trends in health promotion via antioxidant supplements have generated extensive demand in extracting biologically active substances from a variety of plant materials , primarily from polyphenolic - rich plants .
conventional solid - liquid extraction with organic solvents has commonly been operated ; however , the production of undesirable residues during solvent removal has been reported and suggested as the cause of chronic toxicity of the final products . according to the legal limitations of conventional solvents for food and pharmaceutical uses , supercritical fluid extraction ( sfe ) has been introduced as an alternative operation .
this technology utilizes the extraction fluid at temperature and pressure above its critical value , where distinct liquid and gas phases do not exist .
carbon dioxide is the most frequently used solvent in supercritical extraction due to its low cost characteristics and being easily available in high purity form , nontoxic , noninflammable , and nonexplosive .
in addition , the solvent power of co2 can be varied over a wide range by changing the operational conditions .
supercritical co2 ( sc - co2 ) extraction has proved effective in the separation of desired compounds without leaving toxic residues in extracts . however , the commercial application of supercritical fluid technology remained restricted [ 1 , 2 ] .
phenolic compounds are of particular interest for applications in the areas of functional foods and nutraceuticals due to their high antioxidant activities and abundance in the plant kingdom .
arg . , or ma mao , is a native plant classified in the family phyllanthaceae , genus antidesma .
antidesma thwaitesianum has become an attractive fruit for production in scale of industry since recent studies have shown that it contains polyphenolic compounds , especially catechin and proanthocyanidins , and possesses antioxidant ability to protect human breast epithelial cells against oxidative damage [ 35 ] .
however , the concern of organic solvent usage in these studies ( i.e. , methanol , chloroform , acetone , ethyl acetate , and hexane ) hindered its advancement .
the present study , therefore , investigated the feasibility of applying sc - co2 extraction technology to overcome this drawback .
the study outcomes may add more values to antidesma thwaitesianum fruit , hasten its development as health supplements or pharmaceutical products , and promote the transfer of supercritical fluid technology to industry .
aluminum chloride hexahydrate ( alcl36h2o ) , ferrous sulfate heptahydrate ( feso47h2o ) , and 2,2-azino - bis(3-ethylbenzothiazoline-6-sulphonic acid ) ( abts ) were obtained from fluka ( darmstadt , germany ) .
all other chemicals were purchased from sigma - aldrich co. ( st louis , mo , usa ) .
ripe fruits of antidesma thwaitesianum ( harvested in august - september 2013 ) were obtained from doi kham food products co. , ltd .
( royal project , chiang mai , thailand ) . the whole fruit was cleaned and washed under running tap water and rapidly stored at 20c .
the powdered sample was kept at 4c in an air tight container protected from light until extracted .
extraction was conducted using a laboratory scale supercritical fluid extractor unit ( model sfe100 , thar technologies inc .
the extraction protocol was set up according to the study of yilmaz et al .   with slight modification .
briefly , 100  g of antidesma thwaitesianum powder was loaded into the vessel and the supercritical extraction was carried out using the co2 at a constant operating temperature of 40c , pressure of 30  mpa , flow rate of 30  g / min , and the dynamic extraction time of 1  h. a 20  ml of 80% ethanol was used as cosolvent .
the extract was collected in a preweighed collection glass vial and kept at 20c ready for analysis . 

antidesma thwaitesianum powder was continuously shaken with 80% ethanol at room temperature for 4  h , centrifuged at 1000  g ( avanti 30 , beckman coulter , ca , usa ) for 15  min , and then supernatant was separated by filtration through whatman no .
antidesma thwaitesianum powder was extracted with distilled water for 4  h at room temperature .
the extract was centrifuged at 1000  g for 15  min , filtered through whatman no .
1 filter paper , rinsed with distilled water , and then used for analysis . the percent yield was calculated from the ratio between the total extract mass obtained ( g ) and the raw material mass loaded for extraction ( g ) on a dry weight basis .
each extraction procedure was done in triplicate , and the average of the three replicate was taken .
briefly , appropriate dilutions of samples were oxidized with folin - ciocalteu reagent for 3  min . then
, the reaction was neutralized with 7.5% sodium carbonate solution and allowed to stand at 50c for 15  min .
the absorbance was measured at 760  nm and the total polyphenol content in each sample was calculated based on a standard curve , which was prepared using gallic acid and expressed as mg of gallic acid equivalent per 100  g of extract in dry weight ( mg gae/100  g dry wt ) .
the aluminum chloride colorimetric method as described by lin and tang   was used to determine the total flavonoid content .
briefly , the reaction mixture containing the extracted solution , 95% ethanol , 10% alcl36h2o , 1  m ch3cook , was incubated at room temperature for 40  min .
the absorbance was read at 415  nm and the flavonoid content was expressed as mg of quercetin equivalents per 100  g of extract in dry weight ( mg qe/100  g dry wt ) .
briefly , the assay mixture ( extract solution , 77  mg of feso47h2o in 500  ml of hcl : n - butanol = 2  :  3 ) was incubated for 40  min at 95c .
the absorbance was read at 540  nm after cooling and results were expressed as mg of catechin equivalents per 100  g of extract in dry weight ( mg ce/100  g dry wt ) .
the extract solution of antidesma thwaitesianum was centrifuged at 17000  g , 4c for 60  min , and the supernatant obtained was directly analyzed by high - performance liquid chromatography with a diode array detector and a mass spectrometry detector ( hplc - dad / msd ) using an hp 1100 liquid chromatograph equipped with ms detectors ( agilent technology , ca , usa ) .
a c18 column 150  4.6  mm ( 5  m ) ( lichrocart , merck , germany ) was maintained at 40c , with a flow rate of 1  ml / min and detector fixed at 270  nm .
the eluents were h2o acidified to ph 4.0 by 10  mm ammonium formate and formic acid ( a ) and acetonitrile ( b ) . the following linear solvent gradient was applied : 100% a in 5  min , from 100% a to 80% a in 5  min , and a plateau of 10  min , to 60% a in 40  min .
after separation , the hplc effluent was delivered into a single quadrupole mass spectrometer ( agilent technologies , usa ) via orthogonal atmospheric pressure ionization - electrospray ( api - es ) interface mode at 100700 m / z and step size at 0.2 .
the optimum electrospray ionization ( esi ) conditions were as follows : ionization mode , positive ( 4,000  v ) , negative ( 3,500  v ) ; nebulizer pressure , 60  psi ; drying gas flow rate , 13  l / min ; drying gas temperature , 320c .
the abts decolorization assay was performed according to the method described by luximon - ramma et al .
, based on the ability of an antioxidant to scavenge the preformed abts radicals relative to that of the standard trolox .
results were expressed in mg trolox equivalents / g of extract in dry weight ( mg te / g dry wt ) .
the dpph radical scavenging assay was done according to the method of brand - williams et al . ,
based on the reduction of the stable radical , dpph , to the formation of a nonradical form in the presence of hydrogen donating antioxidant .
dpph values were calculated using a linear regression equation of trolox standards , and the relative scavenging capacity of the extracts was expressed as mg trolox equivalent / g of extract in dry weight ( mg te / g dry wt ) .
the reaction mixture was read at 740  nm every 5  min in 1st h , every 10  min in 2nd h , and every 20  min in 3rd h. results were expressed as mg trolox equivalents / g of extract in dry weight ( mg te / g dry wt ) .
all the assays were performed in triplicate , and the results were expressed as mean  sd from the three sets of observations .
comparisons were performed by one way anova followed by fisher 's least significant difference ( lsd ) test using the spss 16.0 software ( spss inc . , chicago , il , usa ) .
this research work examined the use of sc - co2 extraction technology to extract antidesma thwaitesianum fruits .
the extraction yield , phytochemical contents , and antioxidant activity of the sc - co2 extract were evaluated .
the efficiency of the sc - co2 extraction systems was compared to those of the conventional ethanol and water extraction methods .
the results indicated that the best features of antidesma thwaitesianum fruit extract were obtained by means of sc - co2 extraction . in the present study , supercritical fluid extraction , a clean technology ,
it is observed that extraction using sc - co2 gains more product yield about 1.5-fold than that of ethanol ( 17.87  0.55 and 11.50  1.08% , resp . ,
p < 0.05 ) and almost 3 times higher ( p < 0.05 ) in the case of water ( 7.67  0.87% ) .
the extract from sc - co2 also exhibited the highest contents of total phenolic , total flavonoid , and total proanthocyanidin , about 2 - 3-fold higher ( all p < 0.05 ) when compared with the other extracting solvents ( figure 1 ) .
previous studies have shown that the levels of yield as well as phenolic compounds can be altered by processing conditions and the extracting solvent .
co2 at supercritical conditions can effuse through solids like a gas and dissolve materials like a liquid ; thus , it has a higher diffusivity as well as lower density , viscosity , and surface tension compared to ethanol and water . in this regard ,
the favorable transport properties of sc - co2 allow deeper penetration into solid plant matrix and more efficient and faster extraction than conventional organic solvents , causing higher extraction yield and phytochemical contents as observed in this study .
the rise in extraction yield by sc - co2 may also be the result of interaction between extraction temperature and pressure .
this suggestion is based on evidence showing that both factors can increase the extraction yield by increasing the solvent power and selectivity of sc - co2 .
although raising operation pressure and temperature could possibly gain more yield , the extraction process in this study was set at constant temperature of 40c and pressure of 30  mpa , as this condition is considered suitable for thermolabile compound to avoid degradation .
another reason for getting the higher amounts of yield and polyphenols by sc - co2 extraction in the present experiment , particularly over that of water extraction , may lie in the use of ethanol as cosolvent . because co2 is nonpolar and has some limitation for extracting of polar compounds ,
ethanol has widely been used for this purpose because water is not as miscible with co2 as alcohol , while methanol is environmentally hazardous and toxic to human health .
several lines of evidence have indicated that ethanol can enhance extraction yield by increasing the solubility of polar solutes , inducing changes in the structure of cellular matrix via intracrystalline and osmotic swelling , and breaking analyzed matrix binding by competing with polar interaction between matrix and compounds to be extracted .
in addition , increasing the amount of ethanol in the supercritical fluid has been shown to decrease the extraction time and the consumption of co2 . because of the complex nature of polyphenols , the antioxidant activities of plant extracts can not be evaluated by only a single method . in this study , the abts , dpph , and frap assays were used to determine the antioxidant potentials of antidesma thwaitesianum extracts and the results are shown in figure 2 .
the data clearly demonstrated the significantly greater ( p < 0.05 ) scavenging activities of the extract from sc - co2 against abts and dpph radicals than those of the ethanol and water .
ferric reducing antioxidant power also showed similar profile as the radical scavenging activities in which the greatest ability ( p < 0.05 ) was detectable from the sc - co2 extract . hplc - dad / ms assay was used to further characterize the phenolic compounds responsible for the antioxidant activity of antidesma thwaitesianum fruit extract .
however , the main focus of this study was on catechins as they are ubiquitous constituents of vascular plants and frequent components of traditional herbal remedies .
most importantly , catechin has previously been reported to be the major phenolic compound in the extracts of antidesma thwaitesianum by conventional solvent extraction methods . using catechin as a standard reference , the content of catechin identified in the sc - co2 extract was approximately 2 times and almost 5 times higher ( p < 0.05 ) than that recorded from the ethanol and water extracts , respectively ( figures 3 and 4 ) . in general , the radical scavenging property and antioxidant activity of plant extracts are positively correlated with their phytochemical contents . applying this concept to the present results ,
it is reasonable to conclude that the greatest extraction yield as well as the highest amounts of phytochemicals ( phenolics , flavonoids , and proanthocyanidins , primarily catechin ) is responsible for the greatest antioxidant potentials of the sc - co2 product .
in addition , previous study demonstrated that the extract obtained by conventional organic solvent extraction can undergo oxidative transformations during solvent removal . from this viewpoint ,
supercritical extraction uses pure gas and the co2 processing creates a medium without oxygen where oxidation reactions can be avoided , thus allows the recovery of the extract with high purity , completely free of solvent , and retains their antioxidant potency .
the current investigation demonstrates that both quantity and quality of the antidesma thwaitesianum fruit extract are improved by supercritical co2 extraction .
these findings underscore the efficiencies of supercritical technology over traditional solvent extraction methods . due to the legal restrictions in food technologies and the increasing demands for safe and powerful natural antioxidants ,
it is suggested that supercritical co2 extraction is a promising technology for extraction and purification of antioxidant compounds from plant materials including antidesma thwaitesianum .
 supercritical fluid extraction ( sfe ) has increasingly gained attention as an alternative technique for extraction of natural products without leaving toxic residues in extracts . 
 antidesma thwaitesianum muell . 
 arg . 
 ( phyllanthaceae ) , or ma mao , has been reported to exhibit antioxidant health benefits due to its phenolic constituents . to determine whether sfe technique could impact on phenolic contents and associated antioxidant potentials , ripe fruits of antidesma thwaitesianum ( phyllanthaceae ) 
 were extracted using supercritical carbon dioxide ( sc - co2 ) and conventional solvents ( ethanol , water ) . 
 the results showed that the sc - co2 extract contained significantly higher yield , total phenolic , flavonoid , and proanthocyanidin contents than those obtained from ethanol and water . 
 it also demonstrated the greatest antioxidant activities as assessed by abts radical cation decolorization , dpph radical scavenging , and ferric reducing antioxidant power ( frap ) assays . 
 further analysis using high - performance liquid chromatography with diode array and mass spectrometry detectors ( hplc - dad / msd ) revealed the presence of catechin as a major phenolic compound of antidesma thwaitesianum ( phyllanthaceae ) , with the maximum amount detected in the sc - co2 extract . 
 these data indicate that sfe technology improves both quantity and quality of antidesma thwaitesianum fruit extract . 
 the findings added more reliability of using this technique to produce high added value products from this medicinal plant . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions