Isolation of Alkaloids and Anti-tumor Activity of the Crude Methanolic Extract of Algerian Cytisus purgans

In this study, two known quinolizidine alkaloids which are sparteine and lupanine were isolated from the methanolic extract of the plant Cytisus purgans of Algerian flora by open column chromatography. These two compounds were identified on the basis of their spectral data (GC/ MS, IR, MS, 1H and 13C). The anti-tumor activity of the crude methanolic extract of the aerial parts of the plant was also evaluated invitro against human breast cancer (MDA-MB-231) and human lung cancer (A549) cell lines using MTT assay.


INTRODUCTION
The Family of Fabaceae is a cosmopolitan family with about 700 genera and 18000 species, and is one of the most important families, is characterized by a large number of derived traits 1 .This large Family is characterized by an impressive phytochemical diversity.Polyphenols (especially flavonoids and tannins) are common, but from a pharmaceutical perspective various types of alkaloids are probably the most interesting and pharmaceutically relevant groups of compounds 2 .In the genera Genista and Cytisus (both commonly called broom), as well as Laburnum, quinolizidine alkaloids, including cytisine and sparteine, are common 3 .
Cytisus is an ornamental genus, it is presented by about 50 species mainly Mediterranean, is native to central and south Europe, Canaries Isles and North Africa.The common name ' broom ' may have been given to the plant because of its growth habit.Six species represented in Algeria, the more abundant is Cytisus trifolus L'her.Other rare species which are Cytisus balansae Boiss.Et Reut.(Cytisu spurgans), Cytisus fontanesi Spach (Ouarsenis, Bibans…), Cytisus sessilifolius L. (Babor) and Cytisus boeticus Webb.(North of Oran).Cytisus balansae Boiss.(Cytisus purgans) is a rare species of Algeria (Aures, Mahdids, and Lella Khadidja) 4 .
Cytisus purgans is a shrub always more or less ramifies, a trifoliolate and persistent leaves and a yellow flowers, native to central France, north central Spain and Portugal, Algeria and southern Morocco 5 .
The antibacterial activity of the MeOH extract of the plant Cytisus purgans was assayed in vitro by agar disc method against three bacterial species.Cytisus purgans exhibited antibacterial activity towards allbacteria strains.The maximum antibacterial activity was shown by Staphylococcus aureus followed by Pseudomonas aeruginosa and Escherichia coli, respectively 6 .
Quinolizidine alkaloids represent about 2% of the known alkaloids from plants 7 , and most of them were initially noticed as constituents of plants poisonous to humans and livestock.However, accumulated evidence indicates that some of them exhibit potentially useful pharmacological properties like anti-cancer, anti-bacterium, anti-virus, anti-inflammation and pain relief 8 .
The sparteine has been used as an oxytocic drug and is of interest because of itsantiarrhythmic effect and inhibitory effect of natural killer cell growth 9 .
Species which contain lupanine must consider potentially toxic, and unsuitable for forage 10 .
Many plants of the genus Cytisus are consumed as infusions due to its beneficial effects, for example, Cytisus multiflorius have terapeutic properties enclosing diuretic, anti-inflammatory, antihypertension and antidiabetic effects 11 .

Plant collection and identification
Cytisus purgans was collected on June 2013 at Aures Mountains (2328m of altitude) at Khenchela province in the north east of Algeria,and identified by ProfessorMohamed Kaabech, Laboratory of biodiversity and phytogenetic resources, University of Ferhat Abbas, Sétif, Algeria.A voucher specimen is deposited at the herbarium of laboratory of organic chemistry and phytochemistry of University Mohamed Boudiaf of M'sila, Algeria.

Extraction and isolation of alkaloids
The air-dried aerial parts of plant were cut into small pieces and were extracted with 99% methanol three times at room temperature.The combined extracts were concentrated, acidified with 5% hydrochloric acid and then, extracted with diethyl ether three times.The aqueous layer was made alkaline with 25% ammonium hydroxide to pH (9-10) and extracted with chloroform six times.The chloroform extracts were combined and dried over anhydrous sodium sulfate and evaporated to dryness in vacuo to give crude alkaloid mixture 12 .
Column chromatography was performed on a classic 30 cm long x 4 cm diameter glass column packed with 130 g of silica gel 60 (70-230 mesh).The ethyl acetate solution of 1 g of alkaloid fraction was applied to the top of column by use of a pipette.Elution was started with toluene, then with solvent mixtures with increasing polarities: toluene: ethylacetate:acetone and toluene: acetone: diethylamine.Finally, all 145 fractions (obtained by column chromatography) were monitored by analytical TLC (above mentioned solvent mixtures).
Fraction 141 was rechromatographed on preparative TLC.Mobile phase used for elution was chloroform:methanol:acetone=85:10:5 w/w.After elution and drying of preparative TLC plate, by scraping the layers and additional desorption from methanol, sparteine and lupanine were isolated.Purity of compounds was confirmed on analytical TLC with the same mobile phase mentioned above.Also, gas chromatographic/mass spectrometric analyses onAgilent 6890/5973 GC/MSsystem confirmed purity of isolated compounds.

Ant-tumor activity of the crude methanolic alkaloids extract Preparation of stock solutions
Stock solutions of the alkaloids plant extract was made in dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL, filtered through a 0.22 mm Millipore filter before use, and diluted by a nutrient medium to various working concentrations, so the final concentration of DMSO in culture medium never exceeded 0.5% (v/v).

Cell cultures
Human breast cancer (MDA-MB-231) and human lung cancer (A549) cell lines, obtained from Human Tumor Cell Bank (HTB) of the American Type Culture Collection (ATCC), were used in this study.MDA-MB-231 cells were grown in RPMI1640 and A549 cells were grown in DMEM medium.Both media were supplemented with 10 % fetal bovine serum, penicillin/streptomycin and L-glutamine.Cells were maintained in a monolayer culture into the 25 cm 2 tissue culture flasks (Nunclon TM ).The cells were prepared for experiments using trypsinization procedure with trypsin/EDTA.

MTT cell viability assay
Plant alkaloids extract was tested for it effect on viability of two different human tumor cell lines.MDA-MB-231 and A549 cells were seeded in 96-well plates (Sarstedt TM ) at 3x10 3 cells in 200 µL of appropriate tissue culture medium per well.Every concentration of examined compound was tested in triplicate, and incubated at 37ºC in a humidified 5% CO 2 atmosphere.Control cells contained the appropriated amount of DMSO.Culture medium with corresponding concentration of investigated plant extract, but without cells, was used as a blank.
The viability of cultured cells was determined by assaying the reduction of MTT to formazan.In brief, cells were treated with different dilutions of alkaloids (0.001, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25 and 0.5 mg/mL) or cultivated in the cell culture medium containing the appropriate amount of DMSO (control).After 6h, 24 h and 48h of incubation, the culture medium was removed, and MTT solution was added.After additional 4 h of incubation, medium with MTT was removed and DMSO (150µl/well) was added to dissolve the formazan crystals.The plates were shaken for 10 minutes.Absorbance was measured at 595 nm with a multiplate reader.The results are presented as relative to the control value (untreated cells) 13 .

RESULTS AND DISCUSSION
The crude MeOH extract of aerial parts of Cytisus purgans was subjected to open column chromatography; two known quinolizidine alkaloids were isolated which are sparteine and lupanine (figure 1).These two compounds were identified on the basis of their spectral data(GC/MS, IR, MS, 1 H and 13 C).
The IR spectrum of compound 1 showed multiple bands at ~2935 cm -1 and at ~2816 cm - 1 suggested quaternary nitrogen and combination overtones, embodying the C-H stretching the Bohlman's bands characteristic of quinolizidine-type alkaloids 14 .
The peaks at ~1650 cm -1 and at ~1400 cm - 1 indicating the presence of a conjugated carbonyl group and C-N stretching band, respectively 15 .
The IR spectrum revealed, also, the presence of an intense peak at 1022 cm -1 due tothe C-O stretching band.
It is known also that the fragment at m/z 98 (64.75 %) arises from ring A following the fragmentation pattern of the sparteine-type alkaloids 17 .

Anti-tumor activity
We investigated in vitro anti-tumor activity of the crude methanolic extract of alkaloids of cytisuspurgans on two different cell lines (human breast cancer, MDA-MB-231 and human lung cancer, A549 cell lines).The cell viability of studied human cancer cells, 6h, 24h and 48h after treatment with increasing concentrations of the plant extract is presented in Figure 2. Numerical data illustrating cell viability were obtained from MTT assays and presented as percentage of control.Tested plant extract is showed no anti-tumor effects toward of both human cancer cell lines.Neither increasing the dose as well as prolongation of the incubation time with the tested plant extract did not lead to a statistically significant increase of anti-tumor effect.
It is known that cytisus species accumulate sparteine-type quinolizidine alkaloids as basic constituents 12 , this phenomenon is interesting from theviewpoints of chemotaxonomy of leguminous plants and biosynthesis of quinolizidine alkaloids 23 .
Quinolizidine alkaloids can be divided into more than six structural groups, although, previous studies demonstrate that matrine-type quinolizidine alkaloids, for example,mediate growth inhibition in different types of cancer cells 13 , but sparteine-type quinolizidine alkaloids showed more antibacterian and antiviral effects 24,25   To the best of our knowledge, for the first time, we herein report the alkaloid profile of Cytisus purgans of Algerian flora, as well as the anti-tumor activity.

CONCLUSIONS
Cytisus purgans is a rare species of Algerian Flora.The phytochemical study of this plant indicate the presence of two known quinolizidine alkaloids: sparteine and lupanine, these two compounds were isolated from the methanolic extract of the plant and identified using different spectroscopic methods.
In the other side the test of the crude methanolic extract of alkaloids against two human cancer cells which are: breast and lung cancer, showed that the plant not exhibited any anti-tumor effect.
Future studies may show more about the benefits of this plant which accumulate quinolizidine alkaloids.