bamboo leaves have been used in traditional chinese medicine for treating fever and detoxification for over 1000 years . it was found that extract of bamboo leaves has multiple biological activities , such as cancer prevention [ 1 , 2 ] , anti - free radical , and antioxidation [ 3 , 4 ] , and can be used as a pharmaceutical intermediate and food additive . a new product named bamboo - leaf flavonoid has appeared on the market . the main functional components of the extract from bamboo leaf are flavone c - glycosides including isoorientin , isovitexin , orientin , and vitexin ( figure 1 ) , which have been used as marker compounds for the determination of commercial products of bamboo - leaf flavonoids . bamboo is an important renewable resource and more than 1250 bamboo species , belonging to 75 genera , are distributed all over the world . hence , to investigate and determine these flavone c - glycosides in the leaves of different species of bamboo is needed . thin - layer chromatography ( tlc ) chemical screening is simple , fast , and of low cost in identification of chemical ingredient from plant extracts [ 7 , 8 ] . many papers about tlc chemical screening have been published in recent years [ 9 , 10 ] . analytical methods for determination of the c - glycosylflavone isomer pairs orientin / isoorientin and vitexin / isovitexin , including hplc , hpce , hptlc [ 13 , 14 ] , and hplc - ms , have been reported . however , the reported hplc chromatographic conditions need to be optimized to obtain a good resolution of adjacent peaks . furthermore , hplc analysis for selected bamboo species in this study was not reported . the work aimed to develop a two - step procedure to quickly survey a large number of bamboo leave samples ( 51 species in 17 genera ) for the presence of flavone c - glycosides and to optimize and validate an hplc method for the simultaneous quantitation of marker compounds in different bamboo leaves and its commercial products . the leaves of fifty - one bamboo species were collected from five regions in china in 2007 and authenticated by professor jiusheng peng from jiangxi academy of forestry , professor yulong ding from nanjing forestry university , and professor qirong guo from international centre for bamboo and rattan , respectively . bamboo leaves were dried in the shade , ground to powder , and stored below 20c . the hplc - grade acetonitrile and methanol were purchased from fisher scientific ( usa ) . glacial acetic acid ( 99.7% ) was purchased from sigma - aldrich ( usa ) . water was purified with an ultrapure water system ( purelab plus , pall , usa ) . commercial products of bamboo - leaf flavonoids labeled with total content of flavonoids ( 30% ) and antioxidant potency were obtained from jinhua beikang biochemical development co. , ltd . the standard compounds of isoorientin , orientin , isovitexin and vitexin were purchased from shanghai winherb medical s & t development co. , ltd . isoorientin and isovitexin stock solutions were prepared in methanol at the concentration of 370  g / ml and 480  g / ml . orientin and vitexin stock solutions were prepared in methanol / water ( 90/10 , v / v ) at the concentration of 480  g / ml and 440  g / ml . the standard solutions were prepared by transferring appropriate volumes of stock solutions of each compound to a 10  ml volumetric flask and diluting to volume with methanol / water ( 60/40 , v / v ) . a series of standard solutions with concentrations 0.01100  g / ml were prepared . the dried bamboo leaf powder ( 1.0  g ) was extracted by percolation with 20  ml of methanol and then sonicated for 30  min at room temperature . thin - layer chromatography was performed on the plates precoated with silica gel 60 f254 ( merck , germany ) . samples ( 4  l ) were applied to the plate as bands of 8  mm wide by means of a camag ( switzerland ) linomat v sample applicator . the plate was developed in an automatic developing chamber ( adc2 ) ( camag , switzerland ) using ethyl acetate  :  formic acid  :  water ( 82  :  9  :  9 , v / v / v ) as the mobile phase . after development , plates were dried and derivatisation was carried out by immersion of the plates in a solution of np reagent ( diphenylborinic acid 2-aminoethyl ester ( 1.0  g ) in ethyl acetate ( 200  ml ) and peg 400 ( 20  g ) in dichloromethane ( 400  ml ) . dried bamboo leaves powder ( 1.0  g ) was accurately weighed and transferred in a 150  ml conical flask with glass stopper , and 60% aqueous methanol ( 15  ml ) was added [ 3 , 17 ] . the solution was stirred for 12  h and sonicated for 30  min at room temperature and then centrifuged at 5000  rpm for 5  min . the residue was further extracted with 60% methanol ( 2  15  ml ) as mentioned above . all the extracted solutions were combined in a 50  ml volumetric flask and diluted to the mark . an aliquot of 20  l of solution was injected for hplc and lc / ms analysis . the hplc - uv / dad analyses were carried out on a waters alliance 2695 ( milford , ma , usa ) system connected to a model 2996 ( dad ) . the separation was performed using a c18 column ( 4.6  250  mm , 5  m , ymc pack r&d ods - a , ymc japan ) at 30c . the solvent system consisted of a mixture of water with 0.5% ( v / v ) glacial acetic acid and acetonitrile ( 85/15 , v / v ) at a flow rate of 1.0  ml / min . the lc - ms analysis was conducted using an agilent 1100 system equipped with a photodiode array and multiple wavelength detector . the column and the chromatographic condition were the same as those used for the hplc analysis . the lc effluent was split using a t - splitter to produce a flow of 0.25  ml / min . the identification of isoorientin , isovitexin , orientin , and vitexin was performed on the quadrupole mass spectrometer equipped with electrospray ionization ( esi - ms ) in the negative mode . capillary voltage was 3.5  kv , and the drying gas was set at 350c . the hplc method was validated in terms of linearity , limit of detection ( lod ) , limit of quantitation ( loq ) precision , specificity , and accuracy according to the international conference on harmonization ( ich ) guidelines . the calibration curves were constructed by plotting peak area versus concentrations with the help of the empower software . the standard solutions of the analytes were diluted with 60% methanol to yield a series of appropriate concentrations . lod and loq were estimated experimentally by injecting a series of dilute solutions with known concentrations until the signal - to - noise ratio for the standards reached a 3  :  1 ratio for lod and 10  :  1 for loq . intraday and interday variations were chosen to determine the precision of the hplc method . for intraday precision , three different concentrations of standard solutions ( 5 , 15 , and 50  g / ml ) were determined in one day and expressed as relative standard deviation ( rsd ) . for interday precision , the peaks for the marker compounds from sample solutions were confirmed by comparing the retention time and uv spectra with the reference standards . the peaks were considered pure when there was a coincidence between the two spectral sections . the extract of bamboo leaves used for recovery studies was preanalyzed and spiked with the mixed standards at high , middle , and low concentration levels . three replicates were performed for the test and variations were expressed by standard deviation ( sd ) . to optimize the tlc chromatographic conditions , different mobile phases and derivatisation regents were evaluated . the mobile phase ethyl acetate  :  formicacid  :  water ( 82  :  9  :  9 , v / v / v ) gave a good seperation of the markers . satisfactory derivatisation efficiency was obtained using diphenylborinic acid 2-aminoethyl ester and peg 400 as derivatisation regents . after derivatisation , flavone c - glycosides including isoorientin , isovitexin , orientin , and vitexin showed the four characteristic fluorescent bands on the tlc plates with the colour of yellow , green , yellow and green , respectively . the flavone c - glycosides , especially isoorientin , were found in most of these species of bamboo tested . identification of isoorientin , isovitexin , orientin , and vitexin in the different species of bamboo was carried out by the same retention factors ( r 
 f ) and similar colour to the standards compounds . the flavone c - glycosides were found in 40 of 51 species of bamboo examined . tlc screening method proved to be an efficient strategy for the rapid screening of flavone c - glucosides . among the fifty - one species of bamboo , b. multiplex cv . pubescens , and p. acuta showed strong band intensity as to flavone c - glycosides on the tlc plates , so they were examined further by hplc . to achieve a good chromatographic resolution , different types of chromatographic columns such as xterra rp18 ( 4.6  250  mm , 5  m ; waters usa ) , zorbax sb - c18 ( 4.6  250  mm , 5  m ; agilent usa ) , and ymc - pack r&d ods - a ( 4.6  250  mm , 5  m ; ymc japan ) were tested . to enhance the resolution and eliminate the peak tailing of the marker compounds , the best results were obtained using a ymc - pack r&d ods - a column at 30c , with acetonitrile and water containing 0.5% ( v / v ) glacial acetic acid as the mobile phase and isocratic elution ( 1.0  ml / min ) was applied ( figure 3(a ) ) . standard solutions of isoorientin , orientin , vitexin , and isovitexin were prepared at a concentration range of 0.2100  g / ml . the nine - point calibration curves of the analytes showed good linearity ( r 
   0.9997 ) with given concentration ranges . the parameters of linearity and range were summarized in table 2 , and lod and loq values showed the adequate sensitivity of the proposed method . the rsd values for both parameters ranged from 0.05 to 1.56% ( table 3 ) . the recovery values of the four flavone c - glycosides are given in table 4 . average recoveries ranged from 88.44% to 105.83% , confirming the accuracy of the method . the four flavone c - glycosides in bamboo leaves were identified by comparing their uv / dad and ms spectra with those of the standards ( figures 3 and 4 ) . the result of peak purity test showed that there was no coelution to interfere with the quantitation of marker compounds .   the four flavone c - glycosides from bamboo leaves were detected . the retention time , maximum wavelength , and characteristic fragment ions for individual peaks are presented in table 5 .   the optimized method was applied to determinate the four marker compounds in different samples . the concentration ranges of isoorientin , isovitexin , orientin , and vitexin in three bamboo leaves samples were 1.002.78 , 00.31 , 00.07 , and 0.200.68  mg / g , respectively . a total of four flavone c - glycosides were found in the leaf of p. heterocycla cv . pubesceus , which has been used as a raw material to produce commercial products of bamboo - leaf flavonoids . in summary , this paper describes a two - step procedure for the rapid screening of flavone c - glucosides in a large number of samples and quantitative analysis of four marker compounds . the proposed procedure proved to be specific and sensitive and can be regarded as an alternative method to detect and quantify flavone c - glucosides in bamboo leaves and its commercial product .
a strategy for analyzing flavone c - glucosides in the leaves of different species of bamboo was developed . 
 firstly , the flavone c - glycosides were extracted from the bamboo leaves ( 51 species in 17 genera ) with methanol and chromatographed on silica gel 60 plates in automatic developing chamber ( adc2 ) , and a qualitative survey using simple derivatization steps with the np reagent was carried out . 
 the flavone c - glycosides were found in 40 of 51 species of bamboo examined . 
 secondly , an hplc method with photodiode array and multiple wavelength detector was optimized and validated for the simultaneous determination of flavone c - glycosides , including isoorientin , isovitexin , orientin , and vitexin in the leaves of three species of bamboo and the flavone c - glycosides were confirmed by lc / ms . 
 the optimized hplc method proved to be linear in the concentration range tested ( 0.2100  g / ml , r 
 2  0.9997 ) , precise ( rsd  1.56% ) , and accurate ( 88106% ) . 
 the concentration ranges of isoorientin , isovitexin , orientin , and vitexin in three bamboo leaves samples were 1.002.78 , 00.31 , 00.07 , and 0.200.68  mg / g , respectively . 
 the proposed method was validated to be simple and reliable and can be a tool for quality control of bamboo leaf extract or its commercial products .