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.