The anthocyanin content of elderberries strongly correlates to the antioxidant potential of the fruit, which may confer health-promoting properties, which is one reason why elderberries are used in supplements and value-added products. Elderberry is also used as a source of natural food colorants due to the levels of anthocyanins. Understanding the levels of anthocyanins in the blueelderberry grown in hedgerows is critical towards establishing this native fruit as an additional and more sustainable elderberry. The average TMA measured in blue elderberry ranged from 34.2 ± 9.7 to 113.4 ± 18.2 mg CGE per 100 g FW in 2018 and from 43.1 ± 11.5 to 121.5 ± 11.5 mg CGE per 100 g FW in 2019 . TMA was variable between hedgerows in both years of harvest, with relative standard deviation values between 16% and 30%, yet there was not a significant difference in the overall average TMA between 2018 and 2019 . Furthermore, most hedgerows were not significantly different from the other hedgerows harvested that year despite significant differences in TMA values found between farms in both years . Regarding the age of the elderberry shrub, hedgerows 2 and 14 had two of the three highest concentrations of TMA in 2019 . This suggests that blue elderberries can be harvested from plants as young as two years without a significant loss of TMA concentrations. TMA values for the blue elderberries are lower than those found in other elderberry subspecies. In European elderberries, TMA levels range from 170 ± 12 to 343 ± 11 with an average of 239 ± 94 mg CGE per 100 g FW 49.
A study of American elderberry grown in Ohio showed a range from 354 ± 59 to 595 ± 26 mg CGE per 100 g FW.52 In the present study, plastic plants pots bare root prerooted cuttings of American elderberries were planted, along with blue elderberries, on Farm 1 in 2018, and three shrubs were harvested in 2019. These American elderberries had an average TMA value of 263 ± 5.4 mg CGE per 100 g FW, which is more similar to what has been observed in other studies on this subspecies. This suggests it is a subspecies difference contributing to the lower anthocyanin concentration in the blue elderberry and not the difference in growing conditions. Compared to other berries, blue elderberries have similar levels of anthocyanins as raspberries, but lower levels than blueberries and blackberries 100 . The lower concentration of anthocyanins in the blue elderberry may require adjustment of levels used in supplements, food and beverages for optimal performance or health benefit, or as natural coloring agents.In addition to anthocyanins, elderberries contain other phenolic compounds, such as flavonols and phenolic acids, which also contribute to the health promoting properties of elderberry. Phenolic compounds are responsible for organoleptic properties and can help protect foods against lipid oxidation. Therefore, TPC can be useful for making approximate comparisons, for example, between varieties of the same fruit, between similar fruits or in the evaluation of a processing step . It is important to note that the TPC assay is a non-selective assay and is easily impacted by extraction conditions and interfering substances, such as ascorbic acid and reducing sugars. Although there is no evidence that the beneficial effects of polyphenol-rich foods can be attributed to the TPC of a food, it can be a useful measure for making general comparisons with other studies in the literature which reported these values but should be supported by quantitative HPLC data. Herein, the range of TPC measured in the blue elderberries was from 514 ± 41 to 791 ± 34 mg GAE per 100 g FW in 2018 and from 459 ± 50 to 695 ± 41 mg GAE per 100 g FW in 2019 . TPC in the blue elderberries was significantly higher in 2018 than in 2019 .
While there were significant differences found between the farms in both years , most hedgerows were not significantly different than most other hedgerows in the given year when evaluated together . Although the farms in this study were near each other and experience similar climates, there can still be differences in growing conditions for each hedgerow, such as water availability, which has been shown to influence the levels of phenolics in blueberries 101 and strawberries 102 . Hedgerows 2 and 14 were not significantly different from other hedgerows in 2019, indicating that the blue elderberries can be harvested early in the plant’s lifetime, which allows farmers to earn an early return on the investment of establishing hedgerows. These comparisons show that blue elderberries from hedgerows are a rich source of phenolic compounds.Phenolic compounds were identified and quantified in the blue elderberry based upon retention time, absorbance spectra and authentic standards when available. Concentrations for samples from 2018 are presented in Table 4, while samples from 2019 are presented in Table 5. Two peaks with significant area were observed in the HPLC chromatograms at 6.96 min and 11.70 min that did not correlate to standards or library matching. Both compounds eluted between the retention time of gallic acid and protocatechuic acid. The first eluting compound had a maximum absorbance at 300 nm while the second compound had a maximum absorbance at 280 nm. These peaks were collected individually and further evaluated by accurate mass quadrupole time-of-flight tandem mass spectrometry . TOF acquires mass spectral data by pulsing ions entering the flight tube in an orthogonal beam, therefore full spectra are collected. The data captured is accurate enough to determine the elemental composition therefore allowing identification without standards 104 . The two compounds were tentatively identified using high mass accuracy as 5-hydroxypyrogallol hexoside, a tetrahydroxybenzene , and protocatechuic acid dihexoside . Accurate mass was especially helpful since commercial standards for these compounds are not available. 5- HPG hexoside was identified by its fragmentation pattern , showing a precursor ion [MH]- at m/z 303.0723 and product ion [M-hexose-H]- at m/z 141.0186 . This compound was one of the most abundant phenolic compounds in the blue elderberry. While no evidence of5-HPG glycoside was found in the literature, the aglycone has shown to have a high radical scavenging activity compared to other simple phenols 105 .The other novel phenolic compound identified was protocatechuic acid dihexoside, also present in relatively high amount in almost all the samples. The precursor ion [M-H]- at m/z 477.1609 fragmented to give product ions corresponding to [dihexoside – H]- at m/z 323.0981 and [M-dihexose -H]- at m/z 153.0562 m/z .
The loss of 324 amu has been identified as the loss of a dihexoside on other phenolic compounds and was proposed to be sophorose or gentiobiose106. PA is a breakdown product of cyanidin-based anthocyanins and has been quantified in elderberry juice during thermal processing 107. PA has been shown to have pharmacological potential in the prevention and/or treatment of neurodegenerative diseases in humans based on in vitro and in vivo studies 108 .Like other elderberry species, blueberry pot rutin was the predominant flavonol and overall had the highest concentration of any of the flavonols measured, with an average of 57.01 ± 17.42 mg per 100 g FW in 2018 and 51.89 ± 25.53 mg per 100 g FW in 2019. These values fall within the range of what has been found in European elderberry 8,42,49,59. Other flavonols identified include isoquercetin , kaempferol-3-rutinoside, and isorhamnetin-3-rutinoside, which was also a major phenolic compound in the berry. Isorhamnetin- 3-rutinoside averaged 28.30 ± 14.03 mg per 100 g FW in 2018 and 24.71 ± 14.83 mg per 100 g FW in 2019, which is higher than what has been found in other subspecies. Overall, the blue elderberry analyzed in the present study has much higher levels of total flavonols as compared to European elderberry. In the American elderberry, the main flavonols are rutin followed by isorhamnetin-3-rutinoside whereas in European elderberries, the main flavonols are rutin followed by isoquercetin. In blue elderberry grown in Slovenia, rutin and isoquercetin were the two predominant flavonols, though the total flavonols in found for the subspecies was similar to the levels found in this study. The predominant anthocyanin present in the blue elderberry is cyanidin-3-sambubioside, like the European subspecies. The average concentration in 2018 was 32.70 ± 10.18 mg per 100 g FW and 29.66 ± 16.81 mg per 100 g FW in 2019. Cyanidin-3,5-diglucoside was the next most concentrated anthocyanin, averaging 20.11 ± 5.63 mg per 100 g FW in 2018 and 19.80 ± 6.92 mg per 100 g FW in 2019. This is unlike European elderberries, in which cyanidin-3-glucoside is typically the second most prominent anthocyanin, except for the Ljubostinja cultivar which has more cyanidin-3,5-diglucoside than cyanidin-glucoside. Cyanidin-3-sambubioside-5-glucoside and cyanidin-3-glucoside were also quantified in the berries. Cyanidin-3,5-diglucoside and cyanidin-3-sambubioside-5-glucoside were not detected in blue elderberries grown in Slovenia,suggesting the growing location impacts the profile of phenolic compounds or perhaps the two samples going by the same name are not, in fact, related. There were no acylated anthocyanins identified in the blue elderberry, like those abundant in the American elderberry. Overall, total anthocyanin concentrations averaged 61.54 ± 16.70 mg per 100 g FW in 2018 and 58.58 ± 22.18 mg per 100 g FW in 2019. The total concentration of anthocyanins in the berries was much lower compared to the other subspecies. Analysis of European elderberries that measured cyanidin-based anthocyanins found an average of 863.8 ± 49.9 mg per 100 g FW 8 . European elderberries grown in different locations at different altitudes had a range of 289.74 ± 66.18 to 792.66 ± 27.97 mg per 100 g FW 6 . In studies on American elderberries, one had an average of 265 ± 74 mg per 100 g FW 49, another had average of 248 ± 83 mg per 100 g FW 18, and a third had an average of 242.7 ± 91.0 mg per 100 g FW 50 . The flavan-3-ols catechin and epicatechin were measured in the elderberry, with epicatechin typically present in higher concentrations. The concentrations found in the present study are similar to those found in others, even across subspecies. Blue elderberry grown in Slovenia had 4.40 ± 0.26 mg per 100 g FW of catechin and 8.49 ± 0.37 mg per 100 g FW of epicatechin. The same study found no catechin present in S. nigra ssp. nigra, but 6.37 ± 0.28 mg per 100 g FW of epicatechin. In a study of European berries growing in different locations at different altitudes, total flavanol concentrations ranged from 1.93 ± 0.22 to 9.67 ± 0.66 mg per 100 g FW 6 . The variability in phenolic and anthocyanin content observed in this study is not surprising, as multiple other studies have shown significant variability in other commercialized elderberry subspecies, even with clonally propagated cultivars. For example, Lee and Finn saw an average of 45% higher anthocyanins in their second harvest of American elderberries grown in Oregon as compared to their first harvest, though the total phenolics only increased an average of 20%. Johnson et al. observed significant changes between subsequent years in anthocyanin and phenolic compound concentrations in juices prepared from American elderberry grown in two locations in Missouri. For example, in the Adams II sample grown in one Missouri location, the quercetin 3-rutinoside content was 298 ± 48 mg L-1 in 2012, 792 ± 143 mg L-1 in 2013, and 47 ± 13 mg L-1 in 2014. In a study of 107 wild American elderberries samples grown in five regions of the eastern United States by Mudge et al. high variability was found in selected flavonoid compounds with an average RSD of 55.3% across samples. Overall, there is a body of evidence demonstrating that elderberry composition can vary year to year or by growing conditions even in clonally-propagated cultivars; therefore, it may be necessary to use standardization techniques for bio-active compounds in order to maintain consistent quality in elderberry products. Blue elderberry grown in California hedgerows has similar levels of sugar, organic acids, and TPC to the European and American elderberry subspecies. Furthermore, the phenolic profile of blue elderberry is similar to European elderberry, in that chlorogenic acid, rutin, and cyanidin-3-sambubioside are the predominant hydroxycinnamic acid, flavonol, and anthocyanin, respectively. However, anthocyanin levels are significantly lower in the blue elderberry compared to European and American subspecies, yet the levels of total flavonols appears to be much higher than the other subspecies.