Qualitative and semi-quantitative assessment of anthocyanins in Tibetan hulless barley from different geographical locations by UPLC-QTOF-MS and their antioxidant capacities

Tibetan hulless barley (“qingke” in Chinese) is a valuable food in Tibet. Purple qingke (PQK) and black qingke (BQK), two special pigmented types of hulless barley, have traditionally been widely cultivated and consumed in Tibet for thousands of years. The composition and contents of anthocyanins of two cultivars are unknown. This study aimed to explore the composition and contents of anthocyanins of two cultivars and their antioxidant capacities. Six anthocyanins were identified by ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry from barley, most of the anthocyanins were acylated by malonyl group. The total anthocyanin contents ranged from 141 to 2,304μg/g in PQK and from 248.7 to 2902.9 μg/g in BQK. Furthermore, qingke has strong antioxidant activity against DPPH, ABTS ̇+, and FRAP. Qingke may be useful for treating or preventing diseases caused by the overproduction of radicals.


Introduction
Anthocyanins are the plant pigments that are responsible for spectacular red, blue, and purple colors of diverse plants. They are commonly found in foods, such as fruits, vegetables, and grains, and are important components of the human diet. Anthocyanins have been shown to have beneficial effects on human health as antioxidants and as anti-inflammatory, anti-diabetic, anti-obesity, and anticancer agents [1][2][3][4][5][6]. Therefore, the regular consumption of anthocyanin-rich food may help to prevent various chronic disorders and improve health. Based on daily food research in America, the daily consumption of anthocyanins was estimated to be 12.5 mg/person/day [7]. The major dietary anthocyanins were delphinidin, malvidin, and peonidin glycosides, which can be found in many plant foods, including purple sweet potatoes, grapes, berries, and wine [8][9][10].
Tibetan hulless barley, which is called "qingke" in Chinese and "ne" in Tibetan, is cultivated in the "Third Pole of the World," the Tibetan Plateau. It has special characteristics of cold resistance and drought resistance, and it is very important for the ecosystem and agriculture in the remote area. It is the staple food for Tibetans and an important feed for livestock in the Tibetan Plateau. Purple qingke (PQK) and black qingke (BQK), two special pigmented types of hulless barley, have traditionally been widely cultivated and consumed in Tibet [11]. Tibetans have consumed qingke as daily food for thousands of years, because it is a rich source of protein, starch, sugar, and fiber [12].
Recently, much attention has been paid to qingke because of its anthocyanin content. Several previous studies have confirmed the high anthocyanin content of qingke and reported various methods to extract anthocyanins from qingke [13][14][15][16]. However, these studies focused on the development of methods for the extraction and the determination of total anthocyanin contents by spectrophotometric method. Only two studies determined the structures of anthocyanins in barley using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) [17]. There is a lack of information on the types and amounts of anthocyanins in qingke grown in Tibet. Therefore, it is important to develop a method to characterize the anthocyanins in qingke.
In this study, a rapid and reliable method to characterize the anthocyanins in PQK and BQK based on ultraperformance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC/Q-TOF-MS) was used. With this method, six anthocyanins were identified and the content of anthocyanins in barley was determined. Furthermore, the antioxidant activities of PQK and BQK were assessed.
Samples of PQK and BQK were collected from 15 different growing sites in Tibet during harvest and then air-dried until the moisture content decreased to approximately 10%. Details of the sample collection sites (altitude, longitude, and latitude) are shown in Table 1. Each sample was ground and sieved through an 80-mesh sieve before analysis.

Extraction procedure
Briefly, 1 g of samples was extracted with 10 mL of methanol (1% formic acid) using a sonicator (Advanced sonic processing systems, Oxford, CT, USA) at 16 kHz with 300 W power for 30 min at room temperature in darkness. The suspension was centrifuged at 10,000 g for 10 min in a Contifuge 28RS centrifuge (Heraeus, South Plainfield, NJ, USA), and then was filtered through a 0.22 μm membrane filter before HPLC analysis.

Semi-quantification of anthocyanin content by HPLC
HPLC analysis was performed on an Agilent 1200 system (Agilent, USA). The Agilent 1200 system was equipped with an Agilent HPLC workstation, a pump, a DAD detector, and a column thermostat. Samples were separated on a ZORBAX Eclipse Plus column (250 mm × 2.1 mm i.d., 5 μm). The HPLC mobile phases were H 2 O with 0.1% TFA (solvent A) and ACN with 0.1% TFA (solvent B). The elution gradient was 12% B to 12% B in 10 min, followed by a gradient to 15% B in 20 min. The flow rate was 1.0 mL/min, the column temperature was 25°C, and the detection wavelength was 525 nm. Cyanidin-3-glucoside was used as the standard to prepare the calibration curve used for semi-quantitative analyses of individual anthocyanins in PQK and BQK. Each individual anthocyanin was expressed as micrograms of cyanidin-3-glucoside equivalents per gram dry sample weight.

Antioxidant activity
DPPH radical scavenging activity was measured by the method of previous report [18]. A volume of 5 μL of extraction sample was reacted with 995 μL of DPPH solution (0.4 mM) in a 96-well plate. The mixture was kept at room temperature in the dark for 30 min, and the absorbance (Abs) at 517 nm was measured by a microplate reader. GA was used as positive control. The calibration curve was established by plotting the DPPH scavenging ratio (A 0 -A t )/A 0 (A 0 and A t represent the absorbance at the initiation and termination of the reaction, respectively) against GA concentration. The results were expressed as milligrams of GA equivalents per gram of dry weight (DW).
The ABTS assay was performed according to the method of Wang et al. [19]. A volume of 5 μL of sample was added to 995 μL of diluted ABTS˙+ and to reacted in the dark at room temperature for 10 min. The mixture was tested under 732 nm. GA was used as a standard compound, and the results were expressed as mg of GA equivalents per g of DW.
The FRAP assay was performed according to the method of Wang et al. [19]. A volume of 5 µL of sample was added to 995 µL of FRAP reagent and allowed to react at room temperature in the dark for 15 min. Absorbance at 593 nm was recorded. GA was introduced as a standard. The content of GA equivalents was used to express the results.

Data analysis
Mean values of each sample were obtained from three replications and used for further analysis. Data were analyzed with SPSS software (version 20) (IBM Corp., Armonk, NY, USA). Compound identification was performed using the METLIN MS database and Food Composition and Method Development Laboratory database (U.S. Department of Agriculture).
Ethical approval: The conducted research is not related to either human or animal use.

HPLC of anthocyanins
The HPLC profiles showing the anthocyanin peaks in PQK and BQK samples are shown in Figure 1. The HPLC chromatograms were similar for the PQK and BQK samples from different locations. Six peaks with large areas were detected in all samples and represented common characteristic peaks. The relative standard deviations of the retention times of peaks were less than 0.9%, confirming that the developed method was robust and suitable for analyses.

Anthocyanin identification
After HPLC separation, the anthocyanins were identified on the basis of the MS data and the molecular ion characteristics for each peak (Figure 2). The m/z ratios for intact anthocyanins and their daughter fragments are listed in Table 2. Peak 1 had an MS ion at m/z 449.1083, with a fragmentation pattern (m/z 287) consistent with the loss of a hexose molecule. The MS fragments with an MS ion at m/z 287 corresponded to cyanidin aglycone moiety. Thus, peak 1 was tentatively identified as cyanidin-3-glucoside [20]. Peaks 2 and 3 had the same molecular ion (m/z 535.1081) and fragmentation pattern, suggesting that they represented isomers of cyanidin (m/z 287) derivatives attached to malonylated glucopyranosyl residues. On the basis of the retention times of peaks 2 and 3, they were tentatively identified as cyanidin 3-O-(3″-O-malonyl-glucoside) and cyanidin 3-O-(6″-O-malonyl-glucoside), respectively [21]. Peak 4 had a characteristic cyanidin aglycone ion (m/z 287), suggesting that it represented cyanidin glycoside. The fragmentation pattern suggested that peak 4 had two malonylated β-glucopyranosyl residues. Thus, peak 4 was identified as cyanidin 3-O-dimalonylglucoside [22]. The intense ion peaks at m/z 605.1129 and 635.1236 detected for compounds 5 and 6 corresponded to C 27 H 25 O 16 and C 28 H 27 O 17 , respectively. The peak 5 at m/z 271 represented pelargonidin, peak 5 was identified as pelargonidin 3-Odimalonylglucoside [23]. The peak 6 ion at m/z 301 indicated that it was peonidin, and peak 6 was identified as peonidin 3-O-dimalonylglucoside [23].
In this study, six anthocyanins were identified in both PQK and BQK. Four of the six anthocyanin aglycones were derivatives of cyanidin, one of the most common naturally occurring anthocyanins. The main structural feature of anthocyanins in PQK and BQK was a malonyl group.  Acylated anthocyanins are more stable than non-acylated anthocyanins. The malonyl structure also stabilizes anthocyanins. A previous study revealed that environmental factors can affect the anthocyanin profile by regulating the activities of enzymes that catalyze polyphenol biosynthesis [24]. The unique geography (high altitude) and harsh climatic conditions (cold, dry, and strong ultraviolet radiation) in the qingke growing regions of Tibet would influence anthocyanin composition. Specifically, the relatively high concentrations of acylated anthocyanins in qingke may protect plants against ultraviolet damage. This trait is very important for the survival of plants exposed to high levels of ultraviolet radiation [25,26].   Table 3 lists the contents of individual anthocyanins detected in PQK and BQK. The composition and total contents varied considerably among samples from different locations ( Table 4). The total anthocyanin contents ranged from 141 to 2,304 µg/g in PQK and from 248.7 to 2902.9 µg/g in BQK. Lin et al. reported that the total anthocyanin contents were 2.69 mg/100 g in blue qingke and 9.55 mg/100 g in BQK [27]. The different contents were on the basis of different analysis methods. This study focused on the individual anthocyanin content. The predominant anthocyanins were cyanidin 3-O-(6″-O-malonyl-β-glucopyranoside) 3 and cyanidin 3-O-(3″,6″di-O-malonyl-β-glucopyranoside) 4, which accounted for nearly 80% of the total anthocyanin content in PQK and BQK. In another study, the total anthocyanin content in colored barley varieties ranged from 3.2 to 1037.8 µg/g [28,29]. The higher range of concentrations detected in this study may be related to differences in the analyzed cultivars and growing conditions. For PQK, the lowest and highest anthocyanin contents were detected in samples from locations L5 and L10, respectively, and the mean anthocyanin content was 995.1 µg/g. For BQK, the lowest and highest anthocyanin contents were detected in samples from locations L13 and L11, respectively, and the mean anthocyanin content was 1267.4 µg/g. Additionally, the total anthocyanin content was consistently higher in BQK than in PQK collected from the same locations.

Correlation analysis
In order to analyze the variations in anthocyanin contents of PQK and BQK among different growing locations (longitude, latitude, and altitude) in Tibet, the correlations between anthocyanins and geographical details (longitude, latitude, and altitude) of the sample collection locations were analyzed ( Table 5). The correlation coefficient of PQK was −0.52, 0.07, and 0.32, and correlation coefficient of BQK was −0.60, 0.04, and 0.08, respectively. For both PQK and BQK, anthocyanin content was negatively correlated with latitude (P < 0.05) and tended to decrease at increasingly southern latitudes.

Antioxidant activity
In this study, the antioxidant activity of qingke was examined, and the results are shown in Table 6. The DPPH result showed that qingke in L3 has the highest antioxidant activity among locations. The ABTS˙+ result showed that PQK in L6 and BQK in L10 have highest antioxidant activity, respectively. The FRAP + result showed PQK in L3 and BQK in L11 have highest antioxidant activity, respectively. The anthocyanins of qingke showed that high antioxidant activity may be related to their structures. As we know, an increase in the number of hydroxyl substitutions on the B ring appeared to be associated with enhanced antioxidant activity. These results on antioxidant activity of anthocyanins imply that qingke may be useful for treating or preventing diseases caused by the overproduction of radicals. The cultivation of qingke rich in acetylated anthocyanins with strong antioxidant activities may be beneficial for Tibetans who are exposed to high ultraviolet radiation.

Conclusions
In this study, we applied UPLC/Q-TOF-MS to identify and quantify the anthocyanins in PQK and BQK samples collected from 15 different geographical locations in Tibet. To  the best of our knowledge, this is the first report on the compositions and contents of anthocyanins in qingke cultivated in Tibet. Six anthocyanins were identified, with cyanidin 3-O-(6″-O-malonyl-β-glucopyranoside) and cyanidin 3-O-(3″,6″-di-O-malonyl-β-glucopyranoside) being the predominant anthocyanins in PQK and BQK. The extraction of qingke exhibited antioxidant properties. The results indicated that specific geographical factors should be used to identify qingke crops with outstanding anthocyanin contents. As well as being a healthy food, the anthocyaninrich qingke varieties cultivated in Tibet have the potential for development into nutritional supplements and healthcare products.