The mechanism proposed was that the elderberry extract blocks viral glycoproteins which prevent the virus from attaching or entering cells to replicate, and increases expression of IL-6, IL-8, and TNF. Inflammatory modulating activity of elderberry and elder flower extracts have been investigated. Results showed that quercetin, rutin, and kaempferol are strong inhibitors of nitric oxide production, and metabolites from phenolic degradation including caffeic acid and 3,4-dihydroxyphenylacetic acid were also strong inhibitors without cytotoxicity. Only a few studies have been done on the bio-activity of S. nigra ssp. canadensis. In one, the fruit was evaluated for anticancer properties, which showed chemo-preventative activity by inducing quinone reductase and inhibiting cyclooxygenase-2, as well as inhibiting ornithine decarboxylase. These activities are attributed to flavonoids and lipophilic compounds. Another study evaluated two Canadian cultivars of this subspecies evaluated the antiproliferative efficacy of the fruit and flowers on glioma and brain endothelial cells and results showed that elderberry and elder flower extracts inhibited the proliferation of cells under normoxic and hypoxic conditions. The elderberry extracts performed the best and the bioactivities were attributed to the synergistic work of cyanidin 3-sambubioside-5-glucoside and rutin content of the berries, tower garden though the rutin concentration in the flowers still had beneficial effects.
Blue elderberry has only been evaluated as antioxidant activity using the ABTS assay, which indicated that this subspecies has 11.62 ± 0.38 mM Trolox kg-1 FW, roughly one third of S. nigra ssp. nigra evaluated in the same study, where all fruit samples were grown in Slovenia. Further work on elucidating the biological activity of this subspecies through in vivo assays and preferably clinical trials should be explored, especially using blue elderberry plants growing in North America to support its use in supplements. Elderflowers have also been evaluated for their biological activities. A review of antioxidant activity in S. nigra ssp. nigra flowers has recently been published 2 , including ABTS, DPPH, FRAP and CUPRAC assays, therefore it will not be re-summarized here. In general, the data showed that elder flower has higher levels of antioxidant activity compared to the elderberry. Similarly, elder flower extracts had higher nitric oxide inhibition compared to elderberry extracts in vitro. Elderflower is anti-diabetic by increasing insulin-dependent glucose uptake, diuretic, treat respiratory infections, antiviral. While phenolic compounds like flavonols are presumed to be the most active compounds, pectic polysaccharides are also bio-active in elderberry and elder flower, inducing complement fixing and macrophage stimulation. Flowers of the blue elderberry have been evaluated for their antioxidant activity using the ABTS assay, which showed they have 44.87 ± 0.54 mM Trolox kg-1 DW, significantly less than flowers of S. nigra ssp. nigra . Aqueous extracts of wild elder flowers of this subspecies were also found to have neuroprotective effects, especially related to Parkinson’s disease, by increasing the antioxidant response mediated by Nrf2 in cortical astrocytes and improving mitochondrial function in neurons. Unfortunately, that study did not include any growing information about the elder flowers or the concentration of the phenolic compounds in the extract, which would have helped other researchers replicate and expand on the results. While there have been promising studies on the impact of elderberry and elder flower extracts to combat illness and disease, more in vivo studies and clinical trials should be performed to better understand the mechanisms of the bio-activity as well as to determine which compounds are responsible for the bio-activity, particularly in the lesser-known subspecies canadensis and cerulea. This can better inform people involved with the cultivation of elderberry to select for varieties that have the compounds of interest. The market for herbal supplements has been growing in the recent decade and immune system-supporting supplements had a huge spike in sales during the COVID-19 pandemic. Elderberry products are a popular option of alternative medicine in hopes of improving and protecting health. Beverages are a popular use of the elderberry, including syrup or other tonics made by soaking the berries in water or alcohol. It can also be found as an ingredient in various kombuchas, juices, energy drinks, wine, and tea. Elderberry is typically mixed with a variety of other ingredients, including but not limited to ginger, honey, echinacea, and other spices. 12 More recent products using elderberry include gummies typically marketed as health supplements, lozenges, tablets, and powdered berries especially as part of a drink mix. Elderberries are also frequently used in jams and jellies. Pomace, the byproduct of juicing, has been studied for its benefits when incorporated into other products just as baked goods. Beyond its potential for bio-active products to benefit consumers, elderberry can also be used as a natural food dye due to the high concentration of anthocyanins , which can be used in place of artificial red or purple food dyes, particularly in acidic foods. Its application in edible films has recently been investigated, explored various biopolymers that could retain the phenolic compounds of elderberry in the film so that they can remain active to protect foods. Active edible films can be an effective solution to reduce plastic packaging and food waste due to spoilage. Cosmetic and skin care applications are also an area of interest, and current products on the market that include elderberry include lip color, toner, face mask, and Epsom salt.Future chapters will focus on evaluating the blue elderberry and elder flower for their composition. Herein, the data available on the other elderberry subspecies of interest are summarized to provide a basis of the expected composition as well as information to compare the subspecies for their composition. Elderberries have a high amount of water, at about 80%. The main sugars in elderberry are glucose and fructose, with some small amounts of sucrose. Sorbitol was also measured, which was very minor compared to the other three sugars and was seen in the highest concentrations in the wild elderberry. Citric acid is the main organic acid in elderberry, with malic acid the next highest acid. Small amounts of shikimic, tartaric, and fumaric acid have been measured in elderberry as well. Only data on European elderberry is available for microconstituents such as vitamins, minerals, fatty acids, and amino acids. Vitamins found in elderberry include various B vitamins, vitamin C, and vitamin E. The main minerals are magnesium, calcium, and potassium. Because studies of these micronutrients have only been performed on the European elderberry, it is important for further work to include other subspecies, including the American and blue elderberry so that better comparisons can be made. An important group of bioactive compounds found in fruit and vegetables is phenolic compounds, which consist of one or more phenolic groups . Types of phenolic compounds include phenolic acids and flavonoids; flavonoids can be further separated into groups such as anthocyanins, flavonols, flavan-3-ols, and flavones. Phenolic compounds may have some biological activity, although bioavailability can be very low. A common, stacking flower pot tower albeit imperfect, way to measure phenolic content of elderberries is using a colorimetric method like Folin-Ciocalteu which can measure a complex that forms between phenolic compounds and molybdenum-tungsten at 765 nm. Because this method measures all reducing agents in the matrix, reducing sugars and ascorbic acid will also react and increase the absorption thus inflating the total phenolic content . Standard curves are typically constructed using gallic acid, hence the units for TPC are gallic acid equivalents . TPC in European elderberries can vary greatly but reported values include 461 ± 121 49 and 683 ± 49. In American elderberry, TPC has been reported to be 390 ± 56 50, 593 ± 70. One study has included blue elderberry grown in Slovenia, which had a TPC of 416 ± 31. However, because of the imprecise nature of this assay, it is important to identify and measure the concentration of each phenolic compound present whenever possible, the results of which is explored in the following sections. Anthocyanins are water-soluble pigments in plants, and they give elderberries their bluepurple hue. Total monomeric anthocyanin content is typically measured using the pH differential method, which takes advantage of the change in light absorption of anthocyanins in solutions with different pH and the unit is typically cyanidin glucoside equivalents . 51 European TMA content of European elderberry can range from 170 ± 12 to 343 ± 11 mg CGE 100 g -1 FW. In American elderberry, the range of TMA content is 354 ± 59 to 595 ± 26 mg CGE 100 g -1 FW 52 and 106 ± 2 to 444 ± 14 mg CGE 100 g-1 FW. Analysis of the phenolic compounds via high performance liquid chromatography with UV-Visible light detection or with mass spectrometry have elucidated a variety of molecules present in the European elderberry. Anthocyanins, a type of flavonoid and popular for their red to blue pigments, are of high interest in elderberry. Most studies have found that cyanidin -based anthocyanins are the dominant type in European and American elderberry, including cyn 3-O-sambubioside -β-Dglucopyranoside) and cyn 3-O-glucoside. Cyn 3-sambubioside-5-glucoside and cyn 3,5- diglucoside are also commonly seen in the elderberry. The American elderberry has a more unique anthocyanin profile with high presence of acylated anthocyanins compared to the European elderberry, including cyn 3-O-coumaroyl-sambubioside-5-O-glucoside , cyn 3-Ocoumoaryl-sambubioside. These acylated anthocyanins may be more stable during processing, but the authors found that cyn 3-O-coumaroyl-sambubioside was the least stable anthocyanin during storage , whereas cyn 3-O-cou-sam-5- O-glu and cyn-3-O-sam-5-O-glu were more stable. 55 Another major type of phenolic compound in elderberry is flavonol glycosides, which include rutin , isorhamnetin 3-O-glucoside or 3-O-rutinoside, and kaempferol 3-O-rutinoside. Rutin has frequently seen to be the most concentrated flavonol in European elderberry, and often the most concentrated phenolic compound of any present. 8 S. nigra ssp. canadensis also contains higher levels of rutin than other flavonols. Other flavonol glycosides present in elderberry include kaempferol and isorhamnetin derivates, such as kaempferol-rutinoside, isorhamnetin-rutinoside, and isorhamnetin-glucoside. Phenolic acids are also present in high amounts in elderberry, including chlorogenic acid isomers , p-coumaric acid, sinnapic acid, cinnamic acid, and ferulic acid. Flavan-3-ols found in elderberry include -catechin, -epicatechin, and procyanidins. Parts of the elderberry plant are known for having toxic compounds called cyanogenic glycosides that, if consumed, can be dangerous due to the release of cyanide. The stems and leaves have the highest concentration of CNGs, followed by unripe berries and flowers, followed by ripe berries and cooked juices. The primary CNG in elderberry is sambunigrin, which is a diastereoisomer of the more commonly known CNG prunisin. Amygdalin is the next most common CNG, though it is not often measured. European elderberry levels of CNGs can vary greatly depending on the growing location, such that concentrations ranged from 0.08 concentrations ranged from 0.08 ± 0.01 to 0.77 ± 0.08 µg g-1 when fruit was evaluated from various altitudes in Slovenia. These concentrations are lower than those detected in elderberry juice, found to be 18.8 ± 4.3 mg kg-1 in raw juice and 10.6 ± 0.7 mg kg-1 in cooked elderberry juice, suggesting that thermal processing can reduce CNG levels in elderberry products. American elderberries have been evaluated for their concentrations of CNGs. These include amygdalin, sambunigrin , linamarin, and dhurrin. Specifically, the Ozone and Ozark genotypes were evaluated, giving better insight into how CNG concentrations may be impacted by plant genetics. While the total concentrations of the four CNGs in the two American elderberry genotypes were somewhat similar , the composition of which CNGs made up that total were quite different: Ozone elderberries had similar levels of amygdalin and sambunigrin while Ozark elderberries had much higher levels of amygdalin than sambunigrin . The flavor profile of elderberries is an important factor in the consumer sensory experience with elderberry products. Two of the most common compounds identified as drivers of elderberry aroma identified in multiple studies of the berries or elderberry juice are β-damascenone and dihydroedulan. Nonanol was also identified as a key volatile compound contributing to the characteristic elderberry aroma, while ethyl-9-decenoate was found to be important for the characteristic elderberry aroma by another study. While these volatile compounds can be key to the unique aroma, they are not typically the most concentrated compounds. Studies have found the most concentrated compounds to be linalyl acetate, linalool, phenylacetaldehyde, benzaldehyde , hexanal, 2- and 3-methyl-1-butanol, nonanal and benzaldehyde.