RETENTION OF SOME FLAVONES AND FLAVANONES IN FLOUR , GRAIN AND BRAN OF SORGHUM DURING STORAGE

Sorghum is associated with several functional benefits because it contains high levels of phenolic compounds, such as the flavonoids, flavones and flavanones. However, these compounds are sensitive to the period and conditions of storage. Thus, this work aimed to evaluate luteolin, apigenin, naringenin and eriodictyol retention in the sorghum genotypes SC319 (flour and grain) and TX430 (flour and bran) stored for 180 days at 4, 25 and 40 °C. These compounds were quantified by a high-performance liquid chromatograph equipped with a diode array detector. There was effect of the storage time, which favored the flavone and flavanone reduction in the evaluated genotypes. The storage temperature influenced the flavones more than the flavanone contents. The retentions ranged from 70.27 to 88.72% for luteolin, 69.04 to 99.15% for apigenin, 56.92 to 88.51% for naringenin and from 77.10 to 93.42% for eriodictyol at the three temperatures at the end of 180 days. Although flavones and flavanones were better preserved at 4 °C (about 88% of retention), at room temperature (25 °C) flavones and flavanones retentions were at least 77% and 85% after 180 days.

In relation to flavanones, research has demonstrated the antioxidant properties (Khan & Dangles, 2014).Sorghum (Sorghum bicolor (L) Moech) is a cereal that stands out due to its nutritional and functional benefits.This grain provides mainly carbohydrates, proteins, fibers, vitamins (especially B complex) (Queiroz et al., 2015) and minerals (magnesium and zinc) (Paiva et al., 2017).In addition, it contains phenolic compounds relevant to human health, such as phenolic acids, condensed tannins, flavones, flavanones and anthocyanins (Dykes & Rooney, 2006).According to in vitro and in vivo preliminary studies, these sorghum compounds are related to the prevention of chronic diseases, including diabetes, cancer, hypertension, obesity and cardiovascular disease (Awika & Rooney, 2004;Cardoso et al., 2014;Moraes et al., 2012).
However, flavonoids are sensitive to the physicochemical environment (Ioannou et al., 2012).
Thus, the period and conditions of storage may influence their contents.Therefore, understanding the effects of storage period and temperature on the levels of sorghum phenolic compounds are important to preserve their properties during shelf life.Besides, information has not been found in the literature regarding the stability and/or behavior of the flavones and flavanones during storage.Thus, the objective of this study was to evaluate the flavone and flavanone retention in flour, grain and bran of sorghum during storage at different temperatures.

Sorghum samples
Two sorghum genotypes were used: the genotype SC319 (grain and flour), with brown pericarp and pigmented testa (Cardoso et al., 2015) and the genotype TX430 (bran and flour), with black pericarp and without pigmented testa (Dykes et al., 2009).
The genotype SC319 was selected among 100 genotypes of a panel with high genetic variability, due to its high total phenolic contents (unpublished data).This genotype was grown in experimental fields of Embrapa Milho e Sorgo, in Sete Lagoas, Minas Gerais, in 2013.After harvesting, the grains were threshed and stored at -18 °C until use.The whole sorghum grains were ground twice in a Hawos mill before storage.
The genotype TX430 was supplied by CQL-Cereal Quality Lab., from Texas A&M University, College Station, TX, USA and was selected due to its high flavonoid content.This sorghum genotype was grown in College Station, TX, in 2013.After harvesting, the grains were milled using a UDY cyclone mill (Model 3010-030, UDY Corporation, Fort Collins, CO).
After storage, all samples were ground in a cyclone mill (Marconi, Piracicaba, São Paulo) to obtain a particle size of 0.5 mm for use in the chemical analysis.
Sorghum genotypes SC319 (grains and (2012) and modified by Cardoso et al. (2014).For extraction, 20 mL of 1% hydrochloric acid in methanol were added to 1 g of sample and stirred for 2 h at 180 rpm.The suspension was then centrifuged (FANEM Excelsa Baby II centrifuge) at 2790 g for 5 min and the supernatant collected and its volume made up to 20 ml with acidified methanol.Subsequently, the extract was placed in an amber bottle and stored at -18 ± 1 °C until analysis (Dykes et al., 2009).The flavones and flavanones were measured at 340 nm and 280 nm, respectively (Cardoso et al., 2014).

Results and Discussion
There was no difference (p>0.05) in flavone content between flour and grain of the SC319 genotype.However, the difference was significant (p≤0.05) between flour and bran of TX430 genotype.
The sorghum bran of the TX430 genotype showed approximately 6 times more luteolin and about 3 times more apigenin than the flour (Figure 1 and Figure 2).
These results are in accordance with other studies that demonstrated that flavonoids, as other phenolic compounds, are located mainly in the pericarp of the grain (Awika et al., 2005;Dykes & Rooney, 2006;Dykes et al., 2009Dykes et al., , 2011;;Moraes et al., 2015).Therefore, storage under 4 °C was more efficient to preserve the luteolin and apigenin contents from grain, flour and bran of sorghum.However, with few exceptions, there was no significant difference in flavone levels between sorghum samples stored under 25 and 40 °C (Fig. 1 and Fig. 2).
There was no difference (p>0.05) in the flavanone contents between flour and grain of the SC319 genotype.However, the difference was significant between flour and bran of the TX430 genotype.The TX430 bran had flavanone concentrations about 5 to 6 times higher than the flour (Fig. 3 and Fig. 4).
There was no significant (p>0.05)effect of temperature and storage period on the naringenin content on the sorghum flour and grain of the SC319 genotype neither on the flour of TX430 genotype, i.e., the level of this compound remained stable during storage for 180 days (Fig. 3 and Fig. 4).In TX430 bran, at each temperature the naringenin content remained stable until the 120 th day (Figure 4).These results indicate that naringenin has a high stability even when stored at 40 °C.The curves of flavanones degradation in sorghum grain, flour and bran had shown the polynomial function at 4 °C, 25 °C and 40 °C, in the same way of flavones.This was similar to behavior of other sorghum flavonoids (Oliveira et al, 2017).
No studies were found in the literature regarding flavanone losses in cereals during storage.The changes of flavanone levels in fruits during storage seem to be different from those observed in sorghum.
Naringenin levels of orange juice increased when stored during 180 days at 4 °C after previous pulsed electric field (PEF) treatments (Agcam et al., 2014).
On the other hand, in minimally processed orange the naringenin concentration did not change during 12 days of cold storage at 4 °C (Plaza et al., 2011).
The effects of temperature, storage time and temperatures x times interaction on eriodictyol content of SC319 genotype (flour and grain) and TX430 bran (Fig. 3 and Fig. 4) were significant.The level of eridioctyol in the flour of TX430 was not influenced by any of these factors during storage (Fig. 4).
For both genotypes the eridioctyol content was similar at 4 and 25 °C, but reduced considerably at 40 °C.At 4 ° C, the level remained stable from the initial to the end of storage, except in the TX430 bran that presented reduced content at the 180 th day of storage.The losses of eridioctyol at 25 °C occurred from the 60 th to 120 th day and from the beginning of    storage to the 60 th day at 40°C.After this period, the content remained stable until the end of the storage (180 days).
Therefore, storage under 4 °C was efficient to preserve the naringenin and eridioctyol contents of sorghum grain and flour of the SC 319 and flour of the TX430.However, for the sorghum bran, there was a decrease in their contents after 120 days, even at 4 °C.Overall, there was no significant difference in flavanone levels between sorghum samples stored at 25 °C and 4 °C.
The sum of flavanones during storage presented behavior similar to that of eriodictyol, maybe because the eriodictyol content was higher than the naringenin content.
Although there were losses in the flavone and flavanone contents, the retention percentages of these compounds were high at the end of storage, as shown in the Table 1.
For each flavonoid, means followed by different lower case letter (among storage temperatures) and by different upper case letter (among types of product) differ significantly by the Tukey test (p≤0.05).
There was no significant difference (p>0.05) in the flavone and flavanone levels between flour and grain of the genotype SC319 and flour and bran of the genotype TX430 (except in the apigenin content).
There was an effect of temperature on the retention rates of all analyzed compounds.(flour and grain: 83.47 and 82.96%, respectively) and in TX430 (flour and bran: 87.90 and 88.72%, respectively).Then, naringenin in SC319 (flour and grain: 70.00 and 64.62%, respectively) and in TX430 (flour and bran: 85.71 and 87.95%, respectively).
There was no significant difference in the retention (%) at 25 and 40 °C, except in the case of eridioctyol for TX430, which was lower at 40 °C.
These results showed that the storage of sorghum flour, grain and bran for 180 days under refrigeration (4 °C) is better to preserve the flavone and flavanone contents than room temperature (25 °C) or higher temperature (40 °C).

Conclusions
The flavone and flavanone contents reduced flour) and TX430 (flour and bran) were placed in individual polypropylene packages with 10 g capacity.Subsequently, the packages were placed in paper bags to protect from light and stored in three BOD Refrigerated Incubators (SOLAB 200/334) for a period of 180 days, at three temperatures (4 °C ± 2 °C, 25 °C ± 2 °C and 40 °C ± 2 °C.Chemical analyses were performed at 0 (zero, T0), 60 (T60), 120 (T120), and 180 (T180) days of storage.Sorghum grains (from the genotype SC319) remained intact during the storage period and were ground before the analytical procedures.The levels of flavones (luteolin and apigenin) and flavanones (naringenin and eriodictyol) were analyzed simultaneously in the sorghum samples, according to the method proposed by Yang et al.

Flavone
and flavanone were identified by comparing of the retention times of the peaks in the sample in relation to standards and confirmed by the addition of the suspect compound to the sample, in duplicate.The R2 of the analytical curves ranged from 0.9939 to 0.9992.The flavones and flavanones concentrations were expressed in µg/g on dry weight basis.The apparent retention of the flavones and flavanones, at the end of storage (180 days), was calculated according to the equation: A completely randomized design in a 2x3x4 factorial was used for flavones and flavanones (2 types of products x 3 temperatures x 4 storage periods).The genotypes were analyzed separately.The data were submitted to analysis of variance (ANOVA) and the means were compared by the Tukey test at 5% of probability, using SISVAR Software, version 5.6 (UFLA, Lavras, MG).

Figure 1 .
Figure 1.Flavones contents on sorghum genotype SC319 (flour and grain) stored for 180 days under three temperatures (4, 25 and 40 °C).Results are expressed in µg/ g of sample on dry weight basis.Means of three replicates.Means with different lowercase letters among the temperatures and uppercase letters among the times are different by Tukey test (p>0.05).

Figure 1 .
Figure 1.Flavones contents on sorghum genotype SC319 (fl our and grain) stored for 180 days under three temperatures (4, 25 and 40 °C).Results are expressed in µg/ g of sample on dry weight basis.Means of three replicates.Means with different lowercase letters among the temperatures and uppercase letters among the times are different by Tukey test (p>0.05).

Figure 2 .
Figure 2. Flavones contents on sorghum genotype TX430 (fl our and bran) stored for 180 days under three temperatures (4, 25 and 40 °C).Results are expressed in µg/ g of sample on dry weight basis.Means of three replicates.Means with different lowercase letters among the temperatures and uppercase letters among the times are different by Tukey test (p<0.05).

Figure 2 .
Figure 2. Flavones contents on sorghum genotype TX430 (flour and bran) stored for 180 days under three temperatures (4, 25 and 40 °C).Results are expressed in µg/ g of sample on dry weight basis.Means of three replicates.Means with different lowercase letters among the temperatures and uppercase letters among the times are different by Tukey test (p<0.05).

( 3 -
deoxyanthocyanins) during storage under 4, 25 and 40 °C.Sorghum has lower water activity compared to fruits and vegetables, and furthermore, the structure of the grain is able to protect the cells from chilling injury, what could explain the greater stability of flavones during storage under 4 °C.At 40 °C, there was a decrease in the total flavones in the sorghum grain and flour of the SC319 genotype from the initial to the 60 th day of storage and then the contents remained stable until the 120 th or 180 th day (Figure1).When comparing the flavone losses at 4 and 25 °C in the flour and grain of this genotype luteolin showed a tendency to be more sensitive to high temperature than apigenin.Cardoso et al. (2015) also verified this trend regarding the effect of dry heat treatment and conventional oven treatment to sorghum flour.In the same way,Allouche et al. (2007) had verified that luteolin from extra virgin olive oil decreased rapidly with the heating time (180 °C for 36 h in a hot air oven), while apigenin showed a slower degradation.The performance of the sum of flavones content was similar to that of luteolin during storage, maybe because the amount of luteolin is about twice as high as apigenin.

Figure 3 .
Figure 3. Flavanones contents on sorghum genotype SC319 (fl our and grain) stored for 180 days under three temperatures (4, 25 and 40 °C).Results are expressed in µg/ g of sample on dry weight basis.Means of three replicates.Means with different lowercase letters among the temperatures and uppercase letters among the times are different by Tukey test (p<0.05).

Figure 3 .
Figure 3. Flavanones contents on sorghum genotype SC319 (flour and grain) stored for 180 days under three temperatures (4, 25 and 40 °C).Results are expressed in µg/ g of sample on dry weight basis.Means of three replicates.Means with different lowercase letters among the temperatures and uppercase letters among the times are different by Tukey test (p<0.05).

Figure 4 .
Figure 4. Flavanones contents on sorghum genotype TX430 (fl our and bran) stored for 180 days under three temperatures (4, 25 and 40 °C).Results are expressed in µg/ g of sample on dry weight basis.Means of three replicates.Means with different lowercase letters among the temperatures and uppercase letters among the times are different by Tukey test (p<0.05).

Figure 4 .
Figure 4. Flavanones contents on sorghum genotype TX430 (flour and bran) stored for 180 days under three temperatures (4, 25 and 40 °C).Results are expressed in µg/ g of sample on dry weight basis.Means of three replicates.Means with different lowercase letters among the temperatures and uppercase letters among the times are different by Tukey test (p<0.05).
Flavones and flavanones retention (%) on flour and grain of the genotypes SC319 and TX430 after stored for 180 days at three temperatures.*Means of three replicates.(% of retention from time 0 to 180).
in both sorghum genotypes during storage for 180 days.The storage temperature (4 o C, 25 o C and 40 °C) influenced the flavones more than the flavanone contents.Although there were losses in the flavone and flavanone content, the retention percentages of these compounds were high at the end of storage.In general, the flavone apigenin and the flavanone eriodictyol were more stable than the flavone luteolin and the flavanone naringenin in storage at 4, 25 and 40 °C.Apigenin was the most preserved compound in the flour and grain of the genotype SC319, with retentions from 92 to 99% at the end of 180 days.Naringenin presented the lowest retention (from 56.92 to 71.25%) in the same materials.The temperature of 4 °C was better to preserve flavones and flavanones in sorghum flour, grain and bran, with about 88% retention at the end of 180 days.

Table 1 .
Flavones and flavanones retention (%) on flour and grain of the genotypes SC319 and TX430 after stored for 180 *Means of three replicates.(% of retention from time 0 to 180).