zhigancao , glycyrrhizae radix et rhizoma praeparata cum melle , originates from the processed dried roots or rhizomes of glycyrrhriza uralensis fisch .
it is most frequently used in traditional chinese medical formulary to harmonize all drugs and detoxify the adverse effects of herbs .
clinically , it treats disorders such as shortness of breath , fatigue , epigastria and abdominal pain , musculoskeletal and smooth muscle cramp and pain , and diarrhea . due to the numerous bioactive compounds in it such as terpenoids , saponins , polysaccharides , and flavonoids ,
zhigancao has been reported to possess anti - inflammatory activities , antioxidative [ 3 , 4 ] , neuroprotective , antiallergic , anticonvulsant activities , and so forth .
two chalcone derivatives in figure 1 , isoliquiritin apioside ( ila ) and isoliquiritin ( il ) , which are important ingredients in zhigancao , are selected to research their pharmacokinetics .
il has been reported to have shown various pharmacological activities such as antiangiogenic effect dependent upon antitube formation , antidepressant - like effects in mice induced by forced swimming and tail suspension , and inhibitory effects on tyrosinase .  
the bioavailability of drugs is the cornerstone for extending their in vitro biological functions to humans in vivo .
the flavonoid glycosides show low oral bioavailability possibly because of the extensive conjugation of the free hydroxyl groups [ 1214 ] or / and glycosides hydrolysis to the aglycones in the intestinal lumen [ 1416 ] .
the pharmacokinetic study of ila and il is essential for us to comprehend the bioavailability of two analytes after an oral administration of zhigancao extrat . to my knowledge , ila and
il quantification method ( lc - ms / ms ) has been performed to study pharmacokinetics of multiple licorice flavonoids after an oral dose of xiaochaihu - tang to rats , but lc - ms / ms method is simply not available in most laboratories . in this study , we established the hplc method which was highly selective and sensitive for simultaneous quantification of ila and il in rat plasma .
the method was used to explore the possible pharmacokinetics of ila and il after an oral administration of zhigancao extract .
isoliquiritin apioside ( ila ) and isoliquiritin ( il ) were purchased from xi'an xiaocao botanical development co. , ltd .
wogonoside ( internal standard , is ) was obtained from national institute for food and drug control ( beijing , china ) .
zhigancao which was produced under the guidance of the theory of traditional chinese medicine science was provided by chifeng xinzhou traditional chinese medicine co. , ltd .
the material was authenticated as the dried roots of g. uralensis by professor ting - guo kang from liaoning university of traditional chinese medicine .
hplc grade acetonitrile and analytical grade chloroform were purchased from shandong yuwang chemical industry co. , ltd .
the water in the study was purified with a milli - q water purification system from millipore ( bedford , usa ) .
powdered herb materials were extracted twice under reflux condition with 70% ethanol ( 1  :  10 , w / v ) for 1  h under 100c . the extract was filtered and evaporated . finally , the residue was dried in vacuum drying oven at 60c to obtain a powder state of zhigancao extract .
the contents of ila and il in the extract were detected by hplc , 33.72 and 28.17  mg / g , respectively .
the liquid chromatography system employed was agilent 1100 with a variable wave length uv detector ( g1314a vwd ) .
, 5  m , dikma technologies , china ) analytical column with a endcapped c18 ods guard column .
the mobile phase composed of water ( containing phosphoric acid 0.1% , v / v ) and acetonitrile ( 72  :  28 , v / v ) was filtered through 0.22  m millipore membrane filter . the flow rate was 1.0  ml / min .
the detection wavelength was set at 360  nm ( 09  min ) and 276  nm ( 912  min ) .
stock solutions of is , ila and il with concentrations of 440 , 384 , and 480  g / ml , respectively , were prepared in methanol , and stored at 4c . the working solution for is was diluted with methanol to get a final concentration of 8.80  g / ml . stock solutions were diluted with methanol to serial standard working solutions at concentrations of 0.60 , 1.20 , 2.40 , 4.80 , 9.60 , 19.2 , and 38.4  g / ml for ila , 0.75 , 1.50 , 3.00 , 6.00 , 12.0 , 24.0 , 36.0 , and 48.0  g / ml for il .
these solutions were then added to blank plasma ( 1  :  10 ) to make standards of 0.0603.84  g / ml for ila and 0.0754.80  g / ml for il .
the quality control ( qc ) samples which were used for the intra- and inter - day accuracy , and precision , extraction recovery and stability study , were prepared in the same way at 0.12 , 0.48 and 3.07  g / ml for ila , and 0.15 , 0.60 and 3.84  g / ml for il . the 200  l of rat plasma was mixed with 20  l is working solution .
after protein was precipitated with 500  l of acetonitrile in a 1.5  ml polypropylene tube by vortexing for 3  min , the sample was centrifuged at 6,677  g for 5  min .
the supernatant was transferred into a 2.0  ml tube and was added with 1,000  l of chloroform .
after vortexing and centrifugation , 20  l of water phase was injected for analysis .
the analysis method was completely validated using spiked rat plasma samples , including selectivity , linearity , intra- and inter - day precision , accuracy and stability , according to the fda guideline for method validation of bioanalytical assays .
the selectivity of the method was demonstrated by comparing chromatograms of blank plasma samples ( without is ) obtained from rats , plasma samples spiked with the analytes and is , and plasma samples after an oral dose .
all blank plasma samples were prepared and analyzed to ensure the absence of interfering peaks .
the linearity of the method was assessed by plotting calibration curves in plasma at seven concentration levels in triplicate on three consecutive days .
the lower limit of quantification ( lloq ) was defined as the lowest concentration of the calibration curve , that was measured with accuracy and precision by analyzing samples in six replicates at the concentration of 0.060  g / ml for ila and 0.075  g / ml for il .
precision and accuracy were evaluated by determining qc samples at three concentrations in six replicates on the same day ( intra - day accuracy and precision ) and three consecutive days ( inter - day accuracy and precision ) .
accuracy was measured by relative error ( re ) and precision was evaluated by intra- and inter - day relative standard deviation ( rsd ) .
the extraction recovery was evaluated by comparing the peak areas of the extracted qc samples at three concentrations in six replicates with those of unextracted standards that represented 100% recovery .
similarly , the recovery of is was evaluated at a single concentration of 0.88  g / ml in the same way .  
the stability of ila and il in rat plasma was evaluated by comparing the mean of back - calculated concentration of the stored qc samples at three concentrations in triplicate with the spiked concentration of analytes .
the qc samples treated as sample preparation were kept at room temperature for 12  h and then the stability was determined .
the freeze - thaw stability was determined afterthree freeze ( 20c , 24  h ) and thaw ( room temperature ) cycles .
long - term stability was assessed by keeping qc samples at 20c for 15 days .
female sprague - dawley rats ( 200  20  g ) were kept in environmental controlled breeding room for 7 days until the experiment .
the rats were fasted for 12  h but allowed water ad libitum before the zhigancao extract was orally administered at a dose of 1.21  g / kg .
orbital venous blood samples ( 0.5  ml ) were collected before dosing , and at 0.5 , 1.0 , 1.5 , 2 , 3 , 4 , 6 , 8 , 12 , 24 , 36 , and 48  h after an administration .
after centrifuging at 6,677  g for 5  min , the plasma samples were obtained and frozen at 20c until analysis .  
hplc analysis procedure was applied to analyze plasma concentration - time profiles of ila and il .
data was processed by noncompartmental method using drug and statistics ( das ) 2.0 software package ( chinese pharmacological society , shanghai , china ) .
the selectivity was evaluated by analyzing blank samples , spiked samples at lloq , and middle qc levels , and actual samples obtained from rats after an oral administration of zhigancao extract .
for all blank samples , ila , il , and is retention windows were free from endogenous interfering peaks .
the calibration curves were linear over the concentration range of 0.0603.84  g / ml for ila and 0.0754.80  g / ml for il by weighted ( 1/x ) linear least - squares regression method .
the res of the back - calculated values of the standards from their nominal values were constantly within 15% for all values , including the lloq .
the lloq measurement showed the respective averages 0.060  g / ml with rsd 14.5% for ila and 0.077  g / ml with rsd 12.3% for il .
typical regression equations were calculated as follows : ila , y = 1.024x  0.004 ( r = 0.9954 ) ; il , y = 1.004x + 0.007 ( r = 0.9968 ) .   the precision and accuracy data was shown in table 1 .
the intra- and inter - day rsd values were lower than 10% , and the re values were within 5% .
the extraction recoveries of ila and il from rat plasma were 79.5  4.2% , 82.5  4.4% , 84.7  4.0% at 1.2 , 4.8 , and 3.07  g / ml and 81.3  4.9% , 82.3  6.6% , 86.4  3.4% at 1.5 , 6.0 , and 3.84  g / ml , respectively .
these results indicated that the extraction method was suitable to extract ila , il , and is from plasma .
as shown in table 2 , stability of analytes showed no significant sample loss over 12  h at room temperature , three freeze - thaw cycles , and 15 days storage condition .  
the developed method was applied in pharmacokinetic study of ila and il in rat plasma after an oral administration of zhigancao extract ( at a dose containing 40.8  mg / kg ila and 34.1  mg / kg il , resp . ) .
the mean plasma concentration - time profiles ( n = 6 ) were shown in figure 3 .
the assay was sensitive enough for the determination of ila and il in rat plasma after an oral administration of zhigancao extract .
in this study , we established an hplc method to simultaneously quantify ila and il in rat plasma . due to the stronger polarity of ila and il ,
the recovery with methanol was comparable to acetonitrile , during both processes the analyte concentration in the plasma was diluted when 2.5-fold organic solvent volume was added , so that ila and il couldnot be detected at a low concentration . yet evaporating supernatant to dryness and then dissolving it in a small amount of solvent to increase the concentration would be time - consuming and would cause the loss of analyte . so removing the organic solvent from the supernatant was employed . when the supernatant was added with chloroform , a clear water phase could be obtained by precipitating the protein with acetonitrile , but no stratification could be observed when methanol was used instead of acetonitrile therefore , plasma samples were processed by precipitating protein with acetonitrile which
wogonoside was selected as the is because of its appropriate retention and extraction recovery . some other compounds such as rutin , baicalin and hesperidin
were also tested with the selected condition above , but without ideal results . due to its satisfactory separation from the analytes ,
wogonoside was selected as the is . because wogonoside couldnot be detected at 360  nm at the concentration 0.88  g / ml , the wavelength was switched to 276  nm at 9  min .  
the extrapolated fraction of the auc0 accounted for only 5 - 6% , which indicated a suitability of the analytical method for ila and il pharmacokinetic study .
these two analytes exhibited consistent tendencies in plasma concentration - time profiles and similar tmax , t1/2z and mrt values after an oral administration of zhigancao extract .
the fact that these two compounds had the similar pharmacokinetic behavior could be tentatively attributed to their having similar structures .  
comparing the pharmacokinetic data of ila with that of il , cmax and auc of ila were lower than that of il although there was more content of the former in zhigancao extract .
it was possible that ila could be hydrolysis to the il in the intestinal tract or transformed into il after being absorbed .
we have developed and validated a selective and sensitive hplc method for simultaneous quantification of ila and il in rat plasma .
the method was successfully applied to the pharmacokinetic study of ila and il in rat plasma after an oral administration of zhigancao extract .
 a sensitive hplc method was developed for the quantitative determination of isoliquiritin apioside ( ila ) and isoliquiritin ( il ) in rat plasma . 
 after protein precipitation with acetonitrile , chloroform was used to separate lipid - soluble impurities from the plasma samples and remove acetonitrile . 
 a chromatography was carried out on diamonsil c18 ( 150  4.6  mm ; 5  m ) analytical column , using a mobile phase consisting of water ( containing phosphoric acid 0.1% , v / v ) ; acetonitrile ( 72  :  28 , v / v ) at a flow rate of 1.0  ml / min . 
 the wavelength - switching technology was performed to determine ila and il at 360  nm and wogonoside ( internal standard , is ) at 276  nm . 
 the calibration curves of ila and il were fairly linear over the concentration ranges of 0.0603.84  g / ml ( r = 0.9954 ) and 0.0754.80  g / ml ( r = 0.9968 ) , respectively . 
 the average extract recoveries of ila , il , and is were all over 80% . 
 the precision and accuracy for all concentrations of quality controls and standards were within 15% . 
 the lower limit of quantification ( lloq ) was 0.060  g / ml for ila and 0.075  g / ml for il . 
 the method was used in pharmacokinetic study after an oral administration of zhigancao extract to rats . 
1. Introduction
2. Experimental
3. Results 
4. Discussion
5. Conclusion