the 2,6-bis ( 1.1-dimethylethyl)-4-methylphenol ( bht ) , also known as butylhydroxytoluene , is a lipophilic ( fat soluble ) organic compound that is primarily used as an antioxidant food additive as well as in cosmetics , pharmaceuticals , jet fuels , rubber , petroleum products , and electrical transformer oil .
bht is prepared by the reaction of p - cresol ( 4-methylphenol ) with isobutylene ( 2-methylpropene ) catalysed by sulfuric acid .
the species mesembryanthemum crystallinum ( family : aizoaceae , order : caryophyllales ) , also termed the common ice plant , has emerged as a model organism in plant molecular physiology .
the plant 's rise to relative fame started with the serendipitous discovery of a stress - inducible switch from c3 photosynthesis to crassulacean acid metabolism ( cam ) [ 5 , 6 ] .
a second major area of interest centres on the plant 's extreme stress tolerance , particularly tolerance to high salinity .
the plant thrives in coastal area under climatic conditions characterized by short , cool , moist winters and long dry summers .
mesembryanthemum crystallinum shows five distinct growth phases during its life cycle ( i.e. , germinating seedlings , juvenile , adult , flowering , and seed forming ) .
historically , physicians used leaf juice to soothe inflammation of the mucous membranes of the respiratory or urinary system . in europe , the fresh juice has been used to treat water retention and painful urination and to soothe lung inflammation . during normal metabolism ,
plants generate reactive oxygen species ( ros ) , including superoxide radical ( o2 ) , hydrogen peroxide ( h2o2 ) , hydroxyl radical ( ho ) , and singlet oxygen ( o2 ) .
ros are overproduced in plants under stress , including drought and desiccation , salt stress , chilling , heat shock , heavy metals , ultraviolet radiation , air pollutants , such as ozone and so2 , mechanical stress , nutrient deprivation , pathogen attack and high light stress . to mitigate the oxidative damage initiated by ros ,
the aim of this study is to select a method for a better extraction of bht from the leaves of the halophyte plant mesembryanthemum crystallinum .
the leaves of the halophyte plant mesembryanthemum crystallinum were collected from their natural biotope in the two periods june 2007 and december 2008 ( figures 1 and 2 ) .
method 1 ( sanches et al . ) 10  g of plant leaves were mixed in the presence of acetonitrile ( 2  ml ) and hexane ( 20  ml ) , after centrifugation 10  minat 1,036  g , organic fraction was suppressed .
the bht concentration of the extract was determined in the different plant growth stages in parallel with the antioxidant activity . 
 
10  g of plant leaves were mixed in the presence of acetonitrile ( 2  ml ) and hexane ( 20  ml ) , after centrifugation 10  minat 1,036  g , organic fraction was suppressed .
the bht concentration of the extract was determined in the different plant growth stages in parallel with the antioxidant activity . 

method 2 ( bouftira et al . ) 25  g of plant leaves were mixed in the presence of methanol 80% containing ascorbic acid 50  mm .
after centrifugation , 14000    g , at 4c , the supernatant was collected . 
 
25  g of plant leaves were mixed in the presence of methanol 80% containing ascorbic acid 50  mm .
after centrifugation , 14000    g , at 4c , the supernatant was collected .
free radical scavenging activity of plant extract was determined by using a stable free radical , ( 1,1-diphenyl-2-picryl - hydrazyl ) dpph .
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 , uk ) .
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 .
a hewlett packard 1100 analytical hplc was used with a gradient elution method ; within 2  min , the mobile phase was acetonitrile - water ( 65  +  35 ) , after which the acetonitrile was raised to 100% within 15  min .
the total runtime of each analysis was 30  min to ensure cleaning of the column between samples .
the flow rate was 1  ml / min and the injection volume 20  l .
bht was identified by comparison of its retention time and uv spectra with those of an injected pure standard in the hplc method .
the concentration of bht was determined in g / ml / g of fresh leaves ( figure 1 ) .
bht spectra were detected at 280  nm by analytical hplc ( figure 2 ) ; the different spectra were compared between the plant growth stage and the bht standard ( 10  mg / ml ) . the results ( figure 3 ) , by using method 1 of extraction showed that the seedlings stage has the highest antiradical activity ( 65,24    0,27 ) . 

figure 4 showed the comparison of antiradical activity between the two methods ( method 1 and 2 ) . at the same plant growth stage ( floraison ) , the extraction by method 1 has higher antiradical activity ( 51  0,82 ) in comparison with method 2 ( 32,33    2,05 ) .
the results showed that mesembryanthemum crystallinum extract at seeds stage has the highest bht concentration . at this period ,
the plant leaves are purple resulting from tolerance to stress of environment . in the seeds stage
the light stress can attribute in part to stimulate the plant for the synthesis of bht .
it was proved that m. crystallinum after irradiation with high light irradiance 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 .
the results of this study showed also that the method of extraction influences the yield of bht .
the method using hexane and acetonitrile is better than that using methanol 80% for bht extraction .
the extraction method must allow complete extraction of the compounds of interest , and it must avoid their chemical modification .
water and aqueous mixture of ethanol , methanol , and acetone are commonly used for plant extraction .
the purple - coloured dpph is a stable free radical , which is reduced to a , a - diphenyl - b - picrylhydrazine ( yellow coloured ) by reacting with an antioxidant .
antioxidants interrupt free radical chain oxidation by donating hydrogen from hydroxyl groups to form a stable end product , which does not initiate or propagate further oxidation of lipids . the data obtained showed that the antioxidant activity was higher at seedlings stage in comparison with the other plant stages .
in fact , m. crystallinum accumulates more bht at floraison stage in comparison with seedlings stage .
the plant accumulates , at seedlings stage , other antioxidant compounds which work in association with bht .   at the floraison stage
, this study also showed that the method of extraction is in relation with the antioxidant activity .
the method using hexane and acetonitrile ( 51    0,82 ) for extraction showed higher antioxidant activity than those using methanol 80% ( 32,33    2,05 ) .
many medicinal plants are investigated for their antioxidant activity in relation with the methods of extraction and the solvent used .  
it was found that higher amounts of polyphenols and anthocyanins are found in methanol and ethanol extracts than in water extracts .
it means that they are more efficient in cell walls which have unpolar character and cause polyphenols to be released from cells .
more useful explanation for the decrease is ascribed to the enzyme polyphenol oxidase , which degrades polyphenols in water extracts whereas in methanol and ethanol they are inactive . in conclusion ,
the results of this study are very important ; we obtained a better method for bht extraction .
 a naturally occurring bht was identified in the leaves of the halophyte plant mesembryanthemum crystallinum . 
 this phenol was extracted in this study by two methods at the different plant growth stages . 
 one of the methods was better for bht extraction ; the concentration of this phenol is plant growth stage dependent . in this study 
 , the floraison stage has the highest bht concentration . 
 the antioxidant activity of the plant extract was not related to bht concentration . 
 the higher antioxidant activity is obtained at seedlings stage . 