Abstract This study examines the phytochemical profiles and antioxidant activities of highland barley varieties with white, blue, and black seed coat colors, focusing on the effects of cooking methods on black barley. Among the varieties, black barley, particularly Xiongzhang type, showed high concentrations of anthocyanins, proanthocyanidins, flavonoids, and phenolics, all of which are associated with enhanced antioxidant activities. Metabolomic profiling revealed significant biochemical diversity across the barley samples closely linked to seed coat color. Cooking methods substantially influenced β-glucan levels and metabolomic profiles. Specifically, frying and baking increased β-glucan content while inducing specific metabolic changes. The findings highlight the nutritional advantages of black barley and the role of cooking techniques in preserving its bioactive compounds. This study provides valuable insights for breeding initiatives and dietary recommendations to enhance the nutritional quality and health benefits of barley. Keywords: Anthocyanins, Β-Glucan, Antioxidants, Metabolomics, Cooking methods, Barley varieties Highlights * • Black barley exhibited enhanced antioxidant activity. * • Metabolomic diversity was strongly linked to seed coat color. * • Cooking methods significantly influenced β-glucan content and metabolome. 1. Introduction Highland barley (Hordeum vulgare L. var. nudum), also known as hulless or naked barley, is a distinctive crop traditionally cultivated in the high-altitude regions of Tibet, Qinghai, and other parts of the Himalayas. Resilient to harsh environmental conditions, it is a vital part of the diet and culture in these areas due to its nutritional benefits ([41]Obadi, Qi, & Xu, 2021). With growing interest in the health-promoting properties of highland barley, understanding its phytochemical composition and the effects of processing methods on these components are essential. Such insights could support the expanded use of highland barley in functional foods and nutraceuticals. Highland barley is renowned for its high dietary fiber content, particularly β-glucan, a soluble fiber associated with numerous health benefits. β-Glucan helps reduce blood cholesterol, improves glycemic control, and supports immune function ([42]Zeng et al., 2020). Studies show that β-glucan from highland barley can significantly lower low-density lipoprotein cholesterol and total cholesterol levels without affecting high-density lipoprotein cholesterol ([43]Obadi, Sun, & Xu, 2021). Additionally, β-glucan intake has been linked to increased insulin sensitivity and a reduced risk of type 2 diabetes ([44]Tosh, 2013). Given these properties, highland barley serves as a valuable dietary component for supporting cardiovascular health and managing metabolic disorders. In addition to being rich in β-glucan, highland barley contains a wide range of phenolic compounds that are potent antioxidants ([45]Goudar, Sharma, Janghu, & Longvah, 2020). These phenolic compounds include phenolic acids, flavonoids, and anthocyanins, and substantially enhance the antioxidant capacity of highland barley and offer protective effects against oxidative stress-related diseases, such as cardiovascular disease, cancer, and neurodegenerative disorders ([46]Scalbert, Manach, Morand, Rémésy, & Jiménez, 2005). By neutralizing free radicals, these compounds reduce oxidative stress and inflammation, which are key factors in the development of many chronic diseases. Responsible for the blue and black pigmentation of highland barley grains, anthocyanins are particularly noted for their anti-inflammatory and anticarcinogenic properties ([47]Tsuda, 2012). These pigments not only enhance the visual appeal of the grains but also contribute to their health benefits, making highland barley an appealing ingredient for health-oriented food products. The seed coat color of barley grains is closely correlated with their phytochemical composition and antioxidant activity ([48]Dang, Zhang, Zhang, Yang, & Xu, 2022). Compared to white varieties, black and blue barley varieties are richer in anthocyanins and typically exhibit higher antioxidant activity ([49]Kofuji et al., 2012; [50]Xia et al., 2024). These variations in seed coat color result from genetic differences that influence the types and concentrations of phytochemicals within the grains. The genetic and biochemical diversity associated with seed coat color highlights the potential of these varieties for developing functional foods with enhanced health benefits (H. [51]Zhao et al., 2022). For instance, with its relatively high levels of anthocyanins and other phenolic compounds, black highland barley could be beneficial in formulations aimed at boosting antioxidant intake. Understanding the relationship between seed coat color and phytochemical content can guide the selection of barley varieties for specific health applications and nutritional interventions. Cooking methods significantly influence the nutritional and phytochemical profiles of highland barley ([52]Wang et al., 2023). Thermal processing can modify the solubility and bioavailability of dietary fibers and phytochemicals, thereby influencing their health benefits(C. [53]Zhao et al., 2019). In particular, steaming and baking can enhance the extraction and solubilization of β-glucan, increasing its availability and potential health benefits. In contrast, frying and germination may degrade β-glucan and other sensitive compounds, reducing their effectiveness (Y.-P. [54]Bai, Zhou, Zhu, & Li, 2021). Depending on the cooking method, these alterations can either enhance or reduce the health-promoting properties of highland barley. Understanding these effects is essential for optimizing cooking methods to preserve or boost the nutritional quality of highland barley. The aim of this study was to 1) compare the phytochemical profiles and antioxidant activities of highland barley varieties with different seed coat colors, 2) investigate the impact of various cooking methods on β-glucan content and metabolomic profiles in black highland barley and 3) identify the distinct metabolic pathways affected by seed coat color and cooking methods via Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. By achieving these objectives, this study aims to provide valuable insights into the nutritional and functional properties of highland barley. This knowledge may support the development of functional foods and dietary guidelines that maximize the health benefits of highland barley. 2. Material and methods 2.1. Plant samples A total of nine barley samples were used in this study, all of which were provided by the Biotechnology Research Institute of the Shigatse City Science and Technology Bureau in Tibet. These samples included seven local varieties and two cultivated varieties, categorized by color as follows: white (Xila22, Zangqing2000, and Gamuguori), blue (Youxi, Quduigamu, and Garu), and black (Sagui, Qujia, and Xiongzhang). The highland barley grains were cleaned and dried in an oven at 50 °C for 12 h, grounded to pass through a 60-mesh screen, and stored at −20 °C for further use. 2.2. Cooking The barley grains were subjected to various traditional cooking methods, including frying, steaming, baking, and germination, with untreated grains serving as a control. Frying: The barley grains were fried using the traditional Tibetan tsampa preparation method. The grains were first selected, cleaned, and dried. In this process, a specific amount of barley was fried in a pan containing fine sand. Once the sand temperature reached 100 °C, the barley grains popped at a rate of over 90 %. The barley and sand were then separated using a sieve. Steaming: The barley grains were steamed according to a traditional method for preparing steamed buns. After selection, cleaning, and drying, the grains were ground and sifted through an 80-mesh sieve. A flour-to-water ratio of 5:4 (g:g) was then shaped and steamed for 30 min until fully cooked. Baking: The barley grains were baked following a traditional method for making cookies. After selection, cleaning, and drying, the grains were ground and passed through an 80-mesh sieve. Water was added to form a dough, which was molded and baked in an oven at 180 °C with top and bottom heat for 15 min until fully cooked. Germination: The selected and cleaned barley grains were placed on specialized germination trays and incubated in a growth chamber at 25 °C for 4 days. After this germination period, the grains were harvested, dried, ground, and passed through a 60-mesh sieve. The samples were then stored at −20 °C for further analysis. 2.3. Β-Glucan content Beta-glucans were measured in highland barley and various food products using an enzymic method known as AOAC Method 995.16 (McCleary & Mugford, 1997), “B-D-Glucan in Barley and Oats, Streamlined. Enzymatic Method”. 2.4. Anthocyanin content The anthocyanin content was determined following the method described in a previous study ([55]Zhu et al., 2015) with minor modifications. One gram of barley flour was mixed with 20 mL of ethanol solution (pH 3.0, 60 % w/w), stirred thoroughly, and extracted by shaking at 120 rpm at 40 °C for 1 h. The mixture was then centrifuged at 14816g for 15 min. Two aliquots of the supernatant (1 mL each) were adjusted to a final volume of 5 mL using potassium chloride buffer (pH 1.0) and sodium acetate buffer (pH 4.5). After allowing the solution to stand for 1 h, the absorbance was measured at 515 nm and 700 nm. The anthocyanin content was calculated using the formula: Anthocyanin content = (A1.0 - A4.5) × MW × 1000/(ε × C), where A1.0 and A4.5 are the absorbance values at 515 nm and 700 nm for the samples in pH 1.0 and pH 4.5 buffers, respectively. MW is the molecular weight of Cyanidin 3-O-glucoside (C3G) (449.2), ε is the extinction coefficient of C3G (26,900), and C is the buffer concentration. 2.5. Proanthocyanidin content The proanthocyanidin content was measured according to the method described in a previous study ([56]Hümmer & Schreier, 2008). One gram of barley flour was mixed with 25 mL of pure methanol and shaken in the dark at 120 rpm for 1 h. The mixture was centrifuged at 14816g for 15 min, and the supernatant was collected. For analysis, 1 mL of the sample solution was mixed with 3 mL of 4 % vanillin-methanol solution, followed by addition of 1.5 mL of concentrated hydrochloric acid. The solution was mixed thoroughly and left to stand at 22 °C for 10 min. A calibration curve was constructed using catechin standard solutions at gradient concentrations. Absorbance (A) was measured at 500 nm against a reagent blank. Linear regression yielded the equation: A = 5.3012C + 0.0023 (R^2 = 0.999), where C is the catechin concentration (mg/mL). Proanthocyanidin content in barley samples was quantified against this curve and expressed as mg per 100 g DW. 2.6. Total flavonoid content The total flavonoid content was determined following the method described in a previous study ([57]He, Liu, & Liu, 2008) with minor modifications. Five grams of barley flour were combined with 100 mL of 50 % ethanol solution and extracted by shaking at 120 rpm at 40 °C for 2 h. The mixture was centrifuged at 14816g for 15 min, and the supernatant was collected and concentrated to a volume of 10 mL under reduced pressure. To proceed with the analysis, 1 mL of the concentrated flavonoid solution was mixed with 1 mL of a 5 % sodium nitrite solution, shaken, and allowed to stand for 5 min. Next, 2 mL of 10 % aluminum nitrate solution was added, and the mixture was shaken and left to stand for 1 h. Finally, 2 mL of 1 mol/L sodium hydroxide solution was added, and the volume was adjusted to 10 mL using 30 % ethanol. Rutin was used as a standard to create the calibration curve: A = 21.332C + 0.0043(R^2 = 0.999)The total flavonoid content was calculated using the formula: ω = (m[1] × V)/(M × V[1]) × 100. Where m[1] represents the total flavonoid mass of the sample solution, calculated from the standard curve (in mg); V is the extraction volume of the sample (in mL); V1 is the volume of the sample used for measurement (in mL); and M is the mass of the sample (in g). 2.7. Total phenolic content Two grams of barley flour were combined with 20 mL of precooled 80 % acetone solution, mixed thoroughly, and extracted by shaking at 120 rpm under nitrogen for 1 h. The mixture was then centrifuged at 14816g for 15 min, and the supernatant was collected. This residue was subjected to two additional extractions, after which all supernatants were combined and concentrated under reduced pressure at 45 °C using a rotary evaporator until dry. To obtain the bound phenolic extract, the remaining barley flour residue was digested with 10 mL of 2 mol/L sodium hydroxide solution for 1 h, neutralized with 2 mol/L hydrochloric acid, and defatted three times with n-hexane. The solution was extracted with 20 mL of ethyl acetate under nitrogen at 120 rpm for 1 h and centrifuged at 14816g for 15 min with the supernatant subsequently collected. This residue was subjected to five additional extractions, and the combined supernatants were concentrated under reduced pressure at 45 °C until dry. The phenolic content in both the free and bound phenol extracts was determined using the Folin-Ciocalteu method. Specifically, 100 μL of the sample extract was combined with 400 μL of distilled water and 100 μL of Folin-Ciocalteu reagent, shaken and left to react for 6 min. Then, 1 mL of 7 % sodium carbonate solution and 1 mL of ultrapure water were added. The mixture was shaken and allowed to stand in the dark for 90 min before measuring the absorbance at 760 nm. The phenolic content was calculated using a standard curve established with gallic acid (A = 0.0046C + 0.0054，R^2 = 0.999), with the total phenolic content determined as the sum of the free and bound phenolic contents. The total phenolic content was calculated using the formula:W = c × V × N/M. Where W is the content of total polyphenols in barley samples, mg/kg; M is the mass of barley sample (g); c is the mass concentration of barley sample (μg/mL); V is the volume of the extracted liquid (mL); N is the dilution factor. 2.8. Oxygen radical absorption capacity (ORAC) assay The ORAC was measured according to the method described in a previous study ([58]Wolfe & Liu, 2008) with some modifications. One gram of barley flour was combined with 25 mL of precooled ethyl acetate and extracted by shaking at room temperature in the dark for 1 h. The mixture was filtered through Whatman filter paper, and the filtrate was collected. This residue was subjected to two additional extractions, and the filtrates were combined and evaporated to dryness under reduced pressure at 35 °C. The fraction was redissolved in 80 % ethanol to obtain the crude extract. The crude extract and Trolox standard were diluted with 75 mmol/L phosphate buffer (pH 7.4) to the appropriate concentrations. To each well of a 96-well fluorescence plate, 20 μL of the sample or Trolox standard solution was added, followed by 20 μL of 75 mmol/L phosphate buffer (pH 7.4) and 20 μL of 70 nmol/L fluorescein sodium solution. The plate was incubated at 37 °C for 20 min. The reaction was initiated by adding 140 μL of 12 mmol/L 2,2′-azobis(2-methyl-propanimidamide) dihydrochloride solution. Fluorescence intensity was measured continuously at an excitation wavelength of 485 ± 20 nm and an emission wavelength of 530 ± 20 nm every 2 min until the fluorescence decayed to the baseline (D. [59]Huang, Ou, Hampsch-Woodill, Flanagan, & Prior, 2002). The ORAC value of the sample was calculated using a standard curve (A = 0.9809C + 1.0345, R^2 = 0.9928) established with Trolox and expressed as μmol Trolox Equivalents (TE) per gram of dry weight (DW). The relative ORAC value was calculated using the formula: [(AUC[sample]-AUC[black])/(AUC[Trolox]-AUC[black])] (molarity of Trolox/molarity of sample). 2.9. Free radical scavenging capacity assay The free radical scavenging capacity was determined using a modified method described in a previous study ([60]Liang, Chang, Liang, Hung, & Hsieh, 2014). One milliliter of the crude extract from section 2.8 was combined with 5 mL of 0.1 mmol/L DPPH ethanol solution, shaken, and allowed to react in the dark at room temperature for 20 min. Absorbance was measured at 517 nm. The DPPH radical scavenging capacity was calculated using the following formula: DPPH radical scavenging capacity = (1 - absorbance of sample/absorbance of blank) × 100 %.) 2.10. Ferric reducing antioxidant power (FRAP) assay The FRAP value was measured using a modified method described in a previous study ([61]Benzie & Strain, 1996). One milliliter of the crude extract from section 2.8was combined with 3 mL of FRAP working solution, shaken thoroughly, and allowed to react in the dark at 37 °C for 30 min. Absorbance was measured at 593 nm. A standard curve was established using Trolox, and the FRAP value was calculated accordingly. The FRAP value was expressed as TE per 100 g of DW. Total antioxidant capacity is calculated using the following formula: (A-A[0])/(A[max]-A[0]) × 100. A[0] refers to the A[593] nm value measured without test sample; A refers to the A[593] nm value measured when the test sample is added; A[max] refers to the maximum value of Trolox measured in the experiment. 2.11. Metal ion chelating capacity (MCC) assay MCC was determined using a modified method described in a previous study ([62]Decker & Welch, 1990). One milliliter of the crude extract from section 2.8 was combined with 3.7 mL of methanol, 0.1 mL of 2 mmol/L FeCl[2]•4H[2]O solution, and 0.2 mL of 2.5 mmol/L ferrozine solution, and the mixture was incubated in the dark for 10 min. Absorbance was then measured at 562 nm. MCC was calculated using the following formula: MCC = (1 − absorbance of sample/absorbance of blank) × 100 %. 2.12. Metabolomic analysis The barley samples used for metabolomic analysis of differences in seed coat color include three groups: white, blue, and black. The white group consists of a mixture of three untreated white barley varieties—Xila22, Zangqing2000, and Gamuguori—blended in equal proportions. The blue group is composed of three untreated blue barley varieties—Youxi, Quduigamu, and Garu—mixed in equal proportions. The black group consists of three untreated black barley varieties—Sagui, Qujia, and Xiongzhang—blended in equal proportions. For the metabolomic analysis of differences arising from different cooking methods, the barley samples include three groups: control, frying, and baking. The control group is a mixture of three untreated black barley varieties—Sagui, Qujia, and Xiongzhang—blended in equal proportions. The frying group is prepared by mixing these same three black barley varieties in equal proportions, followed by frying according to the method outlined in section 2.2. The baking group is composed of the same three black barley varieties mixed in equal proportions and baked according to the procedure described in section 2.2. Whole barley grains were ground into a fine powder using a freeze mill, and ∼ 50 mg of this powder was weighed and extracted with 1 mL of 80 % methanol containing 0.1 % formic acid to enhance extraction efficiency. The mixture was vortexed for 5 min and sonicated at 4 °C for 30 min. After centrifugation at 14816g for 10 min, the supernatant was collected and filtered through a 0.22-μm membrane filter for analysis. Metabolite profiling was performed using a UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a Q Exactive™ Plus mass spectrometer (Thermo Fisher Scientific). Chromatographic separation was achieved with a Hypersil GOLD™ C[18] column (100 mm × 2.1 mm, 1.9 μm) at a flow rate of 0.3 mL/min. The mobile phase consisted of solvent A (0.1 % formic acid in water) and solvent B (0.1 % formic acid in acetonitrile). The gradient elution program was as follows: 0–2 min, 2 % B; 2–5 min, 2–25 % B; 5–15 min, 25–95 % B; 15–18 min, 95 % B; 18–18.5 min, 95–2 % B; and 18.5–20 min, 2 % B. The injection volume was 2 μL. Mass spectrometry data were acquired in both positive and negative ion modes with a spray voltage of 3.5 kV and a capillary temperature of 320 °C. The mass range was set to 100–1500 m/z, and data-dependent acquisition was employed to capture the top 10 most abundant ions for MS/MS fragmentation. Raw data files were processed using Compound Discoverer™ software (Thermo Fisher Scientific) for peak detection, alignment, and quantification. Metabolites were identified by matching against online databases, such as HMDB, METLIN, and KEGG. 2.13. Statistical analyses SAS 9.4 (SAS Inc., Chicago, IL, USA) was used for the statistical analysis of the results. All treatments were performed in triplicate, and the data were expressed as the mean ± standard deviation. Multiple comparisons were performed using Tukey's HSD method with statistical significance set at P < 0.05. Multivariate statistical analyses, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), were performed using SIMCA-P software (Umetrics, Umeå, Sweden). 3. Results and analysis 3.1. Comparative analysis of phytochemical profiles and antioxidant activities in grain varieties with different seed coat colors The phytochemical profiles and antioxidant activities of grain varieties with different seed coat colors (white, blue, and black) were comprehensively analyzed ([63]Table 1). Comprehensive profiling of phytochemicals and antioxidant activities across white-, blue-, and black-grained varieties revealed striking color-dependent trends ([64]Table 1). Anthocyanin content, quantified as cyanidin-3-glucoside, was markedly elevated in black-grained cultivars, with the highest-performing variety exceeding the lowest (white-grained) by over 13-fold. Proanthocyanidins followed an analogous pattern, with black varieties accumulating levels up to 2.4 times greater than their white counterparts. Similarly, total flavonoids and phenolic compounds—assessed as rutin and gallic acid equivalents, respectively—peaked in the black-grained group, demonstrating 1.6- to 1.5-fold increases relative to blue- and white-seeded varieties. Intriguingly, bound phenolics were most abundant in a white-grained accession, contrasting with the predominance of free phenolics in black varieties. Table 1. Analysis of antioxidant components and activities among barley varieties with white, blue, and black seed coat colors. Seed Coat Color Variety Name Anthocyanins (as Cyanidin- 3-Glucoside) mg/100 g DW Proanthocyanidins (mg/100 g DW) Total Flavonoids (as Rutin) (mg/100 g DW) Total Phenolics (as Gallic Acid) (mg/100 g DW) Free Phenolics (as Gallic Acid) (mg/100 g DW) Bound Phenolics (as Gallic Acid) (mg/100 g DW) ORAC (μmTE/g FW) DPPH(%) FRAP (TE/100 g DW) MCC(%) White Xila22 7.65 ± 0.20 g 81.14 ± 2.38c 313.89 ± 6.86fe 323.14 ± 9.14c 164.48 ± 4.65e 158.66 ± 4.49b 79.42 ± 2.25d 39.70 ± 3.22fe 1941.84 ± 148.44d 26.91 ± 3.03 cd White Zangqing2000 17.38 ± 0.30d 69.85 ± 2.14d 369.10 ± 10.69d 377.12 ± 15.31b 199.50 ± 8.10d 177.62 ± 7.21a 83.35 ± 1.89d 37.66 ± 5.25fe 1220.60 ± 201.17e 25.62 ± 2.90d White Gamuguori 11.17 ± 0.22f 59.04 ± 2.04e 332.21 ± 11.81e 330.26 ± 8.90c 185.94 ± 5.01d 144.33 ± 3.89 cd 81.18 ± 2.19d 26.58 ± 4.10f 1606.19 ± 262.78ed 32.03 ± 0.90cbd White verage of varieties 12.07 ± 4.03C 70.01 ± 9.29C 338.40 ± 25.05B 343.51 ± 26.57B 183.30 ± 15.66C 160.20 ± 14.66 A 81.32 ± 2.66C 34.65 ± 7.18C 1589.55 ± 361.51C 28.19 ± 3.71C Blue Youxi 14.95 ± 0.36e 88.03 ± 2.66b 411.89 ± 9.13c 314.27 ± 7.48c 193.59 ± 4.61d 120.68 ± 2.87e 92.69 ± 3.76c 54.85 ± 6.75 dc 3129.20 ± 414.73cb 34.68 ± 4.22cb Blue Quiduigamu 8.43 ± 0.21 g 70.51 ± 1.46d 327.23 ± 6.73e 361.72 ± 15.24b 188.46 ± 7.94d 173.26 ± 7.30a 93.68 ± 3.09c 44.93 ± 6.65de 3263.14 ± 416.98cb 43.81 ± 3.97 A Blue Garu 17.87 ± 0.33d 88.43 ± 3.06b 295.33 ± 9.74f 362.73 ± 9.15b 227.79 ± 5.75cb 134.93 ± 3.40d 95.18 ± 2.46c 65.23 ± 8.99bc 2932.47 ± 473.69cb 35.56 ± 2.76b Blue verage of varieties 13.75 ± 3.96B 82.32 ± 8.72B 344.82 ± 49.94B 346.24 ± 25.20B 203.28 ± 18.55B 142.96 ± 22.75 B 93.85 ± 3.31B 55.00 ± 11.21B 3108.27 ± 456.65B 38.02 ± 5.53 A Black Sagui 42.06 ± 0.92c 81.75 ± 2.21c 437.64 ± 12.24b 377.06 ± 14.84b 234.53 ± 9.23b 142.53 ± 5.61 cd 96.01 ± 6.49c 79.60 ± 7.37a 3506.28 ± 102.57b 33.02 ± 4.98cbd Black Qujia 46.89 ± 0.82b 74.27 ± 1.51d 410.64 ± 15.81c 387.23 ± 10.01b 217.24 ± 5.62c 169.99 ± 4.40a 103.89 ± 2.55b 75.75 ± 7.63ba 2808.50 ± 97.33c 34.42 ± 3.83 cb Black Xiongzhang 102.38 ± 2.25a 142.42 ± 5.18a 478.12 ± 13.65a 477.59 ± 13.17a 324.28 ± 8.94a 153.31 ± 4.23cb 117.39 ± 3.24a 82.98 ± 5.59a 4523.43 ± 164.51a 31.67 ± 2.07cbd Black verage of varieties 63.78 ± 27.41 A 99.48 ± 30.70 A 442.13 ± 31.05 A 413.96 ± 46.97 A 258.68 ± 47.61 A 155.28 ± 12.27 A 105.76 ± 9.88 A 79.44 ± 7.53 A 3612.74 ± 715.20 A 33.04 ± 3.98B [65]Open in a new tab Note:Different uppercase letters within the same column indicate significant differences among mixed samples with different seed coat colors while different lowercase letters indicate significant differences among individual varieties. Four complementary antioxidant assays consistently ranked black-grained cultivars as possessing the highest radical scavenging and reducing capacity. Oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) values in the leading black variety surpassed those of the lowest-performing white variety by 48 % and ≥ 2-fold, respectively. DPPH radical inhibition further corroborated this hierarchy, with the darkest-pigmented variety exhibiting >3-fold greater activity than the least active white accession. Notably, metal chelation capacity diverged from this trend, with blue-grained specimens outperforming both darker- and lighter-colored counterparts. These results collectively highlight the superior phytochemical composition and antioxidant activity of black grain varieties, particularly Xiongzhang, in comparison to blue and white varieties. The significant variations in phytochemical content and antioxidant capacity suggest that black grains offer enhanced health benefits, warranting further investigation into their bioavailability and physiological effects. These findings underscore the potential of incorporating these varieties into dietary regimens to leverage their health-promoting properties. 3.2. Distinct metabolic pathways and phytochemical profiles in highland barley varieties differentiated by bran color 3.2.1. Metabolomic profiling of highland barley grains differentiated by bran color Highland barley varieties were classified into three groups based on bran color: white, blue, and black ([66]Fig. 1A). The altitudinal distribution ([67]Fig. 1B) presents the altitude of the different varieties, ranging from 3700 m to 4300 m. Xiongzhang is cultivated at the highest altitude, while Zangqing2000 is grown at the lowest. The β-glucan content ([68]Fig. 1C) was notably higher in white-bran varieties, with Xila22 exhibiting the highest concentration. β-glucan content variation among the nine barley varieties showed no systematic association with seed coat pigmentation. While some black-grained varieties contained relatively low β-glucan levels, the black variety Sagui accumulated higher concentrations than its white-grained counterpart Zangqing2000, demonstrating that seed color does not predict polysaccharide content. Comprehensive metabolomic analysis of whole-grain highland barley identified 470 distinct metabolites. Metabolite profiling revealed flavonoids as the predominant class (41 %), followed by phenolic acids (25 %) and alkaloids (21 %), collectively representing the major secondary metabolites identified ([69]Fig. 1D). The Venn diagram ([70]Fig. 2E) delineates the overlap of differentially expressed metabolites among white, blue, and black barley varieties, revealing 72 metabolites shared across all comparisons. Notably, flavonoids and alkaloids collectively accounted for 82 % of the differentially abundant metabolites identified. The 72 metabolites common across all comparisons suggest universally affected core metabolic pathways, while the unique metabolites may represent pathways selectively modulated under specific conditions. Principal component analysis ([71]Fig. 1F) demonstrated clear clustering based on bran color, highlights clear differences in the biochemical composition of the varieties based on bran color. The black variety showed clear separation from the white and blue groups. The heatmap ([72]Fig. 1G) displays the relative abundance of identified phytochemicals in each barley variety, with color intensity indicating the concentration levels. White and blue barley share closely related compositional profiles, whereas black barley exhibits marked divergence. The classification of differential metabolites further confirms that both the number and diversity of metabolites are significantly lower in the White_vs_Blue comparison ([73]Fig. 1H). Notably, in the White_vs_Black comparison, black barley is distinguished by a higher abundance of flavonoids, organonitrogen compounds, and indole derivatives, which are present in significantly lower quantities—or entirely absent—in the White_vs_Blue comparison. Fig. 1. [74]Fig. 1 [75]Open in a new tab Comprehensive analysis of β-glucan content and phytochemical diversity in different bran-colored barley varieties. (A) Visual representation of barley varieties categorized by bran colors. (B) Altitude distribution of highland barley. (C) β-Glucan content across the barley varieties, with significant differences denoted by different letters (P < 0.05). (D) Pie chart showing mean values of the major phytochemicals identified in barley varieties. (E) A Venn diagram illustrating the overlap of differentially expressed metabolites among white, blue, and black barley varieties. Intersections represent shared metabolites among the comparisons: White_vs_Blue, White_vs_Black, and Blue_vs_Black. (F) Principal component analysis differentiating the barley varieties based on phytochemical composition, with distinct clustering observed for white, blue, and black bran colors. (G) Heatmap illustrating the relative abundance of identified phytochemicals across the barley varieties, with color intensity indicating the concentration levels (red = high, green = low). (H) Classification of differential metabolites across white, blue, and black varieties. White1–3, Blue1–3, and Black1–3 represent biological replicates of white-, blue-, and black-hulled highland barley, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to