
				 in recent decades , the essential oils and various extracts of plants have been of great interest as they have been the sources of natural products .
the most commonly used antioxidant at the present time are butylated hydroxyanisole ( bha ) , butylated hydroxytoluene ( bht ) , propylgallate ( pg ) , and tert - butyl hydroxyl toluene ( tbhq ) .
however , they are suspected of being responsible for liver damage and carcinogenesis in laboratory animals [ 3 , 4 ] .
therefore , the development and utilization of more effective antioxidant of natural origin are desired .
the antioxidant activity of phenolic compounds is mainly due to their redox properties , which can play an important role in absorbing and neutralizing free radicals , quenching singlet and triplet oxygen , or decomposing peroxidase .
therefore , an investigation of such antioxidative phenolic compounds in edible plants has been conducted to improve our understanding of their dietary value and potential benefits .
pilea microphylla ( pm ) ( family : urticaceae ) is being used as folk medicine to treat several allergies / wounds in and around malaysia peninsular especially penang island .
it is reported to possess antibacterial activity , moderate antioxidant activity , and total phenolic content   and pm is also used for infertility , inflammations , and womb cleanser .
the purposes of this study were to compare the extracting method together and to evaluate pilea microphylla in urticaceae family as new potential sources of natural antioxidant and antimicrobial activities .
the whole part of pilea microphylla were collected in penang island from usm main campus ( universiti sains malaysia ) in march 2008 .
voucher specimens have been deposited at the herbarium of the school of biological sciences , universiti sains malaysia in april 2008 .
the first method ( method i ) of extraction included the using of four solvents by following nonpolar to polar solvents ( by using soxhlet apparatus ) . in this method ,
the solvents used were hexane , chloroform , ethyl acetate , and methanol . the second method ( method ii ) included 5 solvents system ( by using partition technique ) .
for method ii , the dried materials were extracted by using soxhlet extractor with methanol as a solvent for 72 hours at room temperature ( 30c ) .
the methanolic extracts were further partitioned by adding distilled water in a separating funnel and then followed using chloroform , diethyl ether , ethyl acetate , and butanol as described by mellidis et al .
abbreviations for crude extract used in this paper , namely , he i ( hexane extract of method i ) , ce i ( chloroform extract of method i ) , eae i ( ethyl acetate extract of method i ) , me i ( methanol extract of method i ) , me ii ( methanol extract of method ii ) , ce ii ( chloroform extract of method ii ) , dee ii ( diethyl ether extract of method ii ) , eae ii ( ethyl acetate extract of method ii ) and be ii ( butanol extract of method ii ) .
free radical scavenging activity of pilea microphylla ( meoh , chlorofom , diethyl ether , ethyl acetate , n - hexane , and butanol extracts ) were tested using dpph method with final concentration 1000  g / ml ( etanolic dpph ) ( sigma chemical co. , usa ) ( 300  m ) and was used in the reaction mixture . freshly prepared test samples ( 50  l )
were combined with dpph solution ( 150  l ) in a 96 well microtiter plate .
the reaction mixture were incubated for 30 minutes at 37c and the change in absorbance at 515  nm was measured using micro plate reader ( thermo electron corporation , finland ) .
the obtained absorbance values were converted into the percentage of radical scavenging activity using the following equation : 
								 ( 1)radical  scavenging  activity  ( % ) =  100[asa  c100 ] , 
							 where as : absorbance of the sample , ac : absorbance of the negative control .
the amounts of phenolics in the selected medicinal plant extracts were determined with folin - ciocalteu reagent using the method of salvi et al .
a little bit modified .0.5  ml of each sample was dissolved in 1.0  ml dmso ( 3 replicates ) , 1.0  ml of 10% dilution of folin - ciocalteu reagent and after 3 minutes , 3  ml of na2co3 ( 1% , w / v ) were added and the resulting mixture was incubated at room temperature for 2 hours .
the absorbance of all samples was measured at 760  nm using a spectrophotometer(model u-1900 spectrophotometer hitachi high technology corporation 2006).the standard curve was prepared using 0 , 50 , 100 , 150 , 200 , and 250  mg / l .
results were expressed as milligrams of gallic acid equivalent per gram of dry weight ( mg gae / g dry weight ) , which is a common reference compound .
the total flavonoid content was determined using the dowd method ; 5  ml of 2% aluminium trichloride ( alcl3 ) in methanol was mixed with the same volume of the extract solution ( 0.4  mg / ml ) .
absorption readings at 415  nm using perkinelmer uv - vis lambda 25 spectrophotometer were taken after 10 minutes against a blank sample consisting of a 5  ml extract solution with 5  ml methanol without alcl3 .
the total flavonoid content was determined using a standard curve with quercetin ( 0100  mg
total flavonoid content is expressed as  mg of quercetin equivalents ( qe)/g of extract .
antimicrobial activity was determined using disc diffusion following the method described by national committee for clinical laboratory standard ( nccls ) .
the test bacteria was removed aseptically with an inoculating loop and transferred to a test tube containing 5  ml of sterile distilled water .
sufficient inocula were added until the turbidity equaled 0.5 mcfarland ( 10  cfu / ml ) standards ( biomerieux , marcy d'etoile , france ) .
the test tube suspension ( 1  ml ) was added to 1520  ml of nutrient agar or sabouraud dextrose agar before setting aside the seeded agar plate ( 9  cm in diameter ) to solidify for 15 minutes .
three disks of whatman 's no . 1 filter paper , 6  mm in diameter , were used to screen the antimicrobial activity .
each sterile disk was impregnated with 20  l of extract ( corresponding to 100  mg crude extract / ml ) , amoxicillin ( and vancomycin for streptococcus sp . ) , miconazole nitrate ( 30  g / ml , as positive control for bacteria and fungi , respectively ) , or 10% dmso ( v / v ) ( as negative control ) , before it was placed on the surface of the seeded plates .
antibacterial activity was recorded if a zone of growth inhibition around the well of greater than 6  mm was measured .
dilution series were set up with 0.13 , 0.26 , 0.52 , 1.04 , 2.10 , 4.17 , 8.33 , 16.66 , 33.33 , 66.66 , and 133.33  mg / ml of nutrient broth medium .
to each test tube , 0.5  ml of standardized suspension of bacteria were added and incubated at 37c for 24 hours .
the lowest concentration which did not show any growth of the tested microorganism after macroscopic evaluation was determined as the mic .
the minimum bactericidal concentration of the plant extract on the clinical bacterial isolates was done according to the method highlighted in national committee for clinical laboratory standard ( 2002 ) .
briefly , 1  ml that was pipetted from the mixture obtained in the determination of mic stage was streaked out on the nutrient broth for 24 hours .
the least concentration of the extract with no visible growth was taken as the minimum bactericidal concentration .
the preliminary biological evaluation of the crude extract of pilea microphylla was determined using brine shrimp lethality test according to simionatto et al . with some modification .
the brine shrimp ( artemia salina ) lethality assay is considered to be a useful tool for preliminary assessment of cytotoxicity .  
brine shrimp assays have also been used for the analysis of pesticides residues , to monitor the toxicity of organic waste to marine organisms   and active plant constituents .
the brine shrimp eggs were provided by muka head marine research station of the universiti sains malaysia , and were hatched in artificial sea water ( 38  g salt per liter of water ) .
after 24 hours , the hatched nauplii suspension was left to stand for 1 hour without aeration , and then the nauplii were collected by pipetting from middle layer of solution , in which most of nauplii were swimming .
the crude extracts were dissolved in distilled water to various concentrations and the shrimp larvae were placed in to them ( duplicate ) .
sea water without extract was used as a negative control , while potassium dichromate had a lc50 = 20  g / ml as a positive control .
fifteen nauplii were withdrawn through a glass capillary and placed in each vial containing 4.5  ml of brine solution .
final concentration of extracts in each experiment were used 5.000 , 2.500 , 1.250 , 0.625 , 0.312 , 0.156 , and 0.078  mg / ml . 0.5  ml of the plant extract was added to 4.5  ml of brine solution and maintained at room temperature for 24 hours under the light and surviving larvae were counted .
differences were considered statistically significant at the p < .05 level and one way anova .
me i showed the highest antioxidant activity of 69.51  1.03% with the lowest ec50 of 81.32  g / ml , if compared to other extracts .
thus , the extracts were used to screen for the radical scavenging activity at different concentrations .
their content ranged from 9.91  0.33 to 72.10  0.71  mg gallic acid equivalent ( gae)/g of extracts , with an average of 41.00  mg / g .
as shown in the table 1 , ce i had the highest phenolic at 72.10  0.71  mg  gae / g and ce ii had the highest flavonoid contents at 60.14  0.33  mg  qe / g , unless ce i showed higher flavonoid contents after ce ii at 53.44  0.94  mg 
ce i and eae i showed the weakest dpph radical scavenging capability , at only 0.46  0.98 and 4.10  0.14 , respectively . for further study , butanol extract of method ii ( bepm ii ) was selected for get fractionations by paper chromatography ( pc ) .
bepmf4 displayed the highest antioxidant activity than other fractions ( 27.84  1.27 ) .  
generally , if the ratio between total phenolic content and total flavonoid as shown would be more than 1 , it showed an increase at in total phenolic content of the extract ( as shown in last column ) .
the relationship between total phenolic content and antioxidant activity of pm was calculated in both methods of extraction .
the results indicated that when all extracts in method ii were included in the statistical analysis , there was a negative relationship between them ( r = 0.051 ) , unless method i showed a little bit correlation between the antioxidant activity and total phenolic contents ( r = 0.636 ) .
the antimicrobial effects of pm extracts on pathogenic bacteria , fungi and yeasts are presented in table 2 .
in addition , pure methanol ( control ) had no inhibitory effects on pathogenic microbes tested .
the pm extracts at 100  mg / ml concentration were effective on the tested bacteria .
pm extracts gave large inhibition zone on bacillus spizizenii atcc 6633 and micrococcus sp . by using ce
i and ce ii , respectively . according to table 2 , ce ii was effective against on escherichia coli and methicillin resistant staphylococcus aureus ( mrsa ) .
all tested extracts showed no antifungal activity except hexane extract against aspergillus niger usm ai1 showed reduced of ufc , but nor hypha .
the mic and mbc values that were tested on bacteria are listed in table 3 .
the mbcs were defined as the lowest concentration that killed at least 99.9% of the cells in the initial inoculum .
mic values range between 8.3333.33  mg / ml . eae i showed the lowest concentration of mic and mbc values of 8.33  mg / ml against bacillus spizizenii atcc 6633 .  
brine shrimp results presented in ( table 4 ) show that he i and be ii were virtually nontoxic on shrimps , and also they exhibited very low toxicity , giving lc50 values greater than 100  g / ml after 24 hours .
there is a wide range of lc50 values of crude extracts between 23880  g / ml .
the hexane extract of method i ( lc50 3880  g / ml ) exhibited very low toxicity , ( figure 1 ) .

 pilea microphylla is used in traditional medicine to treat bacterial infection .the claims for treatment infertility , inflammation and cleaning of reproductive sex organ of female human .
the results of this screening experiment demonstrated that p. microphylla contained different levels of total phenolic , total flavonoid and possessed diverse antioxidant properties .
also , previous study showed an ethanolic extract of pilea microphylla which was found to be most potent when subjected to detailed free radical scavenging at 23.15  g / ml . otherwise , methanol and ethanol are in same polarity level .
pilea microphylla had a variety of antimicrobial activity against pathogenic microorganisms . the majority of these crude extracts which inhibited growth of bacteria were active against gram - positive bacteria such as b. cereus , b. subtilis , and methicillin resistant staphylococcus aureus , unless both nonpolar and polar extracts in method ii and butanol extract as a polar extract in method i , exhibited low activity against bacillus cereus and bacillus cereus atcc 10876 .
the results seem to indicate that method ii ( sequential partitions ) may be more effective than method i ( by using soxhelt extractor ) .
e. coli , b. subtilis , methicillin resistant staphylococcus aureus ( mrsa ) inhibited by extracts from method ii .
this study has shown that 63.6% of extracts inhibited gram - positive bacteria ( 7 out of 11 ) and 60% of extracts also inhibited gram - negative bacteria ( 9 out of 15 ) .
however , the frequency of antimicrobial activity statistically was 13.13% ( 39 out of 297 ) .
facey et al . found low antibacterial activity against staphylococcus aureus by using mixture of acetone and ethyl acetate of p. microphylla .
the greater resistance of gram - negative bacteria to plant extracts has been documented previously [ 21 , 22 ] and is supported by our results .
these observations are likely to be the result of the differences in cell wall structure between gram - positive and gram - negative bacteria , with the gram - negative outer membrane acting as a barrier to many environmental substances , including antibiotics .
the disc diffusion method showed mic values of 8.33 to 33.33  mg / ml . the pm extracts also could be used to treat most of bacillus sp . ,   escherichia coli , and methicillin resistant staphylococcus aureus ( mrsa ) .  
hexane extract was not toxic against brine shrimp ( lc50 value more than 1  mg / ml ) .
this is impressive and lends support to the suggestion that hexane extract of pm can be used to treat pathogenic microbial infections such as b. cereus that can cause food poisoning symptoms . in conclusion ,
further evaluation of the antibacterial properties of its extracts and elucidation of the components responsible for the activities is warranted .
two of the organisms inhibited by some of the extracts , b. cereus and mrsa , are important food - borne pathogens , raising the possibility of using the active compounds of this plant to prevent food - borne diseases .
however , the application of any compounds to medicine will require safety and toxicity issues to be addressed and further study for purification of the active compound needs to be done for pilea microphylla .
 a total of 9 plant extracts were tested , using two different kinds of extracting methods to evaluate the antioxidant and antimicrobial activities from pilea microphylla ( urticaceae family ) and including toxicity test . 
 antioxidant activity were tested by using dpph free radical scavenging , also total phenolic contents and total flavonoid contents were determined . 
 toxicity assay carried out by using brine shrimps . 
 methanol extract of method i ( me i ) showed the highest antioxidant activity at 69.51  1.03 . 
 chloroform extract of method i ( ce i ) showed the highest total phenolic contents at 72.10  0.71 and chloroform extract of method ii ( ce ii ) showed the highest total flavonoid contents at 60.14  0.33 . 
 the antimicrobial activity of pilea microphylla extract was tested in vitro by using disc diffusion method and minimum inhibitory concentration ( mic ) . 
 the pilea microphylla extract showed antibacterial activity against some gram negative and positive bacteria . 
 the extracts did not exhibit antifungal and antiyeast activity . 
 the hexane extract of method 
 i ( he i ) was not toxic against brine shrimp ( lc50 value was 3880  g / ml ) . 
 therefore , the extracts could be suitable as antimicrobial and antioxidative agents in food industry . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion