To study the cytotoxic activity of P. harmala. Materials and method: The  alkaloids harmine and harmaline have been isolated from a methanolic  extract from the seeds of P.  harmala L. and have been  characterized by  spectroscopic-Mass and NMR methods. The cytotoxicity of the methanolic  extract and both alkaloids has been investigated in the three human cancer  cell lines UACC-62 (melanoma), TK-10 (renal) and MCF-7 (breast) and then  compared to the positive control effect of the etoposide. Results and  conclusion: The methanolic extract and both alkaloids have inhibited the  growth of these three cancer cell-lines and we have discussed possible  mechanisms involved in their cytotoxicity

Berrougui, Hicham

Ettaib, Abdelkader

Herrera González, María Dolores

López Lázaro, Miguel

Mamouchi, Mohamed

Martín Cordero, Carmen

idUS. Depósito de Investigación Universidad de Sevilla

41JOURNAL  OF  NATURAL  REMEDIES1. IntroductionPeganum harmala  L. (Zygophyllaceae), the so-called harmal, grows spontaneously inuncultivated and steppes areas in semiarid andpre-deserted regions in south Spain and South-East Morocco [1]. Harmala alkaloids aredistributed widely in some medicinal plantsand are found endogenously in mammaliantissues. Harmala alkaloids have a widespectrum of pharmacological actions in thecentral nervous system such as tremorogenesis[2], hypothermia [3,4], hallucinogenesis [5,6],central monoamine oxidase inhibition [7-9],convulsive or anticonvulsive actions andbinding to various receptors including 5-HTreceptors and the benzodiazepine binding siteof GABAA receptors [10]. In addition, thesecompounds also have antioxidative [11],platelet aggregation inhibitory andimmunomodulatory effects [12-13]. There arealso some reports concerning thecardiovascular actions of harmala alkaloids,such as systemic arterial blood pressure andtotal peripheral vascular resistance reductionby harmine, or an in vivo vasorelaxant effectCytotoxic activity of methanolic extract andtwo alkaloids extracted from seeds ofPeganum harmala  L.Hicham Berrougui1,2*, Miguel López-Lázaro1, Carmen Martin-Cordero1, MohamedMamouchi2, Abdelkader Ettaib2, Maria Dolores Herrera1.1. Department of Pharmacology School of Pharmacy. Séville, Spain.2. School of Medicine and Pharmacy, UFR (Natural Substances), Rabat, Morocco.AbstractObjective: To study the cytotoxic activity of P. harmala.  Materials and method: The alkaloids harmineand harmaline have been isolated from a methanolic extract from the seeds of P. harmala  L. and havebeen characterized by spectroscopic-Mass and NMR methods. The cytotoxicity of the methanolic extractand both alkaloids has been investigated in the three human cancer cell lines UACC-62 (melanoma), TK-10 (renal) and MCF-7 (breast) and then compared to the positive control effect of the etoposide. Resultsand conclusion:  The methanolic extract and both alkaloids have inhibited the growth of these threecancer cell-lines and we have discussed possible mechanisms involved in their cytotoxicity.Keywords: Peganum harmala, harmine, harmaline, cytotoxicity, TK-10, MCF-7, UACC-62.Corresponding  authorE-mail: hicham.berrougui@usherbrooke.caVol. 5/1 (2005) 41 - 4542of Harman [14]. In addition, we reported in arecent work the vascular relaxant effect of amethanolic extract from seeds of P. harmalaL. (MEP) [15].In the present study we isolated andcharacterize spectroscopycally two alkaloidsfrom such extract and evaluated their cytotoxicactivity in three human cancer cell lines.2. Material and methods2.1 PlantPeganum harmala  L. (Zygophyllaceae) freshseeds were collected from the medium of atlasregion (Morocco), in May 2002 and botanicallyidentified by botanical section of (U.F.R:Naturals Products), Faculty of Medicine andPharmacy. Rabat; where a voucher specimenis preserved.2.2 Extraction of methanolic extract (ME) andnatural compoundFresh and powdered seeds were successivelyextracted in a soxhlet apparatus with Petrol-ether(60-80°C), CHCl3 , EtOAc and MeOH. Theobtained extract was concentrated underreduced pressure to yield dry residues: 0.67,1.42, 2.8, 31% (w/w), respectively. The naturalscompound (Harmaline and Harmine) wereextracted from the last fraction (Methanolicextract); this fraction was chromatographed onsilica gel column, and the elution was startedwith CHCl3 following with CHCl3 containingincreasing amount of MeOH.All fractions obtained from this silica gelcolumn were subjected to TLC (thin layerchromatography) examination using CHCl3-MeOH (9:1). Fractions with similar migrationtimes were combined. Harmine andharmaline were isolated by crystallisationfrom their fractions using in CHCl3 and MeOHrespectively. the compounds were analysedby TLC, 1H-NMR, 13C-NMR (Nuclear MagneticResonance) and mass spectra.2.3 Human tumour cell lineThe following three human cancer cell lineswere used in these experiments: the humanrenal adenocarcinoma (TK-10), the humanbreast adenocarcinoma (MCF-7) and thehuman melanoma (UACC-62) cell lines. Theywere kindly provided by Dr. G. Cragg,Department of NCI, Maryland, USA.The human tumour cytotoxicities weredetermined following protocols established bythe National Cancer Institute, National Instituteof Health [16]. TK-10, MCF-7 and UACC-62cell lines were cultured in RPMI 1640 medium(Biowhittaker) containing 20% fetal calf serum,20mM L-glutamine, 100U/ml penicillin and100 µg/ml streptomycin. All cell lines weremaintained at 37°C in a 5% CO2 atmospherewith 95% humidity.2.4 Cytotoxicity assayFor the assay, cells were detached with 0.1%trypsin-EDTA (Sigma) to make single-cellsuspensions, and viable cells were countedusing a Coulter counter and diluted withmedium to give final concentrations of 15x1045x104 and 100x104 cells/ml for TK-10, MCF-7and UACC-62 respectively. 100µl/well of thesecell suspensions were seeded in 96-wellmicrotiter plates and incubated to allow forcell attachment.After 24 h the cells were treated with the serialconcentrations of compounds or extracts. Theywere initially dissolved in an amount of 100%DMSO (10 mM) and further diluted in mediumto produce 5 concentrations. 100µL/well ofeach concentration was added to the plates toobtain final concentrations of 10-4, 10-5, 10-6,10-7 and 10-8 M for the compounds and 250,25, 2.5, 0.25 and 0.025µg/mL for the extract.The DMSO concentration for the testeddilutions was not greater than 0.25% (V/V), thesame as in solvent control wells. The finalvolume in each well was 200µl. The plates wereincubated for 48 h.Hicham Berrougui et al. / Journal of Natural Remedies, Vol 5/1 (2005) 41 - 4543Sulphorhodamine B method: This colorimetricassay estimates cell number indirectly bystaining total cellular protein with the dye SRB.After incubating 48 h, adherent cell cultureswere fixed in situ by adding 50µl of cold 50%(W/V) trichloroacetic acid (TCA) andincubating for 60 min. at 4ºC. The supernatantis then discarded, and the plates are washedfive times with deionised water and dried.One hundred ml of SRB solution (0.4% wt/volin 1% acetic acid) is added to each microtiterwell and the culture was incubated for 30 minat room temperature. Unbound SRB wasremoved by washing five times with 1% aceticacid. Then the plates were air-dried. Boundstain is solubilized with Tris buffer, and theoptical densities were read on an automatedspectrophotometric plate reader at a singlewavelength of 492 nm.At the end, IC50 values were calculated and atleast three independent experiments werecarried out for each compound or extract. Dataare given as the mean ± SEM.3. Results and discussion3.1. ChemistryHarmine and harmaline were isolated bycrystallisation after silica gel columnseparation. The TLC analysis revealed twospots (blue and violet) under UV light with Rfvalues 0.62 (harmine) and 0.36 (harmaline).This data are in agreement with those reportedin the literature (0.64 and 0.35respectively)[17]. Furthermore, the 1H-NMRand 13C-NMR data illustrated in Table 1revealed that these compounds were harmineand harmaline (Fig 1).B.C    Harmaline          Harmine             (50 MHz, CDCl3) (50 MHz, DMSO)1 161.889 14.9342 - -3 41.839 137.7624 19.908 111.9475 122.212 122.6256 115.754 109.0687 164.575 160.0748 94.049 94.579 - -10 125.029 134.53411 119.038 114.83312 125.549 127.20613 144.201 141.274CH3-1 19.05 20.344CH3O-7 55.704 55.319(d = doublet, dd = doublet doublet, m = multiplet, s = singulet)Table I.1H (A)- and 13C (B)-NMR data of harmaline and harmine in CDCl3 and DMSO, respectively.A.       Harmaline (200 MHz, CDCl3)       Harmine (200 MHz, DMSO)H δ [ppm] multiplicity J [Hz] H δ [ppm] multiplicity J [Hz]N H 11.54 s N H 11.42 sH-8 7.04 d 1,9 H-8 7.00 d 2.1H-6 6.80 dd 1,9 /8,9 H-6 6.83 dd 2.1/8.6H-5 7.42 d 8,9 H-5 8.04 d 8.6H-4 3.14 m H-4 7.79 d 5.3H-3 3.88 m H-3 8.14 d 5.3OCH3 3.83 s OCH 3 3.86 sCH3 2.99 s CH3 2.71 sHicham Berrougui et al. / Journal of Natural Remedies, Vol 5/1 (2005) 41 - 45443.2. Cytotoxicity assayAlthough the role of nitric oxide in tumorbiology remains incompletely understood, itis known to have both tumour promoting andinhibitory effects, presumed to be dependenton its local concentration within the tumor. Thep53 tumor suppressor gene may influence itsproduction, and NO appears to be pivotal inthe angiogenic processs being necessary forsolid malignant tumor development.In addition, nitric oxide has been shown toinduce cytostasis and cell cycle arrest on thehuman breast cancer cell line MDA-MB-231[18,19]. Bearing in mind that the alkaloidsharmine and harmaline have been shown toinduce NO production from cultured rat aorticendothelial cells [14], we considered thepossible anticancer effect of such alkaloids.Therefore, we assayed the cytotoxic activity,on the human cancer cell lines TK-10, MCF-7and UACC-62, of these alkaloids as well asthe plant extract where they were isolated,being the results depicted in Table 2.The antineoplastic agent etoposide was takenas positive control for comparison with thetested compounds. The extract and thealkaloids were active on the three cancer celllines at the recommended USA NationalCancer Institute (NCI), although they were lessactive than etoposide. Harmaline’s activity wasslightly higher than the observed for harmaline,suggesting that the change of β-carboline todihydro-β-carboline might be increasing theanticancer activity.The alkaloids concentrations producing aninhibition of the growth by 50% (IC50) on thethree human cancer cell lines were similar tothe concentrations required for the inductionof NO production by such compounds [14].Therefore, we might think that their anticanceraction is mediated by NO production.Table 2Cytotoxic activity (expressed as IC50 ± SEM values) of harmine, harmaline,methanolic extract where they were isolated, and the positive control (etoposide)on the human cancer cell lines TK-10, MCF-7 and UACC-62.n TK-10 MCF-7 UACC-62ME 3 IC50 (µg/mL) 38.3 ± 2.3 25.3 ± 4.2 26.6 ± 3.1Harmine 3 IC50 (µM) 40.5 ± 6.8 29.3 ±3.5 18.0 ± 0.7Harmaline 3 IC50 (µM) 27.5 ± 3.2 16.4 ± 2.5 11.1 ± 1.5Etoposide 3 IC50 (µM) 9.95 ± 0.08 0.87 ± 0.21 1.13 ± 0.21n: number of independent experiments.(B)Fig 1. Chemical structure of harmine (A)and harmaline (B).(A)Hicham Berrougui et al. / Journal of Natural Remedies, Vol 5/1 (2005) 41 - 4545However, despite the fact that harmine showedless NO production activity that harmaline, itsanticancer activity in our cancer cell lines washigher, suggesting the involvement of othermechanisms in its anticancer action.In this way, Ayoub et al., [20] have reportedthat harmalol (an indole alkaloid from P.harmala ) inhibited the proliferation of K562-Leukaemic tumor cell line by inhibiting DNAsynthesis and cell division. A previous workhas shown that β-carbolines could intercalateinto DNA, this effect may cause inhibition ofDNA topoisomerases leading to a cytotoxiceffect. Accordingly, Sobhani et al., [21] havereported that P. harmala seeds extract inhibithuman DNA topoisomerase I.1. Hmamouchi M. (1999) Les plantes medicinaleset aromatiques Marocaines, Enigma ed, 9954-8007-0-0: 450.2. Lutes J, Lorden JF, Beales M, Oltmans GA.(1988) Neuropharmacol.  27: 849-855.3. Bruinvels J, Sourkes TL. (1968) Eur. J. Pharmacol.4: 31-39.4. Abdelfattah AFM, Matsumoto K, Gammaz HAK,Watanabe H. (1995) Pharmacol. Biochem.Behav.  52 (2) : 421-426.5. O’Hearn E, Molliver ME. (1993) Neuroscience55 (2): 303-310.6. Grella B, Dukat M, Young R, Teiler M, Herrick-Davis, Gauthier CB, Glennon RA. (1998) DrugAlcohol depend.  50: 99-107.7. Nelson DL, Herbet A, Petillot Y, Pichat L,Glowinski J, Hamon M. (1979) J. Neurochem.32: 1817-1827.8. Fuller RW, Wong CJ, Hemrick-Luecke SK.(1986) Life Sci.  38: 409-412.9. May T, Rommelspacher H, Pawlik M. (1991) J.Neurochem. 56: 490-499.10. Rommelspacher H, Bruning G, Susilo R, NickM, Hill R. (1985) Eur. J. Pharmacol.  109:363-371.11. Tse SYH, Mak IT, Dickens BF. (1991)  BiochemPharmacol. 42: 4549-464.12. Saeed SA, Farnaz S, Simjee RU, Malik A. (1993)Biochem. Soc. Trans.  21: 462S.13. Li WK. (1996) J Lanzhou Med Coll.  22: 16-18.14. Shi CC, Liao JF, Chen-Fu C. (2001) Jpn. J.Pharmacol.  85: 299-305.15. Berrougui H, Herrera MD, Marhuenda E, EttaibA, Hmamouchi M. (2002) Therapie.  57: 236-241.16. Monks A, Scudeiro D, Skehan P, Shoemaker R,Paull K, Vistica D, Hose C, Langley J, CrOniseP, Vaigro-wolff A et al. (1991) J. The NationalCancer Institute  83 (11): 757-766.17. Christy M, Zaidi I, Nasir MA. (1995) FitoterapiaLXVI (1): 73-76.18. Pervin S, Singh R, Chaudhuri G. (2001) Proc.Natl. Acad. Sci.  USA. 98(6): 3583-8.19. Brennan PA, Moncada S. (2002) Ann. R. Surg.Engl.  84(2): 75-78.20. Ayoub MT and Rashan LJ. (1991) Phyto-chemistry  30: 1046-1047.21. Sobhani AM, Ebrahimi SA, Mahmoudian M.(2002) J. Pharm. Pharmaceut. Sci.  5(1): 19-23.ReferencesHicham Berrougui et al. / Journal of Natural Remedies, Vol 5/1 (2005) 41 - 45

Cytotoxic activity of methanolic extract and two alkaloids extracted from seeds of Peganum harmala L.

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Cytotoxic activity of methanolic extract and two alkaloids extracted from seeds of Peganum harmala L.

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