This paper describes a fast and efficient procedure to separate and identify isoquinoline alkaloids from methalonic extract of two Algerian Fumaria (Fumariacea) species (Fumaria capreolata L. and Fumariabastardi L.) used in traditional medicine in cases of hepatobiliary disfunction and diarrhoea. Total quinolizidine alkaloid contents were 426 mg/100 g (F. capreolata) and 521 mg/100 g (F. bastardi). Theisoquinoline alkaloids, stylopine, protopine, fumaritine, fumaricine, fumarophycine, fumariline and fumarofine were determined by gas chromatography – mass spectrometry (GC-MS) in aerial parts ofboth Fumaria capreolata and Fumaria bastardi. In the first species, an ester of phtalic acid was identified, and in the second species a peak seems to be a benzophenanthridine, probably a dehydro derivative and three other peaks which were identified as phtalidisoquinoline, one of them seems to bedihydrofumariline. The chemotaxonomic significance of the results is discussed

Adach, S

Chibane, M

Madani, K

Maiza-Benabdesselam, F

Max, H

English

AJOL - African Journals Online

African Journal of Biotechnology Vol. 6 (21), pp. 2487-2492, 5 November, 2007 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2007 Academic Journals      Full Length Research Paper  Determination of isoquinoline alkaloids contents in two Algerian species of Fumaria (Fumaria capreolata and Fumaria bastardi)  Fadila Maiza-Benabdesselam1*, Mohamed Chibane1, Khodir Madani1, Henry Max2 and Sandrine Adach3  1Faculty of Nature and Life Sciences, 3BS Laboratory A. Mira University, Bejaia 06000, Algeria. 2Faculty of Pharmacy, UMR 7565 CNRS, Henri Poincaré University, Nancy 1, Nancy 54001, France. 3Faculty of Sciences, UMR 7565 CNRS, Henri Poincaré University, Nancy 1, Nancy 54001, France.  Accepted 19 October, 2007  This paper describes a fast and efficient procedure to separate and identify isoquinoline alkaloids from methalonic extract of two Algerian Fumaria (Fumariacea) species (Fumaria capreolata L. and Fumaria bastardi L.) used in traditional medicine in cases of hepatobiliary disfunction and diarrhoea. Total quinolizidine alkaloid contents were 426 mg/100 g (F. capreolata) and 521 mg/100 g (F. bastardi). The isoquinoline alkaloids, stylopine, protopine, fumaritine, fumaricine, fumarophycine, fumariline and fumarofine were determined by gas chromatography – mass spectrometry (GC-MS) in aerial parts of both Fumaria capreolata and Fumaria bastardi. In the first species, an ester of phtalic acid was identified, and in the second species a peak seems to be a benzophenanthridine, probably a dehydro derivative and three other peaks which were identified as phtalidisoquinoline, one of them seems to be dihydrofumariline. The chemotaxonomic significance of the results is discussed.  Key words: Fumaria capreolata, Fumaria bastardi, isoquinoline alkaloids, gas chromatography – mass spectrometry, chemotaxonomy.    INTRODUCTION  The genus Fumaria belongs to Fumariaceae family and encompasses about 40 species (Suau et al., 2001). These plants are annual herbs which have wide distribution in the Mediterranean region (Suau et al., 2005). The identification of Fumaria plants is subjective because they have very close morphological properties (Soušek et al., 1999). The chemotaxonomic evaluation of some types of isoquinoline alkaloids supports the differential of plants among these genera (Preininger, 1986). Several techniques have been used for the determination of isoquinoline alkaloids in plant extract (Soušek et al., 1999). However identification of tertiary bases is a problem and a screening tool based on GC- MS (gas chromatography coupled to mass spectrometry)    *Corresponding author. E-mail: fadilamaiza@yahoo.fr. Tel: 0021371449217. Fax: 0021334214762. technique for direct determination of alkaloids content in Fumaria was developed (Suau et al., 2002). The present paper deals with the GC-MS analysis of the alkaloids fra-ction from aerial parts of North East Algerian’s indigenous plants, Fumaria capreolata (L.) and Fumaria bastardi (L.). Those plants are used in Algerian traditional medicine in cases of hepatobiliary disfunction and gastrointestinal disorders. It was reported that the plant has local reputation in Pakistan and India as anthelmintic, antidys-peptic, blood purifier, cholagogue, diuretic, laxative, sedative, tonic and also considered useful to treat abdominal cramps, fever, diarrhoea  as well as syphilis and leprosy (Gilani et al., 2005).   MATERIALS AND METHODS  Plant material  Aerial parts  of  F. capreolata  and  F. bastardi  were  obtained  from2488           Afr. J. Biotechnol.    Table 1. Identification by gas chromatography-mass spectrometry (GC-MS) of isoquinoline alkaloids in F. capreolata and F. bastardi.  Alkaloid type Alkaloid Rt (min) EI-MS m/z (°/°) F. capreolata F. bastardi Protopine Protopine 20.55 148(100)163(20)354(5) + +  Protoberberine 18.96 148(100) 369(4) +  Terahydroprotoberine Stylopine 19.65 148(100)322(30)323(40) + +  Phtalidisoquinoline 23.7 190(100)148(52)320(80) − +  Phtalidisoquinoline 24.9 190(100)148(25) 320(32) − + Spyrobenzylisoquinoline Fumariline 20.92 322(100)336(20)351(25) + +  Fumarophycine 2.11 322(100)337(85) + +  Fumaritine 22.29 192(100)340(20) + +  Fumarofine 30.25 369(100)354(56)192(’34) + +  Fumaricine 32.2 206(100)355(13) + +  Dihydrofumariline 31.2 190(100)148(25)363(20) − + Dehydrobenzophenanthridine  8.65  330(100)331(5)142(85) − + Phtalic acid ester  7.13 149(100)167(25)317(12) + − Total alkaloids (mg/100 g dry weight)        426±23  521± 36  − =component absent; + = component present. Rt = Retention time.    their natural habitats. All specimens were collected in April and June 2005 when they were at the flowering and fruit setting stages. Plants were collected from Bejaia City in the north east of Algeria and identified by the Botany Laboratory of Bejaia University. The identification was confirmed latter by Professor Max Henry of the University of Nancy 1, France. Specimens are deposited in the herbarium of Bejaia University.   Extraction of alkaloids  Extraction was done as described by Suau et al. (2002a). Dried (at 40oC) samples (5 - 6 g) of the aerial parts from several individuals of each population, were powdered and extracted with methanol (100 ml) in a Soxhlet apparatus for 3 h, and then evaporated to 0.5 ml in vacuum. The methanol residue was taken up in 10 ml of 2.5% hydrochloric acid and filtered. The aqueous acid solution was adjusted to pH 8 with concentrated ammonium hydroxide and extracted with dichloromethane (3 x 10 ml). The extracts were dried over magnesium sulphate and the solvent evaporated to afford a crude extract of alkaloids. After evaporation the yield of each fraction was calculated. Dichloromethane, analytical reagent grade and all other solvents were from Sigma Chemical Co.   GC-MS analysis  GC-MS Analysis was performed on a Fisons Trio 1000 mass selective detector with electronic impact ionisation (70 eV). The column used was a HP-1 (15 m x 0.25 mm i.d. and 1 m phase thickness). Helium was used as a carrier gas at 1.0 mL min-1. The injection temperature was 280oC. The column temperature was held initially at 200oC for 8 min, increased to 250oC at 10oCmin-1 and then held at 250oC for 30 minutes. The MS source temperature was operated at 250°C and the transfer line was maintained at 280oC.The mass range scanned was 125 – 450 g and scan rate was 26 scans/s. RESULTS AND DISCUSSION  GC-MS analysis  The results of GC-MS analysis of the different alkaloid extracts are on Table 1 and shows GC-MS experimental data, retention time and main fragments (their respective intensities) for indole alkaloids of F. capreolata and F. bastardi. Individual alkaloids (Figure 1) were identified from their Art values and their MS by comparison of their MNR data with those in literature (Suau et al., 2002a; Suau et al., 2002b; Suau et al., 2005; Zocoler et al., 2005; Sanchez et al., 2005; Yu et al, 1971; Pereira et al., 1999; Essential data of mass spectra, 1983). Some components remained unidentified due to the lack of reference substances and Library spectra. However we can identify protopine and stylopine as main alkaloids in both F. capreolata and F. bastardi. The presence of peaks at 355 m/z (M+, 10%), 340 (M+-CH3, 20%) and 192 (base peak) was consistent with the occurrence of fumaritine, the peak at 369 m/z (100%) seems to be fumarofine. Others spirobenzyliso-quinolines as fumaricine, fumarophycine and fumaritine were identified in the two species. In F. capreolata, the peak at 149 m/z (100%), 167 m/z (30%), 279 m/z (6%) corresponds to a phtalic acid ester and the peak at 330 m/z (100%) observed in F. bastardi extract seems to be a benzophenanthridine, dehydroderivative (331 molecular ion), probably dihydrosanguinarine witch was found in different species of Fumaria by Suau et al. (2002b). The other benzophenanthridine which can correspond to this peak is chelidonine which was detected in the genus Sarcapnos (Suau et al., 2005). Three other peaks appear  Maiza-Benabdesselam et al.        2489    OONOCH3OO3HCO3HCONOCH3OO          Protopine                                                           Cryptonine   NOOOO                           3HCO3HCON CH3OO OO                   Stylopine                                                    Phtalidisoquinoline         Figure 1.  Isoquinoline alkaloids identified in F. capreolata and F. bastardi.    only in F. bastardi. Although these peaks could not be fully characterised, they were identified as phtalidiso-quinoline alkaloids based on the presence of a prominent ion at 190 m/z as base peak (Blaskó et al., 1982); one of them can be identify as dihydrofumariline. However there are several unresolved peaks, perhaps components derived from aporphines (proeminent ion at m/z 340 and 335) (Pereira et al.1999). Algerian species of Fumaria seems to have highest concentrations of alkaloids than Spain species studied by Suau and his collaborators (Suau et al., 2002a). The total alkaloids (mg/100 g dry weight) of F. bastardi and F. capreolata are 521 and 426, respectively, in Algerian species while the amounts founded in the same species from Malaga are 425 and 412, respectively (Suau et al. 2002a). Moreover, phtalidisoquinoleine found in our F. bastardi extract was absent in F. bastardi of Malaga and the spirobenzylisoquinoline alkaloids were different. In the Algerian species we found fumarilline, fumaritine, fumarofine and dehydrofumaritine, while in Fumaria from Malaga, the alkaloids are parfumine, fumariline and fumarophycine in F. capreolata and parfumine, fumariline and parfumidine in F. bastardi. (Suau et al., 2002a). The traditional use of the two species of Fumaria in the North of Algeria in several gastrointestinal diseases and skin diseases can be explained by the fact that the main constituents of the crude extracts are indole alkaloids, a class of substances which a wide range of pharmaco-logical activities, cholinesterase inhibitors, analgesic, anti-inflammatory, stimulant and depressant of the Central Nervous System (CNS) (Zocoler et al., 2005). Figure 2 shows the chromatographic profiles of the main alkaloids founded in the two Algerian species of Fumaria.   Chemotaxonomic significance  Most of the European Fumariacae can be associated with three chemotypes based on alkaloids contents. Chemota-xonomic investigations of several populations of different species of the genus Fumaria resulted in the definition of two different chemotypes based on the presence of either the protoberberine Stylopine (group I) or the protopine Cryptonine (group II) in the plant (Suau  et al., 2002a). The group I species (Fumaria agraria, F. bastardi, F. capreolata and Fumaria sepium) included the tetrahydro-  2490           Afr. J. Biotechnol.    Trio1000-Sandr ine16:09:59U niv. Henr i Poincaré Nancy 117-May-2006feuilles bastardi140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440m/z0100%148134163162 190164 281209 267252237 322295 320 362325355 418373 388 429 438MF01A2 303 (15.636) Scan EI+ 8.63e3  Spectra of Protopine          Trio1000-Sandr ine14:03:58Univ. Henr i Poincaré Nancy 116-May-2006feuilles capreolata140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440m/z0100%148147323206149174165 190322207288234 236 281 320308 341 408368 386 419 430MF01B1 381 (19.146) Scan EI+ 1.25e4  Spectra of Stylopine  Trio1000-Sandr ine14:03:58U niv. Henr i Poincaré Nancy 116-May-2006feuilles capreolata140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440m/z0100%322148134264155 252236218207175179276307305320323440336346 439351 424383353 393 413 446MF01B1 451 (22.297) Scan EI+ 2.18e3  Spectra of fumariline  Trio1000-Sandr ine09:46:51U niv. Henr i Poincaré N ancy 124-May-2006fleurs b astardi140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440m/z0100%206148142137149190160175 197320207233221318252 271 298322332403364352 389377414447448MF02A2 423 (21.037) Scan EI+ 2.45e3  Spectra of fumaricine Maiza-Benabdesselam et al.        2491    Trio1000-Sandr ine09:46:51U niv. Henr i Poincaré Nancy 124-May-2006fleurs b astardi140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440m/z0100%330142127239207144165197181211221272241271242 257 313281287322331356342 444435360 389 401 416 447MF02A2 147 (8.616) Scan EI+ 1.67e3  Spectra of dehydrophenanthridine.  Trio1000-Sandr ine09:46:51Univ. Henr i Poincaré Nancy 124-May-2006fleurs b astardi140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440m/z0100%190148142 159 187320191 319307256200 213 249 274321336334 356 434432410372 397 441MF02A2 511 (24.997) Scan EI+ 2.81e3  Spectra of phtalidisoquinoline   Figure 2. Gas chromatography-mass spectrometry (GC-MS) profiles of methanolic extracts of F. capreolata and F. bastardi.    protoberberine, stylopine, the biogenic precursor of protopine and cryptonine as main alkaloid (Suau et al., 2002b). We can confirm that our species are well identified and belong to the chemotaxonomic group I. Furthermore, Fumariaceae and Papaveraceae are closely related and the evaluation of some type of isoquinoline alkaloids allows the differentiation among these genera. For example Fumaria is rich in spirobenzy- lisoquinolines, while these components are absent from the other genus which contains aporphines and morphi-nanes (Suau et al., 1991, 2002b).    Conclusion  This study shows that the crude extracts of aerial parts of F. capreolata and F. bastardi contain isoquinoline alkaloids which explain their pharmacological properties. Gas Chromatography coupled to Mass spectrometry (GC-MS) is proven to be a valuable tool for the analysis of Fumaria indole alkaloids, but this method is useless for quaternary alkaloids with base peak lower than 125 m/z. However a successful method based on reversed phase HPLC allows the detection of quaternary alkaloids (Soušek  et al, 1999; Gerasimenko  et al., 2001) and exact quantification of Fumaria officinalis isoquinoline alkaloids was been determinate by nonaqueous capillary electrophoresis-electrospray (Sturn  et al., 2005). Soxhlet extraction combined with the GC-MS method is a direct and fast analytical approach for identification of the various tertiary bases present in alkaloid extracts and only a few grams of plant material are required.   ACKNOWLEDGMENTS  This work was supported equally by the Laboratory of Pharmacognosy of the Faculté de Pharmacie de Nancy 1- France and Faculté des Sciences de la Nature et de la Vie, Université de Béjaia, Algérie. We also thank Dr Raphael Suau from the University of Malaga, Spain for his precious help and kindness. 2492           Afr. J. Biotechnol.    REFERENCES  Blask G, Gula DJ, Shamna M (1982). Phtalidisoquinoline alkaloids. J. Nat. 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Determination of isoquinoline alkaloids contents in two Algerian species of Fumaria (Fumaria capreolata and Fumaria bastardi)

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Determination of isoquinoline alkaloids contents in two Algerian species of Fumaria (Fumaria capreolata and Fumaria bastardi)

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