Journal of Biology Research 2014, 3(1): 1-11 eISSN: 2233-0275 pISSN: 2233-1980 http://www.inast.org/jpbr.html

REGULAR ARTICLE

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. 1Unité de Biochimie des Lipides et Interactions avec les Macromolécules, Faculté des Sciences de Tunis, Université de Tunis–El-Manar. Tunisia. 2Laboratoire de Microorganismes et Biomolécules Actives, Faculté des Sciences de Tunis, Université de Tunis–El- Manar. Tunisia. 3Laboratoire de Protéomie et de biopréservation des aliments. ISSBAT. Université de Tunis–El-Manar. Tunisia.

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 Leguminosae, Chamaecrista sect. absus is further divided into four subsections: Absus, Chamaecrista belongs to subtribe Cassiinae Adenophyllum, Otophyllum and Baseophyllum (Caesalpinioideae), and it comprises over 330 (Coutinho et al., 2012). There is also disagreement species, divided into six sections (Torres et al., about sister-group relationships in Cassiinae. One 2011). The genus Chamaecrista, formerly defined hypothesis considers Chamaecrista a clade distinct as subgenus Lasiorhegma (Irwin and from its sister taxa Senna and Cassia (De Souza Barneby, 1982), has been divided into six sections Conceição et al., 2009). However, other studies of very unequal sizes (Absus, Apoucouita, (Herendeen et al., 2003) indicate that Senna and Caliciopsis, Chamaecrista, Grimaldia and Chamaecrista are sister taxa and that Cassia Xerocalyx* ). Placed in the large family Corresponding author: Khaled SEBEI occurs in a distinct clade (Torres et al., 2011). Corresponding author e-mail: [email protected] Tel: +216 98 925 824 Fax: +216 71 573 526

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According to Irwin and Barneby (1982), regarded as the main dietary phenolic compounds Chamaecrista sect. absus is known as another (Manach et al., 2004). These compounds exhibit a synonym: Cassia absus that belongs to family wide range of physiological properties, such as Caesalpiniaceae and commonly known as Chaksu anti-allergic, anti-atherogenic, anti-inflammatory, in the traditional system of medicine. The seeds are antimicrobial, antioxidant, anti-thrombotic, used in the treatment of ophthalmia, and are cardioprotective, and vasodilatory effects considered as attenuant, astringent and (Balasundram, 2006; Falleh et al., 2008). hypotensive. Seeds are also utilized in ringworm The aim of the present study was, firstly, to infestations, in conjunctivitis and other skin identify, by molecular methods, of black seeds infections (Usmanghani et al., 1989). Reported known, for a long time, in Tunisia as “Al-Habba constituents are the alkaloid chaksine (Voelter et Al-Sawdaa”. In the second hand, the objectives of al., 1985) as well as oils, fatty acids, sterols, this work were to determine the biochemical glycosides, amino acids, gum, resin and composition of chamaecrista oil seed, total unsaponifiable matter (Aftab et al., 1996). Cassia polyphenol, flavonoid, condensed tannin contents marginata and Cassia corymbosa, contain palmitic and antibacterial activities of sterols and terpene acid C16 (17.3 and 17.2%), palmitoleic alcohols. C16:1(trace and 7.4%), stearic acid C18 (4.5 and 4.2%), oleic acid C18:1 (14.2 and 14.8%) and MATERILS AND METHODS linoleic acid C18:2 (49.4 and 41.2%) respectively (Hosamani and Sattigeri, 2002). Four fatty acids: Plant material palmitic, stearic, oleic and linoleic acids (along The seeds of Chamaecrista absus var absus with minor percentage of linolenic acid C18:3 in commonly known as Habbat elbaraka or Elhabba Cassia roxburghii and Cassia absus) were essawda in the traditional system of Tunisian folk identified in all five species. The tested seed oil medicine, were procured from the local herbal contained mainly unsaturated fatty acids i.e. market (Souk Elblat) in Tunis, Tunisia. linoleic acid in the range from 45.96% to 60.25% DNA extraction, PCR amplification and and oleic acid from 26.29% to 34.91%. Palmitic Sequencing acid was the most abundant saturated acid. Three Seed being superficially disinfected by shaking in sterols: cholesterol, stigmasterol and -sitosterol 30% (v/v) H2O2 for 5 min and aseptically rinsed were found in all species. -sitosterol was found to several times with sterile water. Total genomic be a major sterol in C. javanica (67.50 %), C. alata DNA was extracted from plant seeds by grounding (64.79 %), C. roxburghii (63.29 %) and C. absus in liquid dinitrogen and digested for 1 hour at 65°C (53.42%) while sigmasterol was major in C. in CTAB extracting buffer (2% w/v CTAB, 100 laevigata (45.34 %) (Ledwani and Oberoi, 2010). mM Tris-HCl, pH 8.0, 20 mM EDTA, 1.4 M NaCl, To our knowledge, no data are available on and 0.2% v/v 2-mercaptoethanol). After the biochemical composition and antioxidant chloroform- isoamylic alcohol (24/1: v/v) DNA activity of Chamaecrista absus var. absus seeds. was resuspended in 50 μl of TE buffer (10 mM However, Hatano et al., (1999) reported that Tris-HCl pH 7.4; 1 mM of EDTA) and stored at phenolic constituents of of the family −20°C. The internal transcribed spacer (ITS) was Leguminosae have been found to have various amplified using the ITS1 and ITS4 primers (White biological or pharmacological actions including et al., 1990). The amplification reactions was radical-scavenging effects, inhibitory effects on performed in a 50-μl volume of reaction mixture enzymes and antimicrobial effects. These authors [1 mM of each primer, 0.2 mM of each dNTP, and revealed the isolation of new compounds related to 2.5 U of Taq polymerase (Promega, Madison, WI) condensed tannins from Cassia nomame and the in a DNA thermal cycler (2400 geneAmp PCR inhibitory effects of phenolic constituents of C. thermocycler; Perkin Elmer, Foster City, CA) with nomame on lipase and isolated six new phenolic the following program: an initial denaturation at glycosides from seeds of Cassia tora, which have 95°C for 2 min, followed by 35 cycles of a 1-min been used as a traditional medicine for eye denaturation at 94°C, a 40-s annealing at either diseases and intestinal disorders in Asian countries. 53°C and a 1-min elongation at 72°C, with a final Phenolic acids, flavonoids, and tannins are elongation step at 72°C for 10 min. Amplification 2

J. Plant Bio. Res. 2014, 3(1): 1-11 products were analyzed in 1.5% agarose gel in with 2,7-dichlorofluorescein and viewed under UV 0.5× TBE buffer (89 mmol l−1 Tris, 89 mmol l−1 light. borate, 2 mmol l−1 EDTA), stained with ethidium Silylation of triterpene alcohols and sterols bromide, and visualized under UV light (Sambrook fraction et al., 1989). The PCR product was then purified An amount of 2 mg of methylsterols residue was using QIAquick Wizard PCR purification Kit mixed with 125 µl of BSTFA (with 1% TMCS), (Promega) according to the manufacturer‟s 125 µl of pyridine and 450 µl of acetone, the instructions, and subjected to cycle sequencing mixture vortexed for about 10 s and heated at 70 using the Taq Dye Deoxy Terminator Cycle °C for 20 min. After silylation reaction, 1.5 ml of Sequencing kit (Applied Biosystems; HTDS, chloroform was added to the mixture and 1 µl of Tunisia) and fragment separation in an ABI the solution was directly injected into a gas PrismTM 3130 DNA sequencer (Applied chromatograph. Biosystems). The ITS rDNA nucleotide sequence Gas chromatography–flame ionisation detection was compared to sequences listed in the GenBank (GC–FID) database using the BLAST program (Altschul et Fatty acids were methylated using the method of al., 1990). Alignment with retrieved related Metcalfe et al. (1966) modified by Lechevallier sequences and phylogenetic analysis were (1966). Methyl esters were analyzed by gas performed using MEGA version 5.1 program chromatography (GC) using an HP 4890 (Tamura et al., 2011) and a maximum-likelihood chromatograph equipped with a flame ionization was constructed with 1000 bootstraps and detector (FID) on a capillary column coated with using ungulata (FJ009818) sequence as supelcowax TM 10 (30 m length, 0.25 id, 0.2 mm the outgroup. The sequence data was submitted to film thickness). Temperatures of column, detector GenBank (NCBI) which have provided a GenBank and injector were: 200°C, 250°C and 260°C, accession number for our nucleotide respectively. sequence: KC817015. RP-HPLC phenols analysis Determination of oil content Colorimetric quantification of total phenolics was Oil content was determined by extracting dry determined, as described by Dewanto et al., material of Chamaecrista absus seeds with (2002). All samples were analyzed in three petroleum ether using a Soxhlet apparatus replications. Phenolic compound analysis was (Harwood, 1984). This extraction takes 4 h at 42°C carried out using a liquid chromatography RP- and repeated three times for only one sample. The HPLC coupled with a UV-vis Multiwavelength extract was dried in a rotary evaporator at 32°C. detector „waters 996 photoperiode Array Detector: Oil was weighed and stored at -10 °C. The oil 190-400 nm. The separation was carried out on content was determined as different in weight of 250×4.6-mm, LD 5-μm symetry shield C18 dried peanut sample before and after the extraction reversed phase column. The mobile phase (AOCS, 1989). consisted of acetonitrile (solvent A) and water Saponification and TLC analysis (80:20 v/v) with 1 % formic acid (solvent B). The Unsaponifiable fraction of lipids was determined flow rate was kept at 1 ml/min. The injection by saponifying 5 g of oil mixed with both 200 µl volume was 20 μl and peaks were monitored at -cholestanol and an ethanolic KOH 12% 275 nm. The peaks were identified by congruent solution; the mixture was heated at 60°C for 1.30 retention times compared with standards. Analyses h. The unsaponifiable matter was extracted, were performed in triplicates. washed, dried over anhydrous Na2SO4 and DPPH radical-scavenging activity evaporated to dryness using N2. The The DPPH· quenching ability of seed extracts was unsaponifiable matter was separated into measured according to Hanato et al. (1988). One subfractions on preparative silica gel thin-layer ml of the extract at different concentrations was plates (silica gel 60G F254) using one-dimensional added to 0.5 mL of a DPPH· methanol solution. TLC with hexane–diethyl ether (6:4, v/v) as the The mixture was shaken vigorously and left developing solvent. The unsaponifiable fraction standing at room temperature for 30 min in the diluted in chloroform was applied on the silica gel dark. The absorbance of the resulting solution was plates. After development, the plate was sprayed then measured at 517 nm. The antiradical activity 3

J. Plant Bio. Res. 2014, 3(1): 1-11 was expressed as IC50 (μg/mL), the antiradical The fractions of lipids were individually tested dose required to cause a 50% inhibition. A lower against a large panel of microorganisms including IC50 value corresponds to a higher antioxidant Staphylococcus aureus (ATCC 6539), activity of plant extract. The ability to scavenge the Pseudomonas aeruginosa (ATCC 15442), DPPH radical was calculated using the following Escherichia coli (ATCC 25922), Enterococcus equation: (1) DPPH· scavenging effect (%) = [(A0 hirae ATCC10541, Listeria ivanovii and L. inocua − A1)/A0]×100; where A0 is the absorbance of the (RBL 30 and RBL 29 respectively), Bacillus control at 30 min, and A1 is the absorbance of the subtilis and B. cereus (168 and ATCC11778 sample at 30 min. All samples were analyzed in respectively). All strains were obtained from three replications. Institut Pasteur de Tunis. The Bacteriological agar Total flavonoids content was from Biokar Diagnostics (Beauvais, France). Total flavonoids were measured using a Nutrient broth (NB) was from Difco (Becton colorimetric assay developed by Dewanto et al. Dickinson, Le Pont de Claix, France). All the other (2002). An aliquot of diluted sample or standard media, used in this study, were manufactured by solution of (+)-catechin was added to 75 μl of Biorad (Marnes-La Coquette, France) and Merck. NaNO2 solution (7%), and mixed for 6 min, before Antibacterial activity is revealed by growth adding 0.15 ml AlCl3 (10%). After 5 min, 0.5 ml of inhibition in the strains to test. This activity is 1 M NaOH solution was added. The final volume observed in solid solid media. In the present work, was adjusted to 2.5 ml, thoroughly mixed, and the we have been using the well diffusion absorbance of the mixture was determined at 510 method described by Perez et al. (1990). nm. Total flavonoids were expressed as mg (+)- Statistical analysis catechin equivalent g−1 DW (mg CE g−1 DW), The data (three replicates) were statistically through the calibration curve of (+)-catechin (0– evaluated using the JMP SAS version 12.6 400 μg ml−1 range). All samples were analyzed in software (Statistical Analysis System). (SAS, three replications. Institute INC, Box 8000, Cary, North Carolina Total condensed tannins 27511, USA). Procyanidins were measured using the modified vanillin assay described by Sun et al., (1998). RESULTS AND DISCUSSION Three milliliters of 4% methanol vanillin solution Molecular analysis and 1.5 ml of concentrated H2SO4 were added to 50 μl of suitably diluted sample. The mixture was For a long time, it was thought that the seeds allowed to stand for 15 min, and the absorbance whose vernacular name in Tunisia and in Arabia is was measured at 500 nm against methanol as a “Elhabba Essawda” or “Habbat Elbaraka” belong blank. The amount of total condensed tannins was to Nigella genus (AL Gaby, 1998). This is the first expressed as mg (+)-catechin equivalent g-1 DW time that a scientific study focused on the (Ksouri et al., 2008). All samples were analyzed in identification of these seeds wish used in the three replications. traditional system of Tunisian folk medicine, also, Analysis of proteins in gastronomy, cosmetic, aromatherapy and Defatted Chamaecrista absus seeds (100 mg) was pharmaceutical fields. In this study we used the successively extracted with 1 ml distilled water, 1 rDNA intergenic spacers ITS to determine the ml 5.0 M NaCl, 1 ml absolute ethanol, and 1 ml phylogenetic position of plant seeds (Baldwin et 0.2 M phosphate buffer (pH 8.0) for the extraction al., 1995). The analysis was inferred from of the albumin, globulin, prolamin, and glutelin, comparative ITS sequences analysis of 19 related respectively. Each extraction was shaken for 20 species based on Maximum likelihood method min in an Eppendorf tube and centrifuged at (Figure 1). The alignment of 662 pb produced a 10,000 g for 6 min. Each protein fraction assay topology with high bootstrap supports (100%), was performed following Bradford‟s method 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) 9972 Chamaecrista speciosa (FJ009839) 86 51 98 Chamaecrista philippi (FJ009838) Chamaecrista dalbergiifolia (FJ009837) 57 Ts 100 Chamaecrista absus (FJ009832) Chamaecrista_sp. (FJ009831) 68 Chamaecrista campestris (FJ009829) 78 Chamaecrista sp.(FJ009830) 100 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; ; fabids; ; ; Caesalpinioideae; Cassieae; Chamaecrista.

Physico-chemical characterization of that total oil content (%) in Cassia absus seeds was Chamaecrista absus seeds 3,5 % and the following physico-chemical Physico-chemical properties of Chamaecrista properties were investigated : moisture (3,91%), absus seeds are summarized in Table 1. The ash (2,68%), refractive index (1,43), saponification moisture content of seeds is 4,26 % and the weight value (160), iodine value (109), unsaponifiable of thousand seeds (WTS) is 23,6g. The seeds of matter (3,8 %) and sterol content in oil (2,9 %). Chamaecrista absus contain 2,28% oil, 10,32 % of Biochemical composition which are unsaponifiable fraction and 2,4 % of 1. Fatty acids composition: which are sterols. The Chamaecrista seeds Fatty acid composition of Chamaecrista absus exhibited relatively high protein contents (295 seed oil is given in Table 2 which shows that mg.g-1 FW). Using adequate buffers, we isolated linoleic, palmitic oleic and linolenic acids account all protein classes in seeds of Chamaecrista absus. for more than 94% of the total fatty acids. The and their content are : 48, 12, 32 and 8 % major saturated fatty acid in C. absus seed oil was respectively for globulins, albumins, prolamins, Table 1: Physico-chemical characteristics of and glutelins. Ledwani and Oberoi (2010) reported Chamaecrista absus var absus seeds (Mean values, n = 3) 5

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Moisture (%) 4,26 ± 0,21 synthesis of tissue hormones, which regulate blood WTS (g) 23,6 ± 1,3 pressure and take part in immunological response Oil content (g.100 g-1) 2,28 ±0,08 (Ledwani and Oberoi, 2010). Linoleic acid has Index acid 1,32 ± 0,04 attracted interest in the scientific community Unsaponifiables (%) 10,32 ± 0,87 because of its potential effects on body Sterol content (%) 2,4 ± 0,1 -1 composition, by reducing body fat mass and Total Proteines (mg.g FW) 295 ± 3,32 increasing lean mass (Hernandez-Dıaz et al., Globulines (%) 48 ± 2,13 2010). Some authors found that some amount of Albumines (%) 12 ± 0,97 linolenic acid is required for good-flavor Prolamines (%) 32 ± 1,57 compounds. This is due to the formation of Glutelines (%) 8 ± 0,23 WTS: Weight of Thousand Seeds. oxidation products, which are important flavour palmitic acid (17,72 %). This study has revealed compounds. The rate of oxidation of fats and oil is that the oil of Chamaecrista absus var absus affected by many factors such as light, exposure to contained linoleic acid at high level 55,68% and it oxygen, the presence of antioxidants (tocopherols) is considered as the major fatty acid. Saturated and the degree of unsaturation of these fatty acids. fatty acids (SFA), monounsaturated fatty acids In fact, PUFAs with a long chain are especially (MUFA) and polyunsaturated fatty acids (PUFA) sought after (Sebei et al., 2007). accounted respectively for 23,51 %, 13,98 % and 2. Sterols composition and triterpene alcohols: 62,73 % of total fatty acids. The PUFA found were Very little information is available on sterols and dominated by essential fatty acids Omega–6 (ω-6) triterpene alcohols composition of Chamaecrista and Omega–3 (ω-3) that is, linoleic acid (18:2n6; absus var absus oil seed. Sterols attract the interest LA) and α-linolenic acid (18:3n3; ALA). The of food chemists because they are of great linolenic acid was found with a rate of 7,05 %. importance for food labeling and The ratio of unsaturated to saturated fatty acids nutrition purposes and they are also characteristic (U/S) was 3,26 and the ratio of linoleic acid to of the genuineness of vegetable oils (Crane et al., linolenic acid (6/3) was 7,89. 2005). Table 3 summarized the sterol compositions of C. absus var absus oil seed. In this study, we Table 2: Fatty acid compositions (% of total fatty found that β-sitosterol, campesterol and acid) of Chamaecrista seeds. stigmasterol were among the major components constituting about 95,35 % of total sterols, β- Fatty acids % sitosterol represented the main component of the phytosterols (63,23 %), followed by campesterol C12 0.06 ± 0,00 (17,45%) and stigmasterol (15,67%). Cholesterol, C14 0.2 ± 0,00 Δ5-Avenasterol and Δ5-24 stigmastadienol were C16 17.72 ± 0,93 present at lower levels. The sterol fractions C16:1 0.13 ± 0,00 obtained from the unsaponifiable oil of the seed C18 4.6 ± 0,11 oils of cassia absus were identified as C18:1 13.58 ± 0,57 C18:2 55.68 ± 2,01 stigmosterol, -sitosterol and cholesterol and the -sitosterol was found to be major sterol (53.42%) C18:3(3) 7.05 ± 0,29 C20 0.58 ± 0,00 (Ledwani and Oberoi, 2010). β-sitosterol is the C20:1 0.27 ± 0,00 sterol marker in extra virgin olive oil and ranges SFA 23,51 ± 1,12 from 75 to 87% of total sterols (Cercaci et al., MUFA 13,98 ± 0,39 2003). Phytosterols have received particular PUFA 62,73 ± 1,98 attention because of their capability to lower serum All values given are means of three determinations. SFA: cholesterol levels in humans and they may have saturated fatty acids; MUFA: monounsaturated fatty acid; beneficial effects against colon cancer (Awad et PUFA: polyunsaturated fatty acids. al., 2000; Hicks and Moreau, 2001). They are also considered to have anti-inflammatory, anti- This study shows that C. absus oil contain high bacterial, anti-ulcerative and antitumor properties rates of linoleic and linolenic acids, important (Beveridge et al., 2002; Herchi et al., 2009). unsaturated fatty acids which can be used in the 6

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We revealed also that cycloartenol,  amyrine DW. Concerning DPPH scavenging activity, a ans 24 methylen-cycloartenol were the major considerable antiradical ability was found (IC50 = components constituting about 76,85 % of total 16,78 μg/ml) (Table 4). Several authors have triterpene alcohols. Triterpene alcohols were found reported a positive and significant relationship to be minor components of the unsaponifiable between the antioxidant components including matter compared to sterols. The major triterpene phenols, polyphenols and tannins, respectively alcohols compounds, at complete maturity of olive, with the reducing power and DPPH radical are 24-methylene cycloartenol and cycloartenol scavenging capacity (Ksouri et al., 2008; Faleh et (Azadmard-Damirchi and Dutta, 2006). al., 2008). Cycloartenol is formed as the first cyclic Irwin and Barnaby (1982) revealed that, in triterpenoid precursor of sterols and is the substrate Chamaecrista absus, the anthraquinones for the first methylation reaction, resulting in 24- chrysophanol and emodin were isolated from the methylene cycloartenol. -Amyrin and roots, and the flavonoids quercetin and rutin were cycloartenol have 2,3(S)-oxidosqualenes as the isolated from the , chrysophanol and emodin same biosynthetic precursor molecule (Sakouhi et have laxative activities. The seeds of Chamaecrista al., 2009). absus contain the flavonoids apigenin, luteolin, 3. Phenols analysis and antioxidant activities: hydnocarpin and iso-hydnocarpin. These The total phenols, total flavonoids and condensed flavonoids showed anti- tumor activities in vitro, tannin contents, are presented in Table 4. Phenolic and some also in vivo. compounds, plant secondary metabolites, are 4. Antibacterial activity capable of direct chain breaking antioxidant action Chamaecrista absus var absus seed oil showed by radical scavenging. In addition to having the antibacterial activity against almost studied strains. potential for independent antioxidant action, We noticed that sterols exhibited antibacterial polyphenols have been suggested to spare essential activities against strains Listeria ivanovii, Listeria antioxidants (Bhalodia et al., 2011). The total inocua, Escherichia coli, staphylococcus aureus, phenolic content of Chamaecrista absus var absus Bacillus subtilis and Bacillus cereus with major seeds was at the rate of 7, 56 mg GAE/g DW. To activity against Listeria ivanovii and Bacillus our knowledge, no data are available on the subtilis. Triterpenic alcohols exhibited antibacterial phenolic content and composition of this specie. activities against strains Listeria ivanovii, Listeria RP-HPLC study of Chamaecrista absus seeds inocua, Escherichia coli, Bacillus subtilis and extract revealed the identification of 8 phenolic Bacillus cereus (Table 5). compounds with p-coumaric acid as the major Antibacterial assay of phenolic compounds of compound (6,98%). The identified compounds the Chamaecrista tora seeds and structurally were : chlorogenic acid, dihydroxyphenylacetic related compounds revealed that several acid, syringic acid, p-coumaric acid, rutin naphthalenes and anthraquinones have trihydrate, nephtoresorinol, trans-2- antibacterial effects on strains of methicillin- dihydroxycinamic acid and dehydrate quercetin. resistant Staphylococcus aureus (Hatano et al., Phenolic compounds have been reported to be 1999). present in all vegetable oils, which is very important for the oxidative stability of the polyunsaturated fatty acids of these oils. CONCLUSIONS Additionally, edible oils rich in natural This study confirmed that the seeds known as antioxidants may play a role in reducing the risk of “Habbat Elbaraka” or “Al-Habba Essawda” chronic diseases. Thus, the oils examined may be belonged to a species of the genus Chamaecrista used in different food applications to provide and that this species has been identified, by genetic nutrition and health benefits (Siger et al., 2008). methods , such as Chamaecrista absus var. absus. Flavonoid content in Chamaecrista absus var This is the first time that a scientific study has been absus seeds was relatively important: 1,113 mg CE focused on the biochemical composition, g−1 DW. Condensed tannins were present in the antioxidant and antibacterial activities of this same abundance than flavonoids : 0,527 mg CE/g 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 2.82 ± 0,15 Campesterol 17.45 ± 0,61 Stigmasterol 15.67 ± 0,71  Sitosterol 63.23 ± 1,98 -5-24, Stigmastadienol 0.35 ± 0,00 -5-Avensterol 0.31 ± 0,00 Triterpene alcohols %  Amyrine 14,54 ± 0,69 Cycloartenol 13,5 ± 0,71 24 methylene -Cycloartenol 48,81 ± 1,31 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) Triterpene alcohols (ID) Listeria ivanovii + + (12 mm) + (10 mm) (RBL30) Listeria inocua + (10 mm) + (8 mm) (RBL29) Escherichia coli + (10 mm) + (9 mm) (ATCC25922) Pseudomonas eruginosa _ _ (ATCC15442) Staphylococcus aureus + (8 mm) _ (ATCC6533) Enterococcus hirae _ _ (ATCC10541) Bacillus subtilis + + (12 mm) + (9 mm) (168) Bacillus cereus + (10 mm) + (10 mm) (ATCC11778) (-) : Inactive; (+) : Inhibition diameter (ID) between 8–10 mm.

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J. Plant Bio. Res. 2014, 3(1): 1-11 that Chamaecrista absus var absus seed are a rich fistula Linn. I. J. of PharmTech Research. source of fatty acids (polyunsaturated fatty acids Vol. 3, No.1, pp 589-599.

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