Qualification of flavonoids of three sorghum bran varieties by untargeted metabolomics

Qualification of flavonoids of three sorghum bran varieties by untargeted metabolomics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Qualification of flavonoids of three sorghum bran varieties by untargeted metabolomics Mariely Cristine Dos Santos, Naoki Tanaka, Shigemitu Kasuga, Kazuhiro Tanabe, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4679263/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Sorghum bicolor is a source of many bioactive components, such as polyphenols. Those components are present especially in its bran, which is often removed in industrial processes through decortication. In that sense, this work aimed to analyze the polyphenol content, especially free flavonoids, from the bran of a newly developed variety compared to other commercially available varieties. The samples were white sorghum TDN® Sorgho, red sorghum Mini Sorgho, and the newly developed red sorghum RILN-156. First, the decortication was done to obtain the bran samples and those were triturated and then sieved. An untargeted metabolomics analysis (with LC/MS and CE/MS) was done to analyze the different components and identify the free flavonoids. For the general quantification analysis, instead of quantifying by target analysis, colorimetric methods were used to validate the metabolomics analysis. For this, the polyphenol content was extracted with 70% methanol. The antioxidant potential was also investigated using a DPPH assay. The results have shown that the flavonoid content was significant in these samples, especially in the newly developed RILN-156, with 19 flavonoids identified. RILN-156 also presented higher antioxidant capacity than the conventional varieties, a promising finding for its use to prevent chronic diseases, which will be further investigated. Biological sciences/Chemical biology/Metabolomics Biological sciences/Biochemistry/Metabolomics Health sciences/Health care/Nutrition Sorghum bicolor metabolomics polyphenol flavonoid DPPH assay Figures Figure 1 Figure 2 Figure 3 1 Introduction Sorghum ( Sorghum bicolor L .) is a drought-tolerant, nutritious cereal crop highly produced worldwide. As the 5th most-produced cereal globally, it is popular in the bioenergy industry and as a feedstock material [ 1 ]. According to their purpose, there are four main types of sorghum: sweet sorghum, grain sorghum, forage sorghum, and bioenergy sorghum [ 2 ]. Dietary-wise, although grain sorghum presents many nutritional traits, it is still overseen as a food product in many places, primarily because of unwanted sensorial characteristics some varieties can present. An example is the bitter taste of dark-colored sorghum varieties [ 3 ]. In this case, the bitter taste is caused by phenolic components, such as tannins, present in the pericarp of this grain, like most of the phenolic components from cereals [ 4 , 5 ]. The pericarp is one of the discarded parts during the process of decortication, and together with the aleurone layer and seed coat, it forms what is called the sorghum bran [ 6 ]. In the last decades, studies have investigated the benefits of sorghum bran, previously considered industrial waste, to make the most of the essential bioactive components in this matrix. Among the beneficial components of sorghum bran, the previously mentioned phenolic compounds are gaining crescent attention [ 7 – 10 ]. Sorghum bran has colored varieties, and each color influences the profile of the grain's phenolic components [ 11 ]. The class of flavonoids is one of the protagonists for the colors of plants, and, as phenolic compounds, they are considered bioactive components due to their antioxidant, antibacterial, anti-inflammatory, and even anticarcinogenic activities, and plenty more health benefits [ 12 ]. With the challenges in unraveling the structure of sorghum and its bioactive components, researchers and farmers are in search of ideal hybrids for different environments and applications, which expands the variations in phenotypic and structural characteristics among the different sorghum [ 13 – 18 ]. Considering these fundamentals, this research aims to compare the differences in the phenolic content, focusing primarily on the qualitative analysis of the flavonoid profile of sorghum bran from three different varieties, including a newly developed variety. 2 Results These results comprehend the main phenotypic differences among the samples chosen. They are followed by the free flavonoids identified by untargeted metabolomics and their distinction according to the sorghum varieties and the growth environment, and finally, the quantification of the polyphenol content by colorimetric methods. 2.1 Comparison among sorghum samples used in this research The phenotypic differences of the three sorghum varieties used for this research are described in this topic. First, white TDN® Sorgho is bigger than the colored sorghums (approximately 48 mm in height). On the other hand, RILN-156 is not only the smallest (around 35 mm in height) but also the roundest and has a more intense color among these three varieties. Mini Sorgho then stands in the middle regarding size (approximately 41 mm) and color. The varieties also present some distinctions regarding secondary color of the grains, presence of pigmented testa, and thickness of the pericarp. Contrary to colored sorghum, TDN® Sorgho does not present a pigmented testa. As for both the red sorghums, although the floury endosperm - the white part in the middle of the grain - was observed to take different sizes in each grain from the same variety, the color of the corny endosperm (secondary color) of both types of grain is slightly different. Mini Sorgho presents a darker secondary color, closer to a brownish purple, while RILN-156 gets similar to a whitish red. By observing many samples of the grain, the thickness of the pericarp was very close between the red sorghum varieties and thinner in the white sorghum. The general characteristics of the crops are that TDN® Sorgho can reach up to 2 meters, Mini Sorgho can reach up to 1.5 to 1.8 meters, and RILN-156 can reach 1.2 to 1.5 meters. The sowing period is the same for all three varieties: April to August in warm areas, May to August in intermediate areas, and May to July in cold areas. Sorghum usually takes 60 to 80 days to reach its maximum height. 2.2 Untargeted metabolomic analysis of sorghum bran By untargeted metabolomics analysis using LC-MS and CE-MS, nineteen flavonoids were successfully identified (Table 1 ), where the flavonols corresponded to 11% of the flavonoids identified among the three sorghum bran varieties. Table 1 Flavonoids identified by LC/MS and CE/MS Mass Compound Name Flavonoid Classification Molecular Formula 579.176 Naringenin 7-neohesperidoside Flavanone C 27 H 32 O 14 447.092 Isoorientin Flavone C 21 H 20 O 11 221.050 Isofraxidin Coumarin C 11 H 10 O 5 347.105 Malvidin Anthocyanidin C 17 H 15 O 7 + 431.095 Apigenin 8-glucoside Flavone C 27 H 30 O 14 303.047 Taxifolin Dihydroflavonol C 15 H 12 O 7 431.091 Apigenin 7-glucoside Flavone C 21 H 20 O 10 283.060 Glycitein Isoflavone C 16 H 12 O 5 287.051 Eriodictyol Flavanone C 15 H 12 O 6 255.068 Liquiritigenin Flavanone C 15 H 12 O 4 269.044 Galangin Flavonol C 15 H 10 O 5 291.084 Epicatechin Flavanol C 15 H 14 O 6 595.173 Saponarin Flavone C 27 H 30 O 15 465.105 Myricetin 3-rhamnoside Flavonol C 21 H 20 O 12 431.130 Formononetin 7-glucoside Isoflavone C 22 H 22 O 9 255.061 Daidzein Isoflavone C 15 H 10 O 4 255.061 Chrysin Flavone C 15 H 10 O 4 301.071 Chrysoeriol Flavone C 16 H 12 O 6 271.057 Baicalein Flavone C 15 H 10 O 5 With the different profiles regarding the concentration of flavonoids, a Principal Component Analysis (PCA) could be carried out, separating the sorghums, and determining the principal components among the flavonoids (Fig. 1 ). Combining the two principal components, F1 and F2, this analysis could comprehend 70.26% of the total variance of the samples. We can observe that three of the four Mini Sorgho samples have similar flavonoid profiles, indicated by their high positive scores on both principal components. Mini Sorgho (2) and one of the sorghum samples, RILN-156 (2), are characterized by distinct flavonoid profiles compared to the other samples analyzed. In a general manner, contrasting with all the TDN® Sorgho samples having similar flavonoid profiles with each other, the RILN-156 samples all presented variations among themselves, where RILN-156 (3) also has a distinct profile, RILN-156 (1) has an average profile that is not strongly influenced by either principal component and RILN-156 (4) has a flavonoid profile characterized by the highest positive scores on both principal components (together with sorghum Mini Sorgho (4)). As for the flavonoid influences in each sorghum, on the biplot with the variables, it can be observed that the TDN® Sorgho samples were strongly associated with Galangin, Malvidin, and Baicalein. Malvidin was the only anthocyanin that could be identified in this analysis. The flavonoid profile from sorghum mini (1), (3) and (4) and RILN-156 (4) is strongly influenced by Apigenin-7-glucoside, Naringenin-7-neohesperidoside, and Apigenin-8-glucoside. Mini Sorgho (2) is associated with flavonoids like Liquiritigenin, similar to RILN-156 (2), which Eriodictyol also influences. As for the last two RILN-156 samples, RILN-156 (1) is associated with Taxifolin but not strongly characterized by any other variables, while RILN-156 (3) is influenced by variables such as Chrysin, Formononetin-7-rhamnoside, and Epicatechin. Furthermore, to better illustrate this, the heatmap below (Fig. 2 ) shows that apart from the Mini Sorgho (2) sample, which presented a very different profile compared to the other sorghum samples, some groups of flavonoids are more abundant in red sorghum than on white sorghum. Groups present differences in concentration among varieties, and groups vary according to the specific sample. Environmental conditions influenced the latter during the plant's growth, an essential factor in the flavonoid profile of plants. Correspondingly, except for flavonols and anthocyanin, red sorghum varieties presented a more significant concentration of flavonoids overall. Besides flavonols, Mini Sorgho and RILN-156 presented a significant concentration of isoflavones and flavones as well. As mentioned, Taxifolin, representing the dihydroflavonols, was also outstanding in one of the RILN-156 samples. According to the concentrations observed by intensity values on untargeted metabolomics, Galangin had the highest concentration among all the flavonoids. In the present work, Galangin prevailed in white sorghum samples. The second most abundant flavonoid was Daidzein. This isoflavone, however, was almost absent in TDN® Sorgho bran, in contrast with the large amount present in the red sorghum samples. Nonetheless, the bran of TDN® Sorgho presented a higher concentration of anthocyanin than the colored sorghum, even though it is a white variety. Despite the variations within the white sorghum samples collected from separate places, they were still higher in anthocyanin content than both colored varieties. On the other hand, Chrysin was higher in colored samples and barely present in the white sorghum bran. Baicalein, Formononetin-7-glucoside and Eriodictyol also had significant concentrations. 2.3 Validation of untargeted metabolomics data by colorimetric methods This session will present the results of the investigation of free phenolic compounds soluble in methanol from the bran of the white sorghum sample (TDN® Sorgho) and the two red sorghum samples (RILN-156 and Mini Sorgho). In this sense, despite both RILN-156 and Mini Sorgho being red sorghum varieties, the color of the polyphenolic extract from RILN-156 is more intense, closer to brown, in contrast to the orangish tone from Mini extract. We can observe in the following table of all the results obtained that Sorghum RILN-156 bran presented interesting values compared to the other two samples. RILN-156 had higher total phenolic content and flavonoid content. Corroborating with the untargeted metabolomics previously presented results, the total anthocyanin content, represented by apigenin and luteolin in sorghum, was the highest in white sorghum. Nevertheless, as expected, tannin was not detected in white sorghum but similar in Mini Sorgho and RILN-156, higher in Mini Sorgho bran. Except for tannin content, the bran of Mini Sorgho had the lowest concentrations. Table 1 Results obtained from the methanol extraction of TDN®, Mini and RILN-156 bran. Analysis TDN® Sorgho Mini Sorgho RILN-156 Total phenolic content (µg.GAE/g) 55.568 42.220 91.840 Total flavonoid content (µg.QE/g) 413.169 185.067 1196.320 Total anthocyanin content Apigenin (µg/g) 4290.5 104.56 335.34 Luteolin (µg/g) 6231.9 151.84 486.7 Total tannin content (mg.CE/g) N.D. 1.722 1.149 DPPH scavenging assay (mM.TE/g) 230 106.364 266.364 3 Discussion The concentration of phenolic compounds can be influenced by the grain’s genotype, which in turn influences the pericarp color and thickness, as well as the presence of colored testa in the grains and the secondary plant color (phenotype) [ 19 ]. The pericarp color comes from the R and Y genes, where when Y is homozygous recessive (rryy or R_yy), there is the presence of a white pericarp, and when R and Y are dominant, the pericarp comes as red (R_Y_). The gene Z influences the thickness, while the presence or absence of pigmented testa is due to B1 and B2 genes (if these are dominant, there is a pigmented testa) [ 20 ]. In the present samples, we can observe that although both red pericarp sorghums present a pigmented testa and similar thickness, the secondary color of the grain is different. The P and Q genes determine the secondary color: red, purple, or tan. Tan sorghums have recessive P genes (ppqq or ppQQ), while red (PPqq) and purple (PPQQ) sorghums have dominant ones. Sorghums presenting red or purple as secondary colors have been reported to have higher phenolic concentrations in comparison with tan-colored sorghum [ 21 ]. By observing the appearance of the sorghum grains cut in half, we could observe that Mini Sorgho has a brownish purple secondary color, while RILN-156 has a whitish red. White sorghum has a tan secondary color. As mentioned in the results section, under the untargeted metabolomics analysis, flavonols were the larger class of flavonoids identified within the three sorghum bran varieties. Flavonols, one of the most abundant classes of flavonoids, represented by quercetin, have been gaining attention in the food and pharmaceutical industries, especially now that the search for natural healthy compounds and functional foods is standing out. Structurally similar to flavones, flavonols are colorless compounds with an extra non-phenolic hydroxyl group at position 3 [ 22 ]. They have a wide range of roles in the plant defense system, including being responsible for the plant-microbe interaction and protection from UV rays and microbial attacks [ 23 ]. Galangin had the highest concentration among all the flavonoids, especially on TDN® Sorgho. Galangin, a 3,5,7-trihydroxyflavone, is usually found in honey and propolis and has been investigated to prevent diverse diseases and human conditions like aging and inflammation [ 24 ]. Despite being abundant in the samples, Galangin has been reported not to be toxic up to 5 g/kg and not to cause any side effects [ 25 ]. After ingestion, Galangin can be metabolized into kaempferol and quercetin, two important antioxidants [ 26 ]. Such traits enable the study of its antiproliferative ability against several types of cancer cells, like esophageal [ 27 ], leukemia [ 28 ], and even skin cancer [ 29 ]. The second most abundant flavonoid was Daidzein. This isoflavone, however, was almost absent in TDN® Sorgho bran in contrast with large amounts present in the red sorghum samples. Primarily found in soybeans, Daidzein has many roles in the biotic and abiotic stress defense mechanism of plants, such as influencing the receptivity of symbiotic root infection, defense against oxidative stress, and so on [ 30 ]. Being chemically similar to mammalian estrogens, Daidzein presents estrogenic properties that can be beneficial by hindering or substituting estrogen and estrogen receptor complex, protecting against diseases related to the control of estrogens, like breast cancer, diabetes, osteoporosis, and cardiovascular disease [ 31 ]. In a study with soybeans, it was noticed that the Daidzein content increased under waterlogging [ 32 ], which also shows that although Daidzein seems to be a flavonoid abundant in colored sorghums, it can also be influenced by conditions such as water stress. In contrast, the concentration of anthocyanin, represented by the anthocyanidin Malvidin, was higher on TDN® Sorgho bran than the colored sorghum bran despite being a white variety. Although, to our knowledge, it has not yet been reported in sorghum, it is not uncommon. Many factors can modify the color of anthocyanins, from genetic traits to environmental conditions. Despite the environmental conditions affecting the exact amount of Malvidin produced, since there were variations within the white sorghum samples collected from separate places, they still had higher anthocyanin content than both colored varieties. One hypothesis would be sorghum's high production of flavones, considered co-pigments. In some plants, flavones and anthocyanins interact inversely proportional ways: the more flavones produced, the fewer anthocyanins are present. Also, anthocyanins' color or intensity will change depending on the amount of hydrogen ions in flavones [ 33 ]. However, we noticed that, in the present work, even when some of the samples of colored varieties presented a similar concentration of flavones as the white varieties, the anthocyanidin content did not increase. Nonetheless, we cannot rule out that a specific flavone could interact with Malvidin. For example, white sorghum bran samples had a smaller Chrysin content than their anthocyanidin concentration. In contrast, red sorghum brans presented a significant amount of the same flavone (Fig. 3 ). Although further investigation would be necessary to confirm the interaction between these two metabolites, it is still interesting to notice this coincidental pattern. Chrysin (5,7-dihydroxyflavone), besides the common properties of flavonoids, has also been studied regarding its antispasmodic and anxiolytic properties [ 34 ]. On the other hand, the anthocyanidin Malvidin is extensively known for helping in the attribution of the color of red grapes and wine [ 35 ]. As Malvidin has been thoroughly investigated, it has been reported to have anticarcinogenic, diabetes-control, cardiovascular-disease-prevention, and brain-function-improvement properties [ 36 ]. Baicalein, which was also slightly significant in the samples, comes from the chrysin biosynthetic pathway and has been investigated for its contribution to preventing cancer and diseases [ 37 ]. Baicalein has also recently been investigated for treating SARS-CoV-2 [ 38 ]. In addition, Chrysoeriol, Taxifolin, and Eriodictyol were most present in RILN-156. That is a promising result for this newly developed inbred line. Although in smaller concentrations, the other identified flavonoids have also been reported to contribute to health in vitro and in Vivo tests, as some of the benefits are cited in online resources. On the validation of the results, using colorimetric methods, RILN-156 bran presented the highest value of phenolic content (91.840 µg.GAE/g of bran), while Mini Sorgho presented 42.220 µg.GAE/g. Usually, white sorghum presents a lower concentration of phenolic compounds than colored sorghum [ 10 ], contrary to what happened in this work (TDN® Sorgho presented 91.840 µg.GAE/g). Despite these colorimetric analyses being used to validate the untargeted metabolomics data, the results were different regarding the red Mini Sorgho bran. Nonetheless, we can observe that both red sorghums presented a significant difference in the extraction of polyphenol content and flavonoids using methanol, 70%. In contrast, Mini Sorgho presented results that were lower than those of TDN® Sorgho. This could be due to the presence of a large concentration of flavonoids and polyphenols that do not have as much affinity with the solvent as the ones present in RILN-156 since the solvent polarity is important in the determination of which components will be extracted [ 39 ]. Likewise, in the determination of total flavonoid contents, RILN-156 once again had the highest concentration (1196.320 µg.QE/g), followed by TDN® Sorgho (413.169 µg.QE/g) and Mini Sorgho(185.067 µg.QE/g). The results obtained by RILN-156 corroborate previous studies that reported that colored sorghums present higher levels of secondary metabolites such as flavonoids [ 40 ]. As for anthocyanin content, luteolin and apigenin were taken as references. Surprisingly, among the free phenolic compounds quantified, TDN® Sorgho was the sample with the highest concentration of both luteolin (6231.9 µg/g) and apigenin (4290.5 µg/g). RILN-156 had a significantly lower concentration (486.7 µg/g and 335.34 µg/g), as well as Mini Sorgho (151.84 µg/g and 104.56 µg/g). This data endorses the one found in untargeted metabolomics, where Malvidin was more abundant on TDN® Sorgho than on colored sorghum bran. While methanol is often employed for the extraction of anthocyanin and the addition of water could improve its yield due to similar polarity, other conditions, such as temperature and time, should be optimized for focusing on anthocyanin extraction [ 41 ], besides the possible interaction with flavones, that could also be one hypothesis to explain the differences in these results for red sorghum. Total condensed tannin was not detected in white sorghum but presented slight variation among the colored sorghum samples. Mini Sorgho bran (1.722 mg.CE/g), followed by RILN-156 (1.149 mg.CE/g), showed a substantial content of tannins. Despite acting as antioxidants, tannins are also considered anti-nutrients due to their possible inhibition of proteins and their influence on the digestibility of some amino acids [ 42 ]. Sorghums that contain tannin are known to be resistant to birds and insects and provide a higher yield, which might influence farmers' choice depending on the application it will be destined for [ 43 ]. For dietary purposes, it is a positive result that the newly developed sorghum obtained a lower tannin content. More tests in vitro would be necessary to imply that the amount of tannin present could contribute to the bioactivity of the grain while not being as abundant as to have a heavy influence on the absorption of the compounds of interest. On DPPH scavenging activity, the colored sorghum samples, Mini Sorgho, and RILN-156, had 106.364 mM.TE/g and 266.364 mM.TE/g respectively, while the white sorghum TDN® Sorgho had 230 mM.TE/g as a scavenging activity. A higher phenolic content means a higher antioxidant activity since phenolics, and flavonoid molecules have been shown to have a high correlation with the antioxidant activity of plant extracts [ 44 ]. Based on this, the DPPH scavenging activity values obtained from the current samples were as high as the total phenolic contents were. For the human body, a component with better antioxidant activity in a food product means a higher chance of reducing the risk of degenerative diseases, such as cancer. Naturally, many factors affect such diseases. However, studies have proven the mechanism of action of antioxidant compounds in heart and respiratory diseases, arthritis, inflammatory diseases, and even Parkinson’s disease [ 45 ]. In this sense, in vitro and in vivo tests with bioactive antioxidant components extracted from sorghum bran have been conducted for more than a decade now [ 46 – 47 ], indicating that sorghum bran, wildly colored varieties, could be a great addition to human diet depending on the bioavailability of such components. As previously reported in other works [ 48 ], we could observe that despite RILN-156 having a more significant amount of polyphenol, other factors considerably affect the polyphenolic profile of the grain. Nevertheless, it is essential to conduct bioavailability tests to ensure that those components can be metabolized by the human body during digestion, as around 80% of sorghum bioactive components are covalently bound to other cell wall compounds [ 49 ]. The recombinant inbred line RILN-156 had a greater antioxidant scavenging potential than the two other varieties analyzed, which corroborated its polyphenolic compounds level and showed a favorable potential for using this inbred line from now on. Nonetheless, depending on the intended use of the chosen grain, white sorghum is also advantageous because it does not present tannins, which consequently highlights the significance of such studies being conducted comparing different varieties. 4 Methods Detailed information about development of the new variety, codification of samples, preparation and flow of experiments, and so-on can be found on supplementary online information. 4.1 Sorghum samples Three varieties of sorghum were used for this study. Two commercially available seeds are white sorghum TDN® Sorgho and red sorghum Mini Sorgho sold by KANEKO SEEDS CO., LTD. The third variety used was red sorghum RILN-156, produced by Shinshu University, Ina City, Nagano Prefecture, Japan. 4.2 Preparation of sorghum bran The seed was milled with a household rice milling machine (SM – 500W, MK-SEIKO CO., LTD., Japan) using the program according to the amount of sample, which takes around 1 minute and 50 seconds to finish, to obtain the bran from each grain. These milling conditions were done three times for each sample. After that, the bran was collected and sieved with a mesh (opening: 250 µm, diameter: 160mm). The remaining larger bran fractions were triturated with a blender (Wonder Blender WB-1, Osaka Chemical) and further sieved with the same mesh mentioned above. The bran was stored in a sealed container until further analysis. 4.3 Untargeted metabolomic analysis of sorghum bran 4.3.1 Sample preparation for Liquid Chromatography coupled with Mass Spectrometer (LC/MS) and Capillary Electrophoresis coupled with Mass Spectrometer (CE/MS) For the untargeted metabolomics analysis, samples grown in different cities of Nagano Prefecture and districts of Nagano City were collected: TDN® Sorgho from three locations (Iizuna Town, Naniai—Nagano City, and Wakaho—Nagano City), Mini Sorgho from four locations (Chikuma City, Shiojiri City, Suzaka City, and Nagano City), and RILN-156 from four locations (Suzaka City, Ueda City, Naniai—Nagano City, and Shiozaki, Nagano City). Approximately 100 mg of samples were transferred to sample disruptor tubes supplied by Yasui Kikai (Osaka, Japan) and shaken with iron cones cooled in liquid nitrogen. Each sample was analyzed twice for the four platforms: CE/MS and LC/MS in both positive and negative polarities. The average of the two data sets was used as the expression value. Quality control (QC), containing all samples, was prepared, and analyzed every six measurements to ensure measurement accuracy. The coefficient of variance (CV%) was calculated for each metabolite using the QCs, and any metabolites with a CV% exceeding 50% were excluded from the data table. 4.3.2 CE/MS analysis and LC/MS analysis CE/MS experiments were conducted using an Agilent CE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany) coupled with an Agilent 6545 QTOF system (Agilent Technologies, Palo Alto, CA, USA). LC/MS analyses were conducted using an Agilent 1290 series UPLC system equipped with a 6545 quadrupole TOF system (Agilent Technologies, Palo Alto, CA, USA) controlled by MassHunter Workstation B.08.01 software. The analytical column employed was a CAPCELL PAK C18 IF, 2.0 mm ID × 50 mm, 2 µm (Osaka Soda, Osaka, Japan). 4.3.3. Data analysis of untargeted metabolomics The raw data obtained from the mass spectrometer were converted to CSV format using MassHunter Export software (Agilent Technologies). The resulting CSV data included information on m/z (mass-to-charge ratio), retention time, and intensity. Principal component analysis (PCA) was performed using the extension XLSTAT 2019 from Microsoft Excel [ 50 ]. 4.4 Validation of untargeted metabolomics data by colorimetric methods 4.4.1 Extraction of polyphenol from sorghum bran For the extraction, 70% methanol was used, as described by Zhang et al. [ 51 ]. The procedure was carried out according to the protocol first described by Awika et al. [ 52 ] and later adapted by Tyagi et al. [ 53 ]: 2.5 g of sorghum bran were added to 20 mL of the solvent (methanol 70%) and shaken for 2 hours at 25 ºC in a 50 mL conical centrifuge tube. The mixture was then left at a -20 ºC freezer overnight for the dispersion of phenolics to occur. Subsequently, the samples were centrifuged at 7000 x g for 10 minutes, and the supernatant was collected. The precipitate was washed twice (10 mL) with methanol 70%, and the supernatant was collected, mixed, and stored at -20 ºC until further analysis. As the colorimetric analyses were conducted without solvent evaporation, the final concentration was corrected according to the dilution of the sample in methanol for the extraction. 4.4.2 Determination of total phenolic content The total phenolic content (TPC) was determined following the Folin-Ciocalteu method [ 54 ]. The assay was conducted on a microplate adapted from Zhang et al. [ 55 ]. The results were expressed in mg of gallic acid equivalent (GAE mg/100g). 4.4.3 Determination of total flavonoid content The total flavonoid content (TFC) was determined using Chang et al.'s aluminum-chloride colorimetric method [ 56 ], adapted to microplates. The results were expressed in mg of quercetin equivalent (QE mg/100g). 4.4.4 Determination of total anthocyanin content The total anthocyanin content (TAC) was determined using the pH differential method [ 57 ]. Absorbance was read at 270 nm. Luteolin and apigenin were used as the standards [ 53 ]. The molar absorption coefficient for apigenin was calculated using the Beer-Lambert Law: 27.38 for apigenin and 19.98 for luteolin. Then the Beer-Lambert Law was rearranged to calculate the apigenin and luteolin content in the samples. 4.4.5 Determination of total condensed tannin Total condensed tannin (TCT) was determined by the butanol-hydrochloric acid method [ 58 ]. The standard curve was done with catechin; the results are expressed in mg of catechin equivalents (CE) per gram of sample. 4.4.6 DPPH scavenging assay The scavenging activity was determined by the DPPH assay, following Enkhtsetseg et al. [ 59 ]’s adaptation on Adedapo et al. [ 60 ]. Trolox was used for the calibration curve, so results were expressed in µmol Trolox equivalents (TE) per g of sample. 4.4.7 Statistical analysis All analyses were conducted in triplicate, and the average and standard deviation were calculated. The analysis of variance (ANOVA) was conducted using the extension XLSTAT 2019 from Microsoft Excel [ 51 ]. Declarations Conflict of Interest The authors have no relevant financial or non-financial interests to disclose. Funding This work was supported by Project for Enhancing the Environment to Create Innovation in Regional Core Universities, the Japan Society for Cabinet Office. Author Contribution Yoshihiko Amano, Naoki Tanaka and Masahiro Mizuno contributed to the study conception and design. Shigemitsu Kasuga has bred and provided a new sorghum variety. Material preparation, and analysis were performed by Mariely Cristine Dos Santos. Data collection by Metabolomics analysis were performed by Kazuhiro Tanabe and Chihiro Hayashi. The first draft of the manuscript was written by Mariely Cristine Dos Santos and all authors commended on previous versions of the manuscript. Yoshihiko Amano read and approved the final manuscript. Acknowledgement We would like to express my gratitude for the helpful suggestions from Shinshu Sustainability Transformation Initiative, Shinshu University while conducting this research. Data Availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. References Tanwar, R. et al. Nutritional, phytochemical and functional potential of sorghum: A review. Food Chemistry Advances 3:100501. https://doi.org/10.1016/j.focha.2023.100501 (2023). Hossain, M. S. et al. Sorghum: A prospective crop for climatic vulnerability, food and nutritional security. Journal of Agriculture and Food Research 8:100300. https://doi.org/10.1016/j.jafr.2022.100300 (2022). Widowati, S., Luna, P. Nutritional and Functional Properties of Sorghum (Sorghum bicolor (L.) Moench)-based Products and Potential Valorisation of Sorghum Bran. IOP Conference Series: Earth and Environmental Science 1024:012031. https://doi.org/10.1088/1755-1315/1024/1/012031 (2022). Salar, R. K., Purewal, S. S., Sandhu, K. S. Relationships between DNA damage protection activity, total phenolic content, condensed tannin content and antioxidant potential among Indian barley cultivars. Biocatalysis and Agricultural Biotechnology 11:201–206. https://doi.org/10.1016/j.bcab.2017.07.006 (2017). Zuo-Yu, Z., Dahlberg, J. Sorghum Methods and Protocols. New York, Ny Springer New York (2019). Janet, A. A., Hema, K. Evaluation of nutritional and functional properties of anatomical parts of two sorghum (Sorghum bicolor) varieties. Heliyon 9:e17296–e17296. https://doi.org/10.1016/j.heliyon.2023.e17296 (2023). Devi, P. S., Kumar, M. S., Das, S. M. DNA Damage Protecting Activity and Free Radical Scavenging Activity of Anthocyanins from Red Sorghum (Sorghum bicolor) Bran. Biotechnology Research International 2012:1–9. https://doi.org/10.1155/2012/258787 (2012). Herald, T. J., Gadgil, P., Tilley, M. High-throughput micro plate assays for screening flavonoid content and DPPH-scavenging activity in sorghum bran and flour. Journal of the Science of Food and Agriculture 92:2326–2331. https://doi.org/10.1002/jsfa.5633 (2012). Shim, T. J. et al. Toxicological Evaluation and Anti-Inflammatory Activity of a Golden Gelatinous Sorghum Bran Extract. Bioscience, Biotechnology, and Biochemistry 77:697–705. https://doi.org/10.1271/bbb.120731 (2013). Xu, J., Wang, W., Zhao, Y. Phenolic Compounds in Whole Grain Sorghum and Their Health Benefits. Foods 10:1921. https://doi.org/10.3390/foods10081921 (2021). Kumari, P. K., Umakanth, A. V., Narsaiah, T. B., Uma, A. Exploring anthocyanins, antioxidant capacity and α-glucosidase inhibition in bran and flour extracts of selected sorghum genotypes. Food Bioscience 41:100979. https://doi.org/10.1016/j.fbio.2021.100979 (2021). Roy, A. et al . Flavonoids a Bioactive Compound from Medicinal Plants and Its Therapeutic Applications. BioMed Research International 2022:1–9. https://doi.org/10.1155/2022/5445291 (2022). Silva, T. C. da et al. Agronomic divergence of sorghum hybrids for silage yield in the semiarid region of Paraiba. Revista Brasileira de Zootecnia 40:1886–1893. https://doi.org/10.1590/s1516-35982011000900007 (2011). Teixeira, A. de M. et al . Intake and digestibility of sorghum (Sorghum bicolor, L. Moench) silages with different tannin contents in sheep. Revista Brasileira de Zootecnia 43:14–19. https://doi.org/10.1590/s1516-35982014000100003 (2014). Pontieri, P. et al. Mineral content in grains of seven food-grade sorghum hybrids grown under Mediterranean Environment. Australian journal of crop science 8 (11): 1550–1559 (2014). Msongaleli, B. M., Tumbo, S. D., Kihupi, N. I., Rwehumbiza, F. B. Performance of Sorghum Varieties under Variable Rainfall in Central Tanzania. International Scholarly Research Notices 2017:1–10. https://doi.org/10.1155/2017/2506946 (2017). Buntin, G. D., Ni, X., Harris-Shultz, K. R., Knoll, J. E., Toews, M. D., Dunn, D. Grain, silage and forage sorghum hybrid resistance to insect and bird damage. In: Mailthot, DJ, Dunn, DH. Jordan, Jr, Day JL (eds), Soybean, Sorghum Grain and Silage, and Summer Annual Forages, Georgia Agricultural Experiment Stations, Annual Publications. 103-9:74–80. (2017). Smale, M. et al. Farm family effects of adopting improved and hybrid sorghum seed in the Sudan Savanna of West Africa. Food Policy 74:162–171. https://doi.org/10.1016/j.foodpol.2018.01.001 (2018). Dykes, L., Seitz, L. M., Rooney, W. L., Rooney, L. W. Flavonoid composition of red sorghum genotypes. Food Chemistry 116:313–317. https://doi.org/10.1016/j.foodchem.2009.02.052 (2009). Rooney, L. W., Miller, F. R. Variation in the Structure and Kernel Characteristics of Sorghum. Proceedings of the International Symposium on Sorghum Grain Quality, International Crop Research Institute for the Semi-Arid Tropics, Hyderabad, 28–31, 11–13. (1982). Dykes, L., Rooney, L. W., Waniska, R. D., Rooney, W. L. Phenolic Compounds and Antioxidant Activity of Sorghum Grains of Varying Genotypes. Journal of Agricultural and Food Chemistry 53:6813–6818. https://doi.org/10.1021/jf050419e (2005). Jong, M. K., Liu, L., Guo, W., Mohsen, M. Chemical structure of flavonols in relation to modulation of angiogenesis and immune-endothelial cell adhesion. 17:165–176. https://doi.org/10.1016/j.jnutbio.2005.06.006 (2006). 28. Aar R. M. et al. Natural flavonols: actions, mechanisms, and potential therapeutic utility for various diseases. Beni Suef Univ J Basic Appl Sci. 12(1). https://doi.org/10.1186/s43088-023-00387-4 (2023). Mak, K. K. et al . Galangin’s potential as a functional food ingredient. Journal of Functional Foods 46:490–503. https://doi.org/10.1016/j.jff.2018.04.054 (2018). Huh, J. et al . The natural flavonoid galangin inhibits osteoclastic bone destruction and osteoclastogenesis by suppressing NF-κB in collagen-induced arthritis and bone marrow-derived macrophages. European Journal of Pharmacology 698:57–66. https://doi.org/10.1016/j.ejphar.2012.08.013 (2013). Merve, B., Basaran, A., Nurşen, B. Galangin as a Plant Phenolic and Usage in Health and Disease. 433–438. https://doi.org/10.1016/b978-0-12-813008-7.00034-5 (2018). Zhang, C. et al . Galangin Enhances Anticancer Efficacy of 5-Fluorouracil in Esophageal Cancer Cells and Xenografts Through NLR Family Pyrin Domain Containing 3 (NLRP3) Downregulation. Medical Science Monitor 27: https://doi.org/10.12659/msm.931630 (2021). Tolomeo, M. et al . Galangin increases the cytotoxic activity of imatinib mesylate in imatinib-sensitive and imatinib-resistant Bcr-Abl expressing leukemia cells. Cancer Letters 265:289–297. https://doi.org/10.1016/j.canlet.2008.02.025 (2008). Benguedouar, L. et al . Ethanolic Extract of Algerian Propolis and Galangin Decreased Murine Melanoma T. Anti-Cancer Agents in Medicinal Chemistry 16:1172–1183. https://doi.org/10.2174/1871520616666160211124459 (2016). Kim, I. S. Current perspectives on the beneficial effects of soybean isoflavones and their metabolites on plants. Food Science and Biotechnology 31:515–526. https://doi.org/10.1007/s10068-022-01070-7 (2022). Alshehri, M. M. et al. Therapeutic Potential of Isoflavones with an Emphasis on Daidzein. Oxidative medicine and cellular longevity 2021:6331630. https://doi.org/10.1155/2021/6331630 (2021). Dong, H. G., Lee S., Ki, Y. J., Hyen, C. C. Assessment of In-Season N Effects on Soil Water Stress to Growth and Yields of Soybean. Han’guk Toyang Biryo Hakhoe Chi 56:463–477. https://doi.org/10.7745/kjssf.2023.56.4.463 (2023). Robinson, G. M., Robinson, R. A survey of anthocyanins. I. Biochem J. 25(5):1687–1705 (1931). Shukla, R., Pandey, V., Vadnere, G. P., Lodhi, S. Role of Flavonoids in Management of Inflammatory Disorders. 293–322. https://doi.org/10.1016/b978-0-12-813820-5.00018-0 (2019). Moreno, J., Peinado, R. Polyphenols. In: Enological Chemistry. Elsevier eBooks, Academic Press, pp. 53–76 (2012) Merecz-Sadowska, A. et al . Food Anthocyanins: Malvidin and Its Glycosides as Promising Antioxidant and Anti-Inflammatory Agents with Potential Health Benefits. Nutrients 15:3016. https://doi.org/10.3390/nu15133016 (2023) Arshad, H. R., et al . The Multifaceted Role of Baicalein in Cancer Management through Modulation of Cell Signalling Pathways. Molecules (Basel Online) 27:8023–8023. https://doi.org/10.3390/molecules27228023 (2022). Dinda, B., Dinda, M., Dinda, S., Utpal, C. D. An overview of anti-SARS-CoV-2 and anti-inflammatory potential of baicalein and its metabolite baicalin: Insights into molecular mechanisms. European journal of medicinal chemistry 258:115629–115629. https://doi.org/10.1016/j.ejmech.2023.115629 (2023). Rodríguez De Luna, S. L., Ramírez-Garza, R. E., Serna Saldívar, S. O. Environmentally Friendly Methods for Flavonoid Extraction from Plant Material: Impact of Their Operating Conditions on Yield and Antioxidant Properties. The Scientific World Journal 2020:1–38. https://doi.org/10.1155/2020/6792069 (2020). Ajay, P. R. et al . Large-Scale Non-Targeted Metabolomics Reveals Antioxidant, Nutraceutical and Therapeutic Potentials of Sorghum. Antioxidants (Basel) 10:1511–1511. https://doi.org/10.3390/antiox10101511 (2021). Tena, N., Asuero, A. G. Up-To-Date Analysis of the Extraction Methods for Anthocyanins: Principles of the Techniques, Optimization, Technical Progress, and Industrial Application. Antioxidants 11:286. https://doi.org/10.3390/antiox11020286 (2022) Asropi, N. E., Novita, D. D. Physical Quality and Tannin Content of Sorghum (Sorghum bicolor L.) at Different Temperature and Soaking Immersion. IOP Conference Series: Earth and Environmental Science 1024:012068. https://doi.org/10.1088/1755-1315/1024/1/012068 (2022). Lubadde, G., Ebiyau, J., Aru, C., Andiku, J., Wandulu, M., Ugen, M. Sorghum production handbook for Uganda. Uganda: Resources Research Institute of the National Agricultural Research Organisation (NaSARRI-NARO) (2019) Aryal, S. et al . Total Phenolic Content, Flavonoid Content and Antioxidant Potential of Wild Vegetables from Western Nepal. Plants 8:96. https://doi.org/10.3390/plants8040096 (2019). Zhang Y. J. et al. Antioxidant Phytochemicals for the Prevention and Treatment of Chronic Diseases. Molecules 20:21138–21156. https://doi.org/10.3390/molecules201219753 (2015). Burdette, A. et al. Anti-Inflammatory Activity of Select Sorghum (Sorghum bicolor) Brans. Journal of Medicinal Food 13:879–887. https://doi.org/10.1089/jmf.2009.0147 (2010). Wu, L. et al. Chemical Characterization of a Procyanidin-Rich Extract from Sorghum Bran and Its Effect on Oxidative Stress and Tumor Inhibition in Vivo. Journal of Agricultural and Food Chemistry 59:8609–8615. https://doi.org/10.1021/jf2015528 (2011). Nagy, R. et al . Assessment of Bioactive Profile of Sorghum Brans under the Effect of Growing Conditions and Nitrogen Fertilization. Agriculture 13:760–760. https://doi.org/10.3390/agriculture13040760 (2023) Dykes, L., Rooney, L. W. Sorghum and millet phenols and antioxidants. Journal of Cereal Science 44:236–251. https://doi.org/10.1016/j.jcs.2006.06.007 (2006). Addinsoft. XLSTAT statistical and data analysis solution. Long Island, NY. https://www.xlstat.com.2019 . Zhang, W. et al. Polyphenol Profile and In Vitro Antioxidant and Enzyme Inhibitory Activities of Different Solvent Extracts of Highland Barley Bran. Molecules 28:1665–1665. (2023) https://doi.org/10.3390/molecules28041665 Awika, J. M., Rooney, L. W., Waniska, R. D. Anthocyanins from black sorghum and their antioxidant properties. Food Chemistry 90:293–301. https://doi.org/10.1016/j.foodchem.2004.03.058 (2005). Tyagi, V., Saravanan, C., Wang, Y., Bhattacharya, B. Solvent Dependency of Sorghum Bran Phytochemicals Acting as Potential Antioxidants and Antibacterial Agents. Food Technology and Biotechnology 59: https://doi.org/10.17113/ftb.59.01.21.6878 (2021). Singleton, V. L., Orthofer, R., Lamuela-Raventós, R. M. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Oxidants and Antioxidants Part A 299:152–178. https://doi.org/10.1016/s0076-6879(99)99017-1 (1999). Zhang, Q. et al . A Simple 96-Well Microplate Method for Estimation of Total Polyphenol Content in Seaweeds. Journal of Applied Phycology 18:445–450. https://doi.org/10.1007/s10811-006-9048-4 (2006). Chang, C., Yang, M. H., Wen, H. M., Chern, J. C. Estimation of total flavonoid content in propolis by two complementary colometric methods. Journal of Food and Drug Analysis, 10(3):178–182 (2002) Lee, J., Robert, W. D., Ronald, E. W. Determination of Total Monomeric Anthocyanin Pigment Content of Fruit Juices, Beverages, Natural Colorants, and Wines by the pH Differential Method: Collaborative Study. Journal of AOAC International, 88(5) (2005) Makkar, H. P. S., Bluemmel, M., Borowy, N. K., Becker, K. Quantification of tannins in tree foliage: A laboratory manual for the FAO/IAEA coordinated research project on "Use of nuclear and related techniques to develop simple tannin assays for predicting and improving the safety and efficiency of feeding ruminants on tanniniferous tree foliage." International Atomic Energy Agency (IAEA), 26. (2000) Enkhtsetseg, E. et al. Screening Study on Antioxidant Activity of Plants Grown Wildly in Mongolia. Food science and technology research 20:891–897. https://doi.org/10.3136/fstr.20.891 (2014) Adedapo, A. A., Jimoh, F. O., Afolayan, A. J., Masika, P. J. Antioxidant properties of the methanol extracts of the leaves and stems of Celtis africana . Rec. Nat. Prod. 3:23–31. (2009). Additional Declarations No competing interests reported. Supplementary Files SIQualificationofflavonoidsofthreesorghumbranvarietiesbyuntargetedmetabolomicsdsmc.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4679263","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":331649842,"identity":"3defdbbe-9232-4a46-8d8a-8bb2a80be4ba","order_by":0,"name":"Mariely Cristine Dos Santos","email":"","orcid":"","institution":"Shinshu University","correspondingAuthor":false,"prefix":"","firstName":"Mariely","middleName":"Cristine Dos","lastName":"Santos","suffix":""},{"id":331649843,"identity":"8e72e7a8-1c51-4aa4-b608-3a9e85ac3ec4","order_by":1,"name":"Naoki Tanaka","email":"","orcid":"","institution":"Shinshu 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09:31:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4679263/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4679263/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61329576,"identity":"320cbd6a-4324-449b-ac5a-6a1babf8377f","added_by":"auto","created_at":"2024-07-29 14:38:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":440429,"visible":true,"origin":"","legend":"\u003cp\u003eGraphic plots of PCA with the two axes totalizing 70.26% of the variances, where\u003cstrong\u003e \u003c/strong\u003ethe plot on the left represents observations from principal component analysis, and the plot from the right represents the\u003cstrong\u003e \u003c/strong\u003ebiplot of active observations and variables.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4679263/v1/237eb8a65149a95c0f9d71ab.png"},{"id":61330425,"identity":"d03c3f8d-d1c2-40ac-9ecc-d59c110461ad","added_by":"auto","created_at":"2024-07-29 14:46:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":262879,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of flavonoids identified by untargeted metabolomics analysis.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4679263/v1/e6d52fcfc3cba1a29c56eb97.png"},{"id":61329574,"identity":"b1a65f23-7848-40fb-bdff-71a08173bf87","added_by":"auto","created_at":"2024-07-29 14:38:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":200406,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplot graph comparing the concentration of Malvidin and Chrysin in the samples.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4679263/v1/68d1f09ad9d66df982b7299f.png"},{"id":64922749,"identity":"ec6fece1-1bbe-446c-b1bc-937cf4a982cc","added_by":"auto","created_at":"2024-09-20 12:05:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1571557,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4679263/v1/46745f37-6c48-47ab-9b4d-77a4cb7f4bd0.pdf"},{"id":61329577,"identity":"997359c3-852e-4927-afe9-a28505b80c3d","added_by":"auto","created_at":"2024-07-29 14:38:27","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":4876582,"visible":true,"origin":"","legend":"","description":"","filename":"SIQualificationofflavonoidsofthreesorghumbranvarietiesbyuntargetedmetabolomicsdsmc.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4679263/v1/fdf9c385ef3a0a4c26e5de12.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Qualification of flavonoids of three sorghum bran varieties by untargeted metabolomics","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eSorghum (\u003cem\u003eSorghum bicolor L\u003c/em\u003e.) is a drought-tolerant, nutritious cereal crop highly produced worldwide. As the 5th most-produced cereal globally, it is popular in the bioenergy industry and as a feedstock material [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. According to their purpose, there are four main types of sorghum: sweet sorghum, grain sorghum, forage sorghum, and bioenergy sorghum [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Dietary-wise, although grain sorghum presents many nutritional traits, it is still overseen as a food product in many places, primarily because of unwanted sensorial characteristics some varieties can present. An example is the bitter taste of dark-colored sorghum varieties [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In this case, the bitter taste is caused by phenolic components, such as tannins, present in the pericarp of this grain, like most of the phenolic components from cereals [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The pericarp is one of the discarded parts during the process of decortication, and together with the aleurone layer and seed coat, it forms what is called the sorghum bran [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the last decades, studies have investigated the benefits of sorghum bran, previously considered industrial waste, to make the most of the essential bioactive components in this matrix. Among the beneficial components of sorghum bran, the previously mentioned phenolic compounds are gaining crescent attention [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Sorghum bran has colored varieties, and each color influences the profile of the grain's phenolic components [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The class of flavonoids is one of the protagonists for the colors of plants, and, as phenolic compounds, they are considered bioactive components due to their antioxidant, antibacterial, anti-inflammatory, and even anticarcinogenic activities, and plenty more health benefits [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith the challenges in unraveling the structure of sorghum and its bioactive components, researchers and farmers are in search of ideal hybrids for different environments and applications, which expands the variations in phenotypic and structural characteristics among the different sorghum [\u003cspan additionalcitationids=\"CR14 CR15 CR16 CR17\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Considering these fundamentals, this research aims to compare the differences in the phenolic content, focusing primarily on the qualitative analysis of the flavonoid profile of sorghum bran from three different varieties, including a newly developed variety.\u003c/p\u003e"},{"header":"2 Results","content":"\u003cp\u003eThese results comprehend the main phenotypic differences among the samples chosen. They are followed by the free flavonoids identified by untargeted metabolomics and their distinction according to the sorghum varieties and the growth environment, and finally, the quantification of the polyphenol content by colorimetric methods.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Comparison among sorghum samples used in this research\u003c/h2\u003e \u003cp\u003eThe phenotypic differences of the three sorghum varieties used for this research are described in this topic. First, white TDN\u0026reg; Sorgho is bigger than the colored sorghums (approximately 48 mm in height). On the other hand, RILN-156 is not only the smallest (around 35 mm in height) but also the roundest and has a more intense color among these three varieties. Mini Sorgho then stands in the middle regarding size (approximately 41 mm) and color. The varieties also present some distinctions regarding secondary color of the grains, presence of pigmented testa, and thickness of the pericarp. Contrary to colored sorghum, TDN\u0026reg; Sorgho does not present a pigmented testa. As for both the red sorghums, although the floury endosperm - the white part in the middle of the grain - was observed to take different sizes in each grain from the same variety, the color of the corny endosperm (secondary color) of both types of grain is slightly different. Mini Sorgho presents a darker secondary color, closer to a brownish purple, while RILN-156 gets similar to a whitish red. By observing many samples of the grain, the thickness of the pericarp was very close between the red sorghum varieties and thinner in the white sorghum.\u003c/p\u003e \u003cp\u003eThe general characteristics of the crops are that TDN\u0026reg; Sorgho can reach up to 2 meters, Mini Sorgho can reach up to 1.5 to 1.8 meters, and RILN-156 can reach 1.2 to 1.5 meters. The sowing period is the same for all three varieties: April to August in warm areas, May to August in intermediate areas, and May to July in cold areas. Sorghum usually takes 60 to 80 days to reach its maximum height.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Untargeted metabolomic analysis of sorghum bran\u003c/h2\u003e \u003cp\u003eBy untargeted metabolomics analysis using LC-MS and CE-MS, nineteen flavonoids were successfully identified (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e), where the flavonols corresponded to 11% of the flavonoids identified among the three sorghum bran varieties.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFlavonoids identified by LC/MS and CE/MS\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMass\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCompound Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavonoid Classification\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMolecular Formula\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e579.176\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNaringenin 7-neohesperidoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavanone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e447.092\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsoorientin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e221.050\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsofraxidin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCoumarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e11\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e347.105\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMalvidin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnthocyanidin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e431.095\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApigenin 8-glucoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e303.047\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTaxifolin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDihydroflavonol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e431.091\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApigenin 7-glucoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e283.060\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlycitein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIsoflavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e287.051\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEriodictyol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavanone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e255.068\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLiquiritigenin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavanone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e269.044\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGalangin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavonol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e291.084\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEpicatechin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e595.173\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaponarin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e15\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e465.105\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMyricetin 3-rhamnoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavonol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e431.130\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFormononetin 7-glucoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIsoflavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e255.061\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDaidzein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIsoflavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e255.061\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChrysin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e301.071\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChrysoeriol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e271.057\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBaicalein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWith the different profiles regarding the concentration of flavonoids, a Principal Component Analysis (PCA) could be carried out, separating the sorghums, and determining the principal components among the flavonoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Combining the two principal components, F1 and F2, this analysis could comprehend 70.26% of the total variance of the samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe can observe that three of the four Mini Sorgho samples have similar flavonoid profiles, indicated by their high positive scores on both principal components. Mini Sorgho (2) and one of the sorghum samples, RILN-156 (2), are characterized by distinct flavonoid profiles compared to the other samples analyzed. In a general manner, contrasting with all the TDN\u0026reg; Sorgho samples having similar flavonoid profiles with each other, the RILN-156 samples all presented variations among themselves, where RILN-156 (3) also has a distinct profile, RILN-156 (1) has an average profile that is not strongly influenced by either principal component and RILN-156 (4) has a flavonoid profile characterized by the highest positive scores on both principal components (together with sorghum Mini Sorgho (4)).\u003c/p\u003e \u003cp\u003eAs for the flavonoid influences in each sorghum, on the biplot with the variables, it can be observed that the TDN\u0026reg; Sorgho samples were strongly associated with Galangin, Malvidin, and Baicalein. Malvidin was the only anthocyanin that could be identified in this analysis. The flavonoid profile from sorghum mini (1), (3) and (4) and RILN-156 (4) is strongly influenced by Apigenin-7-glucoside, Naringenin-7-neohesperidoside, and Apigenin-8-glucoside. Mini Sorgho (2) is associated with flavonoids like Liquiritigenin, similar to RILN-156 (2), which Eriodictyol also influences. As for the last two RILN-156 samples, RILN-156 (1) is associated with Taxifolin but not strongly characterized by any other variables, while RILN-156 (3) is influenced by variables such as Chrysin, Formononetin-7-rhamnoside, and Epicatechin.\u003c/p\u003e \u003cp\u003eFurthermore, to better illustrate this, the heatmap below (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) shows that apart from the Mini Sorgho (2) sample, which presented a very different profile compared to the other sorghum samples, some groups of flavonoids are more abundant in red sorghum than on white sorghum. Groups present differences in concentration among varieties, and groups vary according to the specific sample. Environmental conditions influenced the latter during the plant's growth, an essential factor in the flavonoid profile of plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCorrespondingly, except for flavonols and anthocyanin, red sorghum varieties presented a more significant concentration of flavonoids overall. Besides flavonols, Mini Sorgho and RILN-156 presented a significant concentration of isoflavones and flavones as well. As mentioned, Taxifolin, representing the dihydroflavonols, was also outstanding in one of the RILN-156 samples. According to the concentrations observed by intensity values on untargeted metabolomics, Galangin had the highest concentration among all the flavonoids. In the present work, Galangin prevailed in white sorghum samples. The second most abundant flavonoid was Daidzein. This isoflavone, however, was almost absent in TDN\u0026reg; Sorgho bran, in contrast with the large amount present in the red sorghum samples.\u003c/p\u003e \u003cp\u003eNonetheless, the bran of TDN\u0026reg; Sorgho presented a higher concentration of anthocyanin than the colored sorghum, even though it is a white variety. Despite the variations within the white sorghum samples collected from separate places, they were still higher in anthocyanin content than both colored varieties. On the other hand, Chrysin was higher in colored samples and barely present in the white sorghum bran. Baicalein, Formononetin-7-glucoside and Eriodictyol also had significant concentrations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Validation of untargeted metabolomics data by colorimetric methods\u003c/h2\u003e \u003cp\u003eThis session will present the results of the investigation of free phenolic compounds soluble in methanol from the bran of the white sorghum sample (TDN\u0026reg; Sorgho) and the two red sorghum samples (RILN-156 and Mini Sorgho). In this sense, despite both RILN-156 and Mini Sorgho being red sorghum varieties, the color of the polyphenolic extract from RILN-156 is more intense, closer to brown, in contrast to the orangish tone from Mini extract.\u003c/p\u003e \u003cp\u003eWe can observe in the following table of all the results obtained that Sorghum RILN-156 bran presented interesting values compared to the other two samples. RILN-156 had higher total phenolic content and flavonoid content. Corroborating with the untargeted metabolomics previously presented results, the total anthocyanin content, represented by apigenin and luteolin in sorghum, was the highest in white sorghum. Nevertheless, as expected, tannin was not detected in white sorghum but similar in Mini Sorgho and RILN-156, higher in Mini Sorgho bran. Except for tannin content, the bran of Mini Sorgho had the lowest concentrations.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults obtained from the methanol extraction of TDN\u0026reg;, Mini and RILN-156 bran.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eAnalysis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTDN\u0026reg; Sorgho\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMini Sorgho\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRILN-156\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTotal phenolic content (\u0026micro;g.GAE/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55.568\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42.220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e91.840\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTotal flavonoid content (\u0026micro;g.QE/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e413.169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e185.067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1196.320\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTotal anthocyanin content\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApigenin (\u0026micro;g/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4290.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e104.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e335.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLuteolin (\u0026micro;g/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6231.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e151.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e486.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTotal tannin content (mg.CE/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN.D.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.722\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.149\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eDPPH scavenging assay (mM.TE/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e230\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e106.364\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e266.364\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3 Discussion","content":"\u003cp\u003eThe concentration of phenolic compounds can be influenced by the grain\u0026rsquo;s genotype, which in turn influences the pericarp color and thickness, as well as the presence of colored testa in the grains and the secondary plant color (phenotype) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The pericarp color comes from the R and Y genes, where when Y is homozygous recessive (rryy or R_yy), there is the presence of a white pericarp, and when R and Y are dominant, the pericarp comes as red (R_Y_). The gene Z influences the thickness, while the presence or absence of pigmented testa is due to B1 and B2 genes (if these are dominant, there is a pigmented testa) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the present samples, we can observe that although both red pericarp sorghums present a pigmented testa and similar thickness, the secondary color of the grain is different.\u003c/p\u003e \u003cp\u003eThe P and Q genes determine the secondary color: red, purple, or tan. Tan sorghums have recessive P genes (ppqq or ppQQ), while red (PPqq) and purple (PPQQ) sorghums have dominant ones. Sorghums presenting red or purple as secondary colors have been reported to have higher phenolic concentrations in comparison with tan-colored sorghum [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. By observing the appearance of the sorghum grains cut in half, we could observe that Mini Sorgho has a brownish purple secondary color, while RILN-156 has a whitish red. White sorghum has a tan secondary color.\u003c/p\u003e \u003cp\u003eAs mentioned in the \u003cspan refid=\"Sec2\" class=\"InternalRef\"\u003eresults\u003c/span\u003e section, under the untargeted metabolomics analysis, flavonols were the larger class of flavonoids identified within the three sorghum bran varieties. Flavonols, one of the most abundant classes of flavonoids, represented by quercetin, have been gaining attention in the food and pharmaceutical industries, especially now that the search for natural healthy compounds and functional foods is standing out. Structurally similar to flavones, flavonols are colorless compounds with an extra non-phenolic hydroxyl group at position 3 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. They have a wide range of roles in the plant defense system, including being responsible for the plant-microbe interaction and protection from UV rays and microbial attacks [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGalangin had the highest concentration among all the flavonoids, especially on TDN\u0026reg; Sorgho. Galangin, a 3,5,7-trihydroxyflavone, is usually found in honey and propolis and has been investigated to prevent diverse diseases and human conditions like aging and inflammation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite being abundant in the samples, Galangin has been reported not to be toxic up to 5 g/kg and not to cause any side effects [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. After ingestion, Galangin can be metabolized into kaempferol and quercetin, two important antioxidants [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Such traits enable the study of its antiproliferative ability against several types of cancer cells, like esophageal [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], leukemia [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and even skin cancer [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe second most abundant flavonoid was Daidzein. This isoflavone, however, was almost absent in TDN\u0026reg; Sorgho bran in contrast with large amounts present in the red sorghum samples. Primarily found in soybeans, Daidzein has many roles in the biotic and abiotic stress defense mechanism of plants, such as influencing the receptivity of symbiotic root infection, defense against oxidative stress, and so on [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBeing chemically similar to mammalian estrogens, Daidzein presents estrogenic properties that can be beneficial by hindering or substituting estrogen and estrogen receptor complex, protecting against diseases related to the control of estrogens, like breast cancer, diabetes, osteoporosis, and cardiovascular disease [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In a study with soybeans, it was noticed that the Daidzein content increased under waterlogging [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], which also shows that although Daidzein seems to be a flavonoid abundant in colored sorghums, it can also be influenced by conditions such as water stress.\u003c/p\u003e \u003cp\u003eIn contrast, the concentration of anthocyanin, represented by the anthocyanidin Malvidin, was higher on TDN\u0026reg; Sorgho bran than the colored sorghum bran despite being a white variety. Although, to our knowledge, it has not yet been reported in sorghum, it is not uncommon. Many factors can modify the color of anthocyanins, from genetic traits to environmental conditions. Despite the environmental conditions affecting the exact amount of Malvidin produced, since there were variations within the white sorghum samples collected from separate places, they still had higher anthocyanin content than both colored varieties.\u003c/p\u003e \u003cp\u003eOne hypothesis would be sorghum's high production of flavones, considered co-pigments. In some plants, flavones and anthocyanins interact inversely proportional ways: the more flavones produced, the fewer anthocyanins are present. Also, anthocyanins' color or intensity will change depending on the amount of hydrogen ions in flavones [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, we noticed that, in the present work, even when some of the samples of colored varieties presented a similar concentration of flavones as the white varieties, the anthocyanidin content did not increase.\u003c/p\u003e \u003cp\u003eNonetheless, we cannot rule out that a specific flavone could interact with Malvidin. For example, white sorghum bran samples had a smaller Chrysin content than their anthocyanidin concentration. In contrast, red sorghum brans presented a significant amount of the same flavone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Although further investigation would be necessary to confirm the interaction between these two metabolites, it is still interesting to notice this coincidental pattern.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eChrysin (5,7-dihydroxyflavone), besides the common properties of flavonoids, has also been studied regarding its antispasmodic and anxiolytic properties [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. On the other hand, the anthocyanidin Malvidin is extensively known for helping in the attribution of the color of red grapes and wine [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. As Malvidin has been thoroughly investigated, it has been reported to have anticarcinogenic, diabetes-control, cardiovascular-disease-prevention, and brain-function-improvement properties [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBaicalein, which was also slightly significant in the samples, comes from the chrysin biosynthetic pathway and has been investigated for its contribution to preventing cancer and diseases [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Baicalein has also recently been investigated for treating SARS-CoV-2 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In addition, Chrysoeriol, Taxifolin, and Eriodictyol were most present in RILN-156. That is a promising result for this newly developed inbred line.\u003c/p\u003e \u003cp\u003eAlthough in smaller concentrations, the other identified flavonoids have also been reported to contribute to health in vitro and in Vivo tests, as some of the benefits are cited in online resources.\u003c/p\u003e \u003cp\u003eOn the validation of the results, using colorimetric methods, RILN-156 bran presented the highest value of phenolic content (91.840 \u0026micro;g.GAE/g of bran), while Mini Sorgho presented 42.220 \u0026micro;g.GAE/g. Usually, white sorghum presents a lower concentration of phenolic compounds than colored sorghum [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], contrary to what happened in this work (TDN\u0026reg; Sorgho presented 91.840 \u0026micro;g.GAE/g). Despite these colorimetric analyses being used to validate the untargeted metabolomics data, the results were different regarding the red Mini Sorgho bran. Nonetheless, we can observe that both red sorghums presented a significant difference in the extraction of polyphenol content and flavonoids using methanol, 70%. In contrast, Mini Sorgho presented results that were lower than those of TDN\u0026reg; Sorgho. This could be due to the presence of a large concentration of flavonoids and polyphenols that do not have as much affinity with the solvent as the ones present in RILN-156 since the solvent polarity is important in the determination of which components will be extracted [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLikewise, in the determination of total flavonoid contents, RILN-156 once again had the highest concentration (1196.320 \u0026micro;g.QE/g), followed by TDN\u0026reg; Sorgho (413.169 \u0026micro;g.QE/g) and Mini Sorgho(185.067 \u0026micro;g.QE/g). The results obtained by RILN-156 corroborate previous studies that reported that colored sorghums present higher levels of secondary metabolites such as flavonoids [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs for anthocyanin content, luteolin and apigenin were taken as references. Surprisingly, among the free phenolic compounds quantified, TDN\u0026reg; Sorgho was the sample with the highest concentration of both luteolin (6231.9 \u0026micro;g/g) and apigenin (4290.5 \u0026micro;g/g). RILN-156 had a significantly lower concentration (486.7 \u0026micro;g/g and 335.34 \u0026micro;g/g), as well as Mini Sorgho (151.84 \u0026micro;g/g and 104.56 \u0026micro;g/g). This data endorses the one found in untargeted metabolomics, where Malvidin was more abundant on TDN\u0026reg; Sorgho than on colored sorghum bran.\u003c/p\u003e \u003cp\u003eWhile methanol is often employed for the extraction of anthocyanin and the addition of water could improve its yield due to similar polarity, other conditions, such as temperature and time, should be optimized for focusing on anthocyanin extraction [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], besides the possible interaction with flavones, that could also be one hypothesis to explain the differences in these results for red sorghum.\u003c/p\u003e \u003cp\u003eTotal condensed tannin was not detected in white sorghum but presented slight variation among the colored sorghum samples. Mini Sorgho bran (1.722 mg.CE/g), followed by RILN-156 (1.149 mg.CE/g), showed a substantial content of tannins. Despite acting as antioxidants, tannins are also considered anti-nutrients due to their possible inhibition of proteins and their influence on the digestibility of some amino acids [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSorghums that contain tannin are known to be resistant to birds and insects and provide a higher yield, which might influence farmers' choice depending on the application it will be destined for [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. For dietary purposes, it is a positive result that the newly developed sorghum obtained a lower tannin content. More tests in vitro would be necessary to imply that the amount of tannin present could contribute to the bioactivity of the grain while not being as abundant as to have a heavy influence on the absorption of the compounds of interest.\u003c/p\u003e \u003cp\u003eOn DPPH scavenging activity, the colored sorghum samples, Mini Sorgho, and RILN-156, had 106.364 mM.TE/g and 266.364 mM.TE/g respectively, while the white sorghum TDN\u0026reg; Sorgho had 230 mM.TE/g as a scavenging activity. A higher phenolic content means a higher antioxidant activity since phenolics, and flavonoid molecules have been shown to have a high correlation with the antioxidant activity of plant extracts [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Based on this, the DPPH scavenging activity values obtained from the current samples were as high as the total phenolic contents were.\u003c/p\u003e \u003cp\u003eFor the human body, a component with better antioxidant activity in a food product means a higher chance of reducing the risk of degenerative diseases, such as cancer. Naturally, many factors affect such diseases. However, studies have proven the mechanism of action of antioxidant compounds in heart and respiratory diseases, arthritis, inflammatory diseases, and even Parkinson\u0026rsquo;s disease [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In this sense, in vitro and in vivo tests with bioactive antioxidant components extracted from sorghum bran have been conducted for more than a decade now [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], indicating that sorghum bran, wildly colored varieties, could be a great addition to human diet depending on the bioavailability of such components.\u003c/p\u003e \u003cp\u003eAs previously reported in other works [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], we could observe that despite RILN-156 having a more significant amount of polyphenol, other factors considerably affect the polyphenolic profile of the grain. Nevertheless, it is essential to conduct bioavailability tests to ensure that those components can be metabolized by the human body during digestion, as around 80% of sorghum bioactive components are covalently bound to other cell wall compounds [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe recombinant inbred line RILN-156 had a greater antioxidant scavenging potential than the two other varieties analyzed, which corroborated its polyphenolic compounds level and showed a favorable potential for using this inbred line from now on. Nonetheless, depending on the intended use of the chosen grain, white sorghum is also advantageous because it does not present tannins, which consequently highlights the significance of such studies being conducted comparing different varieties.\u003c/p\u003e"},{"header":"4 Methods","content":"\u003cp\u003eDetailed information about development of the new variety, codification of samples, preparation and flow of experiments, and so-on can be found on supplementary online information.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Sorghum samples\u003c/h2\u003e \u003cp\u003eThree varieties of sorghum were used for this study. Two commercially available seeds are white sorghum TDN\u0026reg; Sorgho and red sorghum Mini Sorgho sold by KANEKO SEEDS CO., LTD. The third variety used was red sorghum RILN-156, produced by Shinshu University, Ina City, Nagano Prefecture, Japan.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Preparation of sorghum bran\u003c/h2\u003e \u003cp\u003e The seed was milled with a household rice milling machine (SM \u0026ndash; 500W, MK-SEIKO CO., LTD., Japan) using the program according to the amount of sample, which takes around 1 minute and 50 seconds to finish, to obtain the bran from each grain. These milling conditions were done three times for each sample. After that, the bran was collected and sieved with a mesh (opening: 250 \u0026micro;m, diameter: 160mm). The remaining larger bran fractions were triturated with a blender (Wonder Blender WB-1, Osaka Chemical) and further sieved with the same mesh mentioned above. The bran was stored in a sealed container until further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Untargeted metabolomic analysis of sorghum bran\u003c/h2\u003e \u003cp\u003e \u003cb\u003e4.3.1 Sample preparation for Liquid Chromatography coupled with Mass Spectrometer (LC/MS) and Capillary Electrophoresis coupled with Mass Spectrometer (CE/MS)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor the untargeted metabolomics analysis, samples grown in different cities of Nagano Prefecture and districts of Nagano City were collected: TDN\u0026reg; Sorgho from three locations (Iizuna Town, Naniai\u0026mdash;Nagano City, and Wakaho\u0026mdash;Nagano City), Mini Sorgho from four locations (Chikuma City, Shiojiri City, Suzaka City, and Nagano City), and RILN-156 from four locations (Suzaka City, Ueda City, Naniai\u0026mdash;Nagano City, and Shiozaki, Nagano City).\u003c/p\u003e \u003cp\u003eApproximately 100 mg of samples were transferred to sample disruptor tubes supplied by Yasui Kikai (Osaka, Japan) and shaken with iron cones cooled in liquid nitrogen. Each sample was analyzed twice for the four platforms: CE/MS and LC/MS in both positive and negative polarities. The average of the two data sets was used as the expression value. Quality control (QC), containing all samples, was prepared, and analyzed every six measurements to ensure measurement accuracy. The coefficient of variance (CV%) was calculated for each metabolite using the QCs, and any metabolites with a CV% exceeding 50% were excluded from the data table.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e4.3.2 CE/MS analysis and LC/MS analysis\u003c/h2\u003e \u003cp\u003eCE/MS experiments were conducted using an Agilent CE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany) coupled with an Agilent 6545 QTOF system (Agilent Technologies, Palo Alto, CA, USA). LC/MS analyses were conducted using an Agilent 1290 series UPLC system equipped with a 6545 quadrupole TOF system (Agilent Technologies, Palo Alto, CA, USA) controlled by MassHunter Workstation B.08.01 software. The analytical column employed was a CAPCELL PAK C18 IF, 2.0 mm ID \u0026times; 50 mm, 2 \u0026micro;m (Osaka Soda, Osaka, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e4.3.3. Data analysis of untargeted metabolomics\u003c/h2\u003e \u003cp\u003eThe raw data obtained from the mass spectrometer were converted to CSV format using MassHunter Export software (Agilent Technologies). The resulting CSV data included information on m/z (mass-to-charge ratio), retention time, and intensity. Principal component analysis (PCA) was performed using the extension XLSTAT 2019 from Microsoft Excel [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Validation of untargeted metabolomics data by colorimetric methods\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e4.4.1 Extraction of polyphenol from sorghum bran\u003c/h2\u003e \u003cp\u003eFor the extraction, 70% methanol was used, as described by Zhang et al. [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The procedure was carried out according to the protocol first described by Awika et al. [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] and later adapted by Tyagi et al. [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]: 2.5 g of sorghum bran were added to 20 mL of the solvent (methanol 70%) and shaken for 2 hours at 25 \u0026ordm;C in a 50 mL conical centrifuge tube. The mixture was then left at a -20 \u0026ordm;C freezer overnight for the dispersion of phenolics to occur. Subsequently, the samples were centrifuged at 7000 x g for 10 minutes, and the supernatant was collected. The precipitate was washed twice (10 mL) with methanol 70%, and the supernatant was collected, mixed, and stored at -20 \u0026ordm;C until further analysis. As the colorimetric analyses were conducted without solvent evaporation, the final concentration was corrected according to the dilution of the sample in methanol for the extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e4.4.2 Determination of total phenolic content\u003c/h2\u003e \u003cp\u003eThe total phenolic content (TPC) was determined following the Folin-Ciocalteu method [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The assay was conducted on a microplate adapted from Zhang et al. [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The results were expressed in mg of gallic acid equivalent (GAE mg/100g).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e4.4.3 Determination of total flavonoid content\u003c/h2\u003e \u003cp\u003eThe total flavonoid content (TFC) was determined using Chang et al.'s aluminum-chloride colorimetric method [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], adapted to microplates. The results were expressed in mg of quercetin equivalent (QE mg/100g).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e4.4.4 Determination of total anthocyanin content\u003c/h2\u003e \u003cp\u003eThe total anthocyanin content (TAC) was determined using the pH differential method [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Absorbance was read at 270 nm. Luteolin and apigenin were used as the standards [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The molar absorption coefficient for apigenin was calculated using the Beer-Lambert Law: 27.38 for apigenin and 19.98 for luteolin. Then the Beer-Lambert Law was rearranged to calculate the apigenin and luteolin content in the samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e4.4.5 Determination of total condensed tannin\u003c/h2\u003e \u003cp\u003eTotal condensed tannin (TCT) was determined by the butanol-hydrochloric acid method [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The standard curve was done with catechin; the results are expressed in mg of catechin equivalents (CE) per gram of sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e4.4.6 DPPH scavenging assay\u003c/h2\u003e \u003cp\u003eThe scavenging activity was determined by the DPPH assay, following Enkhtsetseg et al. [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u0026rsquo;s adaptation on Adedapo et al. [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Trolox was used for the calibration curve, so results were expressed in \u0026micro;mol Trolox equivalents (TE) per g of sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e4.4.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll analyses were conducted in triplicate, and the average and standard deviation were calculated. The analysis of variance (ANOVA) was conducted using the extension XLSTAT 2019 from Microsoft Excel [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Project for Enhancing the Environment to Create Innovation in Regional Core Universities, the Japan Society for Cabinet Office.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYoshihiko Amano, Naoki Tanaka and Masahiro Mizuno contributed to the study conception and design. Shigemitsu Kasuga has bred and provided a new sorghum variety. Material preparation, and analysis were performed by Mariely Cristine Dos Santos. Data collection by Metabolomics analysis were performed by Kazuhiro Tanabe and Chihiro Hayashi. The first draft of the manuscript was written by Mariely Cristine Dos Santos and all authors commended on previous versions of the manuscript. Yoshihiko Amano read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to express my gratitude for the helpful suggestions from Shinshu Sustainability Transformation Initiative, Shinshu University while conducting this research.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTanwar, R. \u003cem\u003eet al.\u003c/em\u003e Nutritional, phytochemical and functional potential of sorghum: A review. Food Chemistry Advances 3:100501. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.focha.2023.100501\u003c/span\u003e\u003cspan address=\"10.1016/j.focha.2023.100501\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHossain, M. S. \u003cem\u003eet al.\u003c/em\u003e Sorghum: A prospective crop for climatic vulnerability, food and nutritional security. Journal of Agriculture and Food Research 8:100300. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jafr.2022.100300\u003c/span\u003e\u003cspan address=\"10.1016/j.jafr.2022.100300\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWidowati, S., Luna, P. Nutritional and Functional Properties of Sorghum (Sorghum bicolor (L.) Moench)-based Products and Potential Valorisation of Sorghum Bran. IOP Conference Series: Earth and Environmental Science 1024:012031. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1088/1755-1315/1024/1/012031\u003c/span\u003e\u003cspan address=\"10.1088/1755-1315/1024/1/012031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalar, R. K., Purewal, S. S., Sandhu, K. S. Relationships between DNA damage protection activity, total phenolic content, condensed tannin content and antioxidant potential among Indian barley cultivars. Biocatalysis and Agricultural Biotechnology 11:201\u0026ndash;206. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bcab.2017.07.006\u003c/span\u003e\u003cspan address=\"10.1016/j.bcab.2017.07.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZuo-Yu, Z., Dahlberg, J. Sorghum Methods and Protocols. New York, Ny Springer New York (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJanet, A. A., Hema, K. Evaluation of nutritional and functional properties of anatomical parts of two sorghum (Sorghum bicolor) varieties. Heliyon 9:e17296\u0026ndash;e17296. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.heliyon.2023.e17296\u003c/span\u003e\u003cspan address=\"10.1016/j.heliyon.2023.e17296\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDevi, P. S., Kumar, M. S., Das, S. M. DNA Damage Protecting Activity and Free Radical Scavenging Activity of Anthocyanins from Red Sorghum (Sorghum bicolor) Bran. Biotechnology Research International 2012:1\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2012/258787\u003c/span\u003e\u003cspan address=\"10.1155/2012/258787\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerald, T. J., Gadgil, P., Tilley, M. High-throughput micro plate assays for screening flavonoid content and DPPH-scavenging activity in sorghum bran and flour. Journal of the Science of Food and Agriculture 92:2326\u0026ndash;2331. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jsfa.5633\u003c/span\u003e\u003cspan address=\"10.1002/jsfa.5633\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShim, T. J. \u003cem\u003eet al.\u003c/em\u003e Toxicological Evaluation and Anti-Inflammatory Activity of a Golden Gelatinous Sorghum Bran Extract. Bioscience, Biotechnology, and Biochemistry 77:697\u0026ndash;705. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1271/bbb.120731\u003c/span\u003e\u003cspan address=\"10.1271/bbb.120731\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu, J., Wang, W., Zhao, Y. Phenolic Compounds in Whole Grain Sorghum and Their Health Benefits. Foods 10:1921. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/foods10081921\u003c/span\u003e\u003cspan address=\"10.3390/foods10081921\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumari, P. K., Umakanth, A. V., Narsaiah, T. B., Uma, A. Exploring anthocyanins, antioxidant capacity and α-glucosidase inhibition in bran and flour extracts of selected sorghum genotypes. Food Bioscience 41:100979. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fbio.2021.100979\u003c/span\u003e\u003cspan address=\"10.1016/j.fbio.2021.100979\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoy, A. \u003cem\u003eet al\u003c/em\u003e. Flavonoids a Bioactive Compound from Medicinal Plants and Its Therapeutic Applications. BioMed Research International 2022:1\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2022/5445291\u003c/span\u003e\u003cspan address=\"10.1155/2022/5445291\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilva, T. C. da \u003cem\u003eet al.\u003c/em\u003e Agronomic divergence of sorghum hybrids for silage yield in the semiarid region of Paraiba. Revista Brasileira de Zootecnia 40:1886\u0026ndash;1893. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/s1516-35982011000900007\u003c/span\u003e\u003cspan address=\"10.1590/s1516-35982011000900007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeixeira, A. de M. \u003cem\u003eet al\u003c/em\u003e. Intake and digestibility of sorghum (Sorghum bicolor, L. Moench) silages with different tannin contents in sheep. Revista Brasileira de Zootecnia 43:14\u0026ndash;19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/s1516-35982014000100003\u003c/span\u003e\u003cspan address=\"10.1590/s1516-35982014000100003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePontieri, P. \u003cem\u003eet al.\u003c/em\u003e Mineral content in grains of seven food-grade sorghum hybrids grown under Mediterranean Environment. Australian journal of crop science 8 (11): 1550\u0026ndash;1559 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMsongaleli, B. M., Tumbo, S. D., Kihupi, N. I., Rwehumbiza, F. B. Performance of Sorghum Varieties under Variable Rainfall in Central Tanzania. International Scholarly Research Notices 2017:1\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2017/2506946\u003c/span\u003e\u003cspan address=\"10.1155/2017/2506946\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuntin, G. D., Ni, X., Harris-Shultz, K. R., Knoll, J. E., Toews, M. D., Dunn, D. Grain, silage and forage sorghum hybrid resistance to insect and bird damage. In: Mailthot, DJ, Dunn, DH. Jordan, Jr, Day JL (eds), Soybean, Sorghum Grain and Silage, and Summer Annual Forages, Georgia Agricultural Experiment Stations, Annual Publications. 103-9:74\u0026ndash;80. (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmale, M. \u003cem\u003eet al.\u003c/em\u003e Farm family effects of adopting improved and hybrid sorghum seed in the Sudan Savanna of West Africa. Food Policy 74:162\u0026ndash;171. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodpol.2018.01.001\u003c/span\u003e\u003cspan address=\"10.1016/j.foodpol.2018.01.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDykes, L., Seitz, L. M., Rooney, W. L., Rooney, L. W. Flavonoid composition of red sorghum genotypes. Food Chemistry 116:313\u0026ndash;317. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2009.02.052\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2009.02.052\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRooney, L. W., Miller, F. R. Variation in the Structure and Kernel Characteristics of Sorghum. Proceedings of the International Symposium on Sorghum Grain Quality, International Crop Research Institute for the Semi-Arid Tropics, Hyderabad, 28\u0026ndash;31, 11\u0026ndash;13. (1982).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDykes, L., Rooney, L. W., Waniska, R. D., Rooney, W. L. Phenolic Compounds and Antioxidant Activity of Sorghum Grains of Varying Genotypes. Journal of Agricultural and Food Chemistry 53:6813\u0026ndash;6818. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf050419e\u003c/span\u003e\u003cspan address=\"10.1021/jf050419e\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJong, M. K., Liu, L., Guo, W., Mohsen, M. Chemical structure of flavonols in relation to modulation of angiogenesis and immune-endothelial cell adhesion. 17:165\u0026ndash;176. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jnutbio.2005.06.006\u003c/span\u003e\u003cspan address=\"10.1016/j.jnutbio.2005.06.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e28. Aar R. M. \u003cem\u003eet al.\u003c/em\u003e Natural flavonols: actions, mechanisms, and potential therapeutic utility for various diseases. Beni Suef Univ J Basic Appl Sci. 12(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s43088-023-00387-4\u003c/span\u003e\u003cspan address=\"10.1186/s43088-023-00387-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMak, K. K. \u003cem\u003eet al\u003c/em\u003e. Galangin\u0026rsquo;s potential as a functional food ingredient. Journal of Functional Foods 46:490\u0026ndash;503. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jff.2018.04.054\u003c/span\u003e\u003cspan address=\"10.1016/j.jff.2018.04.054\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuh, J. \u003cem\u003eet al\u003c/em\u003e. The natural flavonoid galangin inhibits osteoclastic bone destruction and osteoclastogenesis by suppressing NF-κB in collagen-induced arthritis and bone marrow-derived macrophages. European Journal of Pharmacology 698:57\u0026ndash;66. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ejphar.2012.08.013\u003c/span\u003e\u003cspan address=\"10.1016/j.ejphar.2012.08.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerve, B., Basaran, A., Nurşen, B. Galangin as a Plant Phenolic and Usage in Health and Disease. 433\u0026ndash;438. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/b978-0-12-813008-7.00034-5\u003c/span\u003e\u003cspan address=\"10.1016/b978-0-12-813008-7.00034-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, C. \u003cem\u003eet al\u003c/em\u003e. Galangin Enhances Anticancer Efficacy of 5-Fluorouracil in Esophageal Cancer Cells and Xenografts Through NLR Family Pyrin Domain Containing 3 (NLRP3) Downregulation. Medical Science Monitor 27: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.12659/msm.931630\u003c/span\u003e\u003cspan address=\"10.12659/msm.931630\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTolomeo, M. \u003cem\u003eet al\u003c/em\u003e. Galangin increases the cytotoxic activity of imatinib mesylate in imatinib-sensitive and imatinib-resistant Bcr-Abl expressing leukemia cells. Cancer Letters 265:289\u0026ndash;297. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.canlet.2008.02.025\u003c/span\u003e\u003cspan address=\"10.1016/j.canlet.2008.02.025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenguedouar, L. \u003cem\u003eet al\u003c/em\u003e. Ethanolic Extract of Algerian Propolis and Galangin Decreased Murine Melanoma T. Anti-Cancer Agents in Medicinal Chemistry 16:1172\u0026ndash;1183. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2174/1871520616666160211124459\u003c/span\u003e\u003cspan address=\"10.2174/1871520616666160211124459\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, I. S. Current perspectives on the beneficial effects of soybean isoflavones and their metabolites on plants. Food Science and Biotechnology 31:515\u0026ndash;526. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10068-022-01070-7\u003c/span\u003e\u003cspan address=\"10.1007/s10068-022-01070-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlshehri, M. M. \u003cem\u003eet al.\u003c/em\u003e Therapeutic Potential of Isoflavones with an Emphasis on Daidzein. Oxidative medicine and cellular longevity 2021:6331630. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2021/6331630\u003c/span\u003e\u003cspan address=\"10.1155/2021/6331630\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong, H. G., Lee S., Ki, Y. J., Hyen, C. C. Assessment of In-Season N Effects on Soil Water Stress to Growth and Yields of Soybean. Han\u0026rsquo;guk Toyang Biryo Hakhoe Chi 56:463\u0026ndash;477. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7745/kjssf.2023.56.4.463\u003c/span\u003e\u003cspan address=\"10.7745/kjssf.2023.56.4.463\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobinson, G. M., Robinson, R. A survey of anthocyanins. I. Biochem J. 25(5):1687\u0026ndash;1705 (1931).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShukla, R., Pandey, V., Vadnere, G. P., Lodhi, S. Role of Flavonoids in Management of Inflammatory Disorders. 293\u0026ndash;322. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/b978-0-12-813820-5.00018-0\u003c/span\u003e\u003cspan address=\"10.1016/b978-0-12-813820-5.00018-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreno, J., Peinado, R. Polyphenols. In: Enological Chemistry. Elsevier eBooks, Academic Press, pp. 53\u0026ndash;76 (2012)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerecz-Sadowska, A. \u003cem\u003eet al\u003c/em\u003e. Food Anthocyanins: Malvidin and Its Glycosides as Promising Antioxidant and Anti-Inflammatory Agents with Potential Health Benefits. Nutrients 15:3016. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/nu15133016\u003c/span\u003e\u003cspan address=\"10.3390/nu15133016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArshad, H. R., \u003cem\u003eet al\u003c/em\u003e. The Multifaceted Role of Baicalein in Cancer Management through Modulation of Cell Signalling Pathways. Molecules (Basel Online) 27:8023\u0026ndash;8023. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules27228023\u003c/span\u003e\u003cspan address=\"10.3390/molecules27228023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDinda, B., Dinda, M., Dinda, S., Utpal, C. D. An overview of anti-SARS-CoV-2 and anti-inflammatory potential of baicalein and its metabolite baicalin: Insights into molecular mechanisms. European journal of medicinal chemistry 258:115629\u0026ndash;115629. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ejmech.2023.115629\u003c/span\u003e\u003cspan address=\"10.1016/j.ejmech.2023.115629\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodr\u0026iacute;guez De Luna, S. L., Ram\u0026iacute;rez-Garza, R. E., Serna Sald\u0026iacute;var, S. O. Environmentally Friendly Methods for Flavonoid Extraction from Plant Material: Impact of Their Operating Conditions on Yield and Antioxidant Properties. The Scientific World Journal 2020:1\u0026ndash;38. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2020/6792069\u003c/span\u003e\u003cspan address=\"10.1155/2020/6792069\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAjay, P. R. \u003cem\u003eet al\u003c/em\u003e. Large-Scale Non-Targeted Metabolomics Reveals Antioxidant, Nutraceutical and Therapeutic Potentials of Sorghum. Antioxidants (Basel) 10:1511\u0026ndash;1511. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/antiox10101511\u003c/span\u003e\u003cspan address=\"10.3390/antiox10101511\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTena, N., Asuero, A. G. Up-To-Date Analysis of the Extraction Methods for Anthocyanins: Principles of the Techniques, Optimization, Technical Progress, and Industrial Application. Antioxidants 11:286. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/antiox11020286\u003c/span\u003e\u003cspan address=\"10.3390/antiox11020286\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsropi, N. E., Novita, D. D. Physical Quality and Tannin Content of Sorghum (Sorghum bicolor L.) at Different Temperature and Soaking Immersion. IOP Conference Series: Earth and Environmental Science 1024:012068. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1088/1755-1315/1024/1/012068\u003c/span\u003e\u003cspan address=\"10.1088/1755-1315/1024/1/012068\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLubadde, G., Ebiyau, J., Aru, C., Andiku, J., Wandulu, M., Ugen, M. Sorghum production handbook for Uganda. Uganda: Resources Research Institute of the National Agricultural Research Organisation (NaSARRI-NARO) (2019)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAryal, S. \u003cem\u003eet al\u003c/em\u003e. Total Phenolic Content, Flavonoid Content and Antioxidant Potential of Wild Vegetables from Western Nepal. Plants 8:96. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants8040096\u003c/span\u003e\u003cspan address=\"10.3390/plants8040096\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y. J. \u003cem\u003eet al.\u003c/em\u003e Antioxidant Phytochemicals for the Prevention and Treatment of Chronic Diseases. Molecules 20:21138\u0026ndash;21156. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules201219753\u003c/span\u003e\u003cspan address=\"10.3390/molecules201219753\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurdette, A. \u003cem\u003eet al.\u003c/em\u003e Anti-Inflammatory Activity of Select Sorghum (Sorghum bicolor) Brans. Journal of Medicinal Food 13:879\u0026ndash;887. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1089/jmf.2009.0147\u003c/span\u003e\u003cspan address=\"10.1089/jmf.2009.0147\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, L. \u003cem\u003eet al.\u003c/em\u003e Chemical Characterization of a Procyanidin-Rich Extract from Sorghum Bran and Its Effect on Oxidative Stress and Tumor Inhibition in Vivo. Journal of Agricultural and Food Chemistry 59:8609\u0026ndash;8615. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf2015528\u003c/span\u003e\u003cspan address=\"10.1021/jf2015528\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagy, R. \u003cem\u003eet al\u003c/em\u003e. Assessment of Bioactive Profile of Sorghum Brans under the Effect of Growing Conditions and Nitrogen Fertilization. Agriculture 13:760\u0026ndash;760. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/agriculture13040760\u003c/span\u003e\u003cspan address=\"10.3390/agriculture13040760\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDykes, L., Rooney, L. W. Sorghum and millet phenols and antioxidants. Journal of Cereal Science 44:236\u0026ndash;251. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jcs.2006.06.007\u003c/span\u003e\u003cspan address=\"10.1016/j.jcs.2006.06.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAddinsoft. XLSTAT statistical and data analysis solution. Long Island, NY. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.xlstat.com.2019\u003c/span\u003e\u003cspan address=\"https://www.xlstat.com.2019\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, W. \u003cem\u003eet al.\u003c/em\u003e Polyphenol Profile and In Vitro Antioxidant and Enzyme Inhibitory Activities of Different Solvent Extracts of Highland Barley Bran. Molecules 28:1665\u0026ndash;1665. (2023) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules28041665\u003c/span\u003e\u003cspan address=\"10.3390/molecules28041665\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAwika, J. M., Rooney, L. W., Waniska, R. D. Anthocyanins from black sorghum and their antioxidant properties. Food Chemistry 90:293\u0026ndash;301. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2004.03.058\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2004.03.058\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTyagi, V., Saravanan, C., Wang, Y., Bhattacharya, B. Solvent Dependency of Sorghum Bran Phytochemicals Acting as Potential Antioxidants and Antibacterial Agents. Food Technology and Biotechnology 59: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.17113/ftb.59.01.21.6878\u003c/span\u003e\u003cspan address=\"10.17113/ftb.59.01.21.6878\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingleton, V. L., Orthofer, R., Lamuela-Ravent\u0026oacute;s, R. M. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Oxidants and Antioxidants Part A 299:152\u0026ndash;178. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0076-6879(99)99017-1\u003c/span\u003e\u003cspan address=\"10.1016/s0076-6879(99)99017-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Q. \u003cem\u003eet al\u003c/em\u003e. A Simple 96-Well Microplate Method for Estimation of Total Polyphenol Content in Seaweeds. Journal of Applied Phycology 18:445\u0026ndash;450. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10811-006-9048-4\u003c/span\u003e\u003cspan address=\"10.1007/s10811-006-9048-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang, C., Yang, M. H., Wen, H. M., Chern, J. C. Estimation of total flavonoid content in propolis by two complementary colometric methods. Journal of Food and Drug Analysis, 10(3):178\u0026ndash;182 (2002)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, J., Robert, W. D., Ronald, E. W. Determination of Total Monomeric Anthocyanin Pigment Content of Fruit Juices, Beverages, Natural Colorants, and Wines by the pH Differential Method: Collaborative Study. Journal of AOAC International, 88(5) (2005)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMakkar, H. P. S., Bluemmel, M., Borowy, N. K., Becker, K. Quantification of tannins in tree foliage: A laboratory manual for the FAO/IAEA coordinated research project on \"Use of nuclear and related techniques to develop simple tannin assays for predicting and improving the safety and efficiency of feeding ruminants on tanniniferous tree foliage.\" International Atomic Energy Agency (IAEA), 26. (2000)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEnkhtsetseg, E. \u003cem\u003eet al.\u003c/em\u003e Screening Study on Antioxidant Activity of Plants Grown Wildly in Mongolia. Food science and technology research 20:891\u0026ndash;897. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3136/fstr.20.891\u003c/span\u003e\u003cspan address=\"10.3136/fstr.20.891\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdedapo, A. A., Jimoh, F. O., Afolayan, A. J., Masika, P. J. Antioxidant properties of the methanol extracts of the leaves and stems of \u003cem\u003eCeltis africana\u003c/em\u003e. Rec. Nat. Prod. 3:23\u0026ndash;31. (2009).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Sorghum bicolor, metabolomics, polyphenol, flavonoid, DPPH assay","lastPublishedDoi":"10.21203/rs.3.rs-4679263/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4679263/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eSorghum bicolor\u003c/em\u003e is a source of many bioactive components, such as polyphenols. Those components are present especially in its bran, which is often removed in industrial processes through decortication. In that sense, this work aimed to analyze the polyphenol content, especially free flavonoids, from the bran of a newly developed variety compared to other commercially available varieties. The samples were white sorghum TDN\u0026reg; Sorgho, red sorghum Mini Sorgho, and the newly developed red sorghum RILN-156. First, the decortication was done to obtain the bran samples and those were triturated and then sieved. An untargeted metabolomics analysis (with LC/MS and CE/MS) was done to analyze the different components and identify the free flavonoids. For the general quantification analysis, instead of quantifying by target analysis, colorimetric methods were used to validate the metabolomics analysis. For this, the polyphenol content was extracted with 70% methanol. The antioxidant potential was also investigated using a DPPH assay. The results have shown that the flavonoid content was significant in these samples, especially in the newly developed RILN-156, with 19 flavonoids identified. RILN-156 also presented higher antioxidant capacity than the conventional varieties, a promising finding for its use to prevent chronic diseases, which will be further investigated.\u003c/p\u003e","manuscriptTitle":"Qualification of flavonoids of three sorghum bran varieties by untargeted metabolomics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 14:38:22","doi":"10.21203/rs.3.rs-4679263/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e31e67f6-7ed8-48e7-acd9-59be47ed9c91","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35113644,"name":"Biological sciences/Chemical biology/Metabolomics"},{"id":35113645,"name":"Biological sciences/Biochemistry/Metabolomics"},{"id":35113646,"name":"Health sciences/Health care/Nutrition"}],"tags":[],"updatedAt":"2024-09-20T11:57:06+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-29 14:38:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4679263","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4679263","identity":"rs-4679263","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}