Most flavonoids are found to have a variety of medical and biological activities such as anti-free radical,antioxidant,antitumor,antiviral,anti-inflammatory,antibacterial,etc. activities and are widely used in food and pharmaceutical fields(Zhang,2008; Wang et al., 2011). Bamboo is a renewable natural resource and a vital component of forest resources(Jiang,2007). Bamboo leaf flavonoids were found in many bamboo species(Guo,2007; Sun,2010; 2012). According to reports,bamboo leaf flavonoids have an obvious fresh-keeping effect on soybean milk(Wei et al., 2012). The compound preservative of bamboo leaf flavonoids can prolong the shelf life of chilled meat and improve preservation effect(Shi et al., 2013).

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., 2010a; 2010b). Most of the researches were focused on a few flavonoids. Bamboo leaves contain a variety of flavonoids(Guo,2007; Sun,2010; 2012; Wang et al., 2013), and those methods of detecting a few flavonoids have proved to be unsuitable for determination of other flavonoids founded simultaneously. It was necessary for a sensitive analytical technique for determining more flavonoids content levels simultaneously.In this paper,thirteen flavonoids(isoorientin,orientin,vitexin,isovitexin,luteolin,apigenin-7-O-glucoside-6″-O-rhamnose,apigenin-7-O- glucopyranoside,tricin-7-O-glucoside,6-trans-(2″-O-α-rhamnopyranosyl)ethenyl-5,7,3′,4′-tetrahydroxyflavone,apigenin,tricin,demethyltorosaflavone,7-methoxytricin)were studied. According to reports,these flavonoids were founded in bamboo leaves, and some of them are common ingredients in bamboo leaves. We presented a simple,rapid and sensitive HPLC method with UV detection for qualitative and quantitative analysis of thirteen flavonoids in bamboo leaves.

Bamboo leaves from ten species of Dendrocalamus,Dendrocalamus latiflorus,D. giganteus,D. strictus,D. membranaceus,D. farinosus,D.minor var. amoenus,D. yunnanicus,D. fugongensis,D. br and isii and D. hamiltonii,were collected in Changning Century Bamboo Garden and Xishuangbanna Tropical Botanical Garden in China. They were dried in shade and smashed with a grinder. The comminution granularity was 60.

KQ-250B-type ultrasonic cleaner(Kunshan Ultrasonic Instrument Co.,Ltd)were used. HPLC was performed with a Waters Corp.(Milford,MA)2695 separations module and 2487 dual wavelength absorbance detector.

Soorientin,orientin,vitexin,isovitexin and apigenin(purity＞98%)were obtained from Shanghai Winherb Medical Technology Co.,Ltd.(Shanghai,China). Luteolin,apigenin-7-O-glucopyranoside-6″-O-rhamnoside,apigenin-7-O-glucopyranoside,tricin-7-O-glucopyranoside,6-trans-(2″-O-α-rhamnopyranosyl)ethenyl-5,7,3′,4′-tetrahydroxyflavone,tricin,demethyltorosaflavone and 7-methoxy-tricin were separated and prepared from bamboo leaves in the State Forestry Administration Key Open Laboratory,International Centre for Bamboo and Rattan(Beijing,China).

Analysis grade solvents were obtained from the Beijing Chemical Works(Beijing,China). HPLC grade solvents were obtained from Fisher Scientific(Pittsburgh,PA).

The stock solution of flavonoids were prepared by putting 3.5 mg isoorientin,3 mg 6-trans-(2″-O-α-rhamnopyranosyl)ethenyl-5,7,3′,4′-tetrahydroxyflavone and 5 mg 11 other flavonoids in 10 mL volumetric flasks respectively,dissolving with different ratios of methanol,ethanol and water, and diluting to volume with methanol. Heated the flasks and dissolved with ultrasound in order to promote dissolution. The mixed st and ard solution of the 13 flavonoids were prepared with 13 flavonoide stock solutions. The concentrations of 13 flavonoids in the mixed st and ard solution were isoorientin 26.25 mg·L^-1,6-trans-(2″-O-α-rhamnopyranosyl)ethenyl-5,7,3′,4′-tetrahydroxyflavone 30 mg·L^-1 and 11 other flavonoids 37.5 mg·L^-1. The mixed st and ard solution was diluted to 0.01-37.5 mg·L^-1 with methanol. Solutions were stored at 4 ℃.

HPLC was performed with a Waters Corp.(Milford,MA)2695 separations module and 2487 dual wavelength absorbance detector. Compounds were separated on a YMC-Pack R&D ODS-A RP C18 column(4.6 mm×250 mm,5 μm particle size). The mobile phases gradient consisted of acetonitrile and 0.5% phosphoric acid,see Tab.1. The injection volume was 10 μL,the flow rate 1.0 mL·min^-1,UV detection wavelength at 340 nm.

Tab.1 Method of gradient elution

Tab.1 Method of gradient elution Time/min Acetonitrile (%) 0.5% phosphoric acid (%) Curve 0 15 85 15 20 80 11 20 20 80 6 50 45 55 6 51 80 20 11 58 15 85 11 65 15 85 11

A series concentration of the mixed st and ard solutions were prepared to assess the linearity. Each of the different concentrations of the solutions was injected using the chromatographic conditions to generate corresponding regression equations. The LOD and LOQ for the flavonoids were measured by duplicate injections of the st and ard solutions based on S/Ns of 3 and 10,respectively. The precision of the method was checked by 5 replicate injections of the mixed st and ard solutions and was expressed as RSD. The intraday precision was studied by injecting the mixed st and ard solutions five times on the same day(0,2,4,8 and 12 h), and the interday precision was studied by injecting the mixed st and ard solutions five times on 5 consecutive days. The results were expressed as RSD. The accuracy of the method was tested by obtaining recovery rates. A st and ard addition recovery test of the method was carried out with a 2 g sample of D. latiflorus,to which was added three concentrations of mixed st and ard solutions. Three replicate spiked samples were extracted,purified and analyzed by HPLC. Average recovery rates were calculated. A blank control was also analyzed.

Samples of 2.0 g dry bamboo leaves were extracted with 70%(v/v)ethanol-water(3×30 mL; each time for 30 min)by ultrasonic extraction in 50 mL centrifuge tubes at 60 ℃. The power of the ultrasonic instrument is 250 W. The extracts were filtered after extracting each time. The filtrates were combined and evaporated to dryness by vacuum distillation. The residues were suspended with 50 mL water in separation funnels. After being extracted with petroleum ether(3×50 mL),the aqueous solutions were evaporated to dryness with rotary evaporator. The residues were dissolved with 70%(v/v)ethanol-water in 10 mL volumetric flasks and adjusted to volume with 70%(v/v)ethanol-water. All the samples were filtrated by 0.45 μm microporous membrane(Bonna-Agela Technologies)before injection. Each bamboo species were set three repetitions.

The structures and polarities of some flavonoids are similar,so it is difficult to separate those flavonoids. Of the various mobile phases tried,acetonitrile and 0.5% phosphoric acid with gradient elution(Tab.1)gave the best resolution of the 13 flavonoids. These flavonoids were also separated from the other components in the sample extracts. A typical chromatogram obtained from the flavonoids st and ards is shown in Fig. 1.

	Fig. 1 Chromatogram of the mixed standards

The HPLC method was validated for linearity,accuracy,LOD,LOQ,intraday and interday precision and recovery. The linear relationships are given in Tab.2. The st and ard curves in the corresponding ranges had good linearity. Accuracy,LOD,LOQ and intraday and interday precision are presented in Tab.3. LOD was in the range of 0.01 to 0.10 mg·kg^-1, and LOQ 0.03 to 0.34 mg·kg^-1. The values of RSD for the accuracy and intraday and interday precision indicated it was safe to analyze the samples within 5 days. Recoveries of flavonoids from spiked D. latiflorus are shown in Tab.4. According to the results,the average recoveries of orientin,6-trans-(2″-O-α-rhamnopyranosyl)ethenyl-5,7,3′,4′-tetrahydroxyflavone,apigenin,tricin,demethyltorosaflavone and 7-methoxy-tricin were 39.76%-68.75%. The average recoveries of the other 7 flavonoids were higher than 70%. The method was found to be feasible for qualitative and quantitative determination of flavonoids in bamboo leaves.

Tab.2 Linear relationships between peak area and concentration^①

Tab.2 Linear relationships between peak area and concentration① Names of standards Retention time/min Regression equation R2 Linear range/ (mg·L-1) Isoorientin 13.23 Y=30 059X-567.86 1.000 0 0.01-350.00 Orientin 15.43 Y=25 947X-720.17 0.999 9 0.02-125.00 Vitexin 20.38 Y=28 472X+834.72 0.999 9 0.02-500.00 Isovitexin 20.68 Y=29 984X+1275.2 0.999 9 0.02-500.00 Luteolin 22.19 Y=28 333X+326.6 0.999 9 0.02-37.50 Apigenin-7-O-glucopyranoside-6″-O-rhamnoside 25.60 Y=20 559X+382.8 0.999 9 0.02-125.00 Apigenin-7-O-glucopyranoside 27.67 Y=19 966X+387.47 0.999 9 0.02-37.50 Tricin-7-O-glucopyranoside 28.65 Y=22 939X+782.65 0.999 9 0.02-37.50 6-trans-(2″-O-α-rhamnopyranosyl) ethenyl-5,7,3′,4′-tetrahydroxyflavone 32.20 Y=10 168X-1 293.7 0.999 6 0.02-30.00 Apigenin 42.45 Y=45 372X-1 039 1.000 0 0.02-37.50 Tricin 43.07 Y=30 388X-574.26 1.000 0 0.02-37.50 Demethyltorosaflavone 44.83 Y=28 411X-1 396.9 1.000 0 0.02-37.50 7-methoxy-tricin 54.67 Y=36 008X-1 071.5 0.999 9 0.02-18.75 ①Y=Peak area and X=compound concentration.

Tab.3 LOD, LOQ and precision for the 13 flavonoids

Tab.3 LOD, LOQ and precision for the 13 flavonoids Names of standards LOD/ (mg·kg-1) LOQ/ (mg·kg-1) Accuracy [RSD (%), n=5] Intraday precision [RSD (%), n=5] Interday precision [RSD (%),n=5] Isoorientin 0.03 0.09 1.90 0.15 0.45 Orientin 0.02 0.06 2.23 0.17 0.44 Vitexin 0.01 0.04 1.93 0.29 3.74 Isovitexin 0.01 0.04 1.53 0.25 5.61 Luteolin 0.01 0.03 1.91 0.20 0.59 Apigenin-7-O-glucopyranoside-6″-O-rhamnoside 0.02 0.07 1.34 0.19 1.99 Apigenin-7-O-glucopyranoside 0.01 0.04 1.06 0.18 1.86 Tricin-7-O-glucopyranoside 0.01 0.03 1.79 0.16 0.98 6-trans-(2″-O-α-rhamnopyranosyl)ethenyl- 5,7,3′,4′-tetrahydroxyflavone 0.10 0.34 2.16 0.24 3.15 Apigenin 0.01 0.04 1.82 0.37 0.88 Tricin 0.01 0.04 1.49 0.67 1.72 Demethyltorosaflavone 0.01 0.04 1.08 0.24 1.50 7-methoxy-tricin 0.01 0.03 2.55 0.49 3.02

Tab.4 Recovery of 13 flavonoids from spiked D. latiflorus

Tab.4 Recovery of 13 flavonoids from spiked D. latiflorus (n=3) Names of standards Original quantity/ (mg·kg-1) Addition quantity/ (mg·kg-1) Amount found/ (mg·kg-1) Recovery (%) RSD (%) Isoorientin 8.06 26.25 31.72 82.75 7.21 8.06 19.69 28.13 88.53 10.20 8.06 13.13 22.49 89.21 11.56 Orientin 99.17 37.50 115.59 62.06 22.14 99.17 28.13 112.79 63.76 25.83 99.17 18.75 115.16 68.75 3.95 Vitexin 45.45 37.50 80.32 76.00 4.28 45.45 28.13 69.25 71.91 1.68 45.45 18.75 61.41 70.65 3.23 Isovitexin 136.55 37.50 211.20 87.15 3.06 136.55 28.13 205.76 88.33 5.16 136.55 18.75 208.32 93.17 3.84 Luteolin 6.38 37.50 48.28 102.57 4.16 6.38 28.13 25.29 67.09 1.10 6.38 18.75 20.13 71.06 7.83 Apigenin-7-O-glucopyranoside-6″-O-rhamnoside 244.69 37.50 308.25 76.20 2.22 244.69 28.13 291.63 73.80 3.38 244.69 18.75 281.68 73.01 2.63 Apigenin-7-O-glucopyranoside 67.22 37.50 107.50 77.72 2.80 67.22 28.13 95.64 74.17 6.21 67.22 18.75 89.08 74.50 9.20 Tricin-7-O-glucopyranoside 37.23 37.50 75.39 80.76 2.35 37.23 28.13 65.09 77.52 10.82 37.23 18.75 54.63 73.23 19.43 6-trans-(2″-O-α-rhamnopyranosyl)ethenyl- 12.19 30.00 27.03 55.97 2.62 5,7,3′,4′-tetrahydroxyflavone 12.19 22.50 21.87 53.63 37.52 12.19 15.00 29.56 88.80 24.13 Apigenin 0.91 37.50 18.42 47.40 1.37 0.91 28.13 14.24 48.28 3.67 0.91 18.75 9.80 48.73 8.19 Tricin 5.38 37.50 23.06 50.59 11.49 5.38 28.13 17.40 48.06 7.02 5.38 18.75 18.35 68.40 13.08 Demethyltorosaflavone 0.00 37.50 19.89 53.05 2.53 0.00 28.13 15.70 55.84 11.54 0.00 18.75 8.33 44.44 5.47 7-methoxy-tricin 5.97 37.50 23.61 50.82 7.97 5.97 28.13 14.74 39.76 12.92 5.97 18.75 17.54 63.30 35.04

The 13 flavonoids in 10 bamboo leaf species of Dendrocalamus were compared by use of the proposed method. The results are shown in Tab.5.The chromatogram of an extract of D. latiflorus is shown in Fig. 2. The results obtained reveal differences in the flavonoid composition of the different bamboo species. Luteolin,6-trans-(2″-O-α-rhamnopyranosyl)ethenyl-5,7,3′,4′-tetrahydroxyflavone and demethyltorosaflavone were not found in some bamboo species, and the other 10 flavonoids were found in all of 10 bamboo species. 12 flavonoids were found in D. latiflorus,D. farinosus,D. yunnanicus and D. hamiltonii; 10 flavonoids were found in D. br and isii; 11 flavonoids were found in the other 5 bamboo species. The concentrations of isoorientin,orientin,vitexin,isovitexin and apigenin-7-O-glucoside-6″-O-rhamnose were more than 100 mg·kg^-1,which were higher than the contents of other flavonoids in 10 bamboo leaves. The contents of apigenin-7-O-glucopyranoside and tricin-7-O-glucoside in most bamboo species were 20-100 mg·kg^-1. The concentrations of luteolin,6-trans-(2″-O-α-rhamnopyranosyl)ethenyl-5,7,3′,4′-tetrahydroxyflavone,apigenin,tricin,demethyltorosaflavone and 7-methoxytricin were less than 20 mg·kg^-1.

Tab.5 The contents of flavonoids in the dry leaves of Dendrocalamus ^①

Tab.5 The contents of flavonoids in the dry leaves of Dendrocalamus ① Compounds Flavonoids contents in the leaves of Dendrocalamus/(mg·kg-1) D. latiflorus D. giganteus D. strictus D. membranceus D. farinosus D.minor var. amoenus D. yunnanicus D. fugongensis D. brandisii D. hamiltonii Isoorientin 9.23± 0.85a 148.60± 11.79b 9.79± 0.08a 55.55± 1.22c 58.39± 0.20c 1 582.60± 2.73d 35.04± 0.11e 55.45± 0.30c 28.23± 1.13e 217.15± 1.47f Orientin 102.47± 6.73a 406.19± 5.64b 20.81± 0.58c 162.85± 3.97d 31.84± 0.64e 389.98± 0.81f 81.55± 0.60g 31.72± 0.57e 28.67± 0.33ce 97.10± 2.68a Vitexin 42.00± 2.00a 893.34± 2.79b 702.81± 0.14c 1 862.60± 1.06d 44.75± 0.08a 286.22± 2.62e 62.09± 0.31f 98.63± 0.56g 357.69± 0.29h 330.12± 1.23i Isovitexin 141.85± 8.71a 143.23± 2.12a 122.82± 1.14b 239.72± 2.28c 352.37± 0.98d 1 996.40± 2.27e 233.87± 0.64c 81.12± 0.40f 144.63± 0.39a 187.07± 0.28g Luteolin 6.38± 0.54a 33.62± 1.68b 9.93± 0.17aef 58.76± 0.89c 8.98± 0.29af 98.93± 2.45d 13.49± 0.18e / / 11.37± 0.63ef Apigenin-7-O- glucopyranoside-6″- O-rhamnoside 263.67± 15.44a 264.19± 4.98a 54.82± 0.74b 37.53± 3.26c 201.18± 1.46d 598.12± 6.30e 206.51± 0.33d 75.34± 1.74f 15.38± 0.15g 24.02± 1.07cg Apigenin-7-O- glucopyranoside 67.22± 2.18a 46.20± 0.91b 48.40± 0.79b 20.73± 1.34c 85.51± 0.52d 96.39± 3.29e 58.49± 0.21f 30.36± 0.51g 19.33± 0.16c 18.36± 0.97c Tricin-7- O-glucopyranoside 37.23± 1.52a 35.43± 1.55a 21.17± 1.28bf 117.51± 2.46c 48.08± 0.36d 89.23± 4.72e 43.05± 0.21d 18.95± 0.86b 16.64± 0.05b 25.55± 1.51f 6-trans-(2″-O-α- rhamnopyranosyl) ethenyl-5,7,3′,4′- tetrahydroxyflavone 12.19± 0.41a / / / 7.06± 0.31b / 23.51± 0.86c 13.78± 0.22a / / Apigenin 0.91± 0.11a 4.96± 0.14b 3.53± 0.01c 5.25± 0.15d 1.65± 0.04e 3.73± 0.05c 0.83± 0.02a 1.05± 0.04a 2.37± 0.03f 0.88± 0.10a Tricin 5.38± 0.62a 7.84± 0.29b 7.57± 0.28b 18.58± 0.52c 3.88± 0.08d 7.86± 0.23b 4.19± 0.09d 5.71± 0.16a 11.95± 0.77e 8.06± 0.13b Demethyltorosaflavone / / / / / / / / / 4.00± 0.04 7-methoxy-tricin 5.97± 0.50af 11.89± 0.39b 8.23± 0.19c 17.19± 0.29d 3.76± 0.12e 6.38± 0.13f 7.44± 0.02g 7.83± 0.07cg 11.75± 0.09b 5.32± 0.01a Total flavonoids ①Values are mean ± S.E. (n=3). Values in a low followed by same letters are not significantly different (P=0.05) according to Duncan’s multiple comparison (DMRT). “/”: Not detected.

	Fig. 2 Chromatogram of an extract of D. latiflorus

The content of apigenin-7-O-glucoside-6″-O-rhamnose was higher than other flavonoids in the leaves of D. latiflorus,at a level of 263.67 mg·kg^-1. In the leaves of D. giganteus,D. strictus,D. membranceus,D. fugongensis,D. br and isii and D. hamiltonii,the concentrations of vitexin were maximum,they were 893.34,702.81,1 862.60,98.63,357.69 and 330.12 mg·kg^-1,respectively. In the leaves of D. farinosus,D.minor var. amoenus and D. yunnanicus,the concents of isovitexin were maximum,they were 352.37,1 996.40,233.87 mg·kg^-1,respectively. In the 10 species of bamboo leaves,the concentrations of total flavonoids were in the range of 419.94 to 5 155.84 mg·kg^-1; the maximum content was found in D.minor var. amoenus.

An HPLC method for simultaneous qualitative and quantitative analysis of 13 flavonoids was established. The method was simple,sensitive and accurate. The results obtained showed that the st and ard curves in the corresponding ranges had good linear relationships. LOD was in the range of 0.01 to 0.10 mg·kg^-1, and LOQ 0.03 to 0.34 mg·kg^-1. The values of RSD for the accuracy and intraday and interday precision indicated it was safe to analyze the samples within 5 days. The average recoveries of orientin,6-trans-(2″-O-α-rhamnopyranosyl)ethenyl-5,7,3′,4′-tetrahydroxyflavone,apigenin,tricin,demethyltorosaflavone and 7-methoxy-tricin from D. latiflorus were 39.76%-68.75%; the average recoveries of the other 7 flavonoids were higher than 70%. Bamboo leaves of ten species of Dendrocalamus were extracted and analyzed. The results obtained reveal differences in the flavonoids of the different bamboo species. In 10 dry bamboo leaves,the concentrations of isoorientin,orientin,vitexin,isovitexin and apigenin-7-O-glucoside-6″-O-rhamnose were generally higher; followed with apigenin-7-O- glucopyranoside and tricin-7-O-glucoside; luteolin,6-trans-(2″-O-α-rhamnopyranosyl)ethenyl-5,7,3′,4′-tetrahydroxyflavone,apigenin,tricin,demethyltorosaflavone and 7-methoxy-tricin had lower contents. The maximum concentration of total flavonoids was found in D.minor var. amoenus,the minimum in D. fugongensis. The proposed method would be useful for the research on flavonoids and chemical utilization of bamboo leave resources.