Journal of Plant 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 Cassia 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 J. Plant Bio. Res. 2014, 3(1): 1-11 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 plants 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 tree was constructed with 1000 bootstraps and detector (FID) on a capillary column coated with using Bauhinia 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.