + – – + – – + Seneciphylline (2)  + – – + + – – Seneciphylline N-oxide (4) +  –  –  PA  PA  –  – Senecivernine (6)  – – – – – – + Integerimmine (7)  – – – – – – + Senkirkine (10) –  –  +  –  –  PA  – Otosenine (13)  PA  + – – – – – Doronine (14)  – + – – – – – 7-Angeloylplatynecine (17)  – – – – – + – Senecioracenine (33)  –  PA  – – – – – 7-Angelylheliotridine (36)  – – + – – – – 7-Angelylretronecine (38)  – – – – –  PA  Neopetasitenine (39)  –  PA  – – – – – Senecionine N-oxide (40) PA  –  –  PA  –  –  –   PYRROLIZIDINE ALKALOIDS FROM Senecio  31 NOOOOHOH140  N-oxide of 1  NOOOOH24 N-oxide of 2OH NOOOOH6OH NOOOOHOH7  NOOOO10OHOCH3 NOOOO13OHOCH3O NOOOO14OCH3ClOHOO NO O17H HOH NO O36  7HOH38  7   NOOOO33OCH3OH O NOOOO39OCH3OAcO Scheme 1. 32 MANDIĆ et al. Previous studies of S. erucifolius resulted in the isolation and identification of 17 PAs (1–11, 22, 24–27 and 37). In the present study, three of the previously identified alkaloids (senecionine, seneciphylline and seneciphylline N-oxide) and an additional two, namely otosenine (13) and senecionine N-oxide (40), are now reported and identified for the first time as alkaloids in S. erucifolius. Four alkaloids were identified from S. othonnae, two of them (otosenine and doronine) were previously reported in this plant. Senecioracenine (33) and neo-petasitenine (39) are alkaloids isolated for the first time from this species. Four PAs, namely senkirkine, neosenkirkine, procerine and 7-angeloylhelio-tridine (10, 12, 28 and 36) were previously isolated from S. wagneri. This invest-tigation confirmed the presence of two alkaloids senkirkine (10) and 7-angeloyl-heliotridine (36). Senecionine (1) and seneciphylline (2) identified in S. subalpinus are two of five previously isolated alkaloids. However, seneciphylline N-oxide (4) and sene-cionine N-oxide (40) are alkaloids new for this plant. One (2) and five (1, 6, 7, 8 and 22) alkaloids were previously isolated from S. carpathicus and S. rupestris, respectively. In this investigation, the presence of seneciphylline (2) as well as senecionine, senecivernine and integerimmine (1, 6 and 7 ) respectively, were confirmed.  Seneciphylline  N-oxide (4) was isolated from S. carpathicus for the first time. One (7-angeloylplatynecine) of nine previously reported alkaloids of S. palu-dosus was identified in the present study. An additional two alkaloids, namely senkirkine (10) and 7-angeloylretronecine (38), had not been previously isolated from this plant. In order to anticipate antitumor action, the antiproliferative activity of some alkaloids (senkirkine and a mixture of senecionine and seneciphylline, which were isolated in sufficient quantities to allow bioassays) against malignant cell lines (human cervix carcinoma HeLa cells, human myelogenous leukemia K562 cells and human melanoma Fem-X cells) and normal human cells (PBMC) was tested. The investigated alkaloids (in concentrations from 0 to 100 μM) exhibited no cytotoxic effects against any of the tested human cancer cells. The antimicrobial action of otosenine, seneciphylline and a mixture of sene-cionine and seneciphylline against Staphylococcus aureus, Escherichia coli, Ba-cillus subtilis, Candida albicans and Aspergillus niger was also, investigated. Only the senecionine-seneciphylline mixture (2:1) exhibited a weak (on the limit of de-tection) inhibitory effect against A. niger at a concentration of 1 mg/ml. CONCLUSIONS Fourteen pyrrolizidine alkaloids were isolated and identified from seven Se-necio species (S. erucifolius, S. othonnae, S. wagneri, S. subalpinus, S. carpathi-cus, S. paludosus and S. rupestris). Seven of them were identified for the first time in the particular species. The results are of chemotaxonomic importance.   PYRROLIZIDINE ALKALOIDS FROM Senecio  33 Senkirkine and a mixture of senecionine and seneciphylline at the investi-gated concentrations did not exhibit antiproliferative effects against the tested hu-man malignant cell lines. However, a mixture of senecionine and seneciphylline exhibited a weak antimicrobial effect against Aspergillus niger. Acknowledgement. The authors acknowledge their gratitude to the Ministry of Science of the Republic of Serbia for financial support (Project No. 142053). ИЗВОД ПИРОЛИЗИДИНСКИ АЛКАЛОИДИ ИЗ СЕДАМ САМОНИКЛИХ БИЉНИХ ВРСТА РОДА Senecio КОЈЕ РАСТУ У CРБИЈИ И ЦРНОЈ ГОРИ БОРИС М. МАНДИЋ1, ДЕЈАН Н. ГОЂЕВАЦ2, ВЛАДИМИР П. БЕШКОСКИ2, МИЛЕНА Р. СИМИЋ3, СНЕЖАНА С. ТРИФУНОВИЋ2, ВЕЛЕ В. ТЕШЕВИЋ1, ВЛАТКА В. ВАЈС2 и СЛОБОДАН М. МИЛОСАВЉЕВИЋ1 1Hemijski fakultet, Studentski trg 16, 11000 Beograd, 2Institut za hemiju, tehnologiju i metalurgiju, Univeryitet u Beogradu, Wego{eva 12, 11000 Beograd i 3Farmaceutski fakultet, Univerzitet u Beogradu, Vojvode Stepe 450,11000 Beograd Један од најбројнијих родова на свету је род Senecio (фамилија Asteraceae). Обухвата око 1100 врста  које  представљају  богат  извор  пиролизидинских  алкалоида.  Најзначајнија изложеност људи и животиња биљним токсинима и карциногенима потиче од биљака које садрже пиролизидинске алкалоиде. У овом раду изоловани су пиролизидински алкалоиди из седам врста рода Senecio (S. erucifolius, S. othonnae, S. wagneri, S. subalpinus, S. carpathicus, S. paludosus и S. rupestris), а њихове структуре одређене на бази спектроскопских података (1H- и 13C-NMR, IR и MS). 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Journal of the Serbian Chemical Society

Pyrrolizidine alkaloids from seven wild-growing Senecio species in Serbia and Montenegro

Pyrrolizidine alkaloids from seven wild-growing Senecio species in Serbia and Montenegro

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