2, 6-Bis (1.1-dimethylethyl)-4-methylphenol (BHT) is a synthetic antioxidant used generally for food, cosmetics and pharmaceuticals. The leaf extract from the halophyte plant, Mesembryanthemumcrystallinum, was fractionated by using semi-preparative HPLC. The different fractions were tested for their antioxidant activity using DPPH method. One fraction exhibited a high level of antioxidant activity.The molecule responsible for this antioxidant activity was identified as 2, 6-bis (1.1-dimethylethyl)-4-methylphenol) by gas chromatography/mass spectroscopy (GC/MS)

Abdelly, C

Bouftira, I

Sfar, S

English

AJOL - African Journals Online

African Journal of Biotechnology Vol. 6 (9), pp. 1136-1139, 2 May 2007 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2007 Academic Journals     Full Length Research Paper  Identification of a naturally occurring 2, 6-bis (1.1-dimethylethyl)-4-methylphenol from purple leaves of the halophyte plant Mesembryanthemum crystallinum  I. Bouftira1,2, C. Abdelly2 and S. Sfar1  1Laboratory of Galenic Pharmacy. Faculty of Pharmacy, Monastir. Tunisia. 2Laboratory of Plant Adaptation to Abiotic Stresses, Center of Biotechnology, Technoparc of Borj Cédria, Tunisia.  Accepted 1 March, 2007  2, 6-Bis (1.1-dimethylethyl)-4-methylphenol (BHT) is a synthetic antioxidant used generally for food, cosmetics and pharmaceuticals. The leaf extract from the halophyte plant, Mesembryanthemum crystallinum, was fractionated by using semi-preparative HPLC. The different fractions were tested for their antioxidant activity using DPPH method. One fraction exhibited a high level of antioxidant activity. The molecule responsible for this antioxidant activity was identified as 2, 6-bis (1.1-dimethylethyl)-4-methylphenol) by gas chromatography/mass spectroscopy (GC/MS).   Key words: BHT, HPLC, GC/MS, antioxidant activity, DPPH   INTRODUCTION  Solar radiation is a prerequisite for life on earth as a major source of energy driving the photosynthetic process (Edreva, 2005). Plants have evolved a range of avoidance and tolerance strategies against excess light and UV-radiation (Jansen, 2002). Light stress may trigger pyrimidine dimerization (Britt, 1996) and cause peroxide-ation of lipids, which inevitably leads to membrane damage by oxygen radical formation (Comporti, 1989; Jansen et al., 1998). Naturally generated reactive oxygen species (ROS) are molecules that can attack cell compo-nents and create several types of biological damage (Hutadilok-Towatana et al., 2006). They play important roles in the pathogenesis of various diseases in human ranging from carcinogenesis to aging (Ames et al., 1993). Many plant species have been investigated in search for novel antioxidant compounds (Pourmorad et al., 2006). It has been mentioned that the antioxidant activity of plant might be due to their phenolic compounds (Cook and Samman, 1996). There are some synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole  (BHA),   commonly   used   in  processed     *Corresponding authors E-mail: m.pillay@yahoo.co.uk. or ibtissem.bouftira@laposte.net. Tel: (0216) 73461000. Fax: (0216) 73461830. foods (Yildrim et al., 2001). However, it was suggested that these compounds have some side effects (Branian, 1975; Ito et al., 1983). The halophyte plant Mesembryanthemum crystallinum L. (ice plant) (Aizoacea family) has been established as an extremely stress tolerant model system (Ibdah et al., 2002). Upon irradiation with high light irradiance, M. crystallinum displays a rapid cell-specific accumulation of plant secondary metabolites in the upper leaf epidermis; a phenomenon that is not detectable with salt or drought stress. M. crystallinum induced betacyanin formation and simultaneous formation of flavonol conjugates (Ibdah et al., 2002). A convenient, rapid and sensitive method for the anti-oxidant screening of plant extracts is a free-radical scave-nging assay using 1, 1 diphenyl-2-picryl-hydrazyl (DPPH) stable radical spectrophotometrically. In the presence of an antioxidant, DPPH radical obtains one more electron and the absorbance decreases (Koleva et al., 2002). The aim of this study was to identify antioxidant mole-cules from purple leaves extract of the halophyte M. crys-tallinum.   MATERIAL AND METHODS  Plant material  Arial parts containing betalain pigment of M. crystallinum were coll-     ected from their native biotope in Monastir (Tunisia) coasts in June 2005.   Extraction   Twenty five grams of freshly leaves were homogenized with 100 ml of 80% aqueous methanol, containing 50 mM ascorbic acid. After centrifugation at 14000 x g, 4°C, 10 min, the supernatants was removed and fractionated by semi-preparative HPLC.    Chromatography  Semi-preparative high performance chromatography (HPLC)  A Shimadzu UV detector HPLC was used to separate leaves extract with a gradient elution at 0 min 10% B, 20 min 50% B, 30 min 50% B, 35 min 80% B, 40 min 80% B and 45 min 10% B; solvent A: water (0.1% formic acid) and B: 30/70 water/acetonitrile (0.1% formic acid). (Injection volume: 20 µl; detection at 540 nm).   Gas chromatography / mass spectroscopy (GC/MS)  A Hewlett 5890 Packard series II gas chromatograph with a detector of mass spectroscopy 5972 series was used to separate the extract fraction which contains the maximum of antiradical activity. A HP-Innowax 30 m x 0.25 mm x 0.25 µm polar columns was used; hold 1 min, 5°/min to 250°C.   Radical scavenging activity  Free radical scavenging activity of plant extract was determined by using a stable free radical, (1, 1-diphenyl-2-picryl-hydrazyl) DPPH (Blois, 1958). DPPH solution was prepared at a concentration of 0.024 mg/ml in ethanol. During assay 1 ml of the crude extract was mixed with 1 ml DPPH solution. The mixture was incubated in the room temperature for 30 min; absorbance was recorded at 517 nm (Cam spec M230/330 UV visible spectrophotometer, United Kingdom). Synthetic butylated hydroxytoluene (BHT) was used as a standard for the investigation of the antiradical activity. The percentage of remaining DPPH•(%DPPH REM) at the steady state was determined as follows: %DPPH REM = 100 CDPPH/CDPPH (t=0). Where CDPPH (t=0) is the initial DPPH concentration and CDPPH is the DPPH concentration at the steady state.   RESULTS AND DISCUSSION  In this study, we investigated the potential antiradical activity of extract from purple leaves of the halophyte plant M. crystallinum. Semi-preparative HPLC was used to obtain different fractions of the plant extract at 540 nm; the six peaks were tested for their antiradical activity (Figure 1). Our results (Table 1) showed a very high anti-oxidant level in fraction 1 (1 ml = 98 ± 0.5 % of DPPH inhibition). Further, it was important to identify the molec-ule present in fraction 1 which seemed to be responsible for the significant anti-oxidative activity observed. The molecule was inactivated in the total extract in the prese-nce of other molecules and therefore it was extracted with  80%  methanol  at  acidic  pH  (addition  of  ascorbic  Bouftira et al.        1137    Table 1. Radical scavenging activity of fraction 1. The results referred to the activity of the synthetic BHT.  Extract Fraction 1 BHT % of DPPH inhibition 98±0.5 90±0.4 Quantity 1 ml 1ml= 0.5 mg      Figure 1. HPLC chromatograms of methanol extract at 540 nm.   acid) with a maximum absorption at 540 nm. As an antio-xidant molecule, it is volatile (total volatilization of the fraction after freeze-drying) with a low molecular mass.  The antioxidant molecule was identified as the 2, 6-bis (1.1-dimethylethyl)-4-methylphenol (BHT) using GC/MS (Figures 2 and 3). Synonyms: 2, 6-Di-tert-Butyl-4-Methy-lphenol; BHT; 2, 6-Di-tert-Butyl-1-Hydroxy-4-Methylbe-nzene; 3, 5-Di-tert-Butyl-4-Hydroxytoluene; 4-Hydroxy-3, 5-Di-tert-Butyltoluene; Dibutylated Hydroxytoluene; 4-Methyl-2, 6-di-t-butyl-phenol. Molecular Weight: 220.18 g/mol. Molecular Formula: C15H24O. Melting Point: 71°C. Boiling Point: 265°C. It was insoluble in water and in pro-pylene glycol, but was freely soluble in alcohol. Most studies on the halophyte plant, M. crystallinum, have focused on its physiological responses to abiotic stresses such as salinity, drought and high irradiance. This plant was found to accumulate bioactive compounds such as betacyanin and flavonol conjugates in response to light stress (Ibdah, 2002; Ibdah et al., 2002) and phenolic compounds (Ibdah et al., 2002). In our study, fractionation  of   the   methanol  extract  from  the  purple  1138           Afr. J. Biotechnol.      Figure 2. Mass spectra corresponding to fraction 1. The molecule identified by GC/MS was the 2,6-bis (1.1-dimethylethyl)-4-methylphenol.       Figure 3. The 2, 6-bis (1.1-dimethylethyl)-4 methylphenol extracted from Mesembryanthemum crystallinum leaf tissue.     leaves of the ice plant demonstrated the presence of a phenol compound recognized as a butylated hydroxyt-oluene (BHT). To investigate the potential antioxidant activity of different fractions, we used the DPPH stable free radical method. It is a rapid method to survey the antioxidant activity of a specific compound or plant extra-cts (Koleva et al., 2002). The synthetic BHT was used as a standard; 0.5 mg/ml was needed for the scavenging activity by 90 ± 0.4%. Fraction 1 contained more than 0.5 mg/ml of BHT due to a very high potential for DPPH inhibition (1 ml = 98 ± 0.5 % of DPPH inhibition). The synthetic (BHT) is generally used as a chemical antioxidant and a food additive. It is obtained by alkyl-ation of p-cresol with isobutene or by monobutylation of m, p-cresol mixtures. This phenol derivative reacts with free radicals thereby slowing down the rate of autoxid-ation responsible for changes in the food color and taste. Antioxidant butylated hydroxytoluene (BHT) reduces the amount of reactive oxygen species (ROS) and prevents apoptosis in etiolated seedlings, prolongs coleoptile life span, and prevents the appearance of all apoptotic features mentioned (Bakeeva et al., 2001). In addition, BHT induces large structural changes in the organization of all cellular organelles and the formation of new unusual membrane structures in the cytoplasm (Vanyushim et al., 2004). BHT distorts mitosis resulting in the appearance of multi-blade polyploid nuclei and multinuclear cells. In roots of etiolated wheat seedlings, BHT induces differen-tiation of plastids with the formation of chloroplasts. Therefore, ROS controlled by BHT, seems to regulate mitosis, trigger apoptosis, and control plastid differen-tiation and the organization of various cellular structures formed by endoplasmic reticulum (Vanyushim et al., 2004). It has been demonstrated that BHT is not only an antioxidant it but also possesses antiviral activity (Schwarz, 1996). BHT could inactivate herpes simplex and other lipid-coated viruses in vitro (Snipes et al., 1975). Studies have confirmed the activity of BHT against many different human and animal viruses, including such members of the herpes family as CMV (cytomegalovirus) (Kim et al., 1978), pseudorabies (Pirtle et al., 1986) and genital herpes (Richards et al., 1985). BHT appears to inhibit infectivity of HIV (Aloia et al., 1988). This is due to the fact that BHT is a highly potent, membrane-active antioxidant as well as a membrane  fluidizer.  It  is  known      that ROS play a role in the pathogenesis of viral infec-tions including RNA viruses such as influenza, DNA viruses such as hepatitis B, and retroviruses such as HIV and it’s been suggested that antioxidants may be useful as therapeutic agents in such infections (Schwarz, 1996).    CONCLUSION  The results of the present study are very important because we have shown that BHT, as an antioxidant, occurs naturally in leaf tissues of the halophyte plant M. crystallinum.   This phenolic compound exhibited a very high antioxidant activity (98 ±0.5 % DPPH inhibitions) as compared to the synthetic BHT which showed relatively less activity (90 ± 0.4 % of DPPH inhibition). Future studies need to focus on the potential medicinal proprieties of the halophyte plants. Because of their relatively high tolerance to ROS, halophyte plants are very important source of antioxidant compounds. Due to the presences of bioactive compo-unds in their tissues, halophyte plants can be used extensively for medical, cosmetic and industrial purposes.   ACKNOWLEDGMENTS  We are grateful to Pr. Ayadi (Center for Biotechnology, Sfax. Tunisia) and Pr. Hammami (Faculty of Medecine, Monastir. Tunisia) for their invaluable assistance.   REFERENCES  Aloia RC, Jensen FC, Curtain CC, Mobley PW, Gordon LM (1988). Lipid composition and fluidity of the human immunodeficiency virus. Proc Natl. Acad. Sci. U SA 85: 900-904. 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Identification of a naturally occurring 2, 6-bis (1.1- dimethylethyl)-4-methylphenol from purple leaves of the halophyte plant Mesembryanthemum crystallinum

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Identification of a naturally occurring 2, 6-bis (1.1- dimethylethyl)-4-methylphenol from purple leaves of the halophyte plant Mesembryanthemum crystallinum

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