The ice pop and juice samples did not undergo extraction due to prior extraction of anthocyanins to make the concentrate used in the formulation but were filThered using the 0.45 µm nylon syringe filters prior to HPLC analysis.Anthocyanins and chlorogenic acid were analyzed by HPLC using the method of Cho and others. Samples were analyzed using a Waters HPLC system equipped with a model 600 pump, a model 717 Plus autosampler, and a model 996 photodiode array detector. Separation was carried out at room temperature using a 4.6 mm × 250 mm Symmetry C18 column preceded by a 3.9 mm × 20 mm Symmetry C18 guard column. The mobile phase was a linear gradient of 5% formic acid and methanol from 2% B to 60% B for 60 min at a flow rate of 1 mL/min. Detection wavelengths of 320 nm and 510 nm were used to monitor chlorogenic acid and anthocyanin peaks, respectively. Individual anthocyanin monoglucosides and acylated anthocyanin derivatives were quantified as delphinidin, cyanidin, petunidin, peonidin, and malvidin glucoside equivalents using external calibration curves of a mixture of authentic standards . Chlorogenic acid was quantified using external calibration curves of an authentic standard . Results are expressed as mg of anthocyanin or chlorogenic acid per g of WBB powder.An analytical Hewlett Packard 1100 series HPLC instrument equipped with an autosampler, binary HPLC pump, large plastic garden pots and UV/Vis detector was used. For HPLC/MS analysis, the HPLC apparatus was interfaced to a Bruker model Esquire-LC/MS ion trap mass spectrometer .
Mass spectral data were collected with the Bruker software , which also controlled the instrument and collected the signal at 520 nm. Typical conditions for mass spectral analysis conducted in positive-ion electrospray mode for anthocyanins and negative-ion electrospray mode for flavonols included a capillary voltage of 4000 V, a nebulizing pressure of 30.0 psi, a drying gas flow of 9.0 mL/min, and a temperature of 300 ◦C. Data were collected in full scan mode over a mass range of m/z 50−1000 at 1.0 s per cycle. Characteristic ions were used for peak assignment. For compounds where chemical standards were commercially available, retention times were also used to confirm the identification of components.The WBB powder used to prepare the products contained at least 22 anthocyanins , which were identified by comparing their mass-to-charge values and elution orders with previous studies. Blueberries are unique in that three different sugars are commonly attached to the five anthocyanidins. This was confirmed in our study; however, we were unable to detect peonidin-3-arabinoside using our HPLC method. We were unable to obtain complete separation of all of the anthocyanins present in the extract due to the complexity of the anthocyanin profile. Peak 15 contained two co-eluting compounds, namely cyanidin-3- galactoside and cyanidin-3- galactoside, and peak 18 was composed of three co-eluting compounds, namely delphinidin-3-rutinoside, cyanidin-3- glucoside, and malvidin-3- galactoside. We were unable to identify peak 17, which appeared to be a delphinidin derivative based on its aglycone m/z of 303, but the molecular ion m/z value was ambiguous. Many of the anthocyanins were present in acylated form. Two of the cyanidin glycosides were acylated with malonic acid, whereas delphinidin, cyanidin, and malvidin galactosides as well as petunidin, peonidin, and malvidin glucosides were acylated with acetic acid moieties.
The total anthocyanin content of the juice decreased with storage time for each storage temperature . The total anthocyanin content of juice stored at 4.4 ◦C and 21 ◦C is shown in Figure 2. After eight weeks of storage, the juice stored at 4.4 ◦C retained 90.7% of total anthocyanins compared with control samples , whereas the juice stored at 21 ◦C retained 69.1%. Concentrations of anthocyanins are known to readily decline during storage of blueberry juice at ambient temperature, but refrigeration is an effective treatment to ameliorate anthocyanin losses. Changes in the major individual anthocyanins in the juice stored at 4.4 ◦C and 21 ◦C over eight weeks of storage are shown in Figure S4. At 4.4 ◦C, peonidin-3-galactoside, cyanidin-3-arabinoside, malvidin-3-galactoside, malvidin-3-glucoside, and malvidin-3- galactoside remained stable over the eight weeks of storage. At 4.4 ◦C, all anthocyanins showed >50% retention, with the minimal percent retention being 57.7% for the unknown delphinidin derivative. This compound, however, did not significantly decrease over storage at 21 ◦C, along with the two co-eluting anthocyanins galactoside + cyanidin-3- galactoside. Besides these two compounds, the percent retention of anthocyanins at 21 ◦C ranged from 59% glucoside to 75.5% .The total anthocyanin content of the gummy product decreased with storage time for each storage temperature . The total anthocyanin content of the gummy product stored at 4.4 ◦C and 21 ◦C is shown in Figure 2. After eight weeks of storage, the gummy product stored at 4.4 ◦C and 21 ◦C retained 43.2% and 50.6%, respectively, of their original total anthocyanin content . Consistent with our findings, levels of total anthocyanins declined in gelatin gels prepared with grape pomace extract over 24 weeks of storage at 21 ◦C, with losses most pronounced in gels exposed to neon light. Maier et al. also reported similar retention of total anthocyanins in gels stored for 24 weeks at 6 ◦C and 24 ◦C.
The lower amount of anthocyanins recovered in the gummies stored at 4.4 ◦C compared to the same product stored at 21 ◦C may be explained by reduced extraction efficiency due to the hardening of the gel at low temperature, as opposed to degradation late during storage. Changes in the major individual anthocyanins in the gummy product stored at 4.4 ◦C and 21 ◦C over eight weeks of storage are shown in Figure S5. At 4.4 ◦C, all the individual anthocyanins decreased with storage time with retentions <50%, except for the two co-eluting anthocyanins galactoside + cyanidin-3- galactoside and malvidin-3-glucoside . The percent retentions of the rest of the anthocyanins at this storage temperature ranged from 29.3% to 49.2%. When stored at 21 ◦C, two anthocyanins did not significantly decrease with time, namely the unknown delphinidin derivative and the two co-eluting compounds galactoside + cyanidin-3- galactoside. For the rest of the anthocyanins, percent retentions ranged from 40% to 71% glucoside. In all the products, the individual anthocyanin loss did not appear to be impacted by the anthocyanidin structure or the type of sugar moiety attached .The distribution of the products according to their individual anthocyanin profile as affected by storage time can be visualized on a PCA scores plot . The first principal component explained 83.9% of the variation with all the individual anthocyanins being positively loaded on PC1. Therefore, PC1 represents the amount of individual anthocyanins. The juice and ice pop samples had high scores on PC1 . The oatmeal bar samples also had positive scores on PC1 for the earlier storage times, whereas the oatmeal bar samples stored at 21 ◦C for eight weeks were the only oatmeal sample to have a negative score. Except for the control samples , all the graham cracker cookie samples had negative scores on PC1, regardless of the storage temperature. Finally, all gummy samples had negative scores on PC1, with scores becoming smaller with storage time. The PCA figure confirmed higher values of anthocyanins in the juice and ice pop samples, as well as, to a lesser extent, the oatmeal bars. The graham cracker cookie and gummy samples did not demonstrate high values for anthocyanins, with a clear loss of anthocyanins with storage time for the gummy samples. Percent polymeric color values typically show an inverse correlation with total anthocyanins during storage of blueberry products, and inverse correlations with each individual anthocyanins in all the products and storage temperature . Higher percent polymeric color values indicate that a higher percentage of anthocyanins are resistant to bleaching in the presence of potassium metabisulfite. Since the sulfonic acid adduct attaches at C4 on the middle heterocyclic ring, raspberry plant pot it is thought that anthocyanin–procyanidin polymers are formed via a direct condensation reaction, resulting in a C4–C8 anthocyanin–procyanidin linkage as the major polymers formed in blueberries during storage. Hence, it is possible that declines in anthocyanins during storage of the blueberry products are not true losses due to degradation, but the conversion of monomeric anthocyanins to anthocyanin–procyanidin polymers. Anthocyanins can be degraded via a hydration reaction, where the flavylium ion is converted to a hemiketal structure, which is rapidly converted to cis-chalcone, which slowly arranges to a trans-chalcone structure. The trans-chalcone structure is highly unstable and rapidly degrades to hydroxybenzoic acid derivatives. However, we do not consider that this reaction was responsible for anthocyanin losses in the blueberry products over storage since we did not observe an increase in phenolic acid derivatives in our HPLC chromatograms at 280 nm .The stability of chlorogenic acid in the four blueberry products stored at 4.4 ◦C and 21 ◦C is shown in Figure 5. Chlorogenic acid was stable in all products over storage regardless of storage temperature,except for the juice and oatmeal bar stored at 4.4 ◦C, where levels significantly decreased . At 4.4 ◦C, the chlorogenic acid content decreased from 4.3 to 3.6 mg/g WBB powder in the juice and from 3.0 to 2.6 mg/g WBB powder in the oatmeal bar. At 21 ◦C, chlorogenic acid in the juice showed a slight increasing trend; however, this change was not statistically significant . Chlorogenic acid was also stable in the ice pop over eight weeks of storage at −20 ◦C , with an average value of 6.5 mg/g WBB powder over storage .
Initial levels of chlorogenic acid were higher in all products stored at 21 ◦C compared with 4.4 ◦C storage, which may be due to the variation in processing the two sets of samples for the storage study, or possible the degradation of chlorogenic acid in the WBB powder used to prepare the products. The WBB powder used to prepare samples for the refrigerated storage study was stored at 15.5 ◦C for three months prior to preparing the samples. Blueberries contain polyphenol oxidase, which can readily oxidize chlorogenic acid. Chlorogenic acid was previously found to be stable in blueberry juice, puree, and canned berries stored for six months at 25 ◦C, but blueberry jams lost 27% of chlorogenic acid over six months of storage at 25 ◦C.The stability of total flavonols in the five blueberry products is shown in Figure 6. Total flavonol levels in the oatmeal bar stored at both temperatures and in the gummy product and graham cracker cookie stored at 4.4 ◦C were stable over eight weeks of storage as well as the juice samples stored at 4.4 ◦C from two to eight weeks . Flavonols in the juice stored at 21◦C were also stable over time despite an upward trend, but the increase was not significant . Consistent with our findings, total flavonol concentrations were found to be relatively stable in blueberry jam, juice, puree, and canned berries over six months of storage at 25 ◦C. However, total flavonol content significantly decreased over storage in the gummy product and graham cracker cookie stored at 21 ◦C. Total flavonol levels declined by 45.7% and 28.5%, respectively, in these products over eight weeks of storage. In the gummy product, the most marked loss occurred from six to eight weeks of storage. Moisture loss during late storage presumably led to hardening of the gummies, resulting in incomplete extraction of the flavonols.Anthocyanins are flavonoids found in various fruits and vegetables as natural plant pigments and are highly valued for their health-promoting attributes, such as promoting intestinal barrier function, prventing cardiovascular disease, and alleviating oxidative stress induced by ages, diabetes and inflammation. Anticancer effects have also been reported for anthocyanin extracts including bilberry, blueberry, cranberry and other berries. Anthocyanin consist of the anthocyanidin aglycone plus one or more glycosides. To date, studies of the anticancer mechanisms of anthocyanins have focused on the anthocyanidin aglycone. The reported studies showed that anthocyanidin could affect signalling pathways related to proliferation and apoptosis of tumour cells, e.g., inhibiting the proinflammatoryNF-κB pathway, targeting the PDK1-PI3K/Akt signalling pathway, enhancing the expression of p21WAF1 and suppressing the expression of cyclin A/B simultaneously. However, the impact of the glycosides of anthocyanins on tumour inhibition has not been well clarified, and data on the influence on the bioactivity of glycosides were diverse or even controversial. On the one hand, Oglycosylation of flavonoids reduces their biological effects. For example, the O-glycosylation of flavonoids dramatically diminished the inhibition on producing NO, expressing iNOS, and activating NF-κB in RAW264.7 macrophages and mouse microglial BV-2 cells.