Contribution to the phytochemical study and evaluation of the anti-cancer potential of Glaucium flavum, a plant used in Algerian traditional medicine

Ce travail de thèse a été mené afin d’évaluer les propriétés anticancéreuses de plantes médicinales algériennes dans le cadre de la recherche de nouvelles molécules à potentialités anticancéreuses. La première partie du travail a consisté à sélectionner des plantes utilisées en médecine traditionnelle locale afin d’en évaluer le pouvoir cytotoxique potentiel vis-à-vis de lignées de cellules cancéreuses mammaires humaines. Le screening de l’activité anticancéreuse in vitro a permis de sélectionner l’extrait de la racine de Glaucium flavum (G. flavum) qui s’est révélé très actif sur les cellules cancéreuses sans pour cela affecter les cellules normales testées. L’analyse par cytométrie de flux a démontré que l’extrait de la plante induisait un arrêt du cycle cellulaire en phase G2/M avec une surexpression de la protéine antiproliférative p21. Cet arrêt du cycle est suivi par une induction de la mort cellulaire par apoptose. Un test in vivo (modèle CAM) a confirmé notre intérêt pour cette plante. En effet, un impact significatif du traitement avec G. flavum a été observé sur la croissance tumorale et l'angiogenèse tumorale associée. Au vu de ces résultats prometteurs, cette plante a fait l’objet d’une étude phytochimique approfondie afin d’identifier le(s) alcaloïde(s) responsable(s) de l’effet anti-cancéreux. La deuxième partie du travail présente l’exploration phytochimique de l’extrait de la racine de G. flavum. A partir de l’extrait dichlorométhane de la racine, nous avons pu isoler un alcaloïde majoritaire, la protopine. Cependant, la protopine purifiée n’a pas permis de reproduire complètement l’effet anticancéreux de l’extrait alcaloïdique total. Nous avons pu isoler et identifier un deuxième alcaloïde majoritaire de la racine, la bocconoline. Nous avons démontré une activité antiproliférative significative de cette molécule sur la croissance des cellules cancéreuses de manière dose-dépendante (avec une IC50 de 7,8 μM) sans pour cela affecter la croissance des cellules non tumorales. La quantification de ces deux alcaloïdes dans G. flavum a révélé que la racine contient 0,84% de protopine et 0,07% de bocconoline (m/m), tandis que la partie aérienne ne contient que 0,08% de protopine et pas de bocconoline. Nos résultats ont confirmé que le principal alcaloïde de la partie aérienne de G. flavum est la glaucine et nous avons montré pour la première fois que la protopine est le principal alcaloïde de la racine. Enfin, nous sommes les premiers à démontrer que l’effet anticancéreux spécifique de l’extrait de racine de G. flavum contre les cellules cancéreuses du sein, peut être attribué, au moins en partie, à la bocconoline/This thesis was conducted in order to evaluate the anticancer properties of Algerian medicinal plants and in the framework of the discovery of new molecules with potential anticancer properties. The first part of this work consisted in the selection of specific plants used in local traditional medicine to assess their cytotoxic activity against human breast cancer cells. The screening in vitro of anticancer activity, allowed us to select the root extract of Glaucium flavum (G. flavum), which proved to be very active on breast cancer cells without affecting normal cells. Flow cytometry analysis showed that the plant extract induced a cell cycle arrest in G2/M phase with an overexpression of the antiproliferative protein p21 in cancer cells. The cell cycle arrest was followed by an induction of apoptosis. Then, an in vivo test (CAM model) confirmed the anticancer effect of this plant. Indeed, we observed a significant effect of G. flavum treatment on tumor growth and tumor-associated angiogenesis. Considering these promising results, this plant has been subjected to a phytochemical investigation to identify phytochemical alkaloids responsible for the anticancer effect. The second part of the work consisted in the phytochemical exploration of G. flavum root extract. We isolated the major alkaloid, protopine, from the dichloromethane root extract. However, purified protopine did not reproduce the full cytotoxic activity of the alkaloid extract on cancer cells. Further, we isolated and purified another major alkaloid of root extract, bocconoline. We demonstrated the significant antiproliferative activity of this molecule on the growth of cancer cells in a dose-dependent manner, with an IC50 value of 7.8 µM. Only a low cytotoxic effect was observed against normal human cells. Quantification of alkaloids in G. flavum revealed that the root part contained 0.84% of protopine and 0.07% of bocconoline (w/w), while the aerial part contained only 0.08% of protopine and no bocconoline was detected. Our results confirmed that the main alkaloid of G. flavum aerial part is glaucine and we report for the first time that protopine is the major root alkaloid. Finally, we are the first to demonstrate a specific anticancer effect of G. flavum root extract against breast cancer cells which can be attributed, at least in part, to bocconoline

Evaluation of the cytotoxic and cytostatic activities of alkaloid extracts from different parts of Peganum harmala L. (Zygophyllaceae)

Introduction Peganum harmala L. (P. harmala) is broadly used in folk medicine for the treatment of various diseases including cancer. The seeds in particularly are employed for therapeutic purposes. The present paper investigates the cytotoxic effects of raw alkaloid extracts from different parts of P. harmala including fruits (TAFr), seeds (TASe), roots (TAR) and aerial parts (TAAp); in order to assess traditional claims about the therapeutic potential of this plant. Methods The cytotoxic effects were evaluated on six malignant cancer cells (A549, U373, Hs683, MCF7, B16F10 and SKMEL-28; A549, U373, MCF7 and SKMEL-28 which are resistant to proapoptotic stimuli) by MTT assay and quantitative videomicroscopy analysis. The main alkaloids were quantified by HPLC in the different parts of plant. Results Total alkaloids of the different parts were cytotoxic towards practically all cancer cell lines with IC50 ranging 1–52 μg/mL after 72 h of treatment. Videomicroscopy analysis indicated that the TAFr, TASe and TAR alkaloids affect A549 lung carcinoma cells behaviour and induce a cytostatic effect whereas TAAp extract was cytotoxic rather than cytostatic. TAAp, TAFr, TASe and TAR treatment induced global growth ratio indexes (GGR) of 0.19, 0.26, 0.3 and 0.62, respectively, after 72 h of treatment. Depending on the organe, harmine, harmaline, harmol and vasicine these range between 2 and 90% w/w of the total alkaloids. Conclusion These data indicate that P. harmala alkaloids extract may support the traditional claims regarding its anticancer uses which could be helpful in providing of new cytotoxic agents against chemo-resistant cancer cells.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Anticancer effect of seeds and roots extract of Peganum harmala L.

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Identification and Quantification of the Main Active Anticancer Alkaloids from the Root of Glaucium flavum

Glaucium flavum is used in Algerian folk medicine to remove warts (benign tumors). Its local appellations are Cheqiq el-asfar and Qarn el-djedyane. We have recently reported the anti-tumoral activity of Glaucium flavum root alkaloid extract against human cancer cells, in vitro and in vivo. The principal identified alkaloid in the extract was protopine. This study aims to determine which component(s) of Glaucium flavum root extract might possess potent antitumor activity on human cancer cells. Quantitative estimation of Glaucium flavum alkaloids was realized by HPLC-DAD. Glaucium flavum effect on human normal and cancer cell viability was determined using WST-1 assay. Quantification of alkaloids in Glaucium flavum revealed that the dried root part contained 0.84% of protopine and 0.07% of bocconoline (w/w), while the dried aerial part contained only 0.08% of protopine, glaucine as the main alkaloid, and no bocconoline. In vitro evaluation of the growth inhibitory activity on breast cancer and normal cells demonstrated that purified protopine did not reproduce the full cytotoxic activity of the alkaloid root extract on cancer cell lines. On the other hand, bocconoline inhibited strongly the viability of cancer cells with an IC50 of 7.8 µM and only a low cytotoxic effect was observed against normal human cells. Our results showed for the first time that protopine is the major root alkaloid of Glaucium flavum. Finally, we are the first to demonstrate a specific anticancer effect of Glaucium flavum root extract against breast cancer cells, which can be attributed, at least in part, to bocconoline

Cytostatic effect of seeds and roots extract of Pegalum harmala L.

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Determination of anti-proliferative effect of roots and seeds extract of Algerian Peganum harmala L.

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Revealing the anti-tumoral effect of Algerian Glaucium flavum roots against human cancer cells.

Glaucium flavum (G. flavum) is a plant from the Papaveraceae family native to Algeria where it is used in local traditional medicine to treat warts. G. flavum root crude alkaloid extract inhibited breast cancer cell proliferation and induced G2/M phase cycle arrest and apoptosis without affecting normal cells, which is a highly awaited feature of potential anti-cancer agents. G. flavum significantly reduced growth and vascularization of human glioma tumors on chicken chorioallantoic membrane (CAM) in vivo. The chromatographic profile of the dichloromethane extract of G. flavum root showed the presence of different constituents including the isoquinoline alkaloid protopine, as the major compound. We report for the first time that G. flavum extract may represent a new promising agent for cancer chemotherapy

Inhibition of myeloperoxidase activity by the alkaloids of Peganum harmala L. (Zygophyllaceae).

Seeds and aerial parts of Peganum harmala L. (Peganum harmala) are widely used in Algeria as anti-inflammatory remedies. Evaluation of Peganum harmala total alkaloids extracts and pure β-carboline compounds as an anti-inflammatory treatment by the inhibition of an enzyme key of inflammatory, myeloperoxidase (MPO) and HPLC quantification of the alkaloids from the different parts of plant.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe