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-glucoside42. 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 109. 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, nft vertical farming 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 49 saw an average of 45% higher anthocyanins in their second harvest of American elderberries grown inOregon as compared to their first harvest, though the total phenolics only increased an average of 20%. Johnson et al. 54 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 54.
In a study of 107 wild American elderberries samples grown in five regions of the eastern United States by Mudge et al. 110 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 bioactive 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. 5-Hydroxypyrogallol hexoside and protocatechuic acid dihexoside were identified for the first time in elderberry, which could potentially serve as markers of this subspecies in products that use blue elderberry. There was considerable variation within and between hedgerows in both harvest years, but this appears to be a common attribute for the elderberry species. Blueelderberries have many ecological benefits for farms when planted in hedgerows, grow well in challenging environments, are not killed by wildfires and can therefore, serve as a sustainable source of an increasingly popular fruit.The elderberry is a deciduous, multi-stemmed shrub or small tree.
It can grow several meters high and in diameter and produces hundreds of clusters of aromatic flowers in the spring, that mature into small berries in summer. The plant grows well in a variety of soils and climates, and is a native of Northern America, Europe, and parts of Asia. While there are many subspecies within Sambucus nigra, the primary subspecies widely grown and commercially cultivated include S. nigra ssp. nigra found across Europe, and the “American” subspecies S. nigra ssp. canadensis, which is native to the eastern regions of North America.56 The blue elderberry , is a drought-tolerant subspecies native to the western region of North America. The blue elderberry grows in riparian ecosystems from southern British Columbia, Canada to northwest Mexico. In California, there have been efforts for more than a decade to increase the levels of blue elderberry planted in hedgerows on farms because of its environmental benefits, such as improving the air, water, and soil quality, as well as providing food and shelter for pollinators. It is now recognized that these mature hedgerow plants can be a source of locally grown elderberries and elderflowers to increase income and sustainability for the farm. However, to date there is no data on the concentration of the aroma or phenolic compounds in the flowers from this hardy heat-tolerant subspecies. The berries, flowers and bark of the elderberry plant have a long history of use by humans as both food and traditional medicine. Seeds have been found in archeological sites that date to the late stone age and their medicinal use is documented in the writings of Theophrastus , Pedanius Dioscorides and Gaius Plinius Secundus . Elderflowers are frequently used in medicinal and herbal teas, tonics, liqueurs, lemonades, and sparkling waters for their subtle and unique floral, fruity, and green aromas and medicinal properties. Infusions of elderflowers have been used in many cultures for the treatment of inflammation, colds, fever, and respiratory illness and for their diuretic and antidiabetic effects. Some studies have found evidence to support their use, such as antimicrobial activity of elderflower extract against Gram-positive bacteria and high vitro antioxidant activity. Much of the interest for using elderflower in health-promoting applications is based on the high content of biologically active phenolic compounds in the flowers. European and American elderflowers contain an array of phenolic compounds, such as phenolic acids , flavonols , flavonol glycosides [isorhamnetin-3-O-rutinoside , rutin ], flavan-3-ols [-catechin, -epicatechin], and flavanones. In European-grown elderflowers, the dominant phenolic acid and flavonol glycoside include chlorogenic acid and rutin, although isoquercetin, vertical tower for strawberries isorhamnetin-3-rutinoside and kaempferol-3-rutinoside are also present. For example, in a study of European elderflowers grown in different locations and altitudes, the dominant class of phenolic compounds were the flavonols, namely rutin , whereas chlorogenic acid levels were lower . This study also found that the flowers contain four times more chlorogenic acid than the leaves or berries. The predominant phenolic compounds identified in elderflower syrup, a traditional herbal beverage, include chlorogenic acid and rutin . There has been only one study on the phenolic profile of the flowers of S. nigra ssp. canadensis which appears to be similar to the European subspecies, in that rutin and chlorogenic acid are the primary flavonol and phenolic acid identified, respectively.
The aroma of the elderflower is derived from the volatile organic compounds in the flower and is an important characteristic to understand for consumer acceptance in applications.To date, only the VOCs of elderflowers from the European subspecies have been studied. The American subspecies S. nigra ssp. canadensis has not yet been investigated. As fresh flowers are highly perishable, many commercial products rely on dry, and in some cases, frozen flowers. Thus, it is important to understand how the organoleptic properties of elderflowers change in response to processing. The VOC profile of tea made with elderflowers of three European cultivars using dynamic headspace sampling revealed compounds important to the characteristic aroma to be linalool, hotrienol, and cis– and trans-rose oxide. Similarly, studies indicate that in fresh and dried flowers analyzed by headspace solid phase microextraction coupled with gas chromatography mass spectrometry , linalool oxides are the main aroma compounds. Linalool oxide has a floral, herbal, earthy, green odor. In hexane extracts of dry elderflowers analyzed via HS-SPME/GC-MS, cis-linalool oxide and 2-hexanone were the primary volatiles. The compound 2-hexanone has a fruity, fungal, meaty, and buttery odor.80 In syrups made from elderflowers, terpene alcohols and oxides were identified as the primary aroma compounds. Studies of the impact of drying on volatiles in the flowers demonstrate that nearly all types of drying change the volatile profile significantly. The aim of this study was to characterize the composition of phenolic compounds and VOCs in flowers of the blue elderberry , and to determine how these compounds change in response to drying and in the preparation of teas. Understanding how the aroma and phenolic compounds compare with current commercially available European and American subspecies will help to establish a role for blue elderflowers in commercial applications such as herbal teas and as a flavoring for beverages, as well as identify unique compositional qualities of this native and underutilized flower.An aqueous mixture of ethanol was used to extract the phenolic compounds from flowers. The optimal mixture of ethanol to water was determined by extracting flowers in 0, 25, 50, 75, and 100% ethanol. Solvents also contained 0.1% HCl and 0.1% ascorbic acid . For each extraction, 0.25 g dry flower material and 25 mL solvent were added to 50 mL Eppendorf tubes. The dry flowers with solvent were homogenized for 1 min at 7000 rpm with a 19 mm diameter probe head in the 50 mL tubes. Homogenized extracts were refrigerated overnight at 4 °C, then centrifuged at 4000 rpm for 7 min . The supernatant was filtered through 0.45 µm PTFE, then diluted 50% with 1.5% phosphoric acid before analysis. Three replicates were made for each extraction condition . Hence, four types of samples were made: fresh whole flowers , dry whole flowers , fresh homogenized flowers , and dry homogenized flowers . Flowers were mixed with the determined optimal extraction solvent and followed the same extraction process as described above, except whole flower samples were not homogenized and instead placed directly into the refrigerator to extract overnight.All sample extracts were analyzed via high performance liquid chromatography using an Agilent 1200 system with diode array detection and fluorescence detection . Separation of phenolic compounds was performed on an Agilent PLRP-S column at 35 °C, using a previously published method. Mobile phase A was 1.5% phosphoric acid in water and mobile phase B was 80% acetonitrile, 20% mobile phase A . The flow was set at 1.00 mL min-1 . The gradient used was as follows: 0 min, 6% B, 73 min, 31% B, 78-86 min, 62% B, 90-105 min 6% B. Most phenolic compounds were detected using a at 280 nm , 320 nm , and 360 nm . Flavan-3-ols were detected using a fluorescence detector . Compounds were quantified using external standard curves employing surrogate standards for each group of phenolic compounds [-catechin for flavan-3- ols, chlorogenic acid for phenolic acids and simple phenols, quercetin for flavonol aglycones, and IR for flavonols]. Standards were prepared at concentrations of 200, 100, 50, 10, and 5 mg L -1 , except IR which included an additional concentration of 500 mg L -1 . Triplicate analyses of each concentration were performed .Compounds were separated using HPLC-DAD-FLD as described above and identified using authentic standards to check retention time and absorption spectra. Several peaks in the chromatograms did not match tR or spectra of authentic standards. Therefore, fractions of these peaks were collected. Fractions were dried and reconstituted in 1% formic acid in water.