Dendroaspis angusticeps and Naja pallida lyophilized powder venoms have been obtained from Latoxan Laboratory. Samples were prepared by dissolving D.angusticeps venom in an ammonium acetate buffer and N.pallida venom in distilled water.

Leaves and berries from I.aquifolium have been harvested in Northern Corsica. Leaves have been crushed and successively extracted with hexane and dichloromethane (Soxhlet apparatus). Berries have also been crushed and successively extracted with hexane, dichloromethane and finally with a dichloromethane/ethyl acetate (50/50, v/v) mixture.

C.villosa flowers have been harvested in Southern Corsica, crushed and successively extracted with hexane, dichloromethane and ethyl acetate.C.villosa roots were collected in the same place then extracted with methanol.

D.angusticeps whole venom was submitted to a FPLC (Fast Protein Liquid Chromatography) molecular exclusion chromatographic column (AKTA Start Protein Purification System Lab, GE Healthcare). Only fractions containing low molecular weight proteins (≤10,000 Da), have been pooled and fractionated further by ion-exchange chromatography on cation-exchanger resin [10].

Fas-II and CTX purification steps were performed in HPLC system (1260 Infinity LC System, Agilent Technologies) equipped with a reverse-phase preparative column.

Plant extracts were submitted to successive column chromatography over silica gel according to [11].

LC/MS experiments were carried out using model 2010 (Shimadzu) apparatus. MALDI-TOF mass spectrometry analyses were performed for identifying the molecular mass of intact main proteins from cation-exchange chromatography, using an AXIMA Performance MALDI-TOF/TOF mass spectrometer (Shimadzu). MS/MS analyses were obtained with Bruker Daltonics Maxis Plus.

All plant extracts and chromatography fractions from this extracts have been dissolved in suitable solvents. After, all 1D and 2D NMR spectra were recorded on a Bruker AVANCE 400 spectrometer. The study was realized using the computerized NMR method developed over the past thirty years by the University of Corsica “Chimie et Biomasse” group, UMR CNRS “Sciences Pour l’environnement”. This method allows individual components identification in natural mixture (essential oil, vegetable oil, bio-oil, and solvent extract) starting from its ¹³C-NMR spectrum by comparing chemical shift values in the mixture spectrum with those of reference spectra compiled in two libraries devoted to each phytochemical classe [12]. The first spectral data library was constructed with spectra recorded in the laboratory while the second one was built with spectral data picked up in recent literature. Each compound was identified by taking into account three parameters that were directly available from the computer program: 1) the number of observed signals with respect to what was expected, 2) the difference between the chemical shift of each signal in the mixture and in the reference (∆δ), and 3) the number of overlapping signals of carbons belonging to two components that fortuitously possess the same chemical shifts [12][13]. If necessary, the occurrence of “false positive” could be avoided by taking into account the relative intensities of the non-overlapped signals of protonated carbons of a given component. A component is considered identified if 50% of its signals are observed and they belong to that compound.

Antibacterial activities have been displayed against six bacterial strains obtained from the Pasteur Institute collection: three Gram-negative bacteria (Escherichia coli CIP 54.8 T, Enterobacter aerogenes CIP 60.86 T and Pseudomonas aeruginosa CIP 103467), three Gram-positive bacteria (Staphylococcus aureus CIP 53.156, Staphylococcus epidermidis CIP 53.124 and Bacillus cereus CIP 78.3). Chloramphenicol have been selected as reference antibiotic. Antibacterial activities have been determined by measuring the Minimum Inhibitory Concentrations (MICs) values using microdilution method in liquid medium. Bacteria in the liquid medium Müeller-Hinton Broth (MHB) was used as the positive control of bacterial growth.

Animals venoms provide peptides with many potential biological activities [2]. However, large quantities of these molecules are difficult to obtain, since they often are a minor venom component [14][15]. This first study, presents purification and analytical steps leading to Fasiculin II (Fas-II) and Cardiotoxin (CTX) purification from Dendroaspis angusticeps and Naja pallida venoms, respectively.

3.1.1. Fasciculin II Isolation

D.angusticeps snake has a lethal venom. The main envenomation symptoms reported are respiratory paralysis, longlasting cardiovascular abnormalities and myocardial necrosis. Fas-II is one of the most potent anticholinesterase polypeptide (61 aa residues) in D.angusticeps venom, making poisoning by green mamba bites extremely severe [16]-[18].

As part of this study, native Fas-II peptide was purified from D.angusticeps snake venom. First, raw venom was fractionated by gel filtration then, fractions containing molecular weight proteins lower than 10.000 Dalton (Da) were fractionated further by ion-exchange chromatography (cation-exchanger). MALDI-TOF (Matrix-Assisted Laser-Desorption/Ionization Time-Of-Flight) mass spectrometry analyses were performed on each fractions obtained by ion-exchange chromatography, in order to highlight which one contained Fas-II: [M + H]⁺ experimental mass = 6745.078 ± 1.5 Da vs. theoretical mass = 6745.412 Da (calculated from Fasc-II aa residues sequency given in literature [19][20]). Fractions with Fas-II was then purified using reverse-phase preparative HPLC (High Performance Liquid Chromatography). After sequencing by LC-MS (Liquid Chromatography-Mass Spectrometry), molecular mass was confirmed and the fraction predominant component was identified as Fas-II [10].

3.1.2. Cardiotoxin Isolation

In the balance of this study, CTX (60 aa polypeptide) was also purified from N.pallida snake venom. CTXs are widely described in literature for their ability to induce systolic cardiac arrest in rodent models, to cause necroses and for their potent cytolytic activity [21][22]. This membrane-disruptive effect probably gives N.pallida venom it antibacterial activity [23].

N.pallida raw venom LC-MS analysis allowed to determine the peak of interest. Indeed, m/z (mass-to-charge ratio) values confirmed CTX molecular mass: experimental mass = 6827 Da vs. molecular mass from literature = 6827.41 Da [24]. Then, CTX was purified using preparative HPLC.

This second study describes chemical composition of wild growing corsican understudied plant species mainly using ¹³C-NMR (Carbon-13 Nuclear Magnetic Resonance). It was realized using the computerized NMR method developed over the past thirty years by the University of Corsica “Chimie et Biomasse” group, UMR CNRS “Sciences Pour l’environnement”. In this study, antibacterial activities of crude plant extracts and chromatography fractions have also been determined.

3.2.1. Phytochemical Constituents in Corsican Ilexaquifolium Linne Leaves and Berries Extracts

Ilex aquifolium Linne (common Holly; Aquifoliaceae), is a shrub or a small tree with persistent foliage and glowing dark green leaves. Flowers that are small and white or pink and odoriferous appear between April and July. Fruits are red berries that grow during the August-December period and well known for their toxicity [25]. I.aquifolium is a decorative tree for Christmas festivities. Leaves have been used in folk medicine for several purposes, such as in intermittent fevers and rheumatisms, for their antipyretic properties, and as astringent, diuretic, and expectorant agents [26]. Various parts of this tree are included in traditional medicinal preparations to treat liver, stomach and intestinal cancers, dropsy, gout, jaundice, malaria, warts, swelling and tumours [27]. This study, describes components contained in the leaves and berries of I.aquifolium. Some of this components are known to show biological activities such as anti-inflammatory, antioxidative and antibacterial properties [28]-[31].

In the first place, I.aquifolium leaves have been extracted with hexane and crude extract was then submitted to successive fractionations by column chromatography. After ¹³C-NMR analysis of hexane extract and all chromatography fractions, ten triterpens were identified with spectral data recorded in laboratory or with spectral data picked up in literature (α-amyrin, β-amyrin, β-sitosterol, pseudotaraxasterol, uvaol, erythrodiol, betulin, lupeol, ursolaldehyde, oleanaldehyde). Using the same procedure, I.aquifolium leaves previously extracted by hexane have been extracted a second time by dichloromethane. Dichloromethane leaves crude extracts and all chromatography fractions were analyzed by ¹³C-NMR to allow the identification of two additional triterpens, oleanolic acid and ursolic acid [11].

Ursolic acid is well known to possess a wide range of biological functions, such as antimicrobial, anti-inflammatory and anti-wrinkle activities [32][33]. Oleanolic acid antioxidative and antifungal activities has been previously reported [34][35]. So, with the aim to achieve the quantitation of both acids displaying therapeutic interests useful in various domains in dichloromethane leaves crude extract, a reliable method was developed and validated (accuracy, precision linearity of measurements, limit of detection and limit of quantitation), using ¹H-NMR (proton Nuclear Magnetic Resonance) [36]. Ursolic and oleanolic acids accounted for 55.3% and 20.8% of the dichloromethane extract, respectively.

In the second place, we determined chemical compositions of I.aquifolium berries extracts. Using the same procedure, berries have been extracted with hexane, followed by dichloromethane then, ¹³C-NMR (2D “bidimensional” NMR sometimes analysis of dichloromethane crude extract and all chromatography fractions allowed identification of nine triterpens previously identified in I.aquifolium leaves extracts (α-amyrin, β-amyrin, oleanolic acid, ursolic acid, lupeol, uvaol, ursolaldehyde, oleanaldehyde, pseudotaraxasterol). We also identified two phenolic derivatives (p-hydroxybenzaldehyde, (p-hydroxyphenyl)acetonitrile) and two lactones (menisdaurilide, aquilegiolide), using spectral data recorded in laboratory or with spectral data picked up in recent literature.

After, berries previously extracted by dichloromethane have been extracted a third time with a dichloromethane/ethyl acetate (50/50, v/v) mixture. Crude extract and chromatography fractions ¹³C-NMR spectra revealed presence of four triterpens (α-amyrin, β-amyrin, oleanolic acid, ursolic acid), three additional phenolic derivatives (vanillic acid, p‐hydroxybenzoic acid, 2-hydroxyphenylacetic acid), six monosaccharides (α and β-D-glucopyranose, α and β-D-fructopyranose, α and β-D-fructofuranose) and four lactones. Among these lactones, two were identified in previous berries extract (menisdaurilide, aquilegiolide, dasycarponilide and 7-epi-griffonilide) [11]. These four latest compounds are for the first time described in I.aquifolium extracts.

3.2.2. Phytochemical Constituents in Corsican Calicotome villosa (Poiret) Link Flowers and Roots Extracts

Calicotome villosa (Poiret) Link (Fabaceae) is a shrub that can reach 2 m in high, with grey-tomentose stems and sharp terminations, villous pods, trifoliate and oval leaves, and yellow and grouped flowers during the spring season [37]. It is very common in the Mediterranean area and particularly in Corsica Island, where it grows near the sea while, the subspecies C.villosa subsp. intermedia is distributed especially in the North of Africa and Spain [38]. C.villosa was used for the treatment of foruncle, cutaneous abscess and chilblain [39]. In Palestine, infusion prepared from flowers is used for the treatment of cardiovascular system and nervous system problems and blood dilution [40]. Roots of this plant are used as corn plasters [41]. Many studies describe aerial parts extracts chemical composition of C.villosa[42]-[44], known as well for biological properties including antibacterial, antipyretic or anti-rheumatism activities [45]-[47]. In contrast, few studies describe roots extracts chemical composition and biological properties [41].

In this study, C.villosa flowers were first extracted with hexane then by dichloromethane. Dichloromethane extract was partitioned by successive column chromatography. After, crude extract and chromatography fractions ¹³C-NMR analysis, a flavone (chrysin) and two glycosylated chrysin derivatives (chrysine 7-((4''-O-acetyl)-O-β-D-glucopyranoside); chrysine 7-((6''-O-acetyl)-O-β-D-glucopyranoside)) [11].

Secondly, flowers previously extracted by dichloromethane have been extracted a third time by ethyl acetate. Crude extracts and chromatography fractions ¹³C-NMR (2D NMR sometimes) spectra revealed presence of five monosaccharides (α and β-D-glucopyranose, β-D-fructopyranose, α and β-D-fructofuranose), four phenolic derivatives (caffeic acid; trans-p-coumaric acid; cis-p-coumaric acid; cis-2,4,5-trihydroxycinnamic acid), six flavonoids (apigenine; luteoline; chrysine 7-O-β-D-glucopyranoside; chrysine 7-((4''-O-acetyl)-O-β-D-glucopyranoside); chrysine 7-((6''-O-malonyl)-O-β-D-glucopyranoside); chrysine 7-((4''-O-acetyl)-(6''-O-malonyl)-O-β-D-glucopyranoside)) and a polyol (pinitol). Cis-configuration phenolic derivatives have spare occurrence as natural products and are for the first time described in C.villosa extracts.

The following section of this study describe chemical composition of a methanol roots extract of C.villosa, which has never been submitted to any chemical composition study. Repetitive column chromatography was carried out on this extract and some of its fractions. Crude extract and all chromatography fractions have been submitted to ¹³C-NMR (2D NMR sometimes) analysis and many compounds have been identified using spectral data recorded in laboratory or with spectral data picked up in recent literature, including two sterols (β‐sitosterol; stigmasterol); a stilbene (trans-resveratrol); seven flavonoids (calycosin; daidzein; genistein; liquiritigenin; isoprunetin; bidwillon B; formononetin) and five pterocarpans (maackiain, homoedudiol, isoneorautenol, 11b-hydroxy-11b,1-dihydromaackiain; 3,4-dihydroxy-8,9-methylenedioxypterocarpan). Among identified compounds, two new pterocarpans (4,9-dihydroxy-3-methoxy-2 dimethylallylpterocarpan and 4,9-dihydroxy-3',3'-dimethyl-2,3-pyranopterocarpan) and a new dihydrobenzofuran derivative (2-(1-methylethenyl)-5-hydroxy-6-carbomethoxy-2,3-dihydro-benzofuran) were also identified using a combination of 1D (unidimensional), 2D NMR and MS/MS (tandem mass spectrometry) [48].

3.2.3. Antibacterial Activities

In order to determine some previously identified compounds antibacterial activities, microbiologic assays were performed, measuring the MICs (Minimum Inhibitory Concentrations) of extracts and chromatography fractions against three Gram-negative bacteria (Escherichia coli, Enterobacter aerogenes and Pseudomonas aeruginosa), three Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis and Bacillus cereus) and using chloramphenicol as reference antibiotic.

In the first instance, I. aquifolium leaves extracts and chromatography fractions antibacterial activities have been determined. Only Gram-positive bacteria were sensitive to extracts and chromatography fractions with high content in ursolic acid and oleanolic acid. Both triterpen acids and chloramphenicol displayed similar antibacterial activities (MIC = 4 and 8 mg∙L⁻¹vs. 2 and 4 mg∙L⁻¹). Following this study, a structure-activity relationship has been displayed. Comparing, ursolic acid, oleanolic acid and three structurally related compounds (ursolaldehyde, α-amyrin, uvaol) MICs, this study show that ursolic acid and oleanolic acid antibacterial activities are attributable to the carboxylic acid group (C28 carbon). Ursolic acid having similar MIC with oleanolic acid indicate that methyl groups (C29 and C30 carbons) are not influencing ursolic acid and oleanolic acid antibacterial activities [11].

In the second instance, we determined antibacterial activities ofC.villosa flowers and roots extracts and also antibacterial activities of chromatography fractions from this extracts. Only methanol roots extract displayed antibacterial activity with MIC lower than 32 mg∙L⁻¹. So, different chromatography fractions and sub-fractions from this extract were submitted to assays. Bidwillon B displayed the highest antibacterial activity with MICs similar with those of chloramphenicol (MIC = 4 mg∙L⁻¹vs. 2 et 4 mg∙L⁻¹), followed by homoedudiol (MIC = 8, 16 mg∙L⁻¹) and daidzein (MIC = 16 mg∙L⁻¹). New pterocarpans and chloramphenicol also displayed similar antibacterial activities against S.aureus and B.cereus (MIC = 8 and 16 mg∙L⁻¹vs. 2 and 4 mg∙L⁻¹). Further investigation into specific targets and mode of action of the identified antibacterial compounds will come soon.