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The seeds of Chamaecrista absus contained 2,28% of oil. ... synonym: Cassia absus that belongs to family .... (Promega) according to the manufacturer‟s.
Journal of Plant Biology Research 2014, 3(1): 1-11

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Phylogenetic identification, phytochemical analysis and antioxidant activity of Chamaecrista absus var. absus seeds Khaled SEBEI1*, Imed SBISSI2, Abdelmajid ZOUHIR3, Wahid HERCHI1, Fawzi SAKOUHI1, Sadok BOUKHCHINA1 Unité de Biochimie des Lipides. Faculté des Sciences de Tunis. Université de Tunis EL Manar. Tunisia. Unité de Biochimie des Lipides et Interactions avec les Macromolécules, Faculté des Sciences de Tunis, Université de Tunis–El-Manar. Tunisia. 2 Laboratoire de Microorganismes et Biomolécules Actives, Faculté des Sciences de Tunis, Université de Tunis–ElManar. Tunisia. 3 Laboratoire de Protéomie et de biopréservation des aliments. ISSBAT. Université de Tunis–El-Manar. Tunisia. 1

ABSTRACT It was thought in Tunisia that the seeds whose vernacular name in Arabic is “El-Habba Al-Sawdaa” or “Habbat Al Baraka” belong to Nigella genus. While sequence analysis of the nuclear ITS1, 5,8S and ITS2 rDNA gene showed that these seeds were identified, affirmatively, such as Chamaecrista absus var. absus [GenBank accession number: KC817015]. The seeds of Chamaecrista absus contained 2,28% of oil. Fatty acid composition showed that linoleic, palmitic, oleic and linolenic acids account for more than 94% of the total fatty acids. We found that β-sitosterol represented the main component of the phytosterols (63,23 %), followed by campesterol and stigmasterol. Cycloartenol,  amyrine and 24 methylen-cycloartenol were the major components constituting about 76,85 % of total triterpene alcohols. The fractions of sterols and triterpene alcohols showed antibacterial activities against many strains with major activity against Listeria ivanovii and Bacillus subtilis. Concerning DPPH scavenging activity, a considerable antiradical ability was found (IC50 = 16,78 μg/ml). Keywords: Chamaecrista absus var absus, Elhabba Essawda, oil, sterols, phenols, antioxidant activity.

INTRODUCTION Chamaecrista belongs to subtribe Cassiinae (Caesalpinioideae), and it comprises over 330 species, divided into six sections (Torres et al., 2011). The genus Chamaecrista, formerly defined as Cassia subgenus Lasiorhegma (Irwin and Barneby, 1982), has been divided into six sections of very unequal sizes (Absus, Apoucouita, Caliciopsis, Chamaecrista, Grimaldia and * Xerocalyx). Placed in the large family Corresponding author: Khaled SEBEI Corresponding author e-mail: [email protected] Tel: +216 98 925 824 Fax: +216 71 573 526

Leguminosae, Chamaecrista sect. absus is further divided into four subsections: Absus, Adenophyllum, Otophyllum and Baseophyllum (Coutinho et al., 2012). There is also disagreement about sister-group relationships in Cassiinae. One hypothesis considers Chamaecrista a clade distinct from its sister taxa Senna and Cassia (De Souza Conceição et al., 2009). However, other studies (Herendeen et al., 2003) indicate that Senna and Chamaecrista are sister taxa and that Cassia occurs in a distinct clade (Torres et al., 2011).

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According to Irwin and Barneby (1982), Chamaecrista sect. absus is known as another synonym: Cassia absus that belongs to family Caesalpiniaceae and commonly known as Chaksu in the traditional system of medicine. The seeds are used in the treatment of ophthalmia, and are considered as attenuant, astringent and hypotensive. Seeds are also utilized in ringworm infestations, in conjunctivitis and other skin infections (Usmanghani et al., 1989). Reported constituents are the alkaloid chaksine (Voelter et al., 1985) as well as oils, fatty acids, sterols, glycosides, amino acids, gum, resin and unsaponifiable matter (Aftab et al., 1996). Cassia marginata and Cassia corymbosa, contain palmitic acid C16 (17.3 and 17.2%), palmitoleic C16:1(trace and 7.4%), stearic acid C18 (4.5 and 4.2%), oleic acid C18:1 (14.2 and 14.8%) and linoleic acid C18:2 (49.4 and 41.2%) respectively (Hosamani and Sattigeri, 2002). Four fatty acids: palmitic, stearic, oleic and linoleic acids (along with minor percentage of linolenic acid C18:3 in Cassia roxburghii and Cassia absus) were identified in all five species. The tested seed oil contained mainly unsaturated fatty acids i.e. linoleic acid in the range from 45.96% to 60.25% and oleic acid from 26.29% to 34.91%. Palmitic acid was the most abundant saturated acid. Three sterols: cholesterol, stigmasterol and -sitosterol were found in all species. -sitosterol was found to be a major sterol in C. javanica (67.50 %), C. alata (64.79 %), C. roxburghii (63.29 %) and C. absus (53.42%) while sigmasterol was major in C. laevigata (45.34 %) (Ledwani and Oberoi, 2010). To our knowledge, no data are available on the biochemical composition and antioxidant activity of Chamaecrista absus var. absus seeds. However, Hatano et al., (1999) reported that phenolic constituents of plants of the family Leguminosae have been found to have various biological or pharmacological actions including radical-scavenging effects, inhibitory effects on enzymes and antimicrobial effects. These authors revealed the isolation of new compounds related to condensed tannins from Cassia nomame and the inhibitory effects of phenolic constituents of C. nomame on lipase and isolated six new phenolic glycosides from seeds of Cassia tora, which have been used as a traditional medicine for eye diseases and intestinal disorders in Asian countries. Phenolic acids, flavonoids, and tannins are

regarded as the main dietary phenolic compounds (Manach et al., 2004). These compounds exhibit a wide range of physiological properties, such as anti-allergic, anti-atherogenic, anti-inflammatory, antimicrobial, antioxidant, anti-thrombotic, cardioprotective, and vasodilatory effects (Balasundram, 2006; Falleh et al., 2008). The aim of the present study was, firstly, to identify, by molecular methods, of black seeds known, for a long time, in Tunisia as “Al-Habba Al-Sawdaa”. In the second hand, the objectives of this work were to determine the biochemical composition of chamaecrista oil seed, total polyphenol, flavonoid, condensed tannin contents and antibacterial activities of sterols and terpene alcohols.

MATERILS AND METHODS Plant material The seeds of Chamaecrista absus var absus commonly known as Habbat elbaraka or Elhabba essawda in the traditional system of Tunisian folk medicine, were procured from the local herbal market (Souk Elblat) in Tunis, Tunisia. DNA extraction, PCR amplification and Sequencing Seed being superficially disinfected by shaking in 30% (v/v) H2O2 for 5 min and aseptically rinsed several times with sterile water. Total genomic DNA was extracted from plant seeds by grounding in liquid dinitrogen and digested for 1 hour at 65°C in CTAB extracting buffer (2% w/v CTAB, 100 mM Tris-HCl, pH 8.0, 20 mM EDTA, 1.4 M NaCl, and 0.2% v/v 2-mercaptoethanol). After chloroform- isoamylic alcohol (24/1: v/v) DNA was resuspended in 50 μl of TE buffer (10 mM Tris-HCl pH 7.4; 1 mM of EDTA) and stored at −20°C. The internal transcribed spacer (ITS) was amplified using the ITS1 and ITS4 primers (White et al., 1990). The amplification reactions was performed in a 50-μl volume of reaction mixture [1 mM of each primer, 0.2 mM of each dNTP, and 2.5 U of Taq polymerase (Promega, Madison, WI) in a DNA thermal cycler (2400 geneAmp PCR thermocycler; Perkin Elmer, Foster City, CA) with the following program: an initial denaturation at 95°C for 2 min, followed by 35 cycles of a 1-min denaturation at 94°C, a 40-s annealing at either 53°C and a 1-min elongation at 72°C, with a final elongation step at 72°C for 10 min. Amplification

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products were analyzed in 1.5% agarose gel in 0.5× TBE buffer (89 mmol l−1 Tris, 89 mmol l−1 borate, 2 mmol l−1 EDTA), stained with ethidium bromide, and visualized under UV light (Sambrook et al., 1989). The PCR product was then purified using QIAquick Wizard PCR purification Kit (Promega) according to the manufacturer‟s instructions, and subjected to cycle sequencing using the Taq Dye Deoxy Terminator Cycle Sequencing kit (Applied Biosystems; HTDS, Tunisia) and fragment separation in an ABI PrismTM 3130 DNA sequencer (Applied Biosystems). The ITS rDNA nucleotide sequence was compared to sequences listed in the GenBank database using the BLAST program (Altschul et al., 1990). Alignment with retrieved related sequences and phylogenetic analysis were performed using MEGA version 5.1 program (Tamura et al., 2011) and a maximum-likelihood tree was constructed with 1000 bootstraps and using Bauhinia ungulata (FJ009818) sequence as the outgroup. The sequence data was submitted to GenBank (NCBI) which have provided a GenBank accession number for our nucleotide sequence: KC817015. Determination of oil content Oil content was determined by extracting dry material of Chamaecrista absus seeds with petroleum ether using a Soxhlet apparatus (Harwood, 1984). This extraction takes 4 h at 42°C and repeated three times for only one sample. The extract was dried in a rotary evaporator at 32°C. Oil was weighed and stored at -10 °C. The oil content was determined as different in weight of dried peanut sample before and after the extraction (AOCS, 1989). Saponification and TLC analysis Unsaponifiable fraction of lipids was determined by saponifying 5 g of oil mixed with both 200 µl -cholestanol and an ethanolic KOH 12% solution; the mixture was heated at 60°C for 1.30 h. The unsaponifiable matter was extracted, washed, dried over anhydrous Na2SO4 and evaporated to dryness using N2. The unsaponifiable matter was separated into subfractions on preparative silica gel thin-layer plates (silica gel 60G F254) using one-dimensional TLC with hexane–diethyl ether (6:4, v/v) as the developing solvent. The unsaponifiable fraction diluted in chloroform was applied on the silica gel plates. After development, the plate was sprayed

with 2,7-dichlorofluorescein and viewed under UV light. Silylation of triterpene alcohols and sterols fraction An amount of 2 mg of methylsterols residue was mixed with 125 µl of BSTFA (with 1% TMCS), 125 µl of pyridine and 450 µl of acetone, the mixture vortexed for about 10 s and heated at 70 °C for 20 min. After silylation reaction, 1.5 ml of chloroform was added to the mixture and 1 µl of the solution was directly injected into a gas chromatograph. Gas chromatography–flame ionisation detection (GC–FID) Fatty acids were methylated using the method of Metcalfe et al. (1966) modified by Lechevallier (1966). Methyl esters were analyzed by gas chromatography (GC) using an HP 4890 chromatograph equipped with a flame ionization detector (FID) on a capillary column coated with supelcowax TM 10 (30 m length, 0.25 id, 0.2 mm film thickness). Temperatures of column, detector and injector were: 200°C, 250°C and 260°C, respectively. RP-HPLC phenols analysis Colorimetric quantification of total phenolics was determined, as described by Dewanto et al., (2002). All samples were analyzed in three replications. Phenolic compound analysis was carried out using a liquid chromatography RPHPLC coupled with a UV-vis Multiwavelength detector „waters 996 photoperiode Array Detector: 190-400 nm. The separation was carried out on 250×4.6-mm, LD 5-μm symetry shield C18 reversed phase column. The mobile phase consisted of acetonitrile (solvent A) and water (80:20 v/v) with 1 % formic acid (solvent B). The flow rate was kept at 1 ml/min. The injection volume was 20 μl and peaks were monitored at 275 nm. The peaks were identified by congruent retention times compared with standards. Analyses were performed in triplicates. DPPH radical-scavenging activity The DPPH· quenching ability of seed extracts was measured according to Hanato et al. (1988). One ml of the extract at different concentrations was added to 0.5 mL of a DPPH· methanol solution. The mixture was shaken vigorously and left standing at room temperature for 30 min in the dark. The absorbance of the resulting solution was then measured at 517 nm. The antiradical activity

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was expressed as IC50 (μg/mL), the antiradical dose required to cause a 50% inhibition. A lower IC50 value corresponds to a higher antioxidant activity of plant extract. The ability to scavenge the DPPH radical was calculated using the following equation: (1) DPPH· scavenging effect (%) = [(A0 − A1)/A0]×100; where A0 is the absorbance of the control at 30 min, and A1 is the absorbance of the sample at 30 min. All samples were analyzed in three replications. Total flavonoids content Total flavonoids were measured using a colorimetric assay developed by Dewanto et al. (2002). An aliquot of diluted sample or standard solution of (+)-catechin was added to 75 μl of NaNO2 solution (7%), and mixed for 6 min, before adding 0.15 ml AlCl3 (10%). After 5 min, 0.5 ml of 1 M NaOH solution was added. The final volume was adjusted to 2.5 ml, thoroughly mixed, and the absorbance of the mixture was determined at 510 nm. Total flavonoids were expressed as mg (+)catechin equivalent g−1 DW (mg CE g−1 DW), through the calibration curve of (+)-catechin (0– 400 μg ml−1 range). All samples were analyzed in three replications. Total condensed tannins Procyanidins were measured using the modified vanillin assay described by Sun et al., (1998). Three milliliters of 4% methanol vanillin solution and 1.5 ml of concentrated H2SO4 were added to 50 μl of suitably diluted sample. The mixture was allowed to stand for 15 min, and the absorbance was measured at 500 nm against methanol as a blank. The amount of total condensed tannins was expressed as mg (+)-catechin equivalent g-1 DW (Ksouri et al., 2008). All samples were analyzed in three replications. Analysis of proteins Defatted Chamaecrista absus seeds (100 mg) was successively extracted with 1 ml distilled water, 1 ml 5.0 M NaCl, 1 ml absolute ethanol, and 1 ml 0.2 M phosphate buffer (pH 8.0) for the extraction of the albumin, globulin, prolamin, and glutelin, respectively. Each extraction was shaken for 20 min in an Eppendorf tube and centrifuged at 10,000 g for 6 min. Each protein fraction assay was performed following Bradford‟s method

The fractions of lipids were individually tested against a large panel of microorganisms including Staphylococcus aureus (ATCC 6539), Pseudomonas aeruginosa (ATCC 15442), Escherichia coli (ATCC 25922), Enterococcus hirae ATCC10541, Listeria ivanovii and L. inocua (RBL 30 and RBL 29 respectively), Bacillus subtilis and B. cereus (168 and ATCC11778 respectively). All strains were obtained from Institut Pasteur de Tunis. The Bacteriological agar was from Biokar Diagnostics (Beauvais, France). Nutrient broth (NB) was from Difco (Becton Dickinson, Le Pont de Claix, France). All the other media, used in this study, were manufactured by Biorad (Marnes-La Coquette, France) and Merck. Antibacterial activity is revealed by growth inhibition in the strains to test. This activity is observed in solid solid media. In the present work, we have been using the well diffusion method described by Perez et al. (1990). Statistical analysis The data (three replicates) were statistically evaluated using the JMP SAS version 12.6 software (Statistical Analysis System). (SAS, Institute INC, Box 8000, Cary, North Carolina 27511, USA).

RESULTS AND DISCUSSION Molecular analysis For a long time, it was thought that the seeds whose vernacular name in Tunisia and in Arabia is “Elhabba Essawda” or “Habbat Elbaraka” belong to Nigella genus (AL Gaby, 1998). This is the first time that a scientific study focused on the identification of these seeds wish used in the traditional system of Tunisian folk medicine, also, in gastronomy, cosmetic, aromatherapy and pharmaceutical fields. In this study we used the rDNA intergenic spacers ITS to determine the phylogenetic position of plant seeds (Baldwin et al., 1995). The analysis was inferred from comparative ITS sequences analysis of 19 related species based on Maximum likelihood method (Figure 1). The alignment of 662 pb produced a topology with high bootstrap supports (100%), indicating that the our species is sister to, Chamaecrista absus var. absus.

(1976). Antibacterial activity detection

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68 Chamaecrista hispidula (FJ009833) 53 Chamaecrista glaucofilix (FJ009834) 83 Chamaecrista rupestrium (FJ009835) 67 Chamaecrista botryoides (FJ009836) 78 Chamaecrista setosa (FJ009842) Chamaecrista cathartica (FJ009841) Chamaecrista urophyllidia (FJ009840) 99 72 Chamaecrista speciosa (FJ009839) 86 98 Chamaecrista philippi (FJ009838) Chamaecrista dalbergiifolia (FJ009837)

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Ts 100 Chamaecrista absus (FJ009832) Chamaecrista_sp. (FJ009831) Chamaecrista campestris (FJ009829)

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68 78 100

Chamaecrista sp.(FJ009830) Chamaecrista anamariae (FJ009826) Chamaecrista jacobinea (FJ009827)

Chamaecrista chapadae (FJ009828) Chamaecrista belemii var. belemii (FJ009825) Chamaecrista cytisoides (FJ009844) Bauhinia ungulata (FJ009818) 0.05

Figure 1: Phylogenetic tree of Chamaecrista absus var absus; GenBank: KC817015.1; Locus: KC817015; 714 bp DNA linear. Chamaecrista absus var absus: internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene, complete sequence; and internal transcribed spacer 2, partial sequence. Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; eudicotyledons; core eudicotyledons; rosids; fabids; Fabales; Fabaceae; Caesalpinioideae; Cassieae; Chamaecrista.

Physico-chemical characterization of Chamaecrista absus seeds Physico-chemical properties of Chamaecrista absus seeds are summarized in Table 1. The moisture content of seeds is 4,26 % and the weight of thousand seeds (WTS) is 23,6g. The seeds of Chamaecrista absus contain 2,28% oil, 10,32 % of which are unsaponifiable fraction and 2,4 % of which are sterols. The Chamaecrista seeds exhibited relatively high protein contents (295 mg.g-1 FW). Using adequate buffers, we isolated all protein classes in seeds of Chamaecrista absus. and their content are : 48, 12, 32 and 8 % respectively for globulins, albumins, prolamins, and glutelins. Ledwani and Oberoi (2010) reported

that total oil content (%) in Cassia absus seeds was 3,5 % and the following physico-chemical properties were investigated : moisture (3,91%), ash (2,68%), refractive index (1,43), saponification value (160), iodine value (109), unsaponifiable matter (3,8 %) and sterol content in oil (2,9 %). Biochemical composition 1. Fatty acids composition: Fatty acid composition of Chamaecrista absus seed oil is given in Table 2 which shows that linoleic, palmitic oleic and linolenic acids account for more than 94% of the total fatty acids. The major saturated fatty acid in C. absus seed oil was Table 1: Physico-chemical characteristics Chamaecrista absus var absus seeds (Mean values, n = 3)

of

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Moisture (%) WTS (g) Oil content (g.100 g-1) Index acid Unsaponifiables (%) Sterol content (%) Total Proteines (mg.g-1 FW) Globulines (%) Albumines (%) Prolamines (%) Glutelines (%)

4,26 ± 0,21 23,6 ± 1,3 2,28 ±0,08 1,32 ± 0,04 10,32 ± 0,87 2,4 ± 0,1 295 ± 3,32 48 ± 2,13 12 ± 0,97 32 ± 1,57 8 ± 0,23

WTS: Weight of Thousand Seeds.

palmitic acid (17,72 %). This study has revealed that the oil of Chamaecrista absus var absus contained linoleic acid at high level 55,68% and it is considered as the major fatty acid. Saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) accounted respectively for 23,51 %, 13,98 % and 62,73 % of total fatty acids. The PUFA found were dominated by essential fatty acids Omega–6 (ω-6) and Omega–3 (ω-3) that is, linoleic acid (18:2n6; LA) and α-linolenic acid (18:3n3; ALA). The linolenic acid was found with a rate of 7,05 %. The ratio of unsaturated to saturated fatty acids (U/S) was 3,26 and the ratio of linoleic acid to linolenic acid (6/3) was 7,89. Table 2: Fatty acid compositions (% of total fatty acid) of Chamaecrista seeds. Fatty acids

%

C12 C14 C16 C16:1 C18 C18:1 C18:2 C18:3(3) C20 C20:1 SFA MUFA PUFA

0.06 ± 0,00 0.2 ± 0,00 17.72 ± 0,93 0.13 ± 0,00 4.6 ± 0,11 13.58 ± 0,57 55.68 ± 2,01 7.05 ± 0,29 0.58 ± 0,00 0.27 ± 0,00 23,51 ± 1,12 13,98 ± 0,39 62,73 ± 1,98

All values given are means of three determinations. SFA: saturated fatty acids; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acids.

This study shows that C. absus oil contain high rates of linoleic and linolenic acids, important unsaturated fatty acids which can be used in the

synthesis of tissue hormones, which regulate blood pressure and take part in immunological response (Ledwani and Oberoi, 2010). Linoleic acid has attracted interest in the scientific community because of its potential effects on body composition, by reducing body fat mass and increasing lean mass (Hernandez-Dıaz et al., 2010). Some authors found that some amount of linolenic acid is required for good-flavor compounds. This is due to the formation of oxidation products, which are important flavour compounds. The rate of oxidation of fats and oil is affected by many factors such as light, exposure to oxygen, the presence of antioxidants (tocopherols) and the degree of unsaturation of these fatty acids. In fact, PUFAs with a long chain are especially sought after (Sebei et al., 2007). 2. Sterols composition and triterpene alcohols: Very little information is available on sterols and triterpene alcohols composition of Chamaecrista absus var absus oil seed. Sterols attract the interest of food chemists because they are of great importance for food labeling and nutrition purposes and they are also characteristic of the genuineness of vegetable oils (Crane et al., 2005). Table 3 summarized the sterol compositions of C. absus var absus oil seed. In this study, we found that β-sitosterol, campesterol and stigmasterol were among the major components constituting about 95,35 % of total sterols, βsitosterol represented the main component of the phytosterols (63,23 %), followed by campesterol (17,45%) and stigmasterol (15,67%). Cholesterol, Δ5-Avenasterol and Δ5-24 stigmastadienol were present at lower levels. The sterol fractions obtained from the unsaponifiable oil of the seed oils of cassia absus were identified as stigmosterol, -sitosterol and cholesterol and the -sitosterol was found to be major sterol (53.42%) (Ledwani and Oberoi, 2010). β-sitosterol is the sterol marker in extra virgin olive oil and ranges from 75 to 87% of total sterols (Cercaci et al., 2003). Phytosterols have received particular attention because of their capability to lower serum cholesterol levels in humans and they may have beneficial effects against colon cancer (Awad et al., 2000; Hicks and Moreau, 2001). They are also considered to have anti-inflammatory, antibacterial, anti-ulcerative and antitumor properties (Beveridge et al., 2002; Herchi et al., 2009).

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We revealed also that cycloartenol,  amyrine ans 24 methylen-cycloartenol were the major components constituting about 76,85 % of total triterpene alcohols. Triterpene alcohols were found to be minor components of the unsaponifiable matter compared to sterols. The major triterpene alcohols compounds, at complete maturity of olive, are 24-methylene cycloartenol and cycloartenol (Azadmard-Damirchi and Dutta, 2006). Cycloartenol is formed as the first cyclic triterpenoid precursor of sterols and is the substrate for the first methylation reaction, resulting in 24methylene cycloartenol. -Amyrin and cycloartenol have 2,3(S)-oxidosqualenes as the same biosynthetic precursor molecule (Sakouhi et al., 2009). 3. Phenols analysis and antioxidant activities: The total phenols, total flavonoids and condensed tannin contents, are presented in Table 4. Phenolic compounds, plant secondary metabolites, are capable of direct chain breaking antioxidant action by radical scavenging. In addition to having the potential for independent antioxidant action, polyphenols have been suggested to spare essential antioxidants (Bhalodia et al., 2011). The total phenolic content of Chamaecrista absus var absus seeds was at the rate of 7, 56 mg GAE/g DW. To our knowledge, no data are available on the phenolic content and composition of this specie. RP-HPLC study of Chamaecrista absus seeds extract revealed the identification of 8 phenolic compounds with p-coumaric acid as the major compound (6,98%). The identified compounds were : chlorogenic acid, dihydroxyphenylacetic acid, syringic acid, p-coumaric acid, rutin trihydrate, nephtoresorinol, trans-2dihydroxycinamic acid and dehydrate quercetin. Phenolic compounds have been reported to be present in all vegetable oils, which is very important for the oxidative stability of the polyunsaturated fatty acids of these oils. Additionally, edible oils rich in natural antioxidants may play a role in reducing the risk of chronic diseases. Thus, the oils examined may be used in different food applications to provide nutrition and health benefits (Siger et al., 2008). Flavonoid content in Chamaecrista absus var absus seeds was relatively important: 1,113 mg CE g−1 DW. Condensed tannins were present in the same abundance than flavonoids : 0,527 mg CE/g

DW. Concerning DPPH scavenging activity, a considerable antiradical ability was found (IC50 = 16,78 μg/ml) (Table 4). Several authors have reported a positive and significant relationship between the antioxidant components including phenols, polyphenols and tannins, respectively with the reducing power and DPPH radical scavenging capacity (Ksouri et al., 2008; Faleh et al., 2008). Irwin and Barnaby (1982) revealed that, in Chamaecrista absus, the anthraquinones chrysophanol and emodin were isolated from the roots, and the flavonoids quercetin and rutin were isolated from the leaves, chrysophanol and emodin have laxative activities. The seeds of Chamaecrista absus contain the flavonoids apigenin, luteolin, hydnocarpin and iso-hydnocarpin. These flavonoids showed anti- tumor activities in vitro, and some also in vivo. 4. Antibacterial activity Chamaecrista absus var absus seed oil showed antibacterial activity against almost studied strains. We noticed that sterols exhibited antibacterial activities against strains Listeria ivanovii, Listeria inocua, Escherichia coli, staphylococcus aureus, Bacillus subtilis and Bacillus cereus with major activity against Listeria ivanovii and Bacillus subtilis. Triterpenic alcohols exhibited antibacterial activities against strains Listeria ivanovii, Listeria inocua, Escherichia coli, Bacillus subtilis and Bacillus cereus (Table 5). Antibacterial assay of phenolic compounds of the Chamaecrista tora seeds and structurally related compounds revealed that several naphthalenes and anthraquinones have antibacterial effects on strains of methicillinresistant Staphylococcus aureus (Hatano et al., 1999).

CONCLUSIONS This study confirmed that the seeds known as “Habbat Elbaraka” or “Al-Habba Essawda” belonged to a species of the genus Chamaecrista and that this species has been identified, by genetic methods, such as Chamaecrista absus var. absus. This is the first time that a scientific study has been focused on the biochemical composition, antioxidant and antibacterial activities of this species seeds. In summary, this study has revealed

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Table 3: Sterols and triterpene alcohols composition (%) of Chamaecrista absus var absus seeds. Sterols

%

Cholesterol Campesterol Stigmasterol  Sitosterol -5-24, Stigmastadienol -5-Avensterol Triterpene alcohols

2.82 ± 0,15 17.45 ± 0,61 15.67 ± 0,71 63.23 ± 1,98 0.35 ± 0,00 0.31 ± 0,00 %

14,54 ± 0,69  Amyrine 13,5 ± 0,71 Cycloartenol 48,81 ± 1,31 24 methylene -Cycloartenol All values given are means of three determinations.

Table 4: Total polyphenol, flavonoid and condensed tannin contents and antioxidant activities (DPPH· scavenging ability and reducing power) of Chamaecrista absus var absus seeds (Mean values, n = 3). Total Phenolic contents (mg GAE/g DW) 7,56 ± 0,32 Flavonoid contents (mg CE/g DW) 1,113 ± 0,08 Tanin contents (mg CE/g DW) 0,527 ± 0,02 DPPH· scavenging activity (IC50 μg/ml) 16,78 ± 0,06

Table 5: Antibacterial activity of sterols and Triterpene alcohols. Sterols (ID) Listeria ivanovii + + (12 mm) (RBL30) Listeria inocua + (10 mm) (RBL29) Escherichia coli + (10 mm) (ATCC25922) Pseudomonas eruginosa _ (ATCC15442) Staphylococcus aureus + (8 mm) (ATCC6533) Enterococcus hirae _ (ATCC10541) Bacillus subtilis + + (12 mm) (168) Bacillus cereus + (10 mm) (ATCC11778) (-) : Inactive; (+) : Inhibition diameter (ID) between 8–10 mm.

Triterpene alcohols (ID) + (10 mm) + (8 mm) + (9 mm) _ _ _ + (9 mm) + (10 mm)

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that Chamaecrista absus var absus seed are a rich source of fatty acids (polyunsaturated fatty acids (3 and 6), unsaponifiable matter which contained an important range of phytosterols and terpene alcohols. We showed an important and a wide range of polyphenol contents, antioxidant capacities and antibacterial activities. These important components and capacities have good nutritional attributes and appear to have a very positive effect on human health. These seeds can be used, also, in feed, cosmetic, aromatherapy and pharmaceutical fields.

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