Being a renewable natural resource and a viable replacement for wood,bamboo is a vital component of forest resources of the world(Jiang,2007). Recently,bamboo resource utilizations are drawing an ever increasing attention from different countries of the world(Yue et al.,2007). Many species of bamboo can be the medicinal plants because of the active components they have(China national group corp of traditional amp; herbal medicine,1994). Bamboo-leaf flavonoids,important components of plant flavones,have a variety of medical and biological activities(Jing,2009). For instance,they are effective in anti-free radical,anti-oxidation,anti-aging,anti-bacteria,anti-inflammatory applications(Wang et al.,2007 ; Yang et al.,2011),and can be used to regulate blood fat,prevent the cardio-cerebrovascular diseases(Liu et al.,2009). As functional components,bamboo-leaf flavonoids also have the utilization in manufacturing various products such as cosmetic,feed additive(Zhang et al.,2004 ; Feng et al.,2011 ; Zhang et al.,2003). Thecomparison of flavonoids in the leaves of three different genera of bamboo is useful for choosing bamboo species to extract natural bamboo-flavonoids and routine quality control of the leaves of different bamboo species. Many methods such as spectrophotometry(SP),high performance liquid chromatography(HPLC),high performance thin layer chromatography(HPTLC)have been reported for the study of bamboo-leaf flavonoids(Guo et al.,2007 ; Wang et al.,2010 ; Sun et al.,2010 ; Cui et al.,2011). HPTLC is simpler,inexpensive,highly efficient and accurate,and it has the ability to analyze 10 to 20 samples simultaneously using a small quantity of organic solvent(Wang et al.,2010 ; Sherma,2002). The purpose of this paper was to compare the flavonoid compounds in three genera of bamboo by HPTLC(Cui et al.,2011).

Eleven bamboos from three genera were studied,namely Bambusa textilis,B. tuldoides,B. chungii and B. multiplex; Dendrocalamus giganteus,D. minor and D. latiflorus; Phyllostachys glauca,P. heteroclada,P. nigra and P. nidularia. These eleven bamboos leaves were collected at random from Kunming of Yunnan,Yongan of Fujian,Changning of Sichuan,Nanjing of Jiangsu and Nanchang of Jiangxi in China.

The flavonoid standards isoorientin,orientin,isovitexin,vitexin and tricin(Purity > 98 %,HPLC)were purchased from Shanghai Winherb Medical Science Co.,Ltd,Shanghai,China. Aluminum trichloride was purchased from Tianjin Guangfu Fine Chemical Research Institute,Tianjin,China. Acetone,methanol,ethyl acetate,methylene chloride,formic acid and ethanol were analytical grade,purchased from Beijing Chemical Works(Beijing,China)and Sinopgarm Chemical Reagent Co.,Ltd(Beijing,China). Water was purified with an ultrapure water system(Purelab Plus; Pall Life Sciences,Ann Arber,MI).

Chromatography was performed on HPTLC plates precoated with silica gel HPTLC-Fertigplatten Nano-DURASIL -20 UV254(10 cm × 20 cm,0.2 mm layer,product No. 812014,Macherey-Nagel,Germany).

Hamilton syringe(Bonaduz,Switzerland),Linomat5 applicator(CAMAG,Switzerland),Automated Multiple Development(AMD2,CAMAG,Switzerland),TLC Scanner3(CAMAG,Switzerland)and Reprostar3(CAMAG,Switzerland)were used in this study.

The stock solution of orientin was prepared by dissolving 5 mg orientin in 7 mL methanol-ethanol-water 2:3:2(v/v)in a 10 mL volumetric flask and diluting to volume with methanol. The stock solution of vitexin was prepared by dissolving 5 mg vitexin in 6 mL methanol-ethanol-water 1:2:3(v/v)in a 10 mL volumetric flask and adjusting to volume with methanol. The stock solution of isoorientin,isovitexin,tricin were prepared in the same way,by dissolving 5 mg isoorientin,isovitexin,tricin respectively in methanol in 10 mL volumetric flasks.

The mixed standard solution of the five flavonoids was prepared by mixing 0.9 mL isoorientin,orientin,isovitexin,vitexin and tricin stock solution respectively in a 5 mL volumetric flask,and stored at 4 ℃ .

Bamboo leaves were dried in shade and samples(20 g)were extracted with 95 %(v/v)ethanol-water(3 × 250 mL; each time for 0.5 h)by ultrasonic extraction. The extracts was obtained after removing the solvent by vacuum distillation,suspended in 1 000 mL water respectively,and extracted with 1 000 mL petroleum ether in separation funnels. After being extracted with petroleum ether,the fraction was subjected to chromatography on a 50 cm × 3 cm column packed with polyamide(Taizhou Si-jia Biochemical Plastic Company,Zhejiang,China)and eluted with 50 %(v/v)ethanol-water. The eluent was collected and enriched in vacuum distillation to give the crude flavonoids of bamboo leaves. The crude flavonoids samples of the eleven bamboo species were made respectively. Sample solutions were prepared by dissolving 50 mg crude flavonoids in 5 mL methanol-ethanol-water 2:1:2(v/v)in a 5 mL volumetric flask.

After being cleaned with methanol and methylene chloride to remove the impurity in the precoat,the HPTLC plates were activated at 105 ℃ . Solutions were applied as 7.0 mm bands by Linomat 5 applicator with a 100 μL Hamilton syringe. Multistage developmentwas performed in AMD2 . The plate was developed in the first step with methanol-ethyl acetate-methylene chloride 20:35:45(v/v)to 50 mm; in the second step with acetone-methanol-ethyl acetate-methylene chloride 5:10:35:50(v/v)to 75 mm; in the third step with acetone-methanol-ethyl acetate-methylene chloride 10:5:30:55(v/v)to 90 mm(Tab. 1). Formic acid was added to each components of the mobile phase,and the concentration of formic acid in each components is 10 %(v/v). The developed plate was dried in air and sprayed with 1 %(w/w)aluminum trichloride in ethanol as the chromogenic reagent. Then the plate was left for derivatization and scanned at 366 nm with TLC Scanner3 controlled by Wincats software. Peak areas were recorded and the images of plates were documented by Digistore 2 software.

Tab.1 Process of the development

Tab.1 Process of the development Process Development distance/mm Acetone Methanol Solvents (%,v/v) Ethyl acetate Methylene chloride StepⅠ 50 0 20 35 45 StepⅡ 75 5 10 35 50 StepⅢ 90 10 5 30 35

A series of standard solutions of isoorientin,orientin,isovitexin,vitexin and tricin(90,175,350,700,1 050,1 400,1 750 ng per bands)were prepared to construct standard curve by replicate(n= 3)plotting peak area against the amount of flavonoid. The quantification of the flavonoids in different samples was calculated.

Instrument precision was checked by replicate(n=10)scanning the bands of isoorientin(270 ng),orientin(270 ng),isovitexin(270 ng),vitexin(270 ng)and tricin(540 ng)ten times and was expressed as coefficient of variation(CV [%]). Therepeatability of standards was assessed by replicate(n=9)analysis of 180 ng bands obtained from standard solutions of isoorientin,orientin,isovitexin,vitexin and tricin,expressed as coefficient of variation(CV [%]). To test the repeatability of sample application which expressed as coefficient of variation(CV [%]),3,4,and 5 μL of each of samplesolution was applied in triplicate to a plate and the peak areas were obtained at last. For accuracy testing this experiment was repeated three times. Limits of detection for the flavonoids were measured by reducingthe amounts of the standard solutions applied to the plates.

The intraday precision was studied by scanning a series concentration(90,135,180 ng)of the standards bands for six times on the same day(2 ^nd h,4 ^th h,6^th h,8^th h,10^th h,12^th h)and the interday precision was studied by scanning a series of concentration(90,135,180 ng)of the standards bands for eight times on different days(1 ^st,2 ^nd,3 ^rd,4 ^th,5 ^th,6 ^th,7 ^th,8 ^th day),which were expressed as coefficient of variation(CV [%]).

The accuracy of the method was tested by obtaining recovery at three concentrations(addition to a preanalyzed sample of of 50 %,100 %and 150 %of the amount present in sample). Average recovery(%)was calculated.

Silica was selected to be the absorbent in this study. The standard components in samples are separated well and resolved from other components and can be quantified. A suitable resolution of the five flavonoids standards(Isoorientin,R_F=0.25,Orientin,R_F=0.32,isovitexin,R_F=0.38,vitexin,R_F=0.47,tricin,R_F=0.88)is obtained. A typical chromatography from the mixed standards is shown in Fig. 1 .

	Fig. 1 Absorption spectrogram obtained from the mixed standards of isoorientin,orientin,isovitexin,vitexin and tricin,scanned at 366 nm

The identification was performed by detecting the UV absorption spectra of the standards and which in the samples extracted from bamboo leaves,and the bands from samples were confirmed with those in mixed standards. The fingerprint of the eleven bamboo species was shown in Fig. 2 .

	Fig. 2 Fingerprint photograph of UV366 detection of bamboo-leaf-flavonoids 1-3,B. textilis; 4-6,B. tuldoides; 7-9,B. chungii; 10-12,B. multiplex; 13-15,D. giganteus; 16-18,D. minor; 19-21,D. latiflorus; 22-24,P. glauca; 25-27,P. heteroclada; 28-30,P. nigra; 31-33,P. nidularia; A-I,Standards.

The method was validated for linearity,precision,repeatability,sensitivity and accuracy(Tab. 2 and 3). The relationships between peak area and amount of theflavonoid were found to be linear in the range of 90 to 1 750 ng per bands. Results from determination of the recovery of the flavonoids at three levels from the different bamboo species are presented in Tab. 4 .

Tab.2 Method validation data for quantification of the five flavonoids

Tab.2 Method validation data for quantification of the five flavonoids Property of the method Isoorientin Orientin Isovitexin Vitexin Tricin Instrument precision(CV[%], n=10) 0.70 0.72 0.52 0.68 0.75 Repeatability of standards(CV[%], n=9) 1.87 1.19 0.96 0.85 1.28 Repeatability of samples(CV[%], n=9) 1.94 0.87 0.93 1.02 1.36 Linearity 0.987 3 0.988 4 0.997 5 0.988 8 0.974 2 Equation y= 13.32 x + 1 267.7 y=10.16 x + 293.04 y=13.39 x + 1 262.7 y=9.14 x + 698.07 y=7.11 x + 2 033.7 Limit of detection/ng 35 40 25 25 25

Tab.3 Results of the intraday and interday precision

Tab.3 Results of the intraday and interday precision Components Concentration /[ng per band] Intraday precision (CV [%]) interday precision (CV [%]) Isoorientin 90 1.26 1.90 135 1.00 1.77 180 1.87 1.74 Orientin 90 1.50 1.96 135 1.45 1.61 180 1.69 1.86 Isovitexin 90 1.75 1.54 135 1.29 0.93 180 1.11 1.42 Vitexin 90 1.29 1.87 135 1.84 1.94 180 1.43 1.96 Tricin 90 1.54 1.57 135 1.95 1.95 180 1.66 1.80

Tab.4 Recoveries of isoorientin,orientin,isovitexin,vitexin and tricin in different concentration

Tab.4 Recoveries of isoorientin,orientin,isovitexin,vitexin and tricin in different concentration

The bamboo-leaf flavonoid sample extracts from the eleven different bamboo species were compared by use of the proposed method. The absorption spectrograms at 366 nm of the samples are shown in Fig. 3. Thequantification of flavonoids in bamboo samples arepresented in Tab. 5. In the eleven species from threebamboo genera,Phyllostachys nigra(Lodd. ex Lindl.)Munro the maximum of 0.132%,and the minimumof 0.015% .

	Fig. 3 Absorption spectrogram at UV366 of the three bamboo genera A: B. textilis; B: B. tuldoides; C: B. chungii; D: B. multiplex; E: D. giganteus; F: D. minor; G: D. latiflorus; H: P. glauca; I: P.heteroclada; J: P. nigra; K: P. nidularia.

Tab.5 Quantification of five flavonoids in bamboo-leaf-flavonoid samples determined by HPTLC method in the eleven bamboo species^①

Tab.5 Quantification of five flavonoids in bamboo-leaf-flavonoid samples determined by HPTLC method in the eleven bamboo species①

Flavonoids in the leaves of three different bamboo genera have been compared by a densitometric HPTLC method. The results would be useful for choosing bamboo species to extract natural bamboo-flavonoids and routine quality control of the leaves of different bamboo species. The HPTLC method was simple,precise,efficient,sensitive and accurate,and it couldbe useful for fingerprint studies and for quality controlof bamboo-leaf-flavonoids and their products.

The results obtained reveal differences in theflavonoid composition of the different bamboo speciesfrom different bamboo genera. The in P. nigra was themaximum in the eleven bamboo species. It wasestablished that isoorientin and isovitexin were thecommon compounds in the bamboo species tested.Orientin was found in the extracts of D. giganteus,D. latiflorus,P. glauca and P. nigra,while vitexin was found in the species of D. giganteus,D. latiflorus and P. nigra,tricin was only found in the extracts of P. glauca. The results that some flavonoids were not found in some extracts of bamboo species might because of the incomplete extraction,low concentration of the flavonoids or the limits of detection.