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 The ophrastus , 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, nursery pots the dominant phenolic acid and flavonol glycoside include chlorogenic acid and rutin, although isoquercetin, isorhamnetin-3-rutinoside and kaempferol-3-rutinoside are also present.
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. Blueelder berries 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.84 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.
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.LC/MS-grade acetonitrile and HPLC-grade hydrochloric acid were purchased from Fisher Scientific . HPLC-grade ethanol and acetonitrile were purchased from Sigma Aldrich . 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. 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, large pots plastic 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 . Phenolics were extracted from fresh and dried flowers that were either whole or homogenized. 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. 116 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. These samples were then subjected to high resolution mass spectrometry using an Agilent 6545 quadrupole time-of-flight mass spectrometer , using conditions previously established for elderberry phenolic compounds. Data were then analyzed using Agilent MassHunter Workstation Qualitative Analysis 10.0 . To tentatively identify compounds, the mass to charge ratio of the precursor and fragment ions were compared to online libraries of compounds and using formula generation for the peaks in the spectra.Volatile compounds were analyzed by headspace solid phase microextraction gas chromatography mass spectrometry . The equilibration and extraction parameters were optimized using ground dry flowers, prepared using a spice grinder, pulsed 25 times . A 1 g sample of ground dry flowers was placed into a 20 mL glass vial and the vial was sealed by a crimp-top cap with a Teflon septa. Various incubation temperatures , equilibration times , and extraction times were evaluated to optimize for the highest total peak area and unique compounds identified from samples. The fiber used for all analyses was a divinylbenzene/carbon wide range/polydimethylsiloxane , 23 Ga, 1 cm length, with 80 µm phase thickness . After extraction, the fiberwas injected into the GC and volatile compounds were desorbed at 250 °C for 5 min. Compounds were then separated on a DB-Wax column . Helium was used as a carrier gas at 1 mL min-1 . A temperature program was used with the following steps: 35 °C for 1 min, 3 °C min- 1 to 65 °C, 6 °C min-1 to 180 °C, 30 °C min-1 to 240 °C, hold at 240 °C for 5 min. Total run time was 37.167 min. Compounds were detected with a single quad, triple axis mass spectrometer . The mass range for acquisition was 30 to 300 m/z. The MS transfer line temperature was 250 °C, the source temperature was 230 °C, and the quad temperature was 150 °C. The electron ionization was set to 70 eV. To have the same volume of headspace in fresh and dry flower samples, 0.5 g of fresh whole flowers or 1.5 g ground dry flowers were placed in the 20 mL clear glass vials. For tea samples, 4 mL tea was placed in 20 mL vials. To each sample, 10 µl of 1-butanol-d9 in methanol was added as an internal standard. Volatile compounds were identified using Agilent Mass Hunter Unknown Analysis , using the NIST17 library requiring an ≥ 80% match and that compounds were identified in at least three of the five to be considered a volatile compound in the samples.