The column used was a PLRP-S 100A 3 µm 150 x 4.6 mm at 35 °C, and the injection volume was 10.0 µl. Mobile phase A was water with 1.5 % phosphoric acid, while mobile phase B was 80%/20% acetonitrile/ mobile phase A. The gradient used was 0 min 6% B, 73 to 83 min 31% B, 90 to 105 min 6% B. The DAD was used to monitor hydroxybenzoic acids at 280 nm, hydroxycinnamic acids at 320 nm, flavonols at 360 nm, and anthocyanins at 520 nm. The FLD was used to monitor flavan-3-ols, with excitation at 230 nm and emission at 321 nm. External calibration curves were prepared using chlorogenic acid for phenolic acids, rutin for flavonols, and cyanidin-3-glucoside for anthocyanins , at the following concentrations: 200, 150, 100, 75, 50, 25, 10, 5, and 2.5 mg/L. Catechin was used to quantify flavan-3-ols and standards were run at 150, 100, 75, 50, 25, 10, 5, 2.5, and 1 mg/L. Compounds were identified based on retention time and spectral comparisons with standards. Information about the linear equations and lower limits of detection and quantitation can be found in Table S1 in the supplementary material. The LLOD was calculated as 3.3 times the standard deviation of the y-intercept of the curve divided the slope, while the LLOQ was calculated as 10 times those values.
Several peaks appeared in the HPLC chromatograms that could not be identified using the above parameters. Chromatographic eluents of these peaks were collected individually and dried under vacuum. These extracts were reconstituted with mobile phase A,round plastic pots and 5 µL were injected into the HPLC- QTOF-MS/MS for accurate mass analysis . A Poroshell 120 EC-C18 column was used at 35 °C. Mobile phase A was 1% formic acid in distilled water, and mobile phase B was 1% formic acid in acetonitrile. The gradient used was 0 min 3% B, 30 min 50% B, 31-32 min 95% B, 33-38 min 3% B. The mass spectrometer was used in negative mode, and the mass range for MS was 100 to 1000 m/z while the range for MS/MS was 20-700 m/z. Collision energies at 10, 20, and 40 V were applied. The drying gas was set to a flow of 12 L/min at 250 °C, while the sheath gas was set to 11 L/min at 350 °C. The nebulizer was set to 40 psig, the capillary voltage was 3500 V, the nozzle was set to 500 V, and the fragmentor was set to 100 V. Data was analyzed using Agilent MassHunter Workstation Qualitative Analysis 10.0 . Tentative identification was achieved by comparing the mass to charge ratio of the precursor and fragment ions to online libraries of compounds as well as using formula generation for the peaks in the spectra. 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 141.0186 . This compound was one of the most abundant phenolic compounds in the blue elderberry. While no evidence of 5-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,hydroponic bucket 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 elder flowers 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 .
Elder flowers 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 elder flowers have been used in many cultures for the treatment of inflammation, colds, fever, and respiratory illness and for their diuretic and anti-diabetic effects.Some studies have found evidence to support their use, such as antimicrobial activity of elder flower extract against Gram-positive bacteria and high vitro antioxidant activity.Much of the interest for using elder flower in health-promoting applications is based on the high content of biologically active phenolic compounds in the flowers. European and American elder flowers 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 elder flowers, 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 elder flowers 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 elder flower 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 elder flower 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 elder flowers 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 elder flowers change in response to processing. The VOC profile of tea made with elder flowers 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 head space 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 elder flowers 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 elder flowers, 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 elder flowers 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. LC/MS-grade acetonitrile and HPLC-grade hydrochloric acid were purchased from Fisher Scientific . Purissimum grade phosphoric acid was purchased from Sigma Aldrich and filtered through 0.45 µm polypropylene filters under vacuum. Ascorbic acid was obtained from Acros Organics . Ultrafiltered water was obtained by a Milli-Q system . Analytical standards of rutin, quercetin, chlorogenic acid, and -catechin were purchased from Sigma Aldrich . A standard of n-butyl-d9 was purchased from CDN Isotopes . Kaempferol-3-O-rutinoside, isorhamnetin-3-O-glucoside, IR, and isoquercetin were purchased from ExtraSynthese . 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.