
 selaginella doederleinii hieron , as a traditional chinese medicine , is a well - known perennial pteridophyte plant growing in south and southwestern china at the low altitude .
it has the function of eliminating wind , scattering frigidity , detumescence , and treating cough . in the reported research ,
different types of compounds from selaginella doederleinii , including biflavonoid , alkaloid , xylogen , sterol , and organic acid , have been exhibited [ 26 ] .
some researches   have proposed that the potential antioxidants in selaginella doederleinii are biflavonoids , including amentoflavone , robustaflavone , heveaflavone , and robustaflavone 4 ,  4-o - dimethyl ether ( see figure 1 ) .
most biflavonoids , just like apigenin and its methoxyl / hydroxyl substituents , have prominent antioxidant characteristics and show a wide range of biological activity , such as cancer chemopreventive properties , anti - inflammatory , antimicroorganism , and antioxidative effects [ 813 ] .
traditional screening for antioxidant activity components from natural plants is a time - consuming , inefficient , and costly method , which can not meet the need of the development of modern scientific research .
therefore , it is necessary to find out more new and efficient online techniques to search the active compounds from natural products .
this study aimed to develop a rapid , efficient , and stable method to screen and identify the main antioxidative components in the extracts from selaginella doederleinii .
the online 2,2-diphenyl-1-picrylhydrazyl - ultra - high performance liquid chromatography - q - time - of - flight mass spectrometry ( dpph - uplc - q - tof / ms ) technique was used for the first time to screen and identify antioxidants from ethyl acetate extract in this study .
nine active biflavonoids were yielded and included amentoflavone ( 1 ) , robustaflavone ( 2 ) , bilobetin ( 3 ) , 4-methoxy robustaflavone ( 4 ) , podocarpusflavone a ( 5 ) , hinokiflavone ( 6 ) , ginkgetin ( 7 ) , putraflavone ( 8) , and heveaflavone ( 9 ) . among them , compounds 3 and 8 in this study were first reported from selaginella doederleinii .
herbal materials were identified as selaginella doederleinii by vice professor yang jianwen from the department of pharmacy in zunyi medical college . a voucher specimen ( 20120923 )
has been preserved in the key laboratory of natural pharmaceutical chemistry , zunyi medical college .
2,2-diphenyl-1-picrylhydrazyl ( dpph ) was purchased from sigma pure chemical industry ( berlin , germany ) .
butylated hydroxytoluene ( bht ) , ascorbic acid , and quercetin were purchased from bdh company ( poole , england ) .
acetonitrile of hplc grade was provided by merck chemical industry ( darmstadt , germany ) .
100.0  g of dried selaginella doederleinii was powdered and extracted three times ( each for 2  h ) with 95% ethanol ( 1.0  l ) .
the extracting solution was filtrated , merged , and centrifuged at 4000  rpm under 5c .
then 18.37  g extracts were finally obtained from selaginella doederleinii , which were then dissolved in warm water and used for liquid - liquid extraction with petroleum ether , ethyl acetate , and n - butyl alcohol successively .
the three fractions were concentrated , dried , and obtained 0.52  g , 4.19  g , and 6.64  g , respectively .
the dpph oxidative assay is widely used for quantitative determination of antioxidant capacity in biological samples and food . in short ,
each of polarity fractions was dissolved in methanol ( 4  mgml in methanol ) and diluted different concentrations sample ( 0.08~0.14  mgml in methanol ) .
1.0  ml of the sample was mingled with 4.0  ml dpph solution ( 0.6  mgml in methanol ) .
the mixture was completely shaken , reacted , and left at room temperature in the dark for 60  min .
the absorbance was measured at 517  nm in an ultraviolet spectrophotometer ( shimadzu uv - vis 160a , japan ) .
the percentage of dpph inhibition was calculated by using the following equation : 
 ( 1)i%=abasab100% , 
 where i is the inhibition percentage , a 
 b is the absorbance of the blank sample , and a 
 s is the absorbance of the test sample .
sample concentration providing 50% inhibition ( ic50 ) was calculated by plotting the inhibition percentage against different concentrations sample .
all samples of the test were run in triplicate and the average value was calculated .
the extract from selaginella doederleinii was mingled with dpph solution of different concentrations ( 0.16 , 0.32 , and 0.48  mml ) at the ratio of 1  :  1 ( w / v ) .
the mixture was completely shaken , produced reaction , and was left at room temperature in the dark for 60  min .
then , the mixture was filtered through a 0.45  m pinhole - membrane filter and injected into the uplc system . for the control sample methanol
the mixture and control samples were analyzed in an acquity uplc system ( waters corp . ,
milford , ma , usa ) equipped with ultraviolet ( uv ) and a beh c18 ( 100  2.1  mm , 1.7  m ) .
formic acid ( 0.1% ) and acetonitrile were , respectively , used as the mobile phases a and b. the solvent gradient was used as follows : 3080% b between 0 and 25  min , 8095%  b between 25 and 27  min , and 95100% b between 27 and 30  min .
the flow rate was set at 0.6  ml / min at 30c and the injection volume of the sample was 5  l . in comparison to the uplc chromatographic profiles of the reaction samples and the control samples ,
the molecular weight and structure of the screened antioxidants were identified by q - tof / ms .
high - purity nitrogen was utilized as a nebulizer , auxiliary gas was set at a 2500  l / h , and flow rate was set at 350  l / h .
argon was used as the collision gas at a flow rate of 50  l / h under 1000 degrees .
electrospray ionization ( esi ) capillary voltage was set at + 3.0  kv for positive ion mode .
the sample cone voltage was set at 33  v and the collision energy ( ce ) was set at 2.5  ev .
the mass scan was in the range of 2001500  m / z and sweeping time was 1  s.
antioxidant activity of petroleum ether , ethyl acetate , and n - buoh fractions was then evaluated by dpph radical scavenging activity . as shown in table 1 ,
compared with bht , ascorbic acid , and quercetin , ethyl acetate fraction has the very good antioxidant activity among different polarity fractions .
therefore , the antioxidants were screened and identified from ethyl acetate fraction of selaginella doederleinii with the online dpph - uplc - q - tof / ms assay . according to the uplc conditions in section 2.4
, the quick analysis results of the potential antioxidants from selaginella doederleinii are shown in figure 2 .
it was concluded that the peak areas ( pas ) of the antioxidant would obviously decrease in the uplc chromatogram after reaction with dpph .
therefore , the untreated and dpph - treated ethyl acetate fraction from selaginella doederleinii were analyzed by the optimized uplc separation conditions as stated . through the comparison of the uplc chromatograms of untreated and dpph - treated samples ,
the pas of nine main peaks were all decreased significantly . and nine peaks were detected and isolated from the sample .
their retention time was 5.25 , 6.19 , 7.28 , 8.32 , 8.81 , 9.27 , 10.92 , 11.47 , and 14.92  min , respectively ( see figure 2 ) .
the results indicated that these nine peaks were all antioxidants existing in the ethyl acetate fraction . in order to estimate the radical scavenging capacity of the nine antioxidants ,
pas of nine peaks of untreated sample were set as 100% and the relative pas of the sample that were reacted with dpph of different concentrations were calculated . as the result , the pas of nine peaks all decreased with the increase of dpph concentration . at
but with the increase of dpph concentration , peak 6 decreased the most among the nine peaks ( see table 2 ) .
the results indicate that peak 6 has the strongest radical scavenging capacity and peaks 5 , 3 , 7 , 8 , 9 , 1 , 4 , and 2 follow suit .
the pure biflavonoid compounds were divided into the three types , which were the amentoflavone - type , robustaflavone - type , and hinokiflavone - type biflavonoids , respectively .
the ionization of these compounds was included in both the positive and negative esi ion modes . the positive ion mode provided more rich , accurate , and specific signals than the negative ion mode .
the characteristic fragment ion peaks observed by the cleavage of [ m+h ] ions of the three types of biflavonoids were shown in table 3 .
the most useful fragmentation about compound 1 produces rda cleavage at position 1/3 of the c - ring on flavonoid part ii .
the fragmentation pathway leads to the [ m+h - c8h6o ] at m / z 421 . at the same time , after [ m+h ] can also lose a h2o molecule and undergo rda cleavage at the same 1/3 position , the fragmentation pathway leads to the [ m+h - h2o - c8h6o ] at m / z 403 . and
then , it further loses a series of co molecules continuously and gives rise to the fragment ions at m / z 375 and 347 . after undergoing the cleavage at position 0/4 of the c - ring on flavonoid part ii , another important pathway can lead to the [ m+h - c9h6o3 ] at m / z 377 , [ m+h - c9h6o3-c2h2o ] at m / z 335 , and [ m+h - c9h6o3-c2h2o - co ] at m / z 307 , continuously .
so compound 1 was determined as amentoflavone on the basis of previous deduction and literature . moreover , the [ c7h6o2 ] at m / z 121 and the [ c7h4o4 ] at m / z 153 are important ions , because they provide information whether there is a substituent at b - ring c4 on flavonoid part ii and at a - ring c7 from flavonoid part i. compounds 3 , 5 , 7 , 8 , and 9 exhibited the quasimolecular ion [ m+h ] at m / z 553.1110 , 553.1086 , 567.1273 , 567.1283 , and 581.1434 , respectively . due to involving the cleavage of the c - ring at positions 1/3 and 0/4 on flavonoid part ii , observations of the five compounds fragmentations cleavage pathway indicate that compounds 3 , 5 , 7 , 8 , and 9 clearly belong to an amentoflavone - type biflavonoid ( see figure 3 and table 3 ) .
a series of prominent ions at m / z 435 , 403 ( base peak ) , 347 , 377 , 335 , and 307 were also found in compound 3 ion mass spectra of [ m+h ] .
what is more , other compound 3 characteristic ions were also found such as [ m - c8h6-c2h2o - c3o2-co ] at m / z 283 , [ c7h4o4]at m / z 153 , and [ c8h7o2 ] at m / z 135 .
the analysis results indicate that no substituent is observed on flavonoid part ii . but a methoxy group at position c4 of the b - ring on flavonoid part i is observed .
so compound 3 was considered a bilobetin . a series of prominent ions at m / z 421 , 403 ( base peak ) , 347
, 377 , 335 , and 307 were observed in compound 5 ion mass spectra of [ m+h ] .
in addition , compound 5 ions were also found such as [ m - c8h6-c2h2o - c3o2-co ] at m / z 283 , [ c7h4o4 ] at m / z 153 , and [ c8h7o2 ] at m / z 135 . according to analysis results above
, no substituent is found on flavonoid part i and a methoxy group is observed at position c4 of the b - ring on flavonoid part ii .
so compound 3 was identified as a podocarpusflavone a. a series of prominent ions at m / z 549 , 417 ( base peak ) , 391 , 361 , and 311 were observed in compound 7 ion mass spectra of [ m+h ] . moreover , other compound 7 ions were also observed such as [ c8h6o4 ] at m / z 167 and [ c7h6o2 ] at m / z 121 .
the results show that no substituent is found on flavonoid part ii . but a methoxy group at position c4 of the b - ring on flavonoid part i and a methoxy group at position c7 of a - ring on flavonoid part i are found .
a series of prominent ions at m / z 435 , 417 ( base peak ) , 389 , 361 , and 321 were observed in compound 8 ion mass spectra of [ m+h ] .
furthermore , other compound 8 ions were also observed such as [ m - c8h6-c2h2o - c3o2-co ] 
 m / z 297 , [ c8h6o4 ] at m / z 167 , and [ c8h7o2 ] at m / z 135 . according to analysis results above ,
it is indicated that this molecule has a methoxy group at position c4 of the b - ring on flavonoid part ii and a methoxy group at position c7 of a - ring on flavonoid part i. so compound 8 was identified as putraflavone .
a series of prominent ions at m / z 549 , 417 ( base peak ) , 389 , 361 , and 335 were observed in compound 9 ion mass spectra of [ m+h ] .
what is more , other compound 9 ions were also observed such as [ m - c8h6-c2h2o - c3o2-co ] at m / z 311 , [ c8h6o4 ] at m / z 167 , and [ c8h7o2 ] at m / z 135 .
the analysis results above indicate that a methoxy group is observed at position c4 of b - ring on flavonoid part ii , a methoxy group is observed at position c7 of the a - ring , and a methoxy group is found at position c4 of the b - ring on flavonoid part i. so compound 9 was considered heveaflavone .
compound 2 exhibited the quasimolecular ion [ m+h ] at m / z 539.0977 . compared with that of amentoflavone - type biflavonoids , they are two important and different fragmentations cleavage pathway .
one important fragmentation involves rda cleavage at position 1/3 of the c - ring on flavonoid part i. the fragmentation pathway gives rise to the [ m+h - c7h4o4 ] at m / z 387 .
getting involved with the cleavage at position 1/4 of the c - ring on flavonoid part i , the other significant pathway arouses the [ m+h - c6h4o3 ] at m / z 413 .
so compound 2 was determined as robustaflavone on the ground of previous deduction and literature .
a series of main ions at m / z 427 , 401 , 309 , and 283 were found in compound 4 ion mass spectra of [ m+h ] . according to the [ m - c6h4o4 ] at m / z 401 and [ m - c5h4o3 ] at m / z 427 of compound 4 ( see figure 4 ) ,
because the typical fragmentation pathway of robustaflavone gets the cleavage at positions 1/4 and 1/3 of c - ring on flavonoid part i , other compound 4 characteristic ions are also found such as [ c7h4o4 ] at m / z 153 .
the analysis results indicate that a methoxy group at position c4 of the b - ring on flavonoid part i is observed .
the most main diagnostic ions are a sequence of ions at m / z 286 , 270 , 254 , and 242 , which root in the rupture of the c - o connection on flavonoid parts i and ii ( see figure 5 ) .
furthermore , the predominant ion at m / z 257 is found by the quasimolecular ion losing flavonoid part i and co , continuously .
so compound 6 was determined as hinokiflavone on the basis of previous deduction and literature . through the above analysis , the major fragmentation of the hinokiflavone - type biflavonoids shows great difference with the first two types of biflavonoids , because two flavonoid parts of the hinokiflavone type are not a c - c bond but a c - o bond .
uplc - dpph radical spiking test combined with q - tof / ms , as a rapid , simple , and economical analysis method with which screening antioxidants from selaginella doederleinii successfully was carried out .
the biflavonoids were structurally identified and divided into the three types , that is , amentoflavone , robustaflavone , and hinokiflavone . among them ,
the identified biflavonoids may contribute via their dpph free radical scavenging potential to the beneficial antioxidation effects of selaginella doederleinii .
it demonstrates that uplc - q - tof / ms assay has a great possibility to become high - advanced method for antioxidants screening and identification of constituents in selaginella doederleinii , and it will be useful to the high - value application of the rich medicinal herbs in china .
 
 2,2-diphenyl-1-picrylhydrazyl - ultra - high performance liquid chromatography - q - time - of - flight mass spectrometry ( dpph - uplc - q - tof / ms ) , as a rapid and efficient means , now was used for the first time to screen antioxidants from selaginella doederleinii . 
 the nine biflavone compounds were screened as potential antioxidants . 
 the biflavones were structurally identified and divided into the three types , that is , amentoflavone - type , robustaflavone - type , and hinokiflavone - type biflavonoids . among the compounds bilobetin ( 3 ) and 
 putraflavone ( 8) were found from selaginella doederleinii for the first time and others including amentoflavone ( 1 ) , robustaflavone ( 2 ) , 4-methoxy robustaflavone ( 4 ) , podocarpusflavone a ( 5 ) , hinokiflavone ( 6 ) , ginkgetin ( 7 ) , and heveaflavone ( 9 ) were identified previously in the plant . moreover , nine biflavones possessed a good antioxidant activity via their dpph free radical scavenging . 
 it demonstrates that dpph - uplc - q - tof / ms exhibits strong capacity in separation and identification for small molecule . 
 the method is suitable for rapid screening of antioxidants without the need for complicated systems and additional instruments . 
1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusions