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 fiber was 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, container vertical farming 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. An alkane series was run under the same chromatographic conditions to determine retention indices. Confirmation of identification was performed by comparing the mass spectra and retention indices with those of standards when possible or literature values when standards were not accessible. Relative response was calculated by normalizing peak area for each compound to the internal standard peak area, and relative peak area was calculated using the relative response of a compound divided by the total peak area of a sample.The phenolic compounds were measured in fresh and dry elderflowers of S. nigra ssp. cerulea, both as whole and as homogenized flowers. The treatments used for this study were chosen to reflect the common ways that elderflowers are used in food and beverage applications and to provide more information on how to best extract the phenolic compounds from the flowers. The moisture content of the elderflowers was determined as 75.6 ± 1.7%. To achieve a consistent dry weight used in extractions, either 1.00 g of fresh flowers or 0.25 g of dry flowers were used. The extraction solvent was optimized to increase extraction efficiency of the main phenolic acids, flavonols and flavan-3-ols which included chlorogenic acid, IR, rutin, and -catechin .
While chlorogenic acid, rutin, and catechin could be extracted in either 50:50% ethanol:water or 25:75% ethanol:water for maximum concentrations, the levels of IR increased with increasing amounts of water in the solvent system. However, in solvents containing ≥ 75% water, the flowers turned brown in color suggesting extensive oxidation. Therefore, it was determined that 50:50% ethanol:water was the optimal solvent for the extraction of the range of phenolic compounds in elderflowers without excess oxidation. A recent study of the effect of organicsolvents on the extraction of phytochemicals from butterfly pea flowers also found that 50:50% ethanol:water had optimal extraction properties for the phenolic compounds in flowers. These results differ from a study on the extract of phenolic compounds from dry, powdered European elderflower, which found water to be the optimal extraction solvent, specifically at 100 °C for 30 mins, as compared to 80:20% ethanol:water or 80:20% methanol:water . 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 . Phenoliccompounds 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 , 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 elderflower levels range from about 0.200 to 0.900 mg g-1 fresh weight,6,115 though higher levels were found in elderflower 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 bio-active 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 elderflowers. For example, hydroponic vertical garden levels are reported as 1.65490 ± 0.10951 mg g-1 fresh weight,6 1.8877 ± 0.2691 mg g-1 fresh weight,114 between 15.70 and 23.90 mg g-1 in dried elderflower tea which varied with cultivar,119 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 elderflower 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 elderflowers 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,56 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 elderflowers of S. nigra ssp. cerulea . Two isomers of 5-caffeoylquinic acid in addition to 3- and 4-caffeoylquinic acid have been identified in elderflower 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.6 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 elderflower was identified as 5-hydroxypyrogallol hexoside . This compound has also been identified in the berries of this subspecies grown in the same locations.39 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 elderflower 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 elderflower 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 elderflower of S. nigra ssp. cerulea are comparable to the levels in the European flowers. However, 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.