
 disporopsis pernyi ( hua ) diels , which belongs to disporopsis genus of liliaceae family , mainly grows in south asia such as vietnam , laos , thailand , and yangtze river basin of china .
it was a well - known traditional chinese medicine which has been widely used for the treatment of abnormal sweating , chronic cough , women postpartum weakness and irregular menstrual cycle , and so forth .
the medical value of disporopsis genus plants has not got much attention until the beginning of 21st century .
the roots of the plant are rich in bioactive substances which are possible to have considerable medicinal value and can be used for hypertension cough , inflammation , and tumor .
study suggests that a total of 4 polyphenolic compounds are found in extract including rutin , luteolin , quercetin , and betulinic acid , and the phenolic compounds contribute significantly to the antioxidant and antimicrobial activities [ 2 , 3 ] .
rutin has both antihypertensive effect and antidiabetes effect [ 4 , 5 ] . luteolin which has anti - inflammatory , antibiosis , and anticancer properties has been used for relieving cough and eliminating phlegm . in addition , it has potential anti - hiv effect [ 6 , 7 ] .
quercetin can be used for relieving cough and eliminating phlegm and for hypertension and hyperlipemia ; also , it has neuroprotective and antiproliferative activities [ 8 , 9 ] .
acid can kill human melanoma cell without hurting healthy cell and inhibit the hiv-1 infection .
additionally , recent study shows that it also has the inhibition effects of cerebroma and leukocythemia [ 10 , 11 ] .
the four compounds are major bioactive constitutes in the extract of disporopsis pernyi ( hua ) diels roots .
so far , there is no report on the content of the 4 polyphenolic compounds in disporopsis pernyi ( hua ) diels .
uplc is a simple and quick tool for the quantitative determination of the bioactive constituents in pharmaceutical industry [ 1214 ] . as rutin , luteolin , quercetin , and betulinic acid
the hplc - grade methanol and acetonitrile used were purchased from caledon ( canada ) and formic acid was obtained from dima company ( beijing , china ) .
the rutin , luteolin , quercetin , and betulinic acid were purchased from the national institute for the control of pharmaceutical and biological products in china .
the purity of the standard compounds was 98% ; their chemical structures are shown in figure 1 .
disporopsis pernyi ( hua ) diels were collected from songtao which is in guizhou province of china .
standard stock solutions of rutin , luteolin , quercetin , and betulinic acid were dissolved in methanol , at concentration of 1.0  mg / ml . all standard solutions were filtered through 0.22  m syringe filter . the extraction was carried out using 5.0  g of powdered roots . it was dissolved in 50  ml of 70% ethanol solution and back - flow for 60  min .
after filter and rotary evaporation to no ethanol smell , 50  ml acetic ether was added and extraction was done three times .
the extract and washing liquid were combined and filtered and then evaporated to dryness under reduced pressure in a rotary evaporator .
the dried extract was dissolved in methanol and diluted to a 5  ml volumetric flask .
the hplc system used was a shimadzu nexera x2 uplc ( kyoto , japan ) chromatograph equipped with a solvent delivery unit ( lc-30ad ) , an autosampler ( sil-30ac ) , a column oven ( cto-20a ) , a degasser ( dgu-20a5r ) , and a photodiode array detector ( spd - m20a ) .
separation was conducted on a shim - pack xr - ods column ( 2.0  75  mm , 1.6  m ; shimadzu cooperation , japan ) .
the column temperature was set at 30c . the mobile phase consisted of water containing 0.1% formic acid ( a ) and acetonitrile ( b ) .
the composition of the mobile phase was 5% ( b ) for 02  min , 5%10% ( b ) for 24.5  min , 10%40% ( b ) for 4.511  min , 40%60% ( b ) for 1113  min , 60%70% ( b ) for 13 - 14  min , 70%80% ( b ) for 1416  min , 80%90% ( b ) for 16 - 17  min , 90% ( b ) for 1720  min , and it was held for 3  min and then reequilibrated to 5% ( b ) until the end of the analysis .
the flow rate was 0.2  ml / min and the injection volume was 5  l .
the detection wavelengths of all standards and samples were in the uv at 210 , 254 , and 280  nm .
the 4 standard compounds were accurately weighed and dissolved in methanol to prepare stock solutions at a concentration of 1.0  mg / ml . stock solutions of the compounds were serially diluted to construct calibration curves .
the diluted concentrations of compounds were plotted against the peak area on the calibration curves and the linearity was measured from the correlation coefficient .
blank samples were analyzed in triplicate and the area of the noise peak was calculated as the response .
the lod and loq were calculated as lod = 3.3  sd / s and loq = 10  sd / s , where sd is the standard deviation of the response and s is the slope of the calibration curve .
the precision was calculated by analyzing sample extracts containing low and high concentrations of the compounds .
the precision was represented by the relative standard deviation ( rsd ) , which was calculated using the equation rsd = ( standard deviation / mean )  100 .
the precision was measured three times in a single day ( intraday precision ) and over three consecutive days ( interday precision ) .
the recovery was calculated as follows : recovery ( % ) = ( ( detected concentration  initial concentration)/spiked concentration )  100 .
we used pda detector in this experiment , which could select each wavelength of chromatogram . in our study , we took into account the question that most of the components we studied also have good absorption at wavelength of 350  nm and 370  nm [ 15 , 17 ] . by comparing the resolution and response at different wavelength , the results showed that the resolution and response of the four components at 350  nm and 370  nm are not as good as the wavelengths 210 , 254 , and 280  nm we chose . also , we chose 210  nm to detect betulinic acid for its good response .
combined with the literature reports , methanol - water , acetonitrile - water , and methanol - acetonitrile - water were examined as mobile phase as well as the type ( formic acid and glacial acetic acid ) and concentration ( 0.01% , 0.05% , and 0.1% ) of the acid . a shim - pack xr - ods column was employed for the simultaneous determination of the 4 compounds , as it has been the most frequently used technique in the chemical analysis of herbal medicines by uplc .
peak resolution and shape of the compounds were considered better indicators when 0.1% formic acid was used as a modifier .
taking peak shape , degree of separation , the symmetrical factor , and other factors into consideration , acetonitrile-0.1% formic acid water solution was determined as gradient elution process .
the linearity of the calibration curve was measured by the correlation coefficient ( r ) , which ranged in value from 0.9992 to 0.9997 for each compound .
the lod and loq were 0.1370.264  g / ml and 0.4560.881  g / ml , respectively ( table 1 ) .
the intra- and interday precision , which were represented by the rsd values , were rsd < 2.0% .
the recoveries of the 4 compounds were in the range 96.2%102.6% , with rsd < 2.0% ( table 2 ) .
the results indicate that the developed analytical method was accurate and precise for the analysis of the 4 compounds in disporopsis pernyi ( hua ) diels .
the method we developed was applied to determine the 4 compounds in disporopsis pernyi ( hua ) diels successfully .
the calculated contents of the four compounds were 5.63  g / g for rutin , 2.51  g / g for luteolin , 3.87  g / g for quercetin , and 2.41  g / g for betulinic acid .
the uplc method mentioned here represented an excellent technique for simultaneous determination of rutin , luteolin , quercetin , and betulinic acid in the extract of disporopsis pernyi ( hua ) diels roots , with good sensitivity , precision , and reproducibility .
the method gives a good resolution among the four components with the analysis time ( 25  min ) .
furthermore , the method can be used as quality control of polyphenolic compounds in disporopsis pernyi ( hua ) diels roots and will play a reference role on the determination of polyphenolic compounds in other medicinal plants or pharmaceutical preparations .
 
 disporopsis pernyi ( hua ) diels , which belongs to genus disporopsis , has been widely used for the treatment of abnormal sweating , chronic cough , and so forth . 
 an ultra - performance liquid chromatography ( uplc ) analysis was developed for the determination of rutin , luteolin , quercetin , and betulinic acid in disporopsis pernyi ( hua ) diels roots . 
 uplc analysis was conducted by using a shim - pack xr - ods column with gradient elution with the mobile phase of acetonitrile and water containing 0.1% formic acid and with a flow rate of 0.2  ml / min , detected at 210 , 254 , and 280  nm . 
 the method was precise , with relative standard deviation < 2.0% . 
 the recoveries for the four components in disporopsis pernyi ( hua ) diels were between 98.5 and 100.9% . 
 the average contents of rutin , luteolin , quercetin , and betulinic acid in roots were 5.63 , 2.51 , 3.87 , and 2.41  g / g , respectively . 
 the method was accurate and reproducible and it can provide a quantitative basis for quality control of disporopsis pernyi ( hua ) diels . 