microbial contamination and side effects of synthetic antioxidants are two important major concerns of food and pharmaceutical industries . increasing propensity for replacing synthetic antioxidant by natural one on one side and development of microbial resistance to existing antibiotics from the other has encouraged researchers toward appraising medicinal plants for dual antioxidant and antimicrobial properties . though , since immemorial time , medicinal plants have been used to treat and prevent various human ailments and they are considered as reservoir of bioactive compounds.1 , 2 till date , biological properties and bioactive compounds of many medicinal plants are not studied . extensive investigation of medicinal plants for biological activities and bioactive compounds is the crucial and the foremost step in development of effective alternative medications . in view of this , in the present study , bergenia ciliata sternb . ( family saxifragaceae ) , a high value plant of the sikkim himalaya , has been investigated for antioxidant , antimicrobial activity and bioactive compounds . it is an important perennial medicinal herb that grows widely in the temperate himalaya between 1500 and 3000  m asl . the plant has been in use as folklore medicine since ancient times for dissolution of kidney and gall bladder stones.4 , 5 in sikkim himalaya , bergenia rhizome is used to treat fractured bones , fresh cuts , wounds , diarrhea , pulmonary infections , vomiting , fever , cough and boils by locales.6 , 7 , 8 
 bergenia is also used for the treatment of heart disease , haemorrhoids , stomach disorders and ophthalmia . in addition , it is accredited with analgesic , antiviral , anti - inflammatory and antimalarial properties.9 , 10 the plant 's virtues , to a large extent , are attributed to its secondary metabolites such as bergenin , gallic acid and catechin which are therapeutic and account for its use in traditional medicine.11 , 12 despite the widespread use of this plant in traditional medicine , the scientific literature with respect to its biological properties is scanty . to the best of our knowledge , so far , antioxidant , antimicrobial properties and the bioactive compounds associated with the leaf of bergenia plant , growing in the sikkim himalaya , have not been investigated . therefore , the present study was conducted with the objectives to ( 1 ) obtain the most effective solvent for extracting the potent bioactive compounds , especially phenolics and flavonoids , ( 2 ) investigate different extracts for antioxidant and antimicrobial activities and , ( 3 ) quantify the amount of bergenin , catechin and gallic acid present in the leaf extracts of b.  ciliata . leaves of b.  ciliata were collected during flowering season ( march 2014 ) from the plants growing in the arboretum of g.b . pant institute of himalayan environment and development ( gbpihed ) , sikkim unit , gangtok , india ( latitude 27 21 35.7n ; longitude 88 37 24.4e ) , situated at the elevation of 2047  m asl . the leaves were washed thoroughly under running tap water and finally with distilled water and dried on blotting paper at room temperature to get consistent weight . 2  g powder was soaked separately in 10  ml of different solvents ( ethyl acetate , methanol and hexane ) for 24  h. the supernatant was transferred into a new tube and the residue was re - extracted twice with 10  ml solvent . each extract was resuspended in the methanol to yield a 50  mg / ml stock solution . the yield of the extraction was calculated from { ( w1/w2 )    100 } , where w1 is the weight of extract after evaporation of solvent and w2 is the dry weight of the plant sample . solutions of each extract ( 100  l ; 1  mg / ml ) were taken individually in test tubes . to this solution , 2.5  ml of 10-fold diluted folin after 3  min , 2.0  ml of 7.5 % na2co3 solution was added and the mixtures were incubated for 30  min . the absorbance of the reaction mixtures was measured at 760  nm by using a spectrophotometer ( uv-1800 , shimadzu , kyoto , japan ) . gallic acid was used as a standard and tpc of bergenia extracts was expressed in milligram gallic acid equivalents ( mg gae / g extract ) . total flavonoid content was determined by the aluminum chloride calorimetric method , with some modifications . then , the sample solution ( 2  ml ) was mixed with 2  ml of 2% alcl3 . after 10  min of incubation at ambient temperature , the absorbance of the solution was measured at 435  nm by using a spectrophotometer ( uv-1800 , shimadzu , kyoto , japan ) . the flavonoid content was expressed as milligram quercetin equivalent ( mg qe / g extract ) . the effect of extracts on 2,2-diphenyl-1-picrylhydrazyl ( dpph ) radical was determined using the method of liyana - pathiranan and shahidi . a solution of 0.135  mm dpph in methanol was prepared and 1.5  ml of this solution was mixed with 1.5  ml of extract in methanol . the reaction mixture was mixed thoroughly and left in the dark at room temperature for 30  min . the ability of sample to scavenge dpph radical was calculated by the following equation : dpph radical scavenging activity ( % )  =  [ ( abs control    abs sample)]/(abs control )    100where abs control is the absorbance of dpph radical  +  methanol ; abs sample is the absorbance of dpph radical  +  extract / standard . ic50 value is the concentration of extracts at which dpph radicals are scavenged by 50% . the lower ic50 value indicates higher radical scavenging capacity and vice versa . the 2,2-azino - bis(3-ethylbenzothiazoline-6-sulfonic acid ) the abts radical cation solution was prepared by mixing equal quantities of abts ( 7  mm ) and ammonium persulfate ( 2.45  mm ) and allowing them to react for 1620  h at room temperature , in dark . the working solution was then prepared by diluting the previous solution with methanol until the absorbance at 734  nm was 0.706    0.02 . plant extracts ( 1.5  ml ) were allowed to react with equal volume of the abts working solution and the absorbance was taken at 734  nm after 7  min using the spectrophotometer . the abts scavenging capacity of the extracts was compared with that of bht and the percentage inhibition was calculated as : abts radical scavenging activity ( % )  =  [ ( abs control    abs sample)]/(abs control )    100where abs control is the absorbance of abts radical  +  methanol ; abs sample is the absorbance of abts radical  +  extract / standard . abts radical scavenging activity of extracts was determined by ic50 value as mentioned above in dpph assay . total eight strains ( 4 bacteria , 2 actinomycetes and 2 fungi ) were taken from microbial culture collection established in the microbiology laboratory of gbpihed institute , almora , india . initially test organisms were grown on the respective media i.e. bacteria and actinomycetes in tryptone yeast extract ( ty ) and fungus on potato dextrose broth ( pd ) in conical flask , at 25c for 24  h. antimicrobial activity was performed on ty and pd agar plates following disc diffusion assay at 25c , 2 days for bacteria and 6 days for actinomycetes and fungus . minimum inhibitory concentration ( mic ) of extracts was determined following the protocol of clinical and laboratory standards institute ( clsi).17 , 18 all of the experiments were performed in triplicate , and results were expressed as diameters ( mm ) of inhibition . detection and quantification of catechin , gallic acid and bergenin were carried out using shimadzu 20 ad , hplc system ( shimadzu , japan ) consisted of uv detector , a binary pump , a 10  l injection loop , and reverse phase c-18 column of dimensions 4.6    250  mm . the mobile phase used for catechin was 75% a ( water  +  0.1% trifluoroacetic acid ) and 25 % b ( methanol ) with a flow rate of 0.8  ml / min . the eluted samples were detected by the uv detector at 280  nm . for gallic acid , mobile phase used was 90 % a ( water  +  1% acetic acid ) and 10% b ( acetonitrile ) with a flow rate of 1.0  ml / min . for analysis of bergenin , elution was carried out with solvent 75% a ( water  +  0.1% trifluoroacetic acid ) and 25% b ( acetonitrile ) as mobile phase having flow rate of 1.0  ml / min . calibration curve was constructed by plotting the peak area ( y ) against concentration in g / ml of standard solutions ( x ) . the standard equation obtained from the curve all the experiments were performed in triplicate and the experimental data were expressed as mean    standard deviation ( sd ) . one - way analysis of variance ( anova ) and duncan 's multiple range tests were carried out to determine significant differences ( p  <  0.05 ) between the means by spss ( version 16.0 ) . results showed that leaf extraction yield of b.  ciliata varied considerably as a function of solvent nature and ranged from 8.3 to 38.2 % with a descending order of methanol  >  ethyl acetate  >  hexane ( table  1 ) . extraction with methanol resulted in the highest amount of total extractable compounds whereas the extraction yield with hexane was small in comparison to other solvents . higher extraction yield in methanol might be due to the fact that it easily penetrates the cellular membrane and extracts the intracellular ingredients from the plant material . moreover , results indicated that the plant contains more of polar substances than the others . earlier reports have also suggested that methanol give higher extraction yield than the other solvents such as acetone , diethyl ether , ethyl acetate and water.19 , 20 the antioxidant activity of medicinal plants , fruits and vegetables has been reported to be positively correlated to their total phenolic contents due to their ability to scavenge free radicals.21 , 22 it becomes , therefore , mandatory to estimate total phenolic compounds present in the bergenia extracts . in the present study , the content of extractable phenolic compounds was determined through a linear gallic acid standard curve ( y  =  2.271x  +  0.18 ; r  =  0.995 ) . the highest content of total phenolic compounds was detected in the bergenia methanolic extract ( 473.4  mg gae / g extract ) followed by ethyl acetate extract ( 249.7  mg gae / g extract ) ( p  <  0.05 ) . in the hexane these results demonstrate clearly that the content of phenolic compounds is dependent on the polarity of the solvent used ; higher the polarity of the solvent , higher the content of phenolic compounds . these results are in agreement with the previous study of singh et  al . that reported methanol as an effective solvent for antioxidant extraction , phenolic compounds in particular . flavonoids are common secondary metabolites present in plants which are responsible for many plant biological activities . the  solvent efficiency on tfc , in ascending order was : hexane  <  methanol  <  ethyl acetate . the highest flavonoid content of 208.4  mg qe / g extract was observed in the extract of ethyl acetate followed by methanol extract ( 89.9  qe / g extract ) . these results are in agreement with the report of hajji et  al . which reported that flavonoid content in extract depend on solvent polarity . the dpph free radical scavenging activity in leaf extracts of b.  ciliata is presented in fig .  1 . the methanolic leaf extract showed excellent free radical scavenging activity ( ic50  =  53.5  g / ml ) . in other two leaf solvent extracts , the free radical scavenging activity in ethyl acetate extract ( ic50  =  2593.3  g / ml ) was superior to that of the hexane extract ( ic50  =  3026.7  g / ml ) . nevertheless , when compared to standard , the bht , all the tested bergenia leaf extracts showed significantly ( p  <  0.05 ) lower dpph radical scavenging activity . these results are indicative of the influence of the solvent on the antioxidant activity of the biological extracts . it has been reported that in plant compounds with different polarity and structure are present that dissolve in specific solvents having similar polarity.24 , 25 the difference in the dpph radical scavenging activity in different solvent extracts implies towards the preference of the solvents for extraction of different types and concentrations of bioactive compounds.24 , 25 strong dpph radical scavenging activity of b.  ciliata methanolic extract can be due to higher content of total phenolic compounds . the free radical scavenging activity of bergenia extracts was also determined using abts radical . significant difference ( p  <  0.05 ) was revealed between abts scavenging capacities of extracts measured as ic50 value ( fig .  2 ) . the highest abts radical scavenging activity was found in methanol extract with an ic50 value of 5.4  g / ml , whereas hexane extract exerted the lowest ability to scavenge abts radical with an ic50 value of 51  g / ml . strong abts radical scavenging ability of b.  ciliata methanol and ethyl acetate extracts can be attributed to the presence of flavonoids . earlier , it has been reported that abts radical scavenging ability of bioactive compounds depends on its molecular weight , structure and presence of number of aromatic rings . the correlation coefficients determined between the antioxidant activities and the total phenolic and flavonoid contents in all the b.  ciliata leaf extracts revealed the presence of significant high correlation ( p  <  0.1 ) . the correlation for dpph ic50 value vs tpc and dpph ic50 value vs tfc were  0.913 and  0.056 , respectively . results indicate clearly that the dpph activities of extracts are mainly due to the total phenolic compounds . negative correlation indicated that with the increase in phenolic and flavonoid content , concentration of extract required to inhibit free radical decreases . the correlation for abts ic50 value vs tpc and abts ic50 value vs tfc were  0.911 and  0.782 , respectively , imply that both the total phenol and flavonoids of b.  ciliata are responsible for abts activities . these differences in correlation between phytochemicals and antioxidant assays could be attributed to the different mechanism of the radical antioxidant reaction . in accordance with this study , li et  al . have also reported high negative linear correlation between the phenolic contents and ic50 values in angelicae sinensis confirming that phenolics are likely to contribute to the antioxidant activity of the extracts . however , presence of other metabolites which have significantly contributed in antioxidant activity of b.  ciliata extracts can not be ruled out . the results of the disc diffusion assay ( table  2 ) revealed that all the extracts tested were effective against the bacteria and actinomycetes studied , but showed no activity against fungi . among the tested extracts , methanol extract was found to be the most effective which showed the highest effects against bacillus megaterium strain with inhibition zone of 9.8  mm , followed by nocardia tenerifensis and bacillus subtilis with inhibition zones of 9.7 and 8.8  mm , respectively . the ethyl acetate extract showed the similar pattern of microbial inhibition with highest inhibition zone of 7.5  mm for b. megaterium , followed by 6.2 and 5.5  mm inhibition zone for n. tenerifensis and b.  subtilis , respectively . in contrary to methanol and ethyl acetate extract , the hexane extract had the greatest inhibitory effects against serratia marcescense ( 5.0  mm ) and b.  subtilis ( 4.7  mm ) . the minimum inhibitory concentration ( mic ) was determined in the methanolic extracts of b. ciliata due to its higher antimicrobial activity in disc diffusion assay against the tested organisms . the methanol extract gave the lowest minimal inhibitory concentration against b.  subtilis with mic of 1250  g / ml ( table  3 ) . the highest antimicrobial activity of methanolic extract could be due to the high contents of phenolic compounds and flavonoids present in the extract . these results are in accordance with a previous study where similar observations were recorded in case of bergenia ligulata leaf extracts . b.  ciliata is reported to contain a wide array of pharmaceutically important bioactive compounds.9 , 29 , 30 among all , three main compounds viz . , bergenin catechin and gallic acid are the most sought after compounds of the plant species . during the past years , efforts have been made for identification and quantification of the major bioactive compounds from bergenia spp.5 , 28 , 30 , 31 , 32 however , till date , b.  ciliata plant of the sikkim himalaya has never been analyzed for secondary metabolites . to the best of our knowledge , this is the first report on this aspect . in the present study , different extracts of b. ciliata were analyzed by hplc for the identification and quantification of bergenin , catechin and gallic acid . standards showed high linearity at tested concentrations with correlation coefficients ( r ) of 0.99 , 0.96 and 0.99 for catechin , gallic acid and bergenin , respectively . the chromatographic peaks of the analytes were confirmed by comparing their retention time with those of the standards . from the standard equation obtained ( table  4 ) , the amount of bergenin , catechin and gallic acid calculated in different extracts is presented in table  5 . bergenin was detected in all the extracts and amount of bergenin was found in the order : methanol  >  ethyl acetate  >  hexane . the methanol extract contained bergenin ( 2.43    0.08% ) in amounts that were orders of magnitude higher than those in the ethyl acetate ( 1.63    0.17% ) and hexane ( 0.0024    0.00% )  extracts . hplc analysis of catechin and gallic acid revealed that these compounds were present in methanol extracts only ( table  5 ) . in this study , the antioxidant and antimicrobial activities of b.  ciliata plant growing in the sikkim himalaya are reported for the first time . the methanolic extracts of b.  ciliata leaves , that were shown to contain the highest total phenolic and flavonoid compounds , exhibited high free radical scavenging activity against dpph and abts radicals . further , hplc analysis of different extracts for catechin , gallic acid , and bergenin showed that methanolic extract contained significantly higher amount of these bioactive compounds .
bergenia ciliata sternb . , commonly known as paashaanbhed , is a well known herb of sikkim himalaya with various pharmaceutical properties . 
 however , scientific exploration of b.  ciliata , growing in the sikkim himalaya , for phytochemicals and pharmacological properties is in infancy . with this view 
 , the present study was undertaken to investigate b.  ciliata leaf extracts for antioxidant , antimicrobial activity and bioactive compounds . 
 three solvents viz . , 
 methanol , ethyl acetate and hexane were used for extraction and the respective leaf extracts were analyzed for total phenolic and flavonoid contents along with the antioxidant and antimicrobial activities . amongst the tested solvents , 
 methanol was found to be the best solvent for extraction with highest total phenolic contents and the lowest ic50 values for the 2,2-diphenyl-1-picrylhydrazyl ( dpph ) and 2,2-azino - bis(3-ethylbenzothiazoline-6-sulfonic acid ) ( abts ) assays . 
 methanol extract also exhibited effective antimicrobial activity , particularly against bacteria and actinomycetes . 
 further , high performance liquid chromatography ( hplc ) analysis revealed that methanolic extract contains the highest amount of all the three analyzed bioactive compounds viz . 
 bergenin , catechin and gallic acid . 
 the current study suggests that the methanol extract of b.  ciliata is a potential source of natural antioxidant and antimicrobial compounds that can be used in food and drug industries .