TLC Screening for Antioxidant Activity of Extracts from Fifteen Bamboo Species and Identification of Antioxidant Flavone Glycosides from Leaves of Bambusa. textilis McClure

Interest in the antioxidant activity of bamboo leaves is growing. To discover new sources of natural antioxidants, a TLC bioautography method combined with a new image processing method was developed to evaluate the antioxidant activity of leaf extracts from 15 different species of bamboo. The results showed that the methanolic extract of Bambusa. textilis McClure possessed the highest antioxidant activity among the selected bamboo species. To rapidly identify the antioxidant compounds, the crude extract of B. textilis McClure was analysed by HPLC-UV, and HPLC-micro-fractionation of the extract was carried out. Based on TLC bioautography-guided fractionation, three antioxidant fractions were isolated from B. textilis McClure by preparative chromatography. These three antioxidant compounds were identified as isoorientin 4''-O-β-D-xylopyranoside (1), isoorientin 2''-O-α-L-rhamnoside (2) and isoorientin (3) according to their UV, MS, and NMR data. The proposed TLC screening method could therefore be an easy way to evaluate the antioxidant activity of bamboo leaves, and the results achieved should prove very helpful for promoting their utilization, as B. textilis McClure can be considered a promising plant source of natural antioxidants.


Introduction
There are more than 1250 bamboo (Graminaceae) species distributed all over the World.Bamboo leaves have been used clinically in some Asian countries in the treatment of fever and hypertension [1,2].The medicinal effects of bamboo leaves are mostly attributed to their antioxidant capacity [3].In addition to its use in traditional medicine, an extract from the leaves of P. nigra var.henonis (antioxidant of bamboo leaves, AOB) is used as a food antioxidant in China [4].Some functional components from bamboo leaves, such as flavonoids, lactones and phenolic acids were reported [5].Antioxidants are a group of compounds which possess the ability to protect the body from damages caused by free radicals.Several studies have been carried out to identify the antioxidants from different species of bamboo, such as Phyllostachys edulis [1], Fargesia robusta [3], and Sasa borealis [6].Antioxidant chlorogenic acid derivatives and flavonoids have been identified from the leaves of P. edulis and F. robusta, respectively [1,3].Thus, the antioxidative compounds from different species of bamboo could be different.Moreover, the antioxidative components in bamboo leaves are not fully known.Therefore, some strategies have to be designed for the screening and identification of antioxidative compounds from bamboo leaves.
A great number of TLC techniques have been developed and successfully applied for qualitative and quantitative analysis of antioxidants [7,8], and the stable free radical 2,2-diphenyl-1picrylhydrazyl (DPPH) was often used as a derivatization reagent for this purpose [9].In the screening of antioxidants, the TLC bioautography assay is the method of choice due to several advantages that include flexibility, simplicity and high throughput [10][11][12].B. textilis McClure is a kind of bamboo species used in traditional medicine in China.A metabolic study previously performed by our group reported that three bioactive compounds from B. textilis McClure were identified [13].To the best of our knowledge, the active antioxidant compounds of B. textilis McClure have not been identified.
In this study, we evaluated the antioxidant activities of 15 different species of bamboo using a TLC bioautography assay.The methanolic extract of Bambusa.textilis McClure showed the strongest antioxidant activity among the selected bamboo species.The main antioxidant compounds of Bambusa.textilis McClure were isolated and identified based on preparative-HPLC, MS, and NMR techniques.

TLC Bioautography Assay
To screen the antioxidant capacity of bamboo leaves, a TLC bioautography method was performed.After separation on TLC plates, the compounds with radical scavenging activity were determined in situ with DPPH reagent.The TLC plate was observed with visible light (Figure 1).
As shown in Figure 1, the samples producing yellowish bands on the red background were considered as antioxidants.Usually, the purple background colour was visualized after spraying the plate with DPPH reagent [14,15].The background color of the plate changed from purple to red after 12 h in darkness.The red background makes the yellowish bands clearly visible, which benefits the next image processing.

Image Processing
After separation of the antioxidant compounds of the bamboo-leaf extracts using thin-layer chromatography (TLC), the derivatization was carried out by the DPPH reagent.The TLC photos were examined with ChemPattern 2 Professional Version (a kind of chromatography fingerprint processing software).Finally, the yellowish bands on the TLC plates were converted to the corresponding chromatographic peaks.The chromatographic response (peak area) is directly related to the antioxidant ability of the extract [16].Hence, total peak area of an extract can be considered as the sum of antioxidant activities of all ingredients from the extract.The aim of image processing is to obtain the total peak area.The baseline of peaks was marked manually as one line from the start to the end (Figure 2).

Comparison of Antioxidant Activity of Selected Bamboo Leaves
The antioxidant activity of bamboo leaves can be compared on the basis of their total peak areas.By statistical analysis, different antioxidant activities of the extracts from 15 species of bamboo leaves are shown in Figure 3.

Screening and Isolation of Antioxidant Compounds
To identify the main antioxidant compounds, the leaf extract of B. textilis McClure was fractionated by HPLC.The HPLC chromatogram is shown in Figure 4.Each fraction of the crude extract was evaluated for its antioxidant capacity using the TLC bioautography assay.Antioxidant compounds were visualized as yellow spots on the TLC plates.The TLC bioautography profile of different fractions indicated that only four fractions (F1, F2, F9 and F11) showed obviously yellow spots against the purple background (Figure 5).As seen from Figure 5, TLC analysis of the fractions F1 and F2 showed a series of yellow bands.On the basis of their UV spectra, the compounds present in fractions F1 and F2 were mainly phenolic acids.It can be found from Figures 4 and 5 that the fractions F1 and F2 contained too many compounds, and each compound was less abundant, thus, the fractions F1 and F2 were not considered further in this study.Fraction F9 showed an single active component (compound 1), while TLC revealed that fraction F11 was still not pure and contained two components.Based on preparative HPLC, fraction F11 was separated into two sub-fractions: F11-1 and F11-2 (corresponding to compounds 2 and 3).Thus a total of three antioxidant compounds (compounds 1, 2 and 3) were obtained from the crude extract of B. textilis McClure by preparative chromatography.

Structure Identification of the Three Antioxidant Compounds
The UV spectra of three antioxidant compounds are shown in Figure 6.The typical UV spectra showed that three antioxidant compounds belong to flavonoids according to their two major absorption peaks at 250-360 nm.The chemical structures of flavonoids are based on a C15 skeleton with two aromatic rings (A and B) and one heterocycle ring C [17].Absorption at 349.8 nm suggested that the compounds belong to the flavone family unsubstituted at 3-position.Peaks (270.1 nm) with shoulders indicated the presence of hydroxyl groups in the B ring.

LC-Q-TOF-MS Analysis
In the LC-Q-TOF-MS analysis, the precursor ions and fragment ions from three compounds were observed.Accurate mass data and isotopic distributions for the precursor and product ions were studied and compared to spectral data of reference compound (Figure 7).
As seen from Figure 7

Acid Hydrolysis of Compound 1 and 2
According to the LC-Q-TOF-MS analysis discussed above, the compounds 1 and 2 were identified as O,C-diglycosides.In O,C-diglycosides, the terminal sugars can be released by partial acid hydrolysis.After acid hydrolysis, the degradation products of compound 1 were analyzed by LC-Q-TOF-MS and compared to an isoorientin standard (Figure 8).
It can be found from Figure 8 that the main degradation product (RT = 14.25 min) of compound 1 has the same retention time as the isoorientin standard.The main degradation product was further identified by comparing its UV spectrum, accurate mass, and retention time with those of isoorientin.By the same method, the main degradation product of compound 2 was also identified as isoorientin.To determine whether a reducing-terminal xylose residue was present in the degradation products, we examined the degradation product solution using LC-Q-TOF-MS with a carbohydrate column.The extracted ion chromatogram (EIC) function of the TOF-MS and retention times can be used to distinguish the sugars (xylose and rhamnose) (Figure 9).
As seen from Figure 9, the extracted ion chromatograms (EIC) of the degradation products of compound 1 has the same retention time and accurate mass as those of a xylose standard.Meanwhile, the extracted ion chromatograms (EIC) of the degradation products of compound 2 has the same retention time and accurate mass as those of a rhamnose standard.These data proved that xylose and rhamnose were the reducing terminal sugars of compound 1 and compound 2, respectively, after acid hydrolysis.Based on the combined UV, MS, and NMR data, compounds 1 and 2 were identified as isoorientin 4''-O-β-D-xylopyranoside (1), and isoorientin 2''-O-α-L-rhamnoside (2), which were also supported by their MS fragmentation pathways (Figure 10).Different types of flavonoids, including flavone C-glycosides, flavone O-glycosides, and O,C-diglycosides, have been identified from different bamboo species [5,19].Of the three identified compounds in this study, isoorientin is a known antioxidant compound that has been found in most bamboo species.Compound 2 (isoorientin 2''-O-α-L-rhamnoside) has been found in Sasa borealis.To the best of our knowledge, this is the first report of the identification of compound 1 (isoorientin 4''-O-β-D-xylopyranoside) from the leaves of Bambusa.textilis McClure.The antioxidant activity of bamboo leaves is partially due to the presence of flavonoids [20].Flavonoids are usually extracted as mixtures from some bamboo leaves and then tested as antioxidants [21].The proposed TLC screening method could be an easy way to evaluate the antioxidant activity of bamboo leaves or other plant extracts prior to isolation.Because of similar structures and polarity, it is also difficult to isolate and identify each antioxidant compound from bamboo leaves.
According to known structure-activity relationships, compounds have antioxidant properties, associated with its structure.Luteolin is known as an antioxidant compound.The isolated compounds 1-3 contain the skeletal structure of luteolin, which could be the main reason for their antioxidant activity.

Materials
The bamboo leaves were collected from different regions in China.The species were authenticated by Jiusheng Peng from Jiangxi Academy of Forestry and Yulong Ding from Nanjing Forestry University.Bamboo leaves were dried in the shade, ground to powder, and stored at −20 °C.The species and sampling sites of bamboo leaves are shown in Table 1.

Standard and Sample Preparation
Standard solutions of isoorientin and orientin were prepared in methanol/water (60/40, v/v) at the concentration of 100 µg/mL.Dried bamboo leaves powder (1.0 g) was mixed with 60% aqueous methanol (20 mL) and subjected to 12 h maceration with constant shaking (120 strokes/min) at room temperature [22].The mixture was then sonicated for 30 min without heating and centrifuged at 5,000 rpm for 5 min.The supernatant was colleted and filtered through 0.45 μm nylon membrane for TLC analysis.

TLC Bioautography Assay
Thin-layer chromatography (TLC) was used to separate the chemical constituents of the bamboo-leaf extracts according to our previously reported method [2].The method employed silica gel 60 F254 precoated plates (Merck, Darmstadt, Germany) as stationary phase and mixture of ethyl acetate-formic acid-water (82:9:9, v/v/v) as mobile phase.Samples (8 μL, equivalent to a dried bamboo leaves weight of 400 µg) were were spotted in the form of bands of 8 mm width using a Camag Linomat 5 applicator (Muttenz, Switzerland).The plate was developed to a distance of 80 mm in an automatic developing chamber (ADC2, Camag).After development, the plates were immersed for 1 s in 0.05% (m/v) DPPH methanolic solution with an automatic immersion device (Camag).After immersion and removal of DPPH excess, the plates were kept in a dark box for 12 h and then photographed under visible light using a Camag Reprostar 3. The original TLC images were exported from winCATs TLC workstation (Camag) and stored as Bitmap (.bmp) files for further image processing.

TLC Images Processing and Evaluation of Antioxidant Activity
These .bmpfiles were examined with ChemPattern 2 Professional Version (a kind of fingerprint chromatography processing software, ChemMind Co. Ltd.Beijing, China).The red-green-blue (RGB) colour images were converted to grayscale intensity imagee [23].The chromatographic peaks are shown as signals, and the integration of all peaks was done manually in triplicate.In semi-quantitative analysis, the total peak areas were used to evaluate the antioxidant activity of the extracts tested.Data were analyzed using one-way analysis of variance (ANOVA) and Duncan's multiple range test (p < 0.05).Results were expressed as area.Each value was the mean of triplicate measurements ± standard deviation (three measurements of each sample).

Preparation of Crude Extract of B. textilis McClure
Powdered leaves (100 g) of B. textilis McClure were extracted three times at room temperature with 1 L of 60% aqueous methanol, assisted by sonication for 30 min and maceration with constant shaking (120 strokes/min) for 12 h.After removing the solvent under reduced pressure and freeze-drying, the combined extracts were concentrated and yielded 16 g of a crude extract.The crude extract was dissolved in 100 mL water/acetonitrile (85/15, v/v) and filtered through a 0.45-μm nylon membrane.

HPLC Micro-fractionation
HPLC micro-fractionation of the crude solution was performed by HPLC at a flow rate of 1.0 mL/min.An HPLC mobile phase of acetonitrile/0.5% acetic acid in water (15:85 v/v) was used.Injection volume was 40 µL and detection wavelength was set at 330 nm.Collection of effluent fractions during HPLC analysis was performed on a Waters Fraction collector III (Milford, MA, USA).Fractions were collected every 1 min (1 mL) in tubes and dried under a N-EVAP-12 nitrogen evaporator (Organomation Associates, Inc., Berlin, MA, USA).Each fraction was subjected to the TLC bioautography assay.

LC-Q-TOF-MS Analysis
Analysis of flavonoids: the qualitative analysis of the isolated compounds was performed using LC-Q-TOF-MS equipped with a jet stream ESI interface.Chromatographic separation was carried out on a C 18 column (250 × 4.6 mm I.D., 5 µm, packed with R&D ODS-A, YMC, Kyoto, Japan) at 30 °C.The mobile phase consisted of phase A (aqueous 0.1% acetic acid) and phase B (acetonitrile) in the ratio 85/15 (v/v).The flow rate was 1.0 mL/min and the T splitter gave a flow rate of about 0.20 mL/min toward the MS detector.The TOF/MS system was operated in positive mode and the operating parameters were as follows: drying gas (N 2 ) flow rate, 10 L/min; drying gas temperature, 350 °C; Nebulizer, 60 psi; capillary voltage, 3,500 V; skimmer voltage, 65 V; fragmentor voltage, 175 V; sheath gas temperature, 350 °C; nozzle voltage, 500 V. Mass spectra were acquired in the m/z range of

Figure 1 .
Figure 1.Typical TLC photography of standards and extracts from the different species of bamboo colorized with 0.05% DPPH.S: Standards are isoorientin and orientin.The different bamboo species (No. 1-15) are listed in Table1.

Figure 2 .
Figure 2.An example of TLC chromatogram and integration of peaks from leaf extracts of B. textilis McClure.

Figure 3 .
Figure 3.Comparison of antioxidant capacities of fifteen species of bamboo.Each value is the mean of triplicate measurements ± standard deviation.Different letters (a-j) on the top of the columns were significantly different (p < 0.05).Different bamboo species (No. 1-15) are listed in Table1.For the detailed protocol for evaluation of antioxidant activity see the Experimental section.

Figure 5 .
Figure 5. TLC photography of standards and fractions of B. textilis McClure leaf extracts colorized with 0.05% DPPH.S: Standards are isoorientin and orientin.Lanes F1-F22 in the picture correspond to fraction F1 to fraction F22, respectively.

Figure 6 .
Figure 6.UV spectra of the three antioxidant compounds.
, three molecular ions at m/z 581.1499 [M+H] + , m/z 595.1655 [M+H] + and m/z 449.1081 [M+H] + were observed.The main MS fragments of compound 1 and 2 were the same as those of an isoorientin standard.Among these fragment ions, the major ions for both compound 1 and 2 were at m/z 299.0554 [M+H−150] + and m/z 329.0662 [M+H−120] + .In addition, losses of some neutral molecules such as H 2 O (18 Da) suggested flavonoids with two hydroxy (-OH) groups in ortho positions.As for compound 1, a fragment at m/z 449.1084 [M+H−132] + , corresponded to loss of one pentose unit (132 Da), with a low-intensity and another fragment at m/z 431.0976 [M+H−132−H 2 O] + with higher intensity, as well as the absence of the aglycone ion is in agreement with an O,C-diglycoside structure.The relative intensity above 90% of the sugar unit is characteristic of di-O,C-glycosides [18].Thus, compound 1 could have a pentose unit in the terminal position of a disaccharide unit.The presence of the ion at m/z 329.0662 [M+H-−132−120] + and the absence of a [M+H−132−60] + fragment indicated a hexose as the C-glycosylation sugar.

Table 1 .
1. Investigated leaves of 15 different species of bamboo.