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The present study aims to identify and quantify the phenolic compounds of Azadirachta indica leaf extract using HPLC-MS and to evaluate the antioxidant, antibacterial (against different Gram-positive and negative bacteria) and in vitro anti-proliferative activities of this extract (against breast, human liver and cervix adenocarcinoma-derived cells). The application of this extract as a natural antioxidant for food preservation was also tested on oil-in-water food emulsions for the first time in the present work in order to determine the use of Azadirachta indica leaves as a natural additive to preserve the food against lipid oxidation and rancidity. The results obtained revealed that 50%-aqueous ethanol leaf extract showed the best extraction yield (25.14%), which was characterized by a high content in phenolic compounds and strong antioxidant activity. Moreover, this leaf extract inhibited the growth of the bacterial strains tested (Staphylococcus aureus, Escherichia coli, Salmonella paratyphi and Micrococcus luteus) and showed better anti-proliferative activity against breast and cervix adenocarcinoma-derived cells than human liver cancer cells after 48 h of treatment. Additionally, Azadirachta indica leaf extract showed almost similar effects as gallic acid solutions (0.25% and 0.5%) in preserving the oxidation of oil-in-water food emulsions and prevented the formation of secondary oxidation products (malondialdehyde) as well. The results obtained suggested that extracts of Azadirachta indica leaves are a potential source of antioxidant and antibacterial compounds and pointed to the potential of these natural extracts as therapeutic agents., Peer Reviewed, Postprint (published version)

In the current work, the leaf and flower extracts of Anthyllis vulneraria were evaluated for their chemical characterization using HPLC-MS and for their radical scavenging capacity toward methoxy radicals produced by a Fenton-type reaction using an electron paramagnetic resonance (EPR) spectroscopy assay. The in vitro antiproliferative activity of these extracts against several human-derived cancer cells (breast: MCF-7; cervical: HeLa; hepatocellular: HepG2) was also evaluated. The results showed that the Anthyllis vulneraria leaf extract was characterized by 17 different phenolic compounds, among which phenolic acids were the most abundant, while its flower extract exhibited higher contents of flavonoids. Furthermore, Anthyllis vulneraria extracts demonstrated a potent radical scavenging activity against methoxy radicals. Both extracts also significantly reduced the viability of the different cancer cell lines. The results of the current study suggested that Anthyllis vulneraria extracts are a promising source of antioxidant compounds with health benefits and pointed to their potential use for treating cancer and developing novel therapeutic agents., Peer Reviewed, Postprint (published version)

For millennia, medicinal plants have been used to prevent and cure diseases. Up to now there is a growing interest in their use in several areas as conducted for Anthyllis in our investigation. Actually the present research aims to investigate the biochemical characterisation of a medicinal plant collected from Tunisia named Woundwort [Anthyllis vulneraria L. (Jones and Turrill 1933)] by determining the mineral content, phenolic compound contents and biological activities of its leaf and flower extracts. On the one hand, the results obtained showed that A. vulneraria accumulated minerals at different amounts with significant differences between leaves and flowers extracts. On the other hand, the data revealed that the hydroethanolic flower extract contained the highest content of total polyphenols, flavonoids and condensed tannin, as it exhibited the strongest antioxidant activity. The flower extract also showed better antibacterial effect than leaf extract. These results support the exploitation of active compounds extracted from the leaves and especially the flowers of A. vulneraria, which can provide new alternatives to the use of certain drugs, additives, among others, as they can be used as structure–activity models for the development of new products., Peer Reviewed, Postprint (published version)

Tesi en modalitat de cotutela: Universitat Politècnica de Catalunya i Université de Tunis El Manar. Aplicat embargament des de la data de defensa fins al dia 29/4/2022., The present work has focused on the study of two edible plants, with attributable medicinal values, named Anthyllis vulneraria L. (A.vulneraria) and Azadirachta indica L. (A.indica) collected in Tunisia and India, respectively. The aim of the work was to determine the chemical composition of A.vulneraria leaf and flower extracts and A.indica leaf extract and to investigate their antioxidant, antibacterial and anti-proliferative (against specific cancer cell lines) activities to design formulations for use in the food, pharmaceutical and cosmetic industries. The chemical composition of the extracts was estimated by spectrophotometric assays and quantified by High Performance Liquid Chromatography coupled to Mass Spectrometry (HPLC­ MS) and diode array (HPLC-DAD). The radical scavenging activity of the extracts was evaluated in vitro by different analytical methods. The antioxidant capacity of A.vulneraria and A.indica samples in oil-in-water emulsions and in raw minced beef was also studied to evaluate their preservative and quality evolution effect, in general, and the shelf life of these food products during the storage period. In addition, the antibacterial activity of the different extracts was tested against pathogenic microorganisms causing food poisoning and the antiproliferative activity was evaluated against several human cancer cell lines. The results obtained revealed that: (1) aqueous EtOH extracts of A.vulneraria and A.indica showed better extraction yields, higher content of phenolic compounds and higher anti-radical activity than pure EtOH extracts. (2) A. vulneraria flowers showed higher biological activities than leaves. (3) In Model Food System, at 0.25% (v/v), A.vulneraria flowers extracted in 50%­ aqueous EtOH protected oil-in-water emulsions against oxidation and prolonged shelf life better than leaf extract and even better than the sample with gallic acid (under the same conditions). Moreover, freeze-dried A.vulneraria flower sample, added at 0., El presente trabajo se ha centrado en el estudio de dos plantas comestibles, con valores medicinales atribuibles, denominadas Anthyllis vulneraria L. (A.vu/neraria) y Azadirachta indica L. (A.indica) recolectadas en Túnez e India, respectivamente. El objetivo del trabajo fue determinar la composición química de los extractos de hojas y flores de A.vulneraria y del extracto de hojas de A.indica e investigar sus actividades antioxidantes, antibacterianas y anti­ proliferativas (frente a específicas líneas celulares cancerígenas) para diseñar formulaciones que permitan su uso en la industria alimentaria, farmacéutica y cosmética. La composición química de los extractos se estimó mediante ensayos espectrofotométricos y se cuantificó per cromatografía líquida de alta resolución acoplada a espectrometría de masas (HPLC-MS) y diodo array (HPLC-DAD). La actividad de eliminación de radicales de los extractos se evaluó in vitro mediante diferentes métodos analíticos. También se estudió la capacidad antioxidante de los extractos en emulsiones de aceite en agua y en carne de vacuno cruda picada para evaluar tanto el efecto conservador y de evolución de la calidad, en general, como la vida útil de estos productos alimentarios durante el periodo de almacenamiento. Además, se probó la actividad antibacteriana de los extractos frente a microorganismos patógenos causantes de intoxicaciones alimentarias. La actividad anti-proliferativa se evaluó frente a varias líneas celulares cancerosas humanas. Los resultados obtenidos revelaron que: (1) los extractos acuosos de EtOH de A.vulneraria y A.indica muestran mejores rendimientos de extracción, mayor contenido de compuestos fenólicos y mayor actividad antiradicalaria que los extractos de EtOH puro. (2) las flores de A. vulneraria presentan actividades biológicas mejor que las hojas. (3) en emulsiones de aceite en agua, el extracto floral de A.vulneraria al 50% de EtOH y concentración 0.25% (v/v) protege frente a la oxidación y prolonga l, Postprint (published version)

Lipid oxidation was considered as a problem in food conservation. The present study aims to investigate the effect of lipopeptides DCS1 on the conservation of food models against lipid oxidation by determining the primary and the secondary oxidation products. Lipopeptides DCS1 are able to preserve the nutritional properties of the emulsion during 23¿days of storage, at a concentration of 0.0125% (w/w of emulsion), by slowing down the formation of hydroperoxides and malondialdehyde (MDA) compounds. The direct incorporation of lipopeptides in ground beef patties at a concentration of 0.5% (w/w of meat) was found to be more effective than gelatin film enriched with lipopeptides (2.5%, w/w of gelatin) as a coating, in inhibiting lipid oxidation. Furthermore, lipopeptides DCS1 are not toxic to human kidney cells HEK293 up to a concentration of 250¿µg/ml. The results indicate that lipopeptides DCS1 are effective for the preservation of fatty foods against lipid oxidation., Peer Reviewed, Postprint (author's final draft)

The antioxidant and antimicrobial effects of pecan shell (PSW), combined with roselle flower (RS) and red pepper (CA) were analyzed in beef patties by several methods during chilled storage for 13 days. Additionally, the antioxidant and antimicrobial activities of PSW, RS and CA extracts were determined. The PSW extract exhibited a higher radical scavenging activity (by the DPPH method) and more total phenolic compounds than RS and CA. RS presented the best antimicrobial capacity. Nine formulations of beef patties were prepared, including a control (CM), a synthetic preservative (CAMPA N.3 (A)) and different combinations of PSW, RS and CA. The bacterial counts of the beef patties with RS (4–5 log colony-forming units (CFU)/g meat) were significantly lower than those of the control sample (CM) (6–7 CFU/g meat) at day 6. The thiobarbituric acid-reactive substance (TBARS) values at day 7 of all treatments were similar to the values of samples containing the synthetic antioxidant and significantly lower than the CM group. The order of stability assessed by the TBARS values were in agreement with the hexanal content. Thus, these results support the hypothesis that the combination of PWS, RS and CA could represent a good natural food preservative., Peer Reviewed, Postprint (published version)

The present work aims to investigate the in vitro antioxidant activities of Enterobacter cloacae C3 lipopeptides, as well as the stability of sunflower oil in water (o/w) emulsion and the conservation of raw beef patties. The C3 lipopeptides were assayed for their antioxidant activity through five different tests. The C3 lipopeptides showed good in vitro antioxidant activities. Lipopeptides C3 exhibited important antioxidant properties in 10% sunflower o/w emulsions during prolonged storage (28 days) at 30°C. The effect of C3 lipopeptides on the oxidative stability of raw beef patties showed effectiveness effect in preventing oxidative degradation of lipids via thiobarbituric acid reactive substance and peroxide methods. Cytotoxicity test using human kidney HEK293 cells showed that studied lipopeptides was nontoxic substances. The results of this study indicate that lipopeptides C3 could be appropriate antioxidant agent in food models as inhibitors of lipid oxidation., Peer Reviewed, Postprint (author's final draft)

The aim of this study was to determine the total polyphenol content, radical scavenging and antimicrobial activities of Azadirachta indica (A. indica) and to evaluate their effect on shelf-life stability of raw beef patties during refrigerated storage at 4 ± 1 °C. During 11 days of storage, the antioxidant effect of A. indica on ground beef meat was investigated by the determination of lipid oxidation, pH, anti-radical activity, color, hexanal content, and microbial growth. The results obtained showed that fresh A. indica leaves and synthetic conservative behaved in the same way and retarded the lipid oxidation of chilled beef patties while increasing their pH (5.40 and 5.45, respectively). It can also be said that A. indica limited the loss of color, reduced the metmyoglobin formation (36.70%) and had a significant effect on bacterial growth and hexanal content. In addition, the results obtained through anti-radical and antimicrobial properties showed proportional values of total polyphenol content and radical scavenging activity of leaf extracts as they showed their antimicrobial effect against some bacteria such as Staphylococcus aureus and Micrococcus luteus, among others. These results support the involvement of A. indica in the food industry as a natural antioxidant that could replace synthetic ones, Peer Reviewed, Postprint (published version)

The main targets of this work were to determine the phenolic content of Anthyllis vulneraria (A. vulneraria) leaves and flowers and to evaluate their antioxidant activity. Total polyphenols and flavonoid content (TPC and TFC, respectively) were determined. Antioxidant capacity was evaluated by the Ferric Reducing Antioxidant Power (FRAP), the Oxygen Radical Absorbance Capacity (ORAC), the Trolox Equivalent Antioxidant Capacity (TEAC) and the diphenyl picrylhydrazyl (DPPH) assays, and by the analysis of primary and secondary oxidation products in oil-in-water emulsions and in raw beef patties during storage. The results revealed that the flowers of the A. vulneraria contained the highest content of total polyphenols and flavonoids and extracts from these tissues exhibited the strongest antioxidant activity, as they were more effective at retarding lipid oxidation in oil-in-water emulsions and raw beef patties than extracts from the leaves which had a potent antioxidant effect only at the beginning of the oxidation process. The results of this study allowed us to obtain a deep knowledge about the properties of A. vulneraria and confirmed the possibility of using its biologically active extracts in the food, cosmetic and pharmaceutical industries, Peer Reviewed, Postprint (published version)

Le stress oxydant, causant plusieurs maladies, suscite la recherche de nouveaux remèdes antioxydants. Dans cette optique, l’étude de l’activité antioxydante des extraits méthanoliques du Tripodion (Anthyllis tetraphylla L.) a été étudiée. Cette plante est issue de la région semi-aride de la Tunisie, précisément du Parc National de Boukornine, et appartient à la famille des Fabacées. Les extraits foliaires et floraux sont obtenus après macération dans le méthanol pendant 24h. Le tri phytochimique, l’activité antioxydante par les tests ORAC et TEAC et l’inhibition de la peroxydation lipidique dans le model d’émulsion huile/eau ont été réalisés. Le tri phytochimique a permis de révéler que ces extraits contiennent des teneurs importantes en polyphénols et flavonoïdes surtout au niveau de l’extrait floral (75.93 mg EAG/g MS et 87.86 EQ mM/L d’extrait, respectivement) qui présente aussi les activités antioxydantes (ORAC et TEAC) les plus élevées (92.36 mM TE /g MS et 0.13 µM TE/ g MS, respectivement). En ce qui concerne la capacité antioxydante de la poudre lyophilisée des feuilles et des fleurs testée dans l’émulsion, formée par l’huile de tournesol dans l’eau, les fleurs ont présenté la valeur la plus faible de l’indice de peroxyde, c’est à dire la meilleure activité antioxydante, tandis que les feuilles n’ont présenté un effet antioxydant puissant qu’au début du processus d’oxydation., Postprint (published version)