The European elderberry is the most frequently used subspecies in commercial elderberry-based products and has been extensively studied for its composition, anthocyanin stability, and health benefits in European black elderberry-based products. S. nigra ssp. canadensis is commonly referred to as the American elderberry, a subspecies native to the eastern and central regions of North America. There are several cultivars of the American elderberry, including “Johns” and “Bob Gordon”. The American elderberry, which is utilized in small-batch products, has also been evaluated for its composition 49,50,52,54 and health-promoting properties . The acreage grown of this subspecies has been increasing rapidly and there is a goal to grow over 2,000 acres by 2025, according to the Midwest Elderberry Cooperative. Currently, there is no information on the chemical composition of the fruit of the blue elderberry . With the recent increase in demand for elderberry, blue elderberry grown in hedgerows may be an additional and valuable source of bioactive phenolics and natural colorants. The objective of this study was to determine the moisture content, soluble solids, pH, titratable acidity, and establish the anthocyanin and phenolic profiles of blue elderberries grown in Northern California to support the use of this robust, nft growing system native crop in commercial products.Hedgerows of S. nigra ssp. cerulea were identified on five farms near Davis, California in Spring 2018 with the assistance of an experienced agronomist at The Cloverleaf Farm .
Farm, hedgerow, and harvest information is presented in Table 1. Blue elderberries were determined to be ripe when the berries in a cyme were deep purple, with or without the white bloom, and had no green berries present. Ripe elderberries were harvested by hand from all four quadrants of the elderberry shrub, totaling approximately 3 kg of elderberries. The berries were placed in clear plastic bags, stored on ice, and transported to the laboratory. A subsample was separated for moisture analysis, while the rest was de-stemmed and stored at -20 °C until analyzed.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 earlyin the plant’s lifetime, which allows farmers to earn an early return on the investment of establishing hedgerows. The TPC in blue elderberry is similar to those found in other elderberry species. 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. 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 141.0 . 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 .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. 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 andmedicinal 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, nft hydroponic system the dominant phenolic acid and flavonol glycoside include chlorogenic acid and rutin, although isoquercetin, 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. 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.Elderflowers were harvested from hedgerows on a farm in Winters, CA in May and June 2021. The latitude and longitude coordinates of the hedgerow are 38.634884, -122.007502. Flowers were harvested between 8 and 10 am and were picked from all sides of the shrub. Picked flowers were placed in plastic bags, immediately put on ice, and transported to the laboratory at the University of California, Davis. Flowers were either dried at 25 °C for 24 h in a dehydrator or analyzed fresh. Once dry, stems were removed, and flowers were stored in oxygen-impermeable aluminum pouches. Triplicate samples of fresh flowers were analyzed for their moisture content by drying 1 g of fresh flowers at 95 °C until a consistent weight was achieved so that the same amount of dry matter could be used for fresh and dry flower analyses. 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.