Elderflowers are used in products as either fresh or, more commonly, as dry flowers and as either whole or homogenized flowers . Therefore, each of these parameters were evaluated resulting in the following types of samples: fresh whole flowers ; dry whole flowers ; fresh homogenized flowers ; dry homogenized flowers . Phenolic compounds were quantified using HPLC-DAD-FLD and information regarding the standard curves can be found in Table 1. Significantly more phenolic compounds were extracted from FHF compared with FWF, DWF and DHF indicating that phenolic compounds are released more readily from the vacuoles during homogenization while the flower is still fresh. There was no significant difference in the sum of all measured phenolic compounds between FWF, DWF, or DHF; however, levels of most phenolics were slightly higher in the DHF, suggesting that homogenization also increases the extraction efficiency in dry flowers. Furthermore, a statistically significant interaction was found between the fresh and dry flowers and homogenization of the sample for most phenolic compounds, due to the uniquely high levels present in the FHF and the absence of an equally high increase in DHF . This trend can also be seen in the totals of each phenolic class , vertical vegetable tower as the FHF were significantly higher than all other sample types, with the exception of total flavan-3-ols in DHF .
The levels of IR were significantly higher in FHF, with a maximum concentration of 78.73 ± 4.84 mg g-1 . This is a significant difference as compared with the European and American subspecies, in which rutin is the predominant phenolic compound in flowers and at much lower concentrations. Levels of IR in European elder flower levels range from about 0.200 to 0.900 mg g-1 fresh weight, though higher levels were found in elder flower tea, ranging from 4.260 to 13.500 mg g -1 . This key difference in the flowers of the blue elderberry provides an opportunity to create unique products for consumers looking for high levels of bioactive phenolic compounds, as studies have shown that IR can induce apoptosis in cancer cells. The other flavonol glycosides found in the flowers include rutin, kaempferol-3-Orutinoside, and isorhamnetin-3-O-glucoside. Rutin ranged from 3.20 ± 0.395 µg g-1 in FWF to 10.01 ± 0.97 mg g-1 in FHF . The concentrations of rutin vary greatly across studies of European elder flowers. For example, levels are reported as 1.65490 ± 0.10951 mg g-1 fresh weight,6 1.8877 ± 0.2691 mg g-1 fresh weight, between 15.70 and 23.90 mg g-1 in dried elder flower tea which varied with cultivar,and 4.14443 ± 0.11469 mg g-1 in a traditional beverage called sabesa which is made from fresh flowers. Herein, kaempferol-3-O-rutinoside ranged from 1.24 ± 0.03 to 3.010 ± 0.37 mg g-1 dry weight, and isorhamnetin-3-O-glucoside ranged from 0.77 ± 0.05 to 1.42 ± 0.40 mg g-1 dry weight . Quercetin was the only flavonol aglycone identified in the flower extracts and was low relative to the flavonol glycosides.
Though this compound may be due to the degradation of a quercetin glycoside, quercetin aglycone has been measured in other elder flower studies, and our results are similar to those reported by Viapiana et al. .The flavan-3-ols monomers found in the flowers include -catechin and -epicatechin, highest in the FHF at 1.110 ± 0.30 and 1.24 ± 0.19 mg g-1 , respectively . Epicatechin, but not -catechin, had an interaction between the fresh and dried and homogenization of the sample, as it was significantly higher in FHF. Proanthocyanin B type was also tentatively identified via HPLC-MS/MS analysis in the flowers, and was present in relatively low quantities in all samples . A procyanidin trimer was identified in elder flowers extracts and beverages by Mikulic-Petkovsek et al. .Chlorogenic acid was identified as the main phenolic acid in the flowers of the blue elderberry, like the flowers of the American elderberry, whereas the predominant phenolic acid in the flowers of the European elderberry is neochlorogenic acid .Neochlorogenic acid and other caffeoylquinic acid isomers were also present in the elder flowers of S. nigra ssp. cerulea . Two isomers of 5-caffeoylquinic acid in addition to 3- and 4-caffeoylquinic acid have been identified in elder flower products. Evaluation of the phenolic content of elder plants grown in different locations and altitudes indicate, in general, that plant material from shrubs at higher altitudes had higher levels of hydroxycinnamic acids and flavonols. The authors postulated that the stress of harsher climates at higher altitudes may have led to the increase in hydroxycinnamic acids and flavonols to cope with the increase in UV radiation. They also reasoned that the high amounts of sun and cool nights may increase the metabolism of phenolic compounds.
The flowers in the present study experience hot, dry summers with cool breezes from the Sacramento-San Joaquin Delta at night, and these conditions may contribute to the unique phenolic profile in this flower. The average day/night temperatures for Davis, California while the flowers were growing were 24/7 °C in April 2021, 28/12 °C in May 2021, and 31/13 °C in June 2021, with less than 3 inches of rain during that time span. A phenolic compound unique to the blue elder flower was identified as 5-hydroxypyrogallol hexoside . This compound has also been identified in the berries of this subspecies grown in the same locations. The concentrations of 5-HPG ranged from 1.26 ± 0.20 mg g-1 in DWF to 2.00 ± 0.72 mg g-1 in FHF . Because no commercial standards exist for this compound, the tentative identification of this compound was determined by high resolution QTOF-MS/MS data. The mass spectrum shows the molecular ion [M-H]- at m/z 303.0728 and fragment ion showing the loss of the sugar molecule [M-hexose-H]- at m/z 141.0199. Although the biological properties of this unique phenolic compound have not yet been investigated, 5-HPG hexoside can serve as a marker for S. nigra ssp. cerulea, especially since it is present in relatively high levels in the flower and berry and is not identified in other elderberry subspecies. Elderflower tea is one of the most traditional and simplest ways that the flowers are used in the preparation of beverages. To make elder flower tea, the flowers are infused in hot water to extract the flavor and biologically active phenolic compounds from the flowers. Recommended steeping times can vary widely, however there are no studies investigating the impact of steep time on the extraction of phenolic compounds in the elder flower tea. To address this, the impact of time on the extraction of phenolic compounds from teas made from dried flowers was evaluated. The profile of phenolic compounds extracted in hot water infusions was similar to the profile obtained in ethanol/water extracts, however the concentrations were lower in the hot water extracts . Phenolic compounds were quantified at 5, 10, 15, and 20 minutes of steep time. Over time, the concentrations of total measured phenolic compounds increased 47% from five minutes . These results suggest that longer infusion times are beneficial for extracting the highest level of compounds. The overall levels of phenolic compounds in elder flower of S. nigra ssp. cerulea are comparable to the levels in the European flowers. However, vertical farm tower variability between studies due to post-harvest conditions, extraction solvent, and analytical method make it challenging to make direct comparisons. For example, in this study, FWF, DWF, and DHF all had about 50-60mg g -1 dry weight, while the FHF had ~120 mg g -1 dry weight. The unique composition of phenolic compounds in these flowers provides an opportunity to make different products for the market, especially nutraceutical or functional food products that take advantage of the high levels of IR. Based on our results, it would be best to use fresh flowers and blend or homogenize the flowers to extract the highest levels of phenolic compounds. Before analyzing samples, method parameters were evaluated to find the optimal equilibrium time, temperature, and the extraction time for headspace VOCs. The optimal parameters for flower samples were determined to be 40 °C, 20 min equilibration , and 30 min extraction with a SPME fiber. The optimized conditions for the tea samples were 40 °C, 30 min equilibration, and 30 min extraction. The profiles of volatile compounds were evaluated in fresh whole, dry ground, and teas made from whole fresh and dry flowers by HS-SPME/GC-MS, and relative levels of identified volatile compounds were calculated.
The fresh flowers were evaluated as whole flowers because homogenizing them can cause oxidation and create artifacts in the volatile headspace profiles. This led to higher variability in the relative peak areas of compounds, but it is believed to be truer to the real headspace VOC profile as compared to a homogenized fresh flower sample. Overall, 25 compounds in the fresh whole flowers, 44 compounds in the dry ground flowers, and 18 compounds in the tea preparations were identified. Table 1S contains the m/z ion of the base peak and the average match factor for each of the compounds identified in the headspace of samples. In the headspace of fresh flower, the most concentrated compounds were pentadecane > methyl eugenol > cis-3-hexenyl acetate > α-farnesene > and cis-3-hexenyl-α- methylbutyrate . The contribution of each compound’s odor to the overall aroma of these elder flowers cannot be determined from the concentration alone, as each compound has its own odor activity and threshold. However, it can be useful to know the characteristic odors of these compounds as a way to understand what comprised the general aroma. These compounds are described to have odors such as waxy; clove, spice; fresh, green, sweet, fruity, apple, pear, melon;wood, sweet; ; fruity, sweet, minty, fresh, and green apple, respectively. In addition to pentadecane, several straight chain hydrocarbons were also present, which may be released from the cuticle of the petal or peduncle of the flower. These include 1-pentadecene, heptadecane, 8- heptadecene and 6,9-heptadcadiene. Flowers also contain 4.6% methyl salicylate a compound with a sweet, minty odor80 that is frequently used as an analgesic in liniments to relieve pain. Methyl salicylate has been identified in several other studies on the volatile profile of elder flowers. The profile of headspace VOCs in elder flowers of S. nigra ssp. cerulea differ significantly from the European elder flowers as linalool oxides and other derivatives predominate in the European flowers, and are absent in the present study. Furthermore, the present study indicates a unique headspace VOC profile in the blue elder flowers because pentadecane and methyl eugenol have been identified as major contributors to the headspace VOC profile. Pentadecane has been identified at trace levels in some European elder flower extracts, however methyl eugenol has not been identified in European elder flowers. Methyl eugenol, which has a clove-like aroma19 , appears to be unique to the S. nigra ssp. cerulea elder flower, and could be a unique volatile marker for this subspecies. Tea made with fresh whole elder flowers presented a slightly different headspace VOC profile as compared to fresh flowers. Although methyl eugenol was still a prominent compound in the headspace , there were also two ketones in relatively high concentrations: 2,2,6-trimethyl-4H-1,3-dioxin-4-one and 4-methyl-2-heptanone . Aroma descriptors were not found for these compounds. In addition, two aldehydes including heptanal and nonanal were also present in the headspace of the tea but not the fresh flowers. Heptanal odor is described to be fresh, fatty, green, and herbal, whereas nonanal is described to have waxy, rose, orange peel or fatty notes. Methyl salicylate comprised only 1.27 ± 0.29% of the headspace volatiles in the tea made from fresh flowers. In the headspace of dry elder flowers, the most concentrated compounds were -3-hexen-1-ol > 1-penten-3-ol > 3-methyl-butanal > heptanal, > isocyanato-methane . In general, the dry flowers contain a wider range of volatiles than the fresh flowers, including more aldehydes, alcohols, alkanes, and other hydrocarbons. Other notable volatiles identified include methyl salicylate , dihydroedulan , which is a driver of typical elderberry aroma, and linalyl acetate which is the only linalool derivate identified any preparations of the elder flowers of S. nigra ssp. cerulea, unlike European elder flowers which typically have high concentrations of linalool derivatives. In tea prepared from dried flowers, the headspace aroma less complex than the dried flowers, but many of the aldehydes were still identified, including nonanal , heptanal , and hexanal .